Pesticidal compositions

FIELD OF THE INVENTION

The invention relates to pesticidal compositions, uses, methods and kits for controlling pests, in particular those comprising or using a compound according to Formula I. Also described are genetically altered plants expressing genes that confer resistance to pests.

BACKGROUND OF THE INVENTION

Plants have evolved diverse strategies to cope with the biotic stress induced by herbivorous pests. Aside from the morphological defence strategies making up the constitutive defence response, physiological defence strategies possess both constitutive and induced properties and involve a series of molecular events and biochemical reactions that promote the accumulation of secondary metabolites to disrupt the feeding and digestion of harmful pests.

In particular, some of the most disruptive pests include weevils, aphids, moths and locusts. Sweet potato weevils (SPWs), Cylas formicarius (Fabricius), are one of the most economically significant pests (e.g. for sweet potato) and are listed as international quarantine pests; SPWs can reproduce multiple generations a year. Aphids are among the most destructive insect pests on cultivated plants in temperate regions. Spodoptera litura (tobacco cutworm) is a serious polyphagous pest in Asia, Oceania, and the Indian subcontinent. Locusts have been feared and revered throughout history - related to grasshoppers, these insects form enormous swarms that spread across regions, devouring crops and leaving serious agricultural damage in their wake.

The development of new pesticidal compositions, uses and methods for controlling pests is therefore of interest, particularly those with improved efficacy. There is also a need to modulate pest resistance in plants for agricultural applications and to develop plants that have increased resistance to pests, particularly for agriculturally and commercially important plants.

SUMMARY OF THE INVENTION In a first aspect of the present invention, there is provided a pesticidal composition, comprising a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof, and an agrochemically acceptable carrier:

<Formula l> wherein:

X ¹ is selected from C(R ² )(R ³ ) and C(=O);

X ² is selected from C(R ⁴ )(R ⁵ ) and C(=O);

R ¹ is selected from hydrogen and optionally substituted alkyl; and

R ² to R ⁵ are independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted alkenoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl.

Preferably, X ¹ is C(R ² )(R ³ ).

In a preferred aspect, X ² is C(R ⁴ )(R ⁵ ). In another preferred aspect, X ² is C(=O).

Preferably, R ¹ is hydrogen.

Preferably, R ² is selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl.

More preferably, R ² is hydrogen. Preferably, R ³ is selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl.

More preferably, R ³ is selected from hydroxy and optionally substituted alkenoyl, preferably hydroxy.

Preferably, R ⁴ is selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl.

More preferably, R ⁴ is hydrogen.

Preferably, R ⁵ is selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl.

More preferably, R ⁵ is selected from hydroxy and optionally substituted alkenoyl, preferably hydroxy.

Preferably, the optionally substituted alkenoyl has a structure according to Formula 1 :

<Formula 1> wherein:

R ^a is selected from hydrogen, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; and represents a connection point to the rest of the compound.

Preferably, R ^a is selected from optionally substituted alkyl and optionally substituted aryl.

More preferably, R ^a is optionally substituted aryl. Even more preferably, R ^a is hydroxyaryl, preferably hydroxyphenyl, more preferably monohydroxyphenyl, dihydroxyphenyl or trihydroxyphenyl, or even more preferably dihydroxyphenyl. Yet even more preferably, the optionally substituted alkenoyl has a structure according to Formula 2: wherein represents a connection point to the rest of the compound. Preferably, the compound has a structure according to Formula 11-1 or Formula 11-2:

<Formula 11-1 > <Formula ll-2> wherein X ² and R ¹ to R ⁵ are as defined herein.

Preferably, the compound has a structure according to Formula 111-1 or Formula HI-2:

<Formula lll-1> <Formula lll-2> wherein X ¹ and R ¹ to R ⁵ are as defined herein. Preferably, the compound has a structure according to any one of Formulae I V- 1 to IV- 4:

<Formula IV-3> <Formula IV-4> wherein R ¹ to R ⁵ are as defined herein.

Preferably, the compound is selected from:

More preferably, the compound is selected from:

Preferably, the composition is for controlling pests at a locus.

More preferably, the locus is a plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

Even more preferably, the plant is selected from sweet potato, tobacco, soybean, wheat and Arabidopsis.

Preferably, the pest is an insect, preferably wherein the insect is selected from the order Coleoptera, Hemiptera, Lepidoptera, Diptera or Orthoptera, more preferably wherein the insect is selected from weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura) and locusts.

In another aspect of the present invention, there is provided a use of a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof; or a pesticidal composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier; for controlling pests at a locus, wherein X ¹ , X ² and R ¹ to R ⁵ are as defined herein. More preferably, the locus is a plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

Even more preferably, the plant is selected from sweet potato, tobacco, soybean, wheat and Arabidopsis.

Preferably, the pest is an insect, preferably wherein the insect is selected from the order Coleoptera, Hemiptera, Lepidoptera, Diptera or Orthoptera, more preferably wherein the insect is selected from weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura) and locusts.

In another aspect of the present invention, there is provided a method of controlling pests at a locus, comprising applying a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof; or a pesticidal composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier; wherein X ¹ , X ² and R ¹ to R ⁵ are as defined herein.

More preferably, the locus is a plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

Even more preferably, the plant is selected from sweet potato, tobacco, soybean, wheat, and Arabidopsis.

Preferably, the pest is an insect, preferably wherein the insect is selected from the order Coleoptera, Hemiptera, Lepidoptera, Diptera or Orthoptera, more preferably wherein the insect is selected from weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura) and locusts.

In another aspect of the present invention, there is provided a kit comprising a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof; or a pesticidal composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier; and instructions for use of the compound or pesticidal composition for controlling pests at a locus, wherein X ¹ , X ² and R ¹ to R ⁵ are as defined herein.

More preferably, the locus is a plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

Even more preferably, the plant is selected from sweet potato, tobacco, soybean, wheat, and Arabidopsis.

Preferably, the pest is an insect, preferably wherein the insect is selected from the order Coleoptera, Hemiptera, Lepidoptera, Diptera or Orthoptera, more preferably wherein the insect is selected from weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura) and locusts.

In another aspect of the present invention, there is provided a genetically altered plant, part thereof or plant cell with increased expression and/or activity of a SPWR1 and or SPWR2 protein, preferably compared to a wild-type or control plant.

In one embodiment, the genetically altered plant, part thereof or plant cell comprises a nucleic acid construct comprising a nucleic acid sequence encoding a SPWR1 protein and/or a SPWR2 protein. Preferably, the SPWR1 nucleic acid encodes a SPWR1 protein as defined in SEQ ID NO: 3 or a functional variant or homologue thereof. Preferably, the SPWR2 nucleic acid encodes a protein as defined in SEQ ID NO: 4 or 7 or a functional variant or homologue thereof. In one embodiment the SPWR1 nucleic acid comprises SEQ ID NO: 1. In one embodiment, the SPWR2 nucleic acid comprises SEQ ID NO: 2 or 9. Preferably the SPWR1 and/or SPWR2 nucleic acid sequence are operably linked to at least one regulatory sequence.

In another embodiment, the genetically altered plant, part thereof or plant cell comprises at least one mutation in at least one gene or promoter encoding a SPWR1 and/or SPWR2 protein.

The mutation may be an insertion or a deletion or a substitution. In one embodiment, the mutation is in the coding region of SPWR1 and increases the transactivation activity of the SPWR1 protein.

In another embodiment, the mutation in the promoter of the SPWR2 gene and increases the expression of the SPWR2 protein.

In another aspect of the present invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding at least one of SPWR1 and SPWR2. Preferably, the SPWR1 nucleic acid encodes a SPWR1 protein as defined in SEQ ID NO: 3 or a functional variant or homologue thereof. Preferably, the SPWR2 nucleic acid encodes a protein as defined in SEQ ID NO: 4 or 7 or a functional variant or homologue thereof. In one embodiment the SPWR1 nucleic acid comprises SEQ ID NO: 1. In one embodiment, the SPWR2 nucleic acid comprises SEQ ID NO: 2 or 9. Preferably, the nucleic acid sequence encoding at least one of SPWR1 or SPWR2 is operably linked to one or more regulatory sequences. Preferably, the regulatory sequence may be a constitutive or strong promoter. The regulatory sequence is the SPWR2 ^N73 promoter as defined in SEQ ID NO: 5 or a functional variant or homologue thereof.

In another aspect of the present invention, there is provided a host cell comprising the nucleic acid construct as described herein.

In another aspect of the present invention, there is provided a genetically altered plant, plant part thereof, or plant cell, wherein said plant is characterised by one or more mutations in the plant genome, where the mutation is the insertion of at least one additional copy of a nucleic acid sequence encoding SPWR1 and/or SPWR2 such that said nucleic acid sequence(s) is operably linked to a regulatory sequence, and wherein preferably the mutation is introduced using targeted genome editing. Preferably, the SPWR1 nucleic acid comprises SEQ ID NO: 1 or a functional variant or homologue thereof. Preferably, the SPWR2 nucleic acid comprises SEQ ID NO: 2 or 9 or a functional variant or homologue thereof.

In another aspect of the present invention, there is provided a use of the nucleic acid construct as described herein to increase the pest resistance of a plant or part thereof. In another aspect of the present invention, there is provided a method of increasing the pest resistance of a plant, plant part thereof, or one or more plant cells, the method comprising introducing and expressing in the plant, part thereof or one or more plant cells the nucleic acid construct as described herein.

In another aspect of the invention, there is provided a method of increasing the pest resistance of a plant, plant part thereof, or one or more plant cells, the method comprising introducing one or more mutations in in at least one gene or promoter encoding a SPWR1 and/or SPWR2 protein.

In another aspect of the present invention, there is provided a method of producing a plant, plant part thereof, or one or more plant cells with increased pest resistance, the method comprising introducing and expressing in the plant, part thereof or one or more plant cells the nucleic acid construct as described herein.

In another aspect of the present invention, there is provided a method of producing a plant, plant part thereof, or one or more plant cells with increased pest resistance, the method comprising introducing one or more mutations in in at least one gene or promoter encoding a SPWR1 and/or SPWR2 protein.

DESCRIPTION OF THE FIGURES

The invention is further described in the following non-limiting figures.

Figure 1 shows natural resistance determination and genetic loci identification for SPWR in sweet potato germplasms. Figure 1(a) is an evaluation of SPWR in the storage tubers of G87, N73, and N28. The SPW adults newly hatched from the 14-day-SPW-pretreated tubers were counted. Data are presented as means ± standard deviations (SD) of nine biological replicates. b,c, Evaluation of SPWR in the leaves of G87, N73, and N28 at three weeks after cutting (3 WAC). Figure 1(b) presents the SPWR phenotype of the detached leaves of G87, N73, and N28 under controlled SPW-feeding conditions. Scale bar = 1 cm. Figure 1(c) shows the statistic SPW feeding area as described in Figure 1(b). Data are presented as means ±SD of nine three biological replicates. Asterisks indicate significant difference compared with G87 (Two-tailed Student’s /-test, * p < 0.05, ** p < 0.01 , *** p < 0.001). Figure 1(d) shows fine-mapping of the SPWR1 and SPWR2 loci. Schematic of fine-mapping for SPWR1 (left) and SPWR2 (right) by recombinants around the SPWR locus in the G87 x N73 F1 populations is shown. Markers used for the fine-mapped intervals (between vertical red lines) and the harbored genes are presented according to the Taizhong 6 reference genome.

Figure 2 shows the functional identification of SPWR1 and SPWR2 alleles. Figure 2(a) shows allelic variations of the SPWR1 candidate gene g35097 in G87 and N73. Four positional variations including three single nucleotide polymorphisms (SNPs, indicated by green digits) and a nucleotide insertion-deletion (indel) in the coding region of SPWR1 gene were shown. UTR, untranslated region. Figure 2(b) shows allelic variations of the SPWR2 gene g29415 promoter in G87 and N73. Double-slashes and green digits mark the 122 bp indel (indell) and ~45 bp indel (indel2) in the SPWR2 promoter region. Diamonds indicate the W-box elements, and green diamonds specifically indicate the W- box element within indell . Figures 2(c), (d) and (e) show analyses of SPWR in the SPWR1 transgenic sweet potatoes. Figure 2(c) presents the SPWR phenotype of the leaves from G87, the overexpression lines of SPWR1 alleles (OE-SPWR1 ^G87 and OE- SPWR1 ^N73 ), and the SPWR1 RNA interference lines (RNAi-SPWR1). Scale bar = 1 cm. Figures 2(d) and (e) show the statistic SPW feeding area as described in Figure 2(c). Figures 2(f), (g) and (h) show analyses of SPWR in the SPWR2 transgenic sweet potatoes. Figure 2(f) presents the SPWR phenotype of the leaves from G87, the overexpression lines of SPWR2 alleles (OE-SPWR2 ^G87 and OE-SPWR2 ^N73 ), and the SPWR2 RNA interference lines (RNAi-SPWR2). Scale bar = 1 cm. Figures 2(g) and (h) show the statistic SPW feeding area as described in Figure 2(f). Data are presented as means ± SD of at least nine biological replicates. Figure 2(i) shows the expression levels of SPWR1 (left) and SPWR2 (right) in the leaves of G87 and N73 plants (3 WAC) under control or SPW treatment. IbTUB was amplified as an internal control. Figure 2(j) shows transient expression assay of SPWRI transcription activation in Arabidopsis protoplasts. p35S:SPWR1 ^G87 , p35S:SPWR1 ^N73 , and p35S:SPWR1 ^G87 with different SNP substitutions of the N73 allele were used as effectors. The mini 35S promoters harboring tandem 4x W-box (W-box) or 4x mutated W-box (mW-box) were used as reporters. W- box is a classic WRKY transcriptional factor binding element. p35S:LUC was used as an internal control. Data are presented as means ± SD of three biological replicates. Asterisks indicate significant difference compared with G87, control, SPWR1 ^G87 , or as shown by line (Two-tailed Student’s /-test, * p < 0.05, ** p < 0.01 , and n.s indicates no significance). Multiple comparison was performed by One-way ANOVA, and statistically significant differences are indicated by different lower-case letters (p < 0.05) (e.g. “a”, b , c ).

Figure 3 shows that SPWR1 regulates the SPWR2 expression by specifically binding to the W-box element. Figure 3(a) shows the expression analysis of SPWR2 gene in the leaves of transgenic plants of OE-SPWR1 ^N73 , OE-SPWR1 ^G87 , and RNAi-SPWR1 (in G87) lines at 3 WAC. Data are presented as means ± SD of three biological replicates. IbTUB was amplified as an internal control. Asterisks indicate significant difference compared with G87 or as shown by line (Two-tailed Student’s /-test, * p < 0.05, ** p < 0.01 , *** p < 0.001). Figure 3(b) shows transient expression assay of SPWR1 regulating SPWR2 genes in Arabidopsis protoplasts. p35S:SPWR1 ^G87 and p35S:SPWR1 ^N73 were used as effectors. Reporter constructs used in the assay were present in upper panel. p35S:LUC was used as an internal control. Multiple comparison was performed by Oneway ANOVA, and statistically significant differences are indicated by different lower-case letters (p < 0.05). Figure 3(c) shows ChIP analysis of SPWR1 binding to the W-box- contained Indel region in SPWR2 promoter. Upper panel shows the 2-kb promoter region of SPWR2. P4 ^G87 _Z ^N73 indicates two pairs of primers used for analyzing DNA fragments of G87 allele and N74 allele, respectively. Lower panel shows ChlP-qPCR analysis. ChIP assay was performed using the leaves of OE-SPWR1 ^N73 and OE-SPWR1 ^G87 plants (3 WAC). FLAG antibody was used to precipitate the SPWR1-FLAG protein (OE- SPWR1 ^G87 ^ ⁷³ FLAG), and IgG acted as the negative control (OE-SPWR1 ^G87 ^ ⁷³ lgG\ Relative enrichment fold was calculated by normalizing the amount of each target DNA against that of the reference DNA of IbTUB, and then against the input DNA, respectively. Data represent mean ± SD of three biological replicates. Asterisks indicate significant difference compared with IgG (Two-tailed Student’s /-test, * p < 0.05, ** p < 0.01). Figure 3(d) shows an EMSA assay of SPWR1 DNA binding. The purified GST- SPWR2 ^G87 (lane 2) and GST-SPWR2 ^N73 (lanes 3-6) proteins were incubated with biotin- labeled W-box DNA (55 bp) in lanes 2-6. The un-labeled W-box and mutated W-box DNA (mW-box) were added as cold competitors in lane 5 (1 Ox), lane 6 (50x), and lane 4 (50x), respectively. Arrow heads indicate the position of shifted bands and free probes, respectively.

Figure 4 shows that SPWR1 and SPWR2 regulate the shikimate-quinate metabolic pathway. Figure 4(a) shows evaluation of applying natural metabolites on SPWR of sweet potato leaves. Crude extracts of metabolites were from the healthy N73 and G87 leaves with a SPW pre-treatment, and then applied to the detached G87 leaves for SPWR evaluation. The SPW feeding area was counted after SPW treatment. Data are presented as means ± SD of nine biological replicates. Figure 4(b) shows a heat map of the expression of typical metabolic genes in the shikimate-phenylalanine (Phe) pathway and quinate-chlorogenic acid (CGA) pathway in sweet potato. The data were processed from two replicates of transcriptomic data of G87, N73, and OE-SPWR1 ^N73 under control (01 and 02) and SPW treatment (T1 and T2), respectively. The scale bar indicates fold changes (Iog2 value). Figure 4(c) shows an overview of the shikimate- quinate metabolic pathway including the shikimate-Phe pathway and quinate-CGA pathway in plants. Red color indicates the synthetic route of quinate derivatives downstream of the SPWR2. Figure 4(d) shows the effect of SPW feeding on the content of shikimic acid, quinic acids, caffeic acid, and chlorogenic acid in the leaves of N73 and G87. Figure 4(e) shows the effect of SPW feeding on the content of quinic acid and chlorogenic acid in different transgenic lines. Data are presented as means ± SD with three biological replicates. Asterisks indicate significant difference compared with G87 or as shown by lines (Two-tailed Student’s /-test, * p < 0.05, ** p < 0.01 , *** p < 0.001 , and n.s indicates no significance).

Figure 5 shows the detection of inhibitory effect of quinate derivative compounds on SPWs and utility evaluation in sweet potato. Figures 5(a) and (b) show the effect of various metabolites treatment on the SPWR of sweet potato leaves. Figure 5(a) presents the SPWR phenotype of the sweet potato leaves with metabolites pretreatment. Prior to SPW feeding, the detached leaves from G87 plants (3 WAC) were applied evenly with 20 pM compounds. Scale bar = 1 cm. Figure 5(b) shows the statistic SPW feeding area as described in Figure 5(a). Data are presented as means ± SD of at least nine biological replicates. Figure 5(c) shows the moving distance statistic of SPWs fed by compounds. After SPWs ingested the compound droplet (10 mM), the distance and trajectory of SPW movement were recorded by time-lapse photography for 1 h and analyzed by After Effects software. Figure 5(d) shows analysis of trypsin activity in SPWs. Healthy SPWs were fed by compound solutions as described above and then collected for enzyme activity determination. Data are presented as means ± SD of at least three biological replicates. Figure 5(e) shows evaluation of yield and SPWR of leaves and tubers in selected F1 individuals under field conditions. Asterisks indicate significant difference compared with control or G87 (Two-tailed Student’s /-test, ** p < 0.01 , *** p < 0.001 , and n.s indicates no significance). Figure 5(f) shows a working model of resistance response regulation to SPW attack in sweet potato.

Figure 6 shows SPWR evaluation of the metabolites on sweet potato leaves. Figures 6(a) and (b) show the effect of applying various metabolites on the splitting detached sweet potato leaves. Figure 6(a) presents the SPWR phenotype of the splitting detached G87 leaves with compound pretreatment. Prior to SPW feeding, the detached leaves from G87 plants at 3 WAC were split into halves, which were applied evenly with 20 pM compounds or mock. Scale bar = 1 cm. Figure 6(b) shows the statistic SPW feeding area as described in Figure 6(a). Figures 6(c) and (d) show the effect of applying protocatechuic acid and gallic acid on the SPWR of sweet potato leaves. Data are presented as means ± SD with three biological replicates. Figure 6(c) presents the SPWR phenotype of the sweet potato leaves with compound pretreatment. Scale bar = 1 cm. Figure 6(d) shows the statistic SPW feeding area as described in Figure 6(c). Data are presented as means ± SD of at least nine biological replicates. Asterisks indicate significant difference compared with mock (Two-tailed Student’s /-test, * p< 0.05, ** p < 0.01 , and n.s indicates no significance).

Figure 7 shows SPWR evaluation of quinate derivative compounds on sweet potato leaves. Figure 7(a) shows the SPWR phenotype of the detached G87 leaves pretreated with different quinate derivative compounds. Dotted box indicates the 1 -hydroxyl of the quinate structure. Figure 7(b) shows the statistic SPW feeding area as described in Figure 7(a). Data are presented as means ± SD of nine biological replicates. Asterisks indicate significant difference compared with mock (Two-tailed Student’s /-test, ** p < 0.01 , *** p < 0.001 , and n.s indicates no significance).

Figure 8 shows SPWR evaluation of metabolites on inactivated sweet potato leaves. Figure 8(a) shows the SPWR phenotype of the inactivated sweet potato leaves. The detached G87 leaves were deactivated by 65°C water for 1 min and cooled down to room temperature for compound pretreatment and SPW treatment. Scale bar = 1 cm. Figure 8(b) shows the statistic SPW feeding area as described in Figure 8(a). Data are presented as means ± SD with three biological replicates. Asterisks indicate significant difference compared with mock (Two-tailed Student’s /-test, * < 0.05, ** <0.01 , and n.s indicates no significance). Figure 9 shows amylase and lipase activity analysis of SPWs. Figure 9(a) shows an analysis of amylase activity in SPWs. Figure 9(b) shows an analysis of lipase activity in SPWs. Healthy SPW adults were directly fed by compound solutions and then collected for enzyme activity determination. Data are presented as means ± SD of at least three biological replicates. Asterisks indicate significant difference compared with control (Two-tailed Student’s /-test, * p < 0.05, n.s indicates no significance).

Figure 10 shows SPWR and quality evaluation of quinic acid-treated G87 tubers under storage conditions. Figure 10(a) shows SPWR evaluation of the postharvest tubers. Prior to SPW feeding, the newly harvested G87 tubers were applied with 20 pM quinic acid. The number of wormholes were recorded and counted from the random equal surface area (40 cm ² ) of tubers by Imaged software. Figure 10(b) shows soluble sugar content of the tubers. Figure 10(c) shows starch content of the tubers. The newly harvested G87 tubers were treated with 20 pM quinic acid under dark conditions at 28°C for 7 d. Data are presented as means ± SD of three biological replicates. Asterisks indicate significant difference compared with control (Two-tailed Student’s /-test, ** p < 0.01 , and n.s indicates no significance).

Figure 11 shows the inhibitory effect of quinic acid on different agricultural pests. Figure 11(a) shows inhibitory activity of quinic acid on aphids {Myzus persicae) feeding. Quinic acid-treated Arabidopsis (Col) and tobacco (Nicotiana tabacum, NT) plants were used anti-insect analysis. The Y-axis represents the number of retained aphids per plant after 7 d of treatment. The experiment was performed with three independent replicates. Figure 11(b) shows inhibitory activity of quinic acid on Spodoptera litura. The Y-axis represents the feeding area of Spodoptera litura on soybean leaves. The experiment was performed with three independent replicates. Figure 11(c) shows a two-choice experiment of quinic acid on locust (Locusta migratoria). The Y-axis represents the consumption of insects in wheat seedlings. Data are shown with six biological replicates. Asterisks indicate significant difference compared with the mock (Student’s /-test, * p < 0.05, ** p < 0.01).

Figure 12 shows transcriptomic analysis of SPWR-related genes in G87 and N73. Figure 12(a) shows quality control of transcriptome of G87, N73, and OE-SPWR1 leaves with SPW treatment or not (control). The two biological replicates for each sample were marked with -1 and -2, respectively. Figure 12(b) shows GO analysis of differentially expressed genes (DEGs) involved in metabolic pathways in N73 compared with G87 under SPW treatment. Squares indicate the number of DEGs enriched in the pathway. The color represents the q-value. Red-boxes indicate shikimate pathway-related metabolisms. Figures 12(c) and (d) show transcriptomic expression of genes located at SPWR1 (Figure 12(c)) and SPWR2 (Figure 12(d)) find-mapping regions under control and SPW treatment. The values represent RPKM value of genes in transcriptome.

Figure 13 shows protein sequence alignment and phylogenetic analysis of SPWR1. Figure 13(a) shows protein sequence alignment of SPWR1 in G87, N73, and N28. Blue box indicates the conserved WRKY domain. Red boxes and phrases indicate the amino acid substitutions and indel site. Sequences were aligned with Clustal X. Figure 13(b) shows a phylogenetic tree showing the predicted SPWR1 homologs in different plant species. NCBI accessions: SPWR1 (Ipomoea batatas'), OM283291; WRKY-like (Ipomoea triloba), XP_031129806.1; WRKY-like (Ipomoea nil), XP_019170409.1; WRKY-like (Nicotiana attenuata), XP_019233122.1; WRKY-like (Nicotiana tabacum), XP_016453841.1; WRKY-like (Solanum tuberosum), XP_006352253.1 ; WRKY-like (Salvia splendens), XP_042022560.1 ; WRKY-like (Sesamum indicum), XP_020551475.1; WRKY-like (Olea europaea var sylvestris), XP_022886946.1 ; WRKY-like (Actinidia rufa), GFZ16403.1; WRKY-like (Theobroma cacao), XP_007040478.2.

Figure 14 shows protein and promoter sequence alignment and phylogenetic analysis of SPWR2. Figure 14(a) shows protein sequence alignment of SPWR1 in G87, N73, and N28. Blue box indicates the conserved DHQS domain. Red boxes and phrases indicate the amino acid substitutions. Figure 14(b) shows promoter sequence alignment of SPWR2 in G87, N73, and N28. Red boxes and phrases indicate the Indels. Blue box indicates the W-box element in Indell. Sequences were aligned with Clustal X. Figure 14(c) shows a phylogenetic tree showing the predicted SPWR2 homologs in different species. NCBI accessions: SPWR2 (Ipomoea batatas), OM283290; DHQS-like (Ipomoea nil), XP_031109196.1 ; DHQS-like (Nicotiana tabacum), XP_016464940.1; DHQS-like (Solanum tuberosum), XP_006340763.1 ; DHQS-like (Solanum lycopersicum), NP_001233863.1; DHQS-like (Olea europaea var. sylvestris), XP_022885746.1 ; DHQS-like (Coffea arabica), XP_027073560.1 ; DHQS-like (Arabidopsis thaliana), NP_001030791.1 ; DHQS-like (Oryza sativa), XP_015612522.1 ; DHQS-like /aroB (Escherichia coli), NP_417848.1. Figure 15 shows the generation and expression identification of the SPWR1 and SPWR2 transgenic sweet potatoes. Figures 15(a) and (b) show gene expression analysis of SPWR1 (Figure 15(a)) and SPWR2 (Figure 15(b)) in different transgenic lines. Figure 15(c) shows the phenotype of SPWR1 and SPWR2 transgenic lines at 3 WAC. Scale bar = 1 cm. Figures 15(d) and (e) show a functional analysis of the SPWR2 promoter in SPWR of transgenic sweet potatoes. Figure 15(d) presents the SPWR phenotype of the pSPWR2 ^N73 :SPWR1 ^G87 and pSPWR2 ^G87 :SPWR1 ^G87 leaves. Scale bar = 1 cm. Figure 15(e) shows the statistic SPW feeding area as described in Figure 15(d). Data are presented as means ± SD of nine biological replicates. Asterisks indicate significant difference (Two-tailed Student’s /-test, ** < 0.01 , *** < 0.001). Multiple comparison was performed by One-way ANOVA, and statistically significant differences are indicated by different lower-case letters (p < 0.05).

DETAILED DESCRIPTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, chemistry, molecular biology, plant biology, recombinant DNA technology and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature.

General Chemical Definitions

The term “hydroxyl” or “hydroxy” as used herein refers to the group -OH.

The term “halo” or “halogen” as used herein refers to any radical of fluorine, chlorine, bromine or iodine.

The term “cyano” as used herein refers to the group -CN. The term “alkyl” as used herein, by itself or as part of another group, refers to both straight and branched chain univalent radicals of up to twelve carbons. For example, an alkyl group may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Non-limiting examples of C1-C12 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, 3-pentyl, hexyl and octyl groups. Preferably, the term "alkyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain univalent radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms. An “optionally substituted alkyl” group may include the substituents as described below for the term “optionally substituted”.

The term “haloalkyl” as used herein, by itself or as part of another group, refers to both straight and branched chain radicals of up to twelve carbon atoms, comprising at least one halogen atom. For example, a haloalkyl group may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the term "haloalkyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, and comprising at least one halogen atom.

For example, a “haloalkyl” group may be a fluoroalkyl or perfluoroalkyl group.

Preferably, a “haloalkyl” group may be a Ci-Ce fluoroalkyl group, or a Ci-Ce perfluoroalkyl group.

Even more preferably, a “haloalkyl” group may be a C1-C4 fluoroalkyl group, or a C1-C4 perfluoroalkyl group. For example, a “haloalkyl” group may include difluoromethyl, trifluoromethyl or pentafluoroethyl.

The term “alkenyl” as used herein, by itself or as part of another group, refers to both straight and branched chain univalent radicals of up to twelve carbons, and which comprise at least one carbon-carbon double bond. For example, an alkenyl group may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the term "alkenyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain univalent radical comprising from two to eight carbon atoms, more preferably two to six carbon atoms and even more preferably two to four carbon atoms, and which comprise at least one carbon-carbon double bond. An “optionally substituted alkenyl” group may include the substituents as described below for the term “optionally substituted”.

The term “alkynyl” as used herein, by itself or as part of another group, refers to both straight and branched chain univalent radicals of up to twelve carbons, and which comprise at least one carbon-carbon triple bond. For example, an alkynyl group may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. For example, the term "alkynyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from two to eight carbon atoms, more preferably two to six carbon atoms and even more preferably two to four carbon atoms, and which comprise at least one carbon-carbon triple bond. An “optionally substituted alkynyl” group may include the substituents as described below for the term “optionally substituted”.

The term “cycloalkyl” as used herein refers to an alkyl group comprising a closed ring comprising from 3 to 8 carbon atoms, for example, 3 to 6 carbon atoms. For example, a cycloalkyl group may contain 3, 4, 5, 6, 7 or 8 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, (cyclohexyl)methyl, and (cyclohexyl)ethyl. An “optionally substituted cycloalkyl” group may include the substituents as described below for the term “optionally substituted”.

The term “cycloalkenyl” as used herein refers to a closed non-aromatic ring comprising from 3 to 8 carbon atoms, for example, 3 to 6 carbon atoms, and which contains at least one carbon-carbon double bond. For example, a cycloalkenyl group may contain 3, 4, 5, 6, 7 or 8 carbon atoms. Non-limiting examples of cycloalkenyl groups include 1- cyclohexenyl, 4-cyclohexenyl, 1 -cyclopentenyl, 2-cyclopentenyl. An “optionally substituted cycloalkenyl” group may include the substituents as described below for the term “optionally substituted”.

The term “heterocyclyl” as used herein refers to a saturated or partially saturated 3 to 7 membered monocyclic, or 7 to 10 membered bicyclic ring system, which consists of carbon atoms and from one to four heteroatoms independently selected from the group consisting of O, N, and S, wherein the nitrogen and sulfur heteroatoms may be optionally oxidised, the nitrogen may be optionally quaternised, and includes any bicyclic group in which any of the above-defined rings is fused to a benzene ring, and wherein the ring may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. Non-limiting examples of common saturated or partially saturated heterocyclyl groups include azetinyl, oxetanyl, tetrahydrofuranyl, pyranyl, piperidinyl, piperazinyl, pyrrolidinyl, imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinyl, pyrazolinyl, tetronoyl and tetramoyl groups. An “optionally substituted heterocyclyl” group may include the substituents as described below for the term “optionally substituted”.

The term “alkoxy” as used herein, by itself or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. For example, an alkoxy group may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the "alkoxy" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, appended to the parent molecular moiety through an oxygen atom. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy. An “optionally substituted alkoxy” group may include the substituents as described below for the term “optionally substituted”.

The term “haloalkoxy” as used herein, by itself or as part of another group, refers to both straight and branched chain radicals of up to twelve carbon atoms, comprising at least one halogen atom and being appended to the parent molecular moiety through an oxygen atom. For example, a haloalkoxy group may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the term "haloalkoxy" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, comprising at least one halogen atom and being appended to the parent molecular moiety through an oxygen atom.

For example, a “haloalkoxy” group may be a fluoroalkoxy or perfluoroalkoxy group.

Preferably, a “haloalkoxy” group may be a Ci-Ce fluoroalkoxy group, or a Ci-Ce perfluoroalkoxy group. Even more preferably, a “haloalkoxy” group may be a C1-C4 fluoroalkoxy group, or a Ci- 04 perfluoroalkoxy group. For example, a “haloalkoxy” group may include difluoromethoxy, trifluoromethoxy or pentafluoromethoxy.

The term “alkanoyl” as used herein by itself or as part of another group, refers to an alkyl group, as defined herein, and appended to the parent molecular moiety through an R ^x - C(=O)O- group via the oxygen atom, where R ^x represents the alkyl group. For example, an alkanoyl group may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 carbon atoms. Preferably, the term "alkanoyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from two to eight carbon atoms, more preferably two to six carbon atoms and even more preferably two to four carbon atoms, and being appended to the parent molecular moiety through an R ^X -C(=O)O- group via the oxygen atom, where R ^x represents the alkyl group. Non-limiting examples of alkanoyl groups include acetoxy, propionyloxy, butyryloxy and pentanoyloxy. An “optionally substituted alkanoyl” group may include the substituents as described below for the term “optionally substituted”.

The term “alkenoyl” as used herein by itself or as part of another group, refers to an alkenyl group, as defined herein, and appended to the parent molecular moiety through an R ^y -C(=O)O- group via the oxygen atom, where R ^y represents the alkenyl group. For example, an alkenoyl group may contain 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 carbon atoms. Preferably, the term "alkenoyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from three to eight carbon atoms, more preferably three to six carbon atoms and even more preferably three or four carbon atoms, and being appended to the parent molecular moiety through an R ^y -C(=O)O- group via the oxygen atom, where R ^y represents the alkenyl group. Non-limiting examples of alkenoyl groups include acryloyloxy, butenoyloxy and pentenoyloxy. An “optionally substituted alkenoyl” group may include the substituents as described below for the term “optionally substituted”.

The term “amino” or “amine” as used herein refers to the group -NH2.

The term “aryl” as used herein by itself or as part of another group refers to monocyclic, bicyclic or tricyclic aromatic univalent radicals containing from 6 to 14 carbon atoms in the ring. Common aryl groups include C6-C14 aryl, for example, Ce-Cw aryl. Non-limiting examples of C6-C14 aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups. An “optionally substituted aryl” group may include the substituents as described below for the term “optionally substituted”.

The term “heteroaryl” as used herein refers to aromatic groups having 5 to 14 ring atoms (for example, 5 to 10 ring atoms) and containing carbon atoms and 1 , 2 or 3 oxygen, nitrogen or sulfur heteroatoms. Examples of heteroaryl groups include thienyl (thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (furanyl), benzofuranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthiinyl, pyrrolyl, including without limitation 2H-pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl (pyridinyl), including without limitation 2-pyridyl, 3-pyridyl, and 4-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, tetrazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4/7-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl, p-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl, phenoxazinyl, pyrazolo[1 ,5-a]pyrimidinyl, including without limitation pyrazolo[1 ,5-a]pyrimidin-3-yl, 1 ,2-benzoisoxazol-3-yl, benzimidazolyl, 2-oxindolyl and 2- oxobenzimidazolyl. Where the heteroaryl group contains a nitrogen atom in a ring, such nitrogen atom may be in the form of an N-oxide, e.g., a pyridyl N-oxide, pyrazinyl N-oxide and pyrimidinyl N-oxide. An “optionally substituted heteroaryl” group may include the substituents as described below for the term “optionally substituted”.

As described herein, compounds may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogen atoms of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisaged by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. For example, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl, hydroxyaryl (e.g. monohydroxyphenyl, dihydroxyphenyl and trihydroxyphenyl), and heteroaryl. Preferably, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C ₁ -C ₆ alkoxy group, a C ₁ -C ₆ haloalkoxy group and a hydroxyphenyl group. More preferably, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from halogen, hydroxy, a C ₁ -C ₄ alkyl group, a C ₁ -C ₄ haloalkyl group, a C1-C4 alkoxy group, a C1-C4 haloalkoxy group, a monohydroxyphenyl group, a dihydroxyphenyl group and a trihydroxyphenyl group. Even more preferably, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from fluoro, chloro, hydroxy, a methyl group, a trifluoromethyl group, a methoxy group, trifluoromethoxy group and a dihydroxylphenyl group. As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined. The term “salt” as used herein refers to salts of the compounds as described herein that are derived from suitable inorganic and organic acids and bases. Examples of salts of a basic group include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, 13282014-1 borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p- toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (CI-C4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate.

Certain compounds of the present disclosure may exist in unsolvated forms as well as solvated forms, including hydrated forms. “Hydrate” refers to a complex formed by combination of water molecules with molecules or ions of the solute. “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent may be an organic compound, an inorganic compound, or a mixture of both. Solvate is meant to include hydrate. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetra hydrofuran, dimethylsulfoxide, and water. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

“Tautomer” means compounds produced by the phenomenon wherein a proton of one atom of a molecule shifts to another atom (See, Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms and Structures, Fourth Edition, John Wiley & Sons, pages 69-74 (1992)). The tautomers also refer to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. Examples include keto-enol tautomers, such as acetone/propen-2-ol, imine-enamine tautomers and the like, ring-chain tautomers, such as glucose/2,3,4,5,6-pentahydroxy- hexanal and the like, the tautomeric forms of heteroaryl groups containing a -N=C(H)- NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Where the compound contains, for example, a keto or oxime group or an aromatic moiety, tautomeric isomerism (‘tautomerism’) may occur. The compounds described herein may have one or more tautomers and therefore include various isomers. A skilled person would recognise that other tautomeric ring atom arrangements are possible. All such isomeric forms of these compounds are expressly included in the present disclosure.

“Isomers” mean compounds having identical molecular formulae but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. “Stereoisomer” and “stereoisomers” refer to compounds that exist in different stereoisomeric forms if they possess one or more asymmetric centres or a double bond with asymmetric substitution and, therefore, may be produced as individual stereoisomers or as mixtures. Stereoisomers include enantiomers and diastereomers. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric centre, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer may be characterised by the absolute configuration of its asymmetric centre and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarised light and designated as dextrorotatory or laevorotatory (i.e. , as (+) or (-)-isomers respectively). A chiral compound may exist as either individual enantiomers or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of ADVANCED ORGANIC CHEMISTRY, 6th edition J. March, John Wiley and Sons, New York, 2007) differ in the chirality of one or more stereocentres.

As used herein, bold bonds (e.g. - ) or hashed bonds (e.g. ) are intended to refer to relative stereochemistry, where the compound is not necessarily enantiomerically pure (e.g. are present as racemic mixtures). Enantiomerically (or substantially enantiomerically) pure compounds are represented with bold wedged bonds (e.g. —— ) or hashed wedged bonds (e.g. ).

The term “deuterated” as used herein alone or as part of a group, means substituted by deuterium atoms. The term “deuterated analogue” as used herein alone or as part of a group, means deuterium atoms substituted in place of hydrogen atoms. The deuterated analogue of the disclosure may be a fully or partially deuterium substituted derivative. In some embodiments, the deuterium substituted derivative of the disclosure holds a fully or partially deuterium substituted alkyl, aryl or heteroaryl group.

The disclosure also embraces isotopically-labelled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that may be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to ² H (deuterium, D), ³ H (tritium), ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ CI, and ¹²⁵ l. Unless otherwise stated, when a position is designated specifically as “H” or “hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition or its isotopes, such as deuterium (D) or tritium ( ³ H). Certain isotopically-labelled compounds of the present disclosure (e.g., those labelled with ³ H and ¹⁴ C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e. , ³ H) and carbon-14 (i.e., ¹⁴ C) and fluorine-18 (i.e., ¹⁸ F) isotopes are useful for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ² H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. I sotopically labelled compounds of the present disclosure may generally be prepared by following procedures analogous to those described in the Schemes and in the Examples herein below, by substituting an isotopically labelled reagent for a non-isotopically labelled reagent.

Compositions

In an embodiment of the present invention, provided herein is a pesticidal composition comprising a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof, and an agrochemically acceptable carrier:

<Formula l> wherein:

X ¹ is selected from C(R ² )(R ³ ) and C(=O);

X ² is selected from C(R ⁴ )(R ⁵ ) and C(=O);

R ¹ is selected from hydrogen and optionally substituted alkyl; and

R ² to R ⁵ are independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted alkenoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl.

It has been found that compounds according to Formula I have been found to have particularly potent broad-spectrum efficacy. This is particularly so against certain pests, for example against a number of insects including weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura., preferably larvae of tobacco cutworm) and locusts. As such, compounds and compositions as described herein have improved efficacy against pests compared to other compounds. Without wishing to be bound by theory, it is postulated that the presence of a free -OH group at the 1 -position of the ring in Formula I instead of other moieties (e.g. caffeoyl ester moieties) is responsible for high efficacy. In addition, it is postulated that the presence of a free -OH group at either the 3-position of the ring in Formula I instead of other moieties (e.g. caffeoyl ester moieties) is responsible for further improvement in efficacy. Accordingly, the combination of free -OH moieties at the 3-position and the 1 -position of the ring in Formula I leads to improved efficacy against pests. In an embodiment, X ¹ may be C(R ² )(R ³ ).

In an embodiment, X ² may be C(R ⁴ )(R ⁵ ). In an alternative embodiment, X ² may be C(=O).

In an embodiment, R ¹ may be hydrogen.

In an embodiment, R ² may be selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl. Preferably, R ² may be hydrogen.

In an embodiment, R ³ may be selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl. Preferably, R ³ may be selected from hydroxy and optionally substituted alkenoyl. More preferably, R ³ may be selected from hydroxy and optionally substituted C3-C8 alkenoyl. Even more preferably, R ³ may be selected from hydroxy and optionally substituted C3- Ce alkenoyl. Yet even more preferably, R ³ may be hydroxy.

In an embodiment, R ⁴ may be selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl. Preferably, R ⁴ may be hydrogen.

In an embodiment, R ⁵ may be selected from hydrogen, hydroxy, halogen, optionally substituted alkyl, optionally substituted alkanoyl and optionally substituted alkenoyl. Preferably, R ⁵ may be selected from hydroxy and optionally substituted alkenoyl. More preferably, R ⁵ may be selected from hydroxy and optionally substituted C3-C8 alkenoyl. Even more preferably, R ⁵ may be selected from hydroxy and optionally substituted C3- Ce alkenoyl. Yet even more preferably, R ⁵ may be hydroxy.

In an embodiment, the optionally substituted alkenoyl (e.g. as defined for R ² to R ⁵ ) may have a structure according to Formula 1 :

<Formula 1> wherein: R ^a is selected from hydrogen, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; and

•~vw represents a connection point to the rest of the compound.

In an embodiment, R ^a may be selected from optionally substituted alkyl and optionally substituted aryl. Preferably, R ^a may be selected from optionally substituted Ci-Cs alkyl and optionally substituted Ce-Cw aryl. More preferably, R ^a may be selected from optionally substituted Ci-Ce alkyl and optionally substituted Ce-Cw aryl.

In a preferred embodiment, R ^a may be optionally substituted aryl. Preferably, R ^a may be optionally substituted Ce-Cw aryl. More preferably, R ^a may be optionally substituted Ce- Cw aryl.

In a more preferred embodiment, R ^a may be hydroxyaryl. Preferably, R ^a may be hydroxyphenyl. More preferably, R ^a may be monohydroxyphenyl, dihydroxyphenyl or trihydroxyphenyl. Even more preferably, R ^a may be dihydroxyaryl.

In an embodiment, the optionally substituted alkenoyl (e.g. as defined for R ² to R ⁵ ) may have a structure according to Formula 2: wherein represents a connection point to the rest of the compound.

In an embodiment, the compound may have a structure according to Formula 11-1 or

Formula 11-2:

<Formula 11-1 > <Formula ll-2> wherein X ² and R ¹ to R ⁵ are as defined herein.

In an embodiment, the compound may have a structure according to Formula 111-1 or Formula III-2:

<Formula 111-1 > <Formula lll-2> wherein X ¹ and R ¹ to R ⁵ are as defined herein.

In an embodiment, the compound may have a structure according to any one of Formulae IV-1 to IV-4:

<Formula IV-3> <Formula IV-4> wherein R ¹ to R ⁵ are as defined herein.

In an embodiment, the compound may be selected from: In an embodiment, the pesticidal composition may be for controlling pests at a locus, wherein the locus is a plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

As used herein, the term “controlling pests” may refer to reducing or preventing pest infestation at a locus. For example, the term “controlling pests” may refer to reducing or preventing pests from feeding at the locus, for example from feeding from a plant, a part thereof or a seed thereof.

In an embodiment, the plant may be selected from sweet potato, tobacco, soybean, wheat and Arabidopsis.

The pest is not particularly limited as the compounds show broad-spectrum activity. However, in a preferred embodiment, the pest may be an insect. Preferably, the insect may be selected from the order Coleoptera, Hemiptera, Lepidoptera, Diptera or Orthoptera. More preferably, the insect may be selected from weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura., preferably larvae of tobacco cutworm) and locusts.

The pesticidal composition as described herein contains a compound as described herein and an agrochemically acceptable carrier.

A carrier in a pesticidal composition as described herein is any material with which the active ingredient is formulated to facilitate application to a surface, or to facilitate storage, transport or handling. A carrier may be a solid or a liquid, including a material which is normally gaseous but which has been compressed to form a liquid.

The pesticidal composition is formulated for agricultural use, in particular for pesticidal use. Agricultural use may encompass any use for cultivating plants, including horticultural uses, vertical farming uses and hydroponic uses. Preferably, the pesticidal composition is formulated for insecticidal use.

Any of the carriers normally used in formulating agrochemical (e.g. pesticidal, insecticidal) compositions may be used. For example, the agrochemically acceptable carrier may comprise a solid carrier and/or a liquid carrier. Suitable solid carriers include natural and synthetic clays and silicates, for example natural silicas such as diatomaceous earths; magnesium silicates, for example talcs; magnesium aluminium silicates, for example attapulgites and vermiculites; aluminium silicates, for example kaolinites, montmorillonites and micas; calcium carbonate; calcium sulfate; ammonium sulfate; synthetic hydrated silicon oxides and synthetic calcium or aluminium silicates; elements, for example carbon and sulfur; natural and synthetic resins, for example coumarone resins, polyvinyl chloride, and styrene polymers and copolymers; solid polychlorophenols; bitumen; waxes; and solid fertilisers, for example superphosphates.

Suitable liquid carriers include water; alcohols, for example isopropanol and glycols; ketones, for example acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone; ethers; aromatic or araliphatic hydrocarbons, for example benzene, toluene and xylene; petroleum fractions, for example kerosene and light mineral oils; chlorinated hydrocarbons, for example carbon tetrachloride, perchloroethylene and trichloroethane. Mixtures of different liquids are often suitable.

Agricultural compositions are often formulated and transported in a concentrated form which is subsequently diluted by the user before application. The presence of small amounts of a carrier which is a surface-active agent facilitates this process of dilution. Thus, at least one carrier in a composition as described herein may be a surface-active agent. For example, the composition may contain at least two carriers, at least one of which is a surface-active agent.

A surface-active agent may be an emulsifying agent, a dispersing agent or a wetting agent; it may be nonionic or ionic. Examples of suitable surface-active agents include the sodium or calcium salts of polyacrylic acids and lignin sulfonic acids; the condensation of fatty acids or aliphatic amines or amides containing at least 12 carbon atoms in the molecule with ethylene oxide and/or propylene oxide; fatty acid esters of glycerol, sorbitol, sucrose or pentaerythritol; condensates of these with ethylene oxide and/or propylene oxide; condensation products of fatty alcohol or alkyl phenols, for example p-octylphenol or p-octylcresol, with ethylene oxide and/or propylene oxide; sulfates or sulfonates of these condensation products; alkali or alkaline earth metal salts, preferably sodium salts, of sulfuric or sulfonic acid esters containing at least 10 carbon atoms in the molecule, for example sodium lauryl sulfate, sodium secondary alkyl sulfates, sodium salts of sulfonated castor oil, and sodium alkylaryl sulfonates such as dodecylbenzene sulfonate; and polymers of ethylene oxide and copolymers of ethylene oxide and propylene oxide.

The compositions as described herein may for example be formulated as wettable powders, dusts, granules, solutions, emulsifiable concentrates, emulsions, suspension concentrates and aerosols. Wettable powders usually contain 25, 50 or 75% w/w of active ingredient and usually contain in addition to solid inert carrier, 3-10% w/w of a dispersing agent and, where necessary, 0-10% w/w of stabiliser(s) and/or other additives such as penetrants or stickers. Dusts are usually formulated as a dust concentrate having a similar composition to that of a wettable powder but without a dispersant, and are diluted in the field with further solid carrier to give a composition usually containing 0.5-10% w/w of active ingredient. Granules are usually prepared to have a size between 10 and 100 BS mesh (1.676 - 0.152 mm), and may be manufactured by agglomeration or impregnation techniques. Generally, granules will contain 0.5-75% w/w active ingredient and 0-10% w/w of additives such as stabilisers, surfactants, slow release modifiers and binding agents. The so-called "dry flowable powders" consist of relatively small granules having a relatively high concentration of active ingredient. Of particular interest in current practice are the water-dispersible granular formulations. These are in the form of dry, hard granules that are essentially dust-free, and are resistant to attrition on handling, thus minimising the formation of dust. On contact with water, the granules readily disintegrate to form stable suspensions of the particles of active material. Such formulations contain 90% or more by weight of finely divided active material, 3-7% by weight of a blend of surfactants, which act as wetting, dispersing, suspending and binding agents, and 1-3% by weight of a finely divided carrier, which acts as a resuspending agent. Emulsifiable concentrates usually contain, in addition to a solvent and, when necessary, co-solvent, 10-50% w/v active ingredient, 2-20% w/v emulsifiers and 0-20% w/v of other additives such as stabilisers, penetrants and corrosion inhibitors. Suspension concentrates are usually compounded so as to obtain a stable, nonsedimenting flowable product and usually contain 10-75% w/w active ingredient, 0.5- 15% w/w of dispersing agents, 0.1-10% w/w of suspending agents such as protective colloids and thixotropic agents, 0-10% w/w of other additives such as defoamers, corrosion inhibitors, stabilisers, penetrants and stickers, and water or an organic liquid in which the active ingredient is substantially insoluble; certain organic solids or inorganic salts may be present dissolved in the formulation to assist in preventing sedimentation or as anti-freeze agents for water. Aerosol recipes are usually composed of the active ingredient, solvents, furthermore auxiliaries such as emulsifiers, perfume oils, if appropriate stabilisers, and, if required, propellants.

The specific choice of a carrier, if any, to be utilised in achieving the desired intimate admixture with the final product may be any carrier conventionally used in insect repellent formulations. The carrier, moreover, may also be one that will not be harmful to the environment. Accordingly, the carrier may be any one of a variety of commercially available organic and inorganic liquid, solid, or semi-solid carriers or carrier formulations usable in formulating insect repellent products. For example, the carrier may include silicone, petrolatum, lanolin or many of several other well-known carrier components.

Examples of organic liquid carriers include liquid aliphatic hydrocarbons (e.g., pentane, hexane, heptane, nonane, decane and their analogs) and liquid aromatic hydrocarbons. Examples of other liquid hydrocarbons include oils produced by the distillation of coal and the distillation of various types and grades of petrochemical stocks, including kerosene oils which are obtained by fractional distillation of petroleum.

Other petroleum oils include those generally referred to as agricultural spray oils (e.g., the so-called light and medium spray oils, consisting of middle fractions in the distillation of petroleum and which are only slightly volatile). Such oils are usually highly refined and may contain only minute amounts of unsaturated compounds. Such oils, moreover, are generally paraffin oils and accordingly may be emulsified with water and an emulsifier, diluted to lower concentrations, and used as sprays. Tall oils, obtained from sulfate digestion of wood pulp, like the paraffin oils, may similarly be used. Other organic liquid carriers may include liquid terpene hydrocarbons and terpene alcohols such as alphapinene, dipentene, terpineol, and the like.

Other carriers include silicone, petrolatum, lanolin, liquid hydrocarbons, agricultural spray oils, paraffin oil, tall oils, liquid terpene hydrocarbons and terpene alcohols, aliphatic and aromatic alcohols, esters, aldehydes, ketones, mineral oil, higher alcohols, finely divided organic and inorganic solid materials. In addition to the above-mentioned liquid hydrocarbons, the carrier may contain conventional emulsifying agents which may be used for causing the compounds to be dispersed in, and diluted with, water for end-use application.

Still other liquid carriers may include organic solvents such as aliphatic and aromatic alcohols, ethers, esters, aldehydes, and ketones. Aliphatic monohydric alcohols include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl alcohols. Suitable alcohols include glycols (such as ethylene and propylene glycol) and pinacols. Suitable polyhydroxy alcohols include glycerol, arabitol, erythritol, sorbitol, and the like. Suitable cyclic alcohols include cyclopentyl and cyclohexyl alcohols.

Conventional aromatic and aliphatic ethers, esters, aldehydes and ketones may be used as carriers, and occasionally are used in combination with the above-mentioned alcohols. Still other liquid carriers include relatively high-boiling petroleum products such as mineral oil and higher alcohols (such as cetyl alcohol). Additionally, conventional or so-called “stabilisers” (e.g., tert-butyl sulfinyl dimethyl dithiocarbonate) may be used in conjunction with, or as a component of, the carrier or carriers comprising the compositions as described herein.

Solid carriers which may be used in the compositions as described herein include finely divided organic and inorganic solid materials.

Suitable finely divided solid inorganic carriers include siliceous minerals such as synthetic and natural clay, bentonite, attapulgite, fuller's earth, diatomaceous earth, kaolin, mica, talc, finely divided quartz, and the like, as well as synthetically prepared siliceous materials, such as silica aerogels and precipitated and fume silicas. Examples of finely divided solid organic materials include cellulose, sawdust, synthetic organic polymers, and the like. Examples of semi-solid or colloidal carriers include waxy solids, gels (such as petroleum jelly), lanolin, and the like, and mixtures of well-known liquid and solid substances which may provide semi-solid carrier products, for providing effective repellency.

The compositions as described herein may be formulated and packaged so as to deliver the product in a variety of forms including as a solution, suspension, gel, film or spray, depending on the preferred method of use. The carrier may be an aerosol composition adapted to disperse the compounds into the atmosphere by means of a compressed gas.

In an embodiment, the compositions as described herein may comprise at least one additional active ingredient. For example, the additional active ingredient may be a herbicide or a pesticide.

Uses and Methods for Controlling Pests

In another embodiment of the present invention, provided herein is a use of a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof; or a pesticidal composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier; for controlling pests at a locus:

<Formula l> wherein X ¹ , X ² and R ¹ to R ⁵ are as defined herein.

In another embodiment of the present invention, provided herein is a method of controlling pests at a locus, comprising applying a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof; or a pesticidal composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier:

<Formula l> wherein X ¹ , X ² and R ¹ to R ⁵ are as defined herein.

As used herein, the term “locus” may refer to a plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

The pest is not particularly limited as the compounds show broad-spectrum activity. However, in a preferred embodiment, the pest may be an insect. Preferably, the insect may be selected from the order Coleoptera, Hemiptera, Lepidoptera, Diptera or Orthoptera. More preferably, the insect may be selected from weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura., preferably larvae of tobacco cutworm) and locusts.

The compound or composition as described herein may be applied at a concentration of about 0.1 pM to about 1 mM. In some cases, the compound or composition as described herein may be applied at a concentration of about 0.5 pM to about 500 pM, preferably about 1 pM to about 200 pM, more preferably about 5 pM to about 100 pM, even more preferably about 10 pM to about 50 pM (e.g. about 20 pM).

The compound or composition as described herein may be applied at least once, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. In cases where the compound or composition as described herein is applied at least twice, the length of time between applications may be between a day to a month (e.g. between 1 to 21 days, preferably between 2 to 14 days, more preferably between 5 to 14 days, and even more preferably around 7 days).

Compounds and compositions described herein can be administered to seeds or plants wherein the control of pests is desired.

As used herein, the term “seed” broadly encompasses plant propagating material such as, tubers cuttings, seedlings, seeds, and germinated or soaked seeds.

The compounds and compositions described herein can be administered to the environment of plants (e.g., soil) wherein the control of pests is desired. A compound or composition as described herein may be supplied to a plant exogenously. The compound or composition may be applied to the plant and/or the surrounding soil through sprays, drips, and/or other forms of liquid application.

The compounds described herein may penetrate the plant through the roots via the soil (systemic action); by drenching the locus of the plant with a liquid composition; or by applying the compounds in solid form to the soil, e.g. in granular form (soil application).

A compound or composition as described herein may be applied to a plant, including plant leaves, shoots, roots, or seeds. For example, compound or composition as described herein can be applied to a foliar surface of a plant.

As used herein, the term "foliar surface" broadly refers to any green portion of a plant having surface that may permit absorption, including petioles, stipules, stems, bracts, flowerbuds, and leaves. Absorption commonly occurs at the site of application on a foliar surface, but in some cases, the applied compound or composition may run down to other areas and be absorbed there.

Compounds or compositions described herein can be applied to the foliar surfaces of the plant using any conventional system for applying liquids to a foliar surface. For example, application by spraying will be found most convenient. Any conventional atomisation method can be used to generate spray droplets, including hydraulic nozzles and rotating disk atomisers. In other instances, alternative application techniques, including application by brush or by rope-wick, may be utilised.

A compound or composition as described herein can be directly applied to the soil surrounding the root zone of a plant. Soil applications may require at most or at least 0.1 to 5 kg per hectare of a compound as described herein on a broadcast basis (rate per treated area if broadcast or banded).

For example, a compound or composition as described herein may be applied directly to the base of the plants or to the soil immediately adjacent to the plants.

In some embodiments, a sufficient quantity of the compound or composition is applied such that it drains through the soil to the root area of the plants. Generally, application of a compound or composition as described herein may be performed using any method or apparatus known in the art, including but not limited to hand sprayer, mechanical sprinkler, or irrigation, including drip irrigation.

A compound or composition as provided herein can be applied to plants and/or soil using a drip irrigation technique. For example, the compound or composition may be applied through existing drip irrigation systems. For example, this procedure can be used in connection with cotton, strawberries, tomatoes, potatoes, vegetables, and ornamental plants.

In other embodiments, a compound or composition as described herein can be applied to plants and/or soil using a drench application. For example, the drench application technique may be used in connection with crop plants and turf grasses.

A compound or composition as described herein may be applied to soil after planting. Alternatively, a compound or composition as described herein may be applied to soil during planting, or may be applied to soil before planting.

For example, a compound or composition as described herein may be tilled into the soil or applied in furrow.

In crops grown in water, such as rice, solid granulates comprising the compounds or compositions described herein may be applied to the flooded field or locus of the crop plants to be treated.

The plant according to the various aspects of the invention described herein may be a monocot or a dicot plant. Non-limiting examples of monocot or dicot plants are given below.

A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, sweet potato, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species.

Also included are biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).

A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, yam, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana or a citrus, such as an orange.

Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.

Most preferred plants are sweet potato, tobacco, soybean, wheat, and Arabidopsis.

Kits

In another embodiment of the present invention, provided herein is a kit comprising a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof; or a pesticidal composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier; and instructions for use of the compound or pesticidal composition for controlling pests at a locus:

<Formula l> wherein X ¹ , X ² and R ¹ to R ⁵ are as defined herein.

In an embodiment, the locus may be a plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

In an embodiment, the plant may be selected from sweet potato, tobacco, soybean, wheat, and Arabidopsis.

The pest is not particularly limited as the compounds show broad-spectrum activity. However, in a preferred embodiment, the pest may be an insect. Preferably, the insect may be selected from the order Coleoptera, Hemiptera, Lepidoptera, Diptera or Orthoptera. More preferably, the insect may be selected from weevils (e.g. sweet potato weevils), aphids, moths (e.g. tobacco cutworm, Spodoptera litura., preferably larvae of tobacco cutworm) and locusts.

Plants with Increased Pest Resistance

In another embodiment of the invention, there is provided a genetically altered plant, plant part or plant cell, wherein the plant is characterised by increased expression and/or activity of at least one SPWR1 (Sweet potato weevils resistance 1) protein and/or at least one SPWR2 (Sweet potato weevils resistance 2) protein.

SPWR1 and SPWR2 activate quinate biosynthesis and contribute to resistance to pests, including weevils (e.g. sweet potato weevils). Evidence suggests that this resistance could also have broad-spectrum use, for example against other insects (e.g. aphids, moths and locusts). Of significant note, when expression of SPWR1 or SPWR2 is suppressed, pest resistance is reduced. However, overexpression of SPWR1 or SPWR2 leads to enhanced pest resistance as shown in Example 1.

Preferably, the expression and/or activity of the SPWR1 protein and/or the SPWR2 protein, compared to a wild-type or control plant. The term “increasing” means an increase in the levels of SPWR1 and/or SPWR2 expression and/or activity by up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a control plant or wild-type plants.

By “increasing the activity of SPWR1” is meant increasing the transactivation activity of SPWR1 compared to the level of transactivation activity in a wild-type or control plant. Such an increase may be up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a control plant or wild-type plants.

In one embodiment the genetically altered plant, plant part thereof or plant cell comprises and expresses a nucleic acid construct comprising a nucleic acid sequence encoding a SPWR1 and/or a SPWR2 nucleic acid. Preferably, the SPWR1 and SPWR2 nucleic acid is operably linked to a regulatory sequence, such as a strong constitutive promoter, such as but not limited to 35S. In one embodiment, the nucleic acid construct is stably incorporated into the plant genome.

In a further embodiment of the present invention, there is provided a host cell or genetically altered plant comprising the nucleic acid construct as described herein.

In an alternative embodiment, the genetically altered plant, part thereof or plant cell comprises at least one mutation in at least one gene encoding the SPWR1 protein and/or in at least one gene encoding the SPWR2 promoter and/or at least one gene encoding the SPWR2 protein. Preferably, the at least one mutation in the SPWR1 nucleic acid increases the transactivation activity of SPWR1 as described above. Preferably the at least one mutation in the promoter of SPWR2 increases the expression of the SPWR2 protein. Preferably the at least one mutation in the SPWR2 nucleic acid increases the DHQS-catalysing activity of SPWR2 as described above.

By “at least one mutation” is meant that where the SPWR1 and/or SPWR2 gene is present as more than one copy or homoeologue (with the same or slightly different sequence) there is at least one mutation in at least one gene. Preferably, all genes are mutated.

In one embodiment, the mutation may be an insertion and/or a deletion and/or a substitution.

In one embodiment, the mutation is at least one mutation in at least one SPWR1 gene. Preferably, said mutation leads to increased transactivation of the SPWR1 protein. Preferably, the mutation leads to at least one substitution in the SPWR1 protein. Preferably the mutation is in the coding region. Preferably, the mutation leads to a substitution next to the WRKY domain (i.e. N-terminal or C-terminal of the WRKY domain as shown in Figure 13) in the SPWR1 protein. More preferably, the mutation leads to an amino acid substitution from Asparagine to Threonine in SPWR1. Even more preferably, the mutation leads to an amino acid substitution from Asparagine to Threonine near the WRKY domain of the SPWR1 protein. Most preferably, the mutation is at position 563 of SEQ ID NO: 6 (or a corresponding position in a homologous sequence) causing an amino acid substitution from Asparagine to Threonine near the WRKY domain of the SPWR1 protein.

In another embodiment, the mutation is in the promoter region of the SPWR2 gene. Preferably, said mutation leads to increased expression of SPWR2. Preferably, the mutation is in the W-box element (comprising sequence: (T)TGAC(C/T) of the promoter region. Preferably, the mutation is an indel in the promoter region of the SPWR2 gene. More preferably, the mutation is a 100 to 200bp, more preferably a 120-130bp, even more preferably a 122 base pair indel in the promoter region of the SPWR2 gene.

In an embodiment, the mutation may be introduced by using mutagenesis or targeted genome editing.

Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. To achieve effective genome editing via introduction of site-specific DNA DSBs, four major classes of customisable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). Meganuclease, ZF, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.

A preferred genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art. In short, CRISPR is a microbial nuclease system involved in defence against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (l-lll) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non- repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.

One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted.

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.

The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5 ' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3.

Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.

Alternatively, more conventional mutagenesis methods can be used to introduce at least one mutation. These methods include both chemical and physical mutagenesis. Examples include T-DNA mutagenesis and targeting induced local lesions in genomes (TILLING). These techniques are well known in the art. Furthermore, rapid high- throughput screening procedures allow the analysis of amplification products for identifying a mutation, as compared to a corresponding non-mutagenised wild type plant. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3.

As used herein, the nucleic acid sequence of SPWR1 comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 1 or 8, preferably SEQ ID NO: 1 or a functional variant or homologue thereof. Preferably, the nucleic acid sequence encodes a SPWR1 polypeptide as defined in SEQ ID NO: 3 or 6, preferably SEQ ID NO: 6.

As used herein, the nucleic acid sequence of SPWR2 comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 2 or 9 or a functional variant or homologue thereof. Preferably, the nucleic acid sequence encodes a SPWR2 polypeptide as defined in SEQ ID NO: 4 or 7.

As used throughout, by “SPWR2 promoter” is meant a region extending at least or approx. About 1.2kbp upstream of the ATG codon of the SPWR2 ORF. In one embodiment, the sequence of the SPRW2 promoter comprises or consists of a nucleic acid sequence as defined in any one of SEQ ID NO: 5 or 10 or a functional variant or homologue thereof.. In one embodiment, the SPWR2 promoter may also include 5’ UTR sequences.

The term “variant” or “functional variant” as used throughout with reference to any of the sequences described herein refers to a variant gene sequence or part of the gene sequence (such as a fragment) which retains the biological function of the full non-variant sequence. For example, a functional variant of SPWR1 has transactivation activity, as described in the Examples. A functional variant of SPWR2 has a functional W-box and/or DHQS-catalysing activity. A functional variant also comprises a variant of the gene of interest, which has sequence alterations that do not affect function, for example in nonconserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence that results in the production of a different amino acid at a given site that does not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

As used in any aspect of the invention described throughout a “variant” or a “functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid or amino acid sequence. The sequence identity of a variant can be determined using any number of sequence alignment programs known in the art. As an example, Emboss Stretcher from the EMBL-EBI may be used: https://www.ebi.ac.uk/Tools/psa/emboss stretcher/ (using default parameters: pair output format, Matrix = BLOSUM62, Gap open = 1 , Gap extend = 1 for proteins; pair output format, Matrix = DNAfull, Gap open = 16, Gap extend = 4 for nucleotides).

Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

Suitable homologues can be identified by sequence comparisons and identification of conserved domains. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example when overexpressed in a plant.

In another embodiment of the present invention, provided herein is a genetically altered plant, plant part thereof, or plant cell, wherein said plant is characterised by one or more mutations in the plant genome, where the mutation is the insertion of at least one additional copy of a nucleic acid sequence encoding SPWR1 and/or SPWR2 nucleic acid such that said nucleic acid sequence(s) is operably linked to a regulatory sequence, and wherein preferably the mutation is introduced using targeted genome editing. Preferably SPWR1 comprises SEQ ID NO: 1 and SPWR2 comprises SEQ ID NO: 2 or a functional variant or homologue or fragment thereof.

In one embodiment of the present invention, provided herein is a use of a nucleic acid construct as described herein to increase the pest resistance of a plant or part thereof.

In one embodiment of the present invention, provided herein is a method of increasing the pest resistance of a plant, plant part thereof, or one or more plant cells, the method comprising introducing and expressing in the plant, part thereof or one or more plant cells a nucleic acid construct as described herein. In an alternative embodiment, there is provided a method of increasing the pest resistance of a plant, plant part thereof, or one or more plant cells, the method comprising introducing at least one mutation described above into at least one gene encoding a SPWR1 protein and/or at least one gene encoding a SPWR2 protein.

In one embodiment of the present invention, provided herein is a method of producing a genetically altered plant, plant part thereof, or one or more plant cells with increased pest resistance, the method comprising introducing and expressing in the plant, part thereof or one or more plant cells a nucleic acid construct as described herein. In an alternative embodiment, there is provided a method of genetically altered plant, plant part thereof, or one or more plant cells with increased pest resistance, the method comprising introducing at least one mutation described above into at least one gene encoding a SPWR1 protein and/or at least one gene encoding a SPWR2 protein.

The nucleic acid constructs of the invention may be introduced into a plant cell using any suitable method known to the skilled person (the term “introduced” can be used interchangeably with “transformation”).

Any of the nucleic acid constructs described herein, may be introduced into said plant through a process called transformation. The term "introduction" or "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.

To select transformed plants, the plant material obtained in the transformation is, in certain embodiments, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker.

In one embodiment of the invention, there is provided a genetically altered plant part of the invention, where the plant part is a seed or grain, Preferably the plant part comprises the nucleic acid construct of the invention stably incorporated into the plant genome or the plant part comprises one or more mutations in the SPWR1 gene and/or the SPWR2 promoter and/or the SPWR2 gene, as described above.

The plant according to the various aspects of the invention described herein may be a monocot or a dicot plant. Non-limiting examples of monocot or dicot plants are given above.

In an embodiment, the plant may be selected from sweet potato, tobacco, soybean, wheat, and Arabidopsis. A control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. Accordingly, in one embodiment, the control plant does not have altered expression of a SPWR1 and/or SPWR2 nucleic acid and/or altered activity of a SPWR1 and/or SPWR2 polypeptide, as described herein. In an alternative embodiment, the plant been genetically modified, as described above. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The invention is now described in the following non-limiting examples.

EXAMPLES

Materials and Methods

Sweet potato materials and growth conditions: The sweet potato materials were vegetatively propagated, and high SPWR germplasms were screened at the experimental station of College of Coastal Agriculture Sciences, Guangdong Ocean University, Zhanjiang, China (21 ° 35' N, 110° 35' E), during the autumn and winter seasons from 2015 to 2017. The selected sweet potato germplasms and F1 population were subsequently planted in the field or under laboratory conditions in South China Botanical Garden of the Chinese Academy of Sciences, Guangzhou, China (23° 16' N, 113° 23' E). All sweet potato plants were then planted in round pots filled with sandy peat soil and placed in a growth chamber (temperature: 28°C, photoperiod: 10 h light/14 h dark, light intensity: -200 pmol m ^-2 s ^-1 ) until tuberization (ca. 9-10 weeks).

For QTL mapping and gene cloning, the F1 population was generated by crossing G87$ x N73J. The recombinant individuals from the population were used for fine-mapping of the SPWR1 and SPWR2 loci.

Evaluation of the SPWR of the leaves and tubers of sweet potato: SPW (Cylas formicarius) were originated from the experimental station of the College of Coastal Agriculture Sciences, Guangdong Ocean University. The damaged storage roots with SPW eggs were placed into a shaded custom-made breeding cage (400 meshes per mm ² ); adults reproduced under 28°C and 60% humidity conditions and were subcultivated every 45 days in growth room.

To evaluate the level of SPWR of sweet potato leaves, SPW adults at the same developmental stage were placed into Petri dishes with 10 insects (five males and five females) in each dish and pre-starved for 24 h. The detached healthy leaves from plants at 3-4 weeks old after cutting (WAC), the petioles of which were wrapped by wet paper towels to prevent wilting, were then placed into the Petri dish for SPW feeding. The treatment without insects was used as the control. Each treatment was performed under 28°C and dark conditions for 4.5 h in parallel Petri dishes, which corresponded to at least nine biological replicates. The treated leaves were placed on an X-ray film viewer to obtain photos for measurements of the feeding areas. The feeding areas of leaves were calculated using Imaged software (http://imagej.nih.gov/ij/). Leaves were then quickly frozen with liquid nitrogen and used in subsequent experiments.

To evaluate the effect of metabolites on SPWR of sweet potato, the leaves or tubers were applied evenly with 20 pM compounds (close to the physiological concentration of quinate in N73 with SPW treatment) or mock solution and allowed to air dry prior to SPW treatment.

Tubers harvested from the same batch that were relatively uniform in size and had the skin intact were used in an experiment to determine the SPWR of sweet potato storage roots. The tubers were placed into a plastic box with small holes for air exchange. Each box contained one tuber and seven SPW adults (three males and four females). After 14 days of treatment under 28°C and dark conditions, the adults were removed, and the boxes were kept under the same conditions for an additional 30 days. The number of adults hatched from the SPW-treated tubers was recorded every day until no new adults appeared, and the newly hatched adults were immediately removed from the boxes to prevent additional eggs from being laid.

Sweet potato genome resequencing, SLAF-sequencing, and genetic map construction: The sequencing and analysis were performed by Biomarker Technologies, Inc. (Beijing, China) and the BMKCloud platform (http://en.biocloud.net/). At least 6 pg of genomic DNA was extracted from the sweet potato leaves of the parents (G87 and N73) and 240 F1 progeny to construct the sequencing libraries. Paired-end sequencing (2 x 125 bp) was performed on an Illumina HiSeq 2500 system per the manufacturer’s instructions (Illumina, Inc., San Diego, USA). The enzyme Rsal (New England Biolabs, Inc., NEB, USA) was used to digest the genomic DNA for SLAF library construction. SLAF marker identification and genotyping were performed following procedures described by Sun et al., PLoS ONE, 8, e58700 (2013). Sequences mapping and SNP/indel calling was performed using SOAP software (Li et al., Bioinformatics, 24, 713-714 (2008); Zhang et al., DNA Res, 22, 183-191 (2015)). The marker codes of the polymorphic SLAFs were analyzed according to population type, and there were five segregation types (ab x cd, ef x eg, hk x hk, Im x ||, and nn x np). Marker loci were partitioned into linkage groups (LGs) based on their locations in the Taizhong 6 genome (Yang et al., Nat. Plant., 3, 696-703 (2017)). The modified logarithms of odds (LOD) scores between markers were calculated to further confirm the robustness of the markers for each LG. A developed HighMap strategy was used to order the SLAF markers and correct genotyping errors within LGs (Liu et al., PLoS ONE, 9, e98855 (2014)).

QTL detection and map-based cloning. QTL analysis was performed by MapQTL Software (MapQTL® 6, Kyazma B.V., Wageningen, Netherlands (2009)) using the SPWR phenotypes of F1 population based on the above SLAF-sequencing data and genetic map. LOD values were determined based on a 1000-permutation test. QTL were significant when LOD values were greater than 3. Fine-mapping was conducted following the methods of a previous study (Royo, Plant Physiol., 177, 1234-1253 (2018)). The indel and SNP markers were developed in the regions of SPWR1 and SPWR2 loci from the resequencing data of N73 and G87. For SPWRI , the F1 recombinants were identified from the enlarged fine-mapping populations using 11 markers; for SPWR2, the F1 recombinants were identified from the enlarged fine-mapping populations using 15 markers. The SPWR of these recombinants, along with the genotypes, was used to delimit the genomic interval containing SPWR1 and SPWR2.

Plasmid construction and sweet potato transformation: For the p35S:SPWR1-FLAG (OE-SPWR1) and p35S:SPWR2-FLAG (OE-SPWR2) constructs, the coding regions of SPWR1 and SPWR1 without the stop codon were amplified from G87 or N73 and cloned into the modified binary vector p35S-FLAG with the pCambia1300 backbone (Novagen), respectively. For the p35S:SPWR1-RNAi (SPWR1-RNAi) and p35S:SPWR2-RNAi (SPWR2-RNAi) constructs, the two complementary specific sequences of SPWR1 or SPWR2 were amplified and cloned into the modified binary vector p35S-RNAi with pCambia1300 backbone (Novagen), respectively. For the pSPWR2N73:SPWR2G87 and pSPWR2G87:SPWR2G87 constructs, ~2.0 kb promoters of SPWR2 were amplified from N73 or G87 and cloned into p35S:SPWR2G87-FLAG to replace the 35S promoter region.

For sweet potato transformation, the single colony of Agrobacterium tumefaciens strain AGL1 with target binary plasmid was selected and further cultured on LB plates at 28°C for 2 days to activate the Agrobacteria. The Agrobacteria colonies were then collected from the plates through centrifugation at 5,000 g for 3 min. After washing with the wash buffer (10 mM MgCh, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), pH 5.6), Agrobacteria were diluted to ODO.5 by infiltration buffer (10 mM MgCh, 10 mM MES, 100 pM acetosyringone, pH 5.6). One mL of Agrobacteria mix liquid was injected into the fresh stem section (15 cm in length) of 4 WAC of sweet potato by syringe. The injected sweet potato stems were then transplanted in soil and selected by hygromycin resistance until tuberization. The vegetative buds sprouted from the transformed stem were then genotyped, phenotyped, and subjected to gene expression analysis.

Gene expression analysis: Total RNA was extracted using the Plant RNA Kit (Omega Bio-tek, Inc., USA) and reverse-transcribed using M-MLV reverse transcriptase (Promega, Inc., USA). Quantitative RT-PCR (qPCR) was performed in triplicate on a Roche LightCycler480 real-time system with the SYBR qPCR Mix (Q711-02, Vazyme, Inc., Nanjing, China) per the manufacturer’s instructions. The relative expression level was normalized to that of IbTUB (g9986) and calculated using the relative quantification method (2-AACt) (Liu et al., Nat. Commun., 7, 12768 (2016)).

Extraction and quantitative analysis of metabolites in sweet potato: The metabolite extraction and quantitative determination method were based on previously described methods with modifications (Liao et al., J. Adv. Res., 24, 2 (2020)). Approximately 100 mg of fresh sweet potato leaves was ground in liquid nitrogen and dissolved with 1 mL of cold methanol. The mixture was extracted for 30 min using an ultrasonic apparatus (DTC-27, 200W, 40kHz; Dingtai, China) and then centrifuged at 5,000 g and 4°C for 10 min. The supernatant was filtered through a 0.22-pm membrane and analyzed using UPLC-QTOF-MS (Waters Corp., Milford, MA, USA) with a UPLC AQUICTY HSS T3 column (100 mm x 2.1 mm, 1.8 pm; Waters) at 35 °C with a flow rate of 0.3 mL/min. Standards of shikimic acid, caffeic acid, chlorogenic acid, quinic acid, and quinate derivative metabolites were purchased from Sigma-Aldrich Co., Ltd., USA and Shanghai Yuanye Biotechnology Co., Ltd., China, and used to identify and quantify the metabolites in the sweet potato samples. Methanol and acetonitrile were purchased from Thermo Fisher Scientific, Inc., USA. Compound direct feeding experiment and analysis of digestive enzyme activity. SPW were placed in a 12-well flat-bottomed cell culture plate (one per well) for 24 h of prestarvation. Ten pl of compound solutions (20 mM) in the buffer (20 pM MES, pH 5.8) was added into the center of the culture wells. After the SPWs made contact with and ingested the compound droplet, the movements of SPWs were recorded by time-lapse photography for 1 h. The distance and trajectory of SPW movements were calculated using After Effects software (Adobe, www.adobe.com/products/aftereffects)

Trypsin, lipase, and amylase activity was measured using various metabolism assay kits (MAK290, MAK046, and MAK009, respectively, Sigma-Aldrich, USA). Healthy SPWs were directly fed by compound solution as described above and then rapidly frozen in liquid nitrogen with PBS buffer (pH 7.4) for full homogenization. After centrifugation, the supernatant was collected for testing. The activity of trypsin, lipase, and amylase in SPW supernatant was determined using fluorometric or colorimetric approaches per standard procedures, and the activity of these enzymes in the samples was determined using standard curves.

DHQS enzyme activity: The enzyme activity of SPWR2 protein was assayed following the method in a previous study (Tahara et al., Planta., 253, 3-18 (2021)). Recombinant 6xHis-SPWR2/DQHS proteins (10 pg mL ^-1 ) were incubated with substrate (10 pg mL ^-1 DHAP) and cofactor (40 pg mL’ ¹ NAD) in 50 mM Tris-HCI buffer (pH 7.5) at 25 °C for 2.5, 5, 10, and 40 min. The reaction was stopped by adding two volumes of malachite green or HCI, and the absorbance was measured at 620 nm for preliminary determination. For qualitative assay of 3-DHQ: reaction mixtures and 3-DHQ standard solution were freeze-dried, and 10 pL of 40 mg mL ^-1 methoxyamine hydrochloride dissolved in pyridine was added to methoxylate ketone groups at 30°C for 90 min. Then, acidic protons were trimethylsilylated in 90 pL MSTFA (containing 1 % TMCS) at 37°C for 30 min (N.B. 3-DHQ is the conventional name for the compound - the dehydro (i.e. oxo) unit is in the 5-position of Formula I as described herein, and the free alcohol is in the 3-position of Formula I as described herein). Samples were analyzed with a GC-MS system (Shimadzu Corporation, Japan), under the following conditions: injection volume, 1 pL; splitless mode; injection temperature, 250°C; column, DB-5 column (30 m x 0.25 mm i.d., 0.25 pm film thickness); the oven temperature program had an initial temperature of 60°C for 1 min, followed by ramping at 10 °C / min to 325 °C, which was then maintained for 10 min; the helium flow rate was 1 mL min ^-1 ; interface temperature, 290°C; ion source temperature, 250°C. 5-DHQ derivative was identified based on a comparison with authentic standards regarding their retention time and mass spectra. 5- DHQ derivative was recognized by characterized fragments m/z of 73, 147, and 507 at 18.36 min.

ChIP assay: ChIP assay was performed as previously described (Liu et al., Nat. Commun., 7, 12768 (2016)). Briefly, the leaves of the 4-week-old transgenic lines (OE- SPWR1) were treated with SPWfor4.5 h and harvested for fixation by 1 % formaldehyde. Nuclei were isolated and chromatin DNA were sonicated with an average size of about 250 bp. The solubilized chromatins were immunoprecipitated (IP) by Protein G Agarose (16-201 , Millipore) with anti-FLAG monoclonal antibody (F3165, Sigma) as IP or IgG (ab172730, Abcom) as control, and the co-IP DNA was recovered at 65°C for 6 h. The recovered DNA was then purified and analyzed by qPCR with SYBR qPCR Mix (Q711- 02, Vazyme). Relative enrichment fold was calculated by normalizing the target DNA fragment against that of a reference DNA IbTUB, and then against the respective input DNA.

Transient expression assay: For the pSPWR2:GUS construct, ~2.0 kb promoter of SPWR2 was amplified from G87 or N73 and cloned into the pHY107 vector harboring a GUS reporter (Liu et al., Nat. Commun., 7, 12768 (2016)). For the pW-box:GUS and pmW-box:GUS constructs, four tandem W-box or mutated W-box (mW-box) with 35S- mini promoter were synthesized and cloned into the pHY107 vector. The pSPWR2N73Aindel1 :GUS, pSPWR2N73Aindel2:GUS, and pSPWR2N73AW-box:GUS constructs were modified from pSPWR2N73:GUS by overlapping PCR, respectively. The different versions of p35S:SPWR1-FLAG constructs were used as effectors, and a construct containing p35S:LUC was used as an internal control to evaluate the protoplast transfection efficiency. Arabidopsis mesophyll protoplasts were prepared, transfected, and cultured as previously described (Liu et al., Nat. Commun., 7, 12768 (2016)). Relative GUS activity was calculated by normalizing the GUS activity against that of LUC, and the data presented were the averages of three biological replicates. RNA-sequencing analysis: The 3-week-old sweet potato G87 and N73 and transgenic lines (SPWR1-0E) were grown in growth chamber under control and SPW-feeding treatment for 4.5 h. Total RNA was extracted from the treated leaves by Plant RNA Kit (R6827, Omega). The sequencing library was constructed using Ultra RNA sample preparation kit (Illumina) and then sequenced using an Illumina HiSeq2500 according to the standard method (Illumina, Inc). Total reads were mapped to the Taizhong 6 genome (pasi3, https://ipomoea-genome.org/download_genome.html). The differentially expressed genes (DEG) were identified by the Cuffdiff with the criteria set (fold change > 1.5) and adjusted FDR (P-value < 0.05). Two independent biological replicates were used for the RNA-sequencing analysis. The gene expression patterns were graphically represented in a heat map by cluster analysis using TBtools software (Chen et al., TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant., 13, 1194-1202 (2020)). Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was conducted according to database (https://www.kegg.jp/kegg/pathway.html).

Statistical analyses: For SPWR evaluation, at least nine biological replicates were analyzed, and the individual numbers are showed in the figure legends. For biochemical and molecular biology analysis, at least three individual leaves were mixed in each sample with three biological replicates, and the numbers of replicates are presented in the figure legends. Standard deviations (SD) and p-value were calculated by Student’s t-test and ANOVA.

Aphid materials and testing methods: Aphids {Myzus persicae) were cultured at 28°C and 70% humidity. Five aphids with same size were inoculated with the leaves of healthy Arabidopsis and tobacco plants (four plants for each group). The control group was treated with either the mock (20 mM MES, pH 5.8), or the quinic acid solution (100 pM quinic acid, 20 mM MES, pH 5.8), every other day. After 7 d, the number of aphids on each plant was counted to evaluate the inhibitory effect. The rate of aphid inhibition (RAI) was calculated to reflect the degree of inhibition. RAI = (Insect number of control plants - Insect number of treated plants) / Insect number of control plants. Spodoptera litura materials and testing methods: Spodoptera litura eggs were incubated at 28°C and 70% humidity. The hatched larvae were fed with the leaves of soybean plants for 48 h and subsequently starved for 12 h prior to experiment. Three pieces of healthy leaves of similar size were applied with either the mock (20 mM MES, pH 5.8), or the quinic acid solution (20 pM quinic acid, 20 mM MES, pH 5.8). After dried, these leaves were placed with three larvaes each petri dish for 24 h. The feeding areas were then measured by an X-ray film viewer using Imaged software (http://imaqei.nih.gov/ii/), which was used to determine the insect inhibitory effect.

Locust materials and testing methods: Locust {Locusta migratoria) eggs were incubated at 28°C and 70% humidity. The second instar nymphs of Locusta migratoria were used in the experiment after 8 h of starvation. Ten-day-old wheat seedlings were applied with either the mock (20 mM MES, pH 5.8), or the quinic acid solution (100 pM quinic acid, 20 mM MES, pH 5.8). After dried, these seedlings were placed with 6 nymphs for 18 h for two-choice experiments. The changed weight of seedlings represents the consumption of locust nymphs that determines the rejection effect of insects.

Example 1 - Weevils

Genetic control of natural resistance to SPWs in sweet potato: To obtain high SPW- resistant materials, a total of 282 sweet potato germplasms were collected and propagated, including 208 germplasms collected from Guangdong, Guangxi, and Hainan Provinces in southern China, where sweet potatoes are often infested with SPW, and 74 cultivars from East Asia for regional comparison. Field screening of sweet potatoes for resistance to SPWs in three consecutive years (2015, 2016, and 2017) was conducted by evaluating damage to the tuber and stem base. One moderate-resistance and two high-resistance accessions from Zhanjiang, Yangjiang, and Maoming in Guangdong were identified from 208 germplasms, while all 74 cultivars from East Asia showed high susceptibility to SPWs. SPW treatment experiments revealed that the postharvest tubers of N73 and N28 were highly resistant to SPWs compared with the susceptible control Guangshu 87 (G87, a major domestic cultivar, China) (Figure 1a). A method for evaluating the level of SPWR was developed by accurately quantifying the leaf damage area under controlled SPW-feeding conditions. This method confirmed that N73 and N28 have high SPWR (Figures 1 b, 1c). Given that there is a strong correlation between the SPWR levels of leaves and tubers, and SPWs generally infest the leaf veins overground prior to proceeding to the tubers in the field, sweet potato leaves were used to determine SPWR.

Due to the self-incompatibility of sweet potato, the F1 population was generated by crossing G87 ($) with N73 (J) to dissect the genetic basis of SPWR. The SPWR levels of F1 progeny (n = 240) were evaluated by determining the feeding area of SPWs; SPWR levels were divided into ten grades and exhibited a skewed normal distribution pattern, which was similar (R ² = 0.8582) in the two independent experiments. Quantitative trait loci (QTL) analysis was next performed by genetic mapping (average distance 0.68 cM) based on high-depth whole-genome resequencing of parents and specific-locus amplified fragment (SLAF) analysis of F1 progeny. Two loci for SPWR were repeatedly identified in the same regions of sweet potato chromosomes 9 and 7 under two independent experimental conditions, suggesting that the SPWR trait in this group is controlled probably by two major genes. The segregation in F1 resistance also indicates the presence of heterozygous dominant genotypes for SPWR in parents, which stems from the high genetic heterozygosity caused by the self-incompatibility of sweet potato. In addition, due to the considerable morphologic difference of leaves between parents, a QTL analysis for leaf shape was also performed and identified an association locus non-overlapping with the SPWR locus on chromosome 7, supporting that the genetic map is reliable and available for QTL.

Map-based cloning and identification of SPWR1 and SPWR2 genes: The two major loci for SPWR, sweet potato weevils resistance 1 (SPWR1 , Chromosome 9) and SPWR2 (Chromosome 7), were mapped to relatively wide genomic regions (0.23 Mb and 0.7 Mb, respectively) in the F1 population based on the Taizhong 6 genome (Yang et al., Nat. Plant., 3, 696-703 (2017)) (Figure 1 d). To identify the genes controlling these loci, a larger G87 x N73 F1 hybrid population (n = 856) was further generated and individual recombinants were detected, in which only a SPWR locus was segregated and another one remained consistent. Fine-mapping analysis narrowed the SPWR1 locus down to a 0.14-Mb region between markers SNP-7.326 and SNP-7.462, which contains 25 predicted genes (Figure 1d). These genes were next screened through sequence comparison and transcriptomic analysis in plants subjected to SPW treatment (Figure 12). Only the gene g35097, which was predicted to encode a conserved WRKY protein, was induced by SPW feeding and possessed nonsynonymous variations (three SNPs and one indel ‘CAC’) in the coding region between parents (Figure 2a, Figure 13a and Figure 12c); this gene was thus considered a candidate gene for SPWR1. The SPWR2 locus was fine-mapped to a 0.07-Mb region containing 16 genes between the markers indel-30.23 and indel-30.30 in the F1 population (Figure 1d). The gene g29415, which encoded a conserved dehydroquinate synthase (DHQS), contained five nonsynonymous SNPs with amino acid changes and two indels in the proximal promoter region between parents (Figure 2b and Figures 14a, 14b), and its expression was induced by SPW feeding (Figure 12d); this gene was thus considered a candidate gene for SPWR2. Further comparative analyses revealed that the genotypes of SPWR1 and SPWR2 are homozygous in N73, but heterozygous in G87 (Figures 2a, 2b), suggesting that the high SPWR in N73 may be conferred by homozygous SPWR1/2. The SPWR allele in N73 was designated as the N73 allele (SPWRN73), and the only allele in G87 as the G87 allele (SPWRG87).

To verify the function of the two candidate genes, the overexpression (OE) and RNA interference (RNAi) transgenic lines were generated in a G87 background using the CaMV35S promoter-driving coding sequence (CDS) of SPWRN73 or SPWRG87 and RNAi target sequence, respectively (Figures 15a-15c). Compared with the G87 control plants, overexpression of the SPWR1G87 and SPWR1 N73 alleles significantly enhanced the resistance to SPWs, and RNAi-SPWR1 showed higher susceptibility to SPWs (Figures 2c-2e), indicating that the SPWR1 gene (g35097) contributes to the SPWR in sweet potato. OE-SPWR1 N73 showed markedly higher SPWR than OE- SPWR1G87; however, the expression level of SPWR1 was similar in these two transformants and in SPW-treated G87 and N73 (Figure 2i and Figure 15a), suggesting that a change in the coding region of the N73 allele of SPWR1 confers high SPWR. The SPWR1 gene encodes a putative WRKY transcription factor, and orthologs of this gene exist in several plant species (Figure 13b). The functional SNPs/indels of SPWR1 were identified by transient expression assays. Rather than the other variations, SNP2563, which causes an amino acid substitution from asparagine to threonine near the WRKY domain in the N73 allele compared with G87, is an essential site that greatly enhances the transactivation activity of the SPWR1 protein (Figures 2a, 2j and Figure 13a). The SPWR response was enhanced or weakened in the transformants of OE-SPWR2 and RNAi-SPWR2, respectively, suggesting that the SPWR2 gene (g29415) mediates the defense response to SPWs (Figures 2f-2h and Figures 15b, 15c). Notably, there was no visible difference in SPWR between OE-SPWR2N73 and OE-SPWR2G87 (Figures 2f, 2g). This observation suggests that the SNPs in the SPWR2 CDS are unlikely the main cause of the changes in SPWR between G87 and N73. Phylogenetic analysis showed that the SPWR2 gene encodes a conserved DHQS protein that acts as a key enzyme catalyzing the conversion of 3-deoxy-D-arabino-heptulosonate-7- phosphate (DAHP) to 3-dehydroquinate (3-DHQ; also known as (1 R,3R,4S)-1 ,3,4- trihydroxy-5-oxocyclohexane-1 -carboxylic acid) in prokaryotes and some eukaryotes (Figure 14c). The enzyme activity assay verified that SPWR2 possesses DHQS- catalyzing activity. No difference in DHQS activity was observed between proteins of the N73 and G87 alleles. SPWR2 expression was significantly higher in N73 than in G87 (Figure 2i). It was then hypothesized that the indels in the SPWR2 promoter region, rather than the SNPs in the CDS, are the cause of differential expression and functional variation in the SPWR2 locus between G87 and N73 (Figure 2b and Figure 14b). Consistent with this hypothesis, the stable transgenic line harboring the SPWR2G87 CDS driven by the native SPWR2N73 promoter (pSPWR2N73:SPWR2G87) in a G87 background showed markedly elevated SPWR2 expression and high SPWR compared with G87 and pSPWR2G87:SPWR2G87 (Figures 15d, 15e). These results indicate that SPWR2 is a functional DHQS gene that increases SPWR in sweet potato in a promoterdependent manner.

Sequence comparative analyses showed that the genotypes of SPWR1 and SPWR2 in N73 are similar to those in N28 (another high SPWR germplasm) (Figures 13a and 14b), suggesting that these two accessions may share a similar resistance regulation in SPWR.

SPWR1 transactivates SPWR2 by specifically binding to the W-box element: Overexpression or RNAi of SPWR1 markedly altered the expression of SPWR2 in sweet potato, suggesting that SPWR1 transcriptionally regulated the SPWR2 gene (Figure 3a and Figure 15a). This observation, coupled with the presence of a W-box, a putative WRKY transcriptional factor binding element, within the 122-bp indell of the SPWR2N73 promoter (Figure 2b and Figure 14b), prompted us to examine the effect of SPWR1 on SPWR2 transcription. The transient expression assay showed that both SPWR1G87 and SPWR1 N73 induced the GUS expression in pSPWR2N73:GUS, but not that in pSPWR2G87:GUS, and SPWR1 N73 had a markedly higher activity (Figure 3b). The induced GUS activity of pSPWR2N73:GUS was abolished by the deletion of indell rather than the addition of G87 indel2 in the SPWR2N73 promoter (Figure 3b). Furthermore, W-box mutation (pSPWR2N73AW-box:GUS) markedly impaired the expression of pSPWR2N73:GUS (Figure 3b). It is indicated that the SPWR1 protein might activate pSPWR2 transcription by binding to the W-box element contained in the indell of the N73-derived promoter. Next, a chromatin immunoprecipitation (ChIP) assay was performed using OE-SPWR1 N73 and OE-SPWR1G87 transgenic lines to study this regulation in vivo (Figure 3c). The SPWR1 N73 protein was highly associated with the indell region (P4) of the SPWR2N73 promoter, and the enrichment of SPWR1G87 in the SPWR2N73 promoter was far lower than that of SPWR1 N73; however, neither SPWR1G87 nor SPWR1 N73 bound to the SPWR2G87 promoter (Figure 3c). Activation of SPWR2 transcription was higher in OE-SPWR1 N73 than in OE-SPWR1G87 (Figure 3a), which suggests that the W-box in the SPWR2N73 promoter is essential for the SPWR1 N73-mediated activation of SPWR2. An electrophoretic mobility shift assay (EMSA) confirmed that SPWR1 can physically bind to the specific W-box element in the SPWR2N73 promoter but not the mutated W-box (Figure 3d). Overall, these findings indicate that SPWR1 directly transactivates SPWR2 expression by binding to the W-box element in a variation-dependent manner.

The SPWR1-SPWR2 regulatory module activates the shikimate-quinate pathway: Substances conferring plant resistance to herbivorous insects include both peptides and metabolites. To characterize the biochemical nature of the resistant factor involved in SPWR, the metabolites were extracted from SPW-fed leaves of N73 and G87. The application of crude extract of metabolites from N73 leaves significantly enhanced the SPWR of G87 leaves compared with the metabolites extracted from G87 leaves (Figure 4a), indicating that the endogenous chemical substances affect the feeding behavior of SPW. Because SPWR2 encodes a DHQS enzyme that catalyzes the early step in the shikimate pathway, the main genes involved in this pathway were analyzed. The expression of these genes was induced by SPW treatment, according to the two replicates in the transcriptomic analysis (Figure 12a), and it was positively correlated with the level of SPWR in G87/N73 and OE-SPWR1 (Figures 4b, 4c). Furthermore, Gene Ontology analysis revealed that many of the genes with up-regulated expression in response to SPW feeding in N73 were involved in shikimate pathway-related phenylpropanoid and phenylalanine metabolism (Figure 12b). The content of diverse derivative metabolites downstream of the SPWR2 biochemical reaction, such as shikimic acid, quinic acid, caffeic acid, and chlorogenic acid, was also highly correlated with the expression of SPWR1 and SPWR2 and the SPWR levels in G87/N73 and transformants (Figures 4d, 4e). These analyses suggest that the shikimate-quinate pathway, activated by the SPWR1-SPWR2 module, might be involved in natural resistance to SPWs in sweet potato.

The presence of 3-hydroxyl and 1 -hydroxyl groups lead to improved SPWR: Several typical metabolites in the shikimate-quinate pathway were next selected and their effects on SPWR were evaluated by exogenous application (Figure 4c). Compared with the mock, treatments of shikimic acid, quinic acid, or chlorogenic acid significantly reduced the area damaged by SPWs on the leaves and tubers of G87. Quinic acid had the highest anti-SPW activity (Figures 5a, 5b), and in particular more than chlorogenic acid. Without wishing to be bound by theory, it was postulated that this is due to the presence of a free alcohol at the 3-position (or 5-position), rather than an ester moiety. By contrast, the effects of caffeic acid and p-coumaric acid on SPWR were negligible (Figures 5a, 5b). A similar result was also obtained using the splitting detached leaf method, in which individual differences between leaves are absent (Figures 6a, 6b). Other metabolites such as protocatechuic acid and gallic acid do not affect SPWR (Figures 6c, 6d). These results indicate that the quinate derivative metabolites from the shikimate-quinate pathway confer resistance to SPW. Next, chlorogenic acid (3- caffeoylquinic acid), a stable chemical with a low rate of in vivo chemical conversion, with its single caffeoyl analogs (1-caffeoylquinic acid, 5-caffeoylquinic acid, and 4- caffeoylquinic acid) and double caffeoyl analogs (1 ,3-dicaffeoylquinic acid, 3,4- dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid) were compared. All chemicals with a 1 -hydroxyl in the quinate structure enhanced the SPWR of sweet potato leaves, whereas the application of 1-caffeoylquinic acid or 1 ,3- dicaffeoylquinic acid which lack a 1 -hydroxyl in the quinate structure had no effect on SPWR (Figures 7a, 7b), suggesting that the 1 -hydroxyl group in quinate derivatives is also responsible for the high SPWR. The inhibitory effect of compounds by directly feeding quinate derivative metabolites to SPWs to eliminate the effect of in vivo chemical conversion was also examined. Consistent with the above observations, quinic acid (and chlorogenic acid), but not 1- caffeoylquinic acid, affected the behavior and movement of SPWs (Figure 5c). Exogenous application on heated leaves (after inactivating plant endogenous enzymes to eliminate the possibility of chemical conversion) further confirmed that these functional metabolites have direct activity against SPWs (Figure 8). Enzyme analysis demonstrated that ingestion of quinic acid and chlorogenic acid significantly repressed the digestive enzyme activity of SPWs, especially the trypsin activity in the gut of SPWs; however, 1-caffeoylquinic acid did not have this effect (Figure 5d and Figure 9). Overall, these observations suggest that quinic acid and quinate derivative metabolites confer natural resistance to SPW.

The evaluation of utility of SPWR genes and quinate metabolites application in sweet potato: Among F1 progeny of N73 (J) and G87 ($), the lines (#167 and #170) containing the functional alleles SPWR1 N73 and SPWR2N73 show high SPW resistance, and their morphological characters were similar to the major cultivar G87. The agricultural traits of these lines were further studied. The quality and yield of these lines were similar to or higher than G87 (Figure 5e), indicating that the SPWR1/2 N73 alleles would be useful for breeding sweet potato varieties with SPWR. The anti-SPW chemical effect during sweet potato postharvest storage was also tested. Spraying a low concentration of quinic acid (20 pmol L' ¹ ) on postharvest sweet potato tubers can protect them from attack by SPWs without compromising the quality (Figure 10). Overall, the findings indicate that the SPWR1-SPWR2 regulatory module could be used to aid the breeding of sweet potato varieties with SPWR. Because of their low cost and low toxicity to mammals (Hue and Low, 2015), quinic acid and its derivatives could be used as agricultural additives for eco-friendly SPW management.

Discussion: SPWs pose a major threat to sweet potato protection globally and induce substantial economic losses annually. However, SPWR germplasms, and the regulatory mechanism underlying SPWR, remain poorly studied. To cope with biotic stress, plants generally recognize invasion signals and produce resistance factors, such as efficient metabolites, to mount a defense response. Two natural SPW-resistant alleles, SPWR1 and SPWR2, were identified using forward genetic approaches that play important regulatory roles in the defense response to SPWs in sweet potato. Under SPW attack, SPWR1 activity is induced and directly promotes the expression of SPWR2; it then subsequently activates the shikimate-quinate pathway. Accumulation of quinate derivative metabolites then confers natural resistance to SPWs (Figure 5f). The work clarifies the regulation of SPW resistance from the genetic level to the metabolic level in sweet potato, which not only aids the understanding of the molecular and biochemical mechanisms of SPWR but also provides information that could be used to promote the breeding of sweet potato varieties with SPWR.

Sweet potato germplasms with SPWR have not been widely used because of the lack of an effective approach for identifying resistant materials and resistance response regulations. Sweet potato germplasms from south China were collected and screened, where infestations of SPW are widespread, and three germplasms with SPWR were selected through 3-year multi-plot field experiments. It was found that N73 and N28, the two germplasms showing the highest SPWR, have the similar haplotypes of SPWR genes and identical resistance mechanisms against SPWs, while the other germplasms do not show obvious resistance. N73 is a common landrace cultivated in Yangjiang, Guangdong. The quality and yield of sweet potato were not affected in selected G87 x N73 F1 individuals that had strong SPW-resistance, indicating that it has high potential to be used in resistance breeding. Whether SPWR1-SPWR2-mediated resistance has a broad-spectrum effect on other pests of sweet potato or in other plants requires further investigation. In any case, the natural allelic variations harbored in N73 and N28 are rare and valued among the existing sweet potato resource, and well worth utilizing in breeding.

Although the genetic basis of SPWR has been a major focus of research for decades, the major genes involved in SPWR and the molecular regulation underlying SPWR have not yet been clarified. A recent genome-wide association study (GWAS) was conducted to investigate the SPWR loci in sweet potato, but no SNPs associated with phenotypes were identified over the significance threshold, suggesting that the small differences in genetic background and resistance between parents or subpopulations may prevent SNP-based genetic clustering. The higher genetic diversity from the non-inbred varieties would be more conducive to GWAS analysis for SPWR. In another study, three simple sequence repeat (SSR) markers for SPWR were identified using a QTL method based on 405 published SSR markers, and a significant association of field phenotype with hydroxycinnamic acid ester concentrations was detected. This suggests that sweet potato populations produced by the crossing of parents with different levels of resistance are available for mapping SPWR loci by QTL. This study of the genetic mechanism underlying SPWR is the first to identify SPW-resistant genes. F1 population was developed using parents with pronounced differences in SPWR for fine-mapping and isolated two major SPWR genes using expression analysis and sequence comparison. There were also notable morphological differences in the leaves of the parents (G87 and N73). Leaf shape was associated with another locus, which appeared to be the same leaf morphology locus examined in a previous study; this finding corroborates the reliability of the genetic map established in this study. Importantly, an efficient and repeatable method for evaluating SPWR was established and a rapid transgenic approach was developed for identifying the function of sweet potato genes, which strongly supports the study regarding the regulatory mechanism of SPWR.

WRKY proteins comprise a plant-specific transcription factor superfamily and contain a conserved WRKY domain with DNA-binding ability. The SPWR1 locus was mapped to the g35097 allele, which encodes a potential WRKY protein, and was classified into a conserved and unknown subfamily that only exists in the Convolvulaceae and Solanaceae. The SNP2563 in SPWR1 is present near the conserved WRKY domain region; it can cause changes in the SPWR1 protein enrichment to SPWR2 DNA (Figures 3c, 3d and Figure 13a), thus producing functional alterations of SPWR1. The effect of the SPWR2 locus on SPWR is closely associated with the expression of the g29415 gene, which depends on whether the indel of its promoter harbors a functional W-box (Figure 2b, Figure 3b and Figure 14b). This W-box element links the SPWR1 protein and the SPWR2 gene. Given that the G87 genome contains both the G87 and N73 alleles of SPWR1 and SPWR2, and only homozygous SPWR1 N73 and SPWR2N73 exist in N73, the N73 alleles might show dosage-dependent incomplete dominance relative to G87 alleles.

Secondary metabolites, including hydroxycinnamic acid esters and chlorogenic acid, play an important role in the natural response to SPW infestation. Caffeoylquinic acid compounds show biological activity in response to biotic stresses, such as inhibiting bacterial growth in millet and mediating resistance to thrips in chrysanthemum, carrot flies in carrots, leaf beetles in Salix integra, and aphids in lettuce. Quinate derivatives could be used in broad-spectrum insecticides for pest control. However, previous studies have not compared the activity of metabolites, nor identified specific chemical structures and functional groups mediating the insecticidal effects of metabolites. In this study, various quinate derivatives were compared and found that the 3-hydroxyl and 1 -hydroxyl of the quinate structure are important functional groups mediating natural SPWR (Figure 5, Figure 6 and Figure 7), suggesting that compounds with 3-hydroxyl and 1 -hydroxyl groups could be used to improve the efficacy of pesticides.

Multiple pest management approaches have been used to reduce the damage to sweet potatoes induced by SPW, and many of these approaches can have deleterious effects on human health and the environment. Molecular markers for SPWR have been developed and breeding materials have been obtained showing high SPWR with agronomic traits similar to those in F1 progeny. This study provides valuable information and insights that could aid the breeding of varieties with SPWR in sweet potato and other polyploid crops with complex genetic backgrounds.

Example 2 - aphids

The aphids {Myzus persicae) were put on the leaves of Arabidopsis and tobacco plants treated 100 pM with quinic acid or mock. The number of aphids stayed on each plant was counted and the rate of aphid inhibition (RAI) was calculated after 7 d of inoculation. The result showed that the aphid density in quinic acid-treated plants (both Arabidopsis and tobacco) was significantly decreased compared with that in control plants (Figure 11a). The average RAI in Arabidopsis and tobacco was 51.39% and 54.41 %, respectively, indicating that quinic acid has a significant inhibitory effect on aphid infestation.

Example 3 - moths

The 2.5-day-old Spodoptera litura larvae were used to feed the leaves of soybean plants pretreated with 100 pM quinic acid or mock, and the feeding leaf area was measured to evaluating the anti-insect effect. A concentration of 100 pM was found to be highly potent, such that the larvae died quickly after feeding. The concentration of quinic acid was therefore decreased to 20 pM for testing purposes. After 2 d of feeding, most of young larvae died in treatment group but not the control group, and the feeding area of the leaves in treatment group was significantly smaller than that in control group (Figure 11 b), suggesting that quinic acid has a remarkable inhibitory effect on Spodoptera litura larvae.

Example 4 - locusts

The second instar nymphs of locust {Locusta migrate ria) were used to selectively feed the leaves of wheat plants pretreated with 100 pM quinic acid or mock, and the feeding weight of plants (insect consumption) was measured for evaluating the anti-insect effect. The results showed that the locust nymphs were inclined to feed on the control plants more than the treated plants (Figure 11c), suggesting that quinic acid has a repellent effect on the locust nymphs.

Examples Summary

Overall, quinate derivatives are relatively low in cost and can be used as active broadspectrum pest control chemicals in eco-friendly insecticides to enhance the management of pests such as weevils, aphids, moths and locusts. Quinate derivatives have been shown to exhibit improved pesticidal activity compared to other structurally similar compounds such as chlorogenic acid. Without wishing to be bound by theory, it is postulated that this is due to the presence of a hydroxyl group at the 3-position instead of an ester moiety. The presence of a 1 -hydroxyl group also appears to be responsible for the observed activity.

In addition, increasing the expression and/or levels of SPWR1 and/or SPWR2 confers resistance to pests by activating quinate biosynthesis. Here, it can be seen that when the expression of SPWR1 or SPWR2 is suppressed, plants are more susceptible to pests. Conversely, when the expression of SPWR1 or SPWR2 is increased, plants exhibit increased pest resistance. SEQUENCE LISTING

SEQ ID NO. 1 (N73-SPWR1-CDS):

ATGGACAGCGCTTATAACGGGGAATACAAGGCTCTACTCAATGAGTTAATCCAAGGG ATGGAAT

GTGCAAAGCAGCTAAGAGTTCATCTCAACTCTGCAGCTTCTTCTGAAACCCAATACT TTTTTCT

GCAGAGGATACTCTCTTCTTATGAGAAAGCCCTGTTGATTCTCAAATGGAGGTTGGT AGGGCAA

TCCCACCCGGTGGCACCACCTCTGCCGGGTGCACCTGAACCTTCCATCTCTCTCGAT AGGAGCC

CCGACATTAACAACAACGGTTTTAAGGAGCAGCAGGACTACAATGTATCAAAAAAGA GAAAGGC

GATGCCAACATGGACAGAACAAGTAAGAGTTGGTGCTGAGAATGGACTTGAAGGCCC TACAGAA

GATGGATATAGTTGGCGAAAATACGGGCAGAAAGACATCCTTGGAGCTAAATATCCC AGGAGCT

ACTACAGATGCACGTTTCGCCTAATGCACAACTGCTGGGCAACTAAGCAAGTGCAGA GATCTGA

TGATGATCCAACAGTATTTGACATCACATACAAGGGGGCACATACATGCACCTTAGC TCCCACC

ACCTCAGTTCCCCCGCTGAGATCGCCTGAAAAACAAGAACTCAAGCAAATCCATCAC CAGAACG

AGAATTTCCAGGCAATGCAATCAAACCAGATGCTAATGAACCTGAGAGCAAGCCTGA GAGTGAA

CACAGATGGATTGGACACAAAGGAAACCGCATTCCCCTTCTCCTTCCCTCCAACATT CTCGGGA

CTTACAGACGAGAATCAACACTTCCAGAGCTCCCAGGTTGATGACAATGCAGTGGCA TTGGGCA

CATACTCACCATCCTTTGTTTCCCCCACCACTCCCGAATCGAACTACTTCTCTGCTT CACAGCA

ACACACAAATGCCTTCAAGGGAGTTCATATATCCTCAGCCAACACATCATCAACAAA CTCTCCA

ATTGGGGGCTTGGACTTGGACTACTCACTTCACCCTGCAACTTTAGACCCAAATTTC CCATTTG

ATACCTTGGAGTTTCTCACATGA

SEQ ID NO. 2 (N73-SPWR2-CDS):

ATGGCTTCCTCTGTCTGTTCACCCAGACATTCCCTCTTTCTTTCTAGCTCCACCAAA TCCCCCC

TGCGTGATTGCCCCGTGCGCTTTCCTGCCTTCAATGCTTTTCATTCAAGGCGGCGTT TAGTCTC

TGCCACCCGACTGAAAGTCAGTGCCAGCTCCCCAGCTCCGGCGGTGGATCAGTCGCC TAGTAGA

GCTGCGTCCCGGGCGCCCACAGTCGTTGAGGTTGATTTGGGTAACCGGAGCTACCCG ATTTACA

TCGGGTCTGGACTCCTTGATCAACCTGAGCTCCTTCAAAGGCATGTTCATGGAAAGA GAGTTCT

TGTTGTGACAAACTCAACCGTTGCTCCATTGTATTTGGATAAAACTATTAGTGCTTT AACAGAT

GGGAATCCTAATGTTAGTGTTGAAAGTGTGATTTTGCCAGATGGAGAGCAGTTTAAG AACATGG

AAAATCTCATGAAAATCTTTGATAAATCGATTGAATCACGATTGGATAGGCGTTGTA CTTTTGT tGCCCTTGGCGGTGGAGTAATTGGTGATATGTGTGGGTATGCTGCTGCTTCATACCTTCG AGGA

GTCAATTTCATACAGATTCCTACAACTGTTATGGCACAGGTAGATTCTTCAGTTGGT GGGAAAA

CTGGCATTAACCACCCACTTGGTAAAAATATGATCGGTGCATTCTACCAACCCCAAT GTGTGCT

TGTAGACACGGATACTTTGAATACCCTACCAGACAGAGAATTAGCTTCTGGTCTTGC TGAGGTT

ATAAAATATGGACTTATTAGAGATGCTGAATTTTTTGAGTGGCAAGAGAAGAATATG CCATCTT TGCTAGCAAGGGACCCAAGTGCATTTGCTTATGCTATCAAACGTTCCTGTGAAAACAAGG CTGA

GGTGGTGTCCTTGGATGAAAAGGAAAGTGGATTAAGGGCAACACTGAACTTGGGCCA CACGTTT

GGCCATGCAATAGAAACTGGTGTTGGCTATGGTCAATGGCTACATGGAGAAGCTGTT GCAGCTG

GAACGGTTATGGCTGTTGATATGTCACATCACTTAGGATGGATTGACGACTTACTAG CACAGCG

TGTTGGTAATATCCTTAAAGAGGCAAAGTTGCCCAATGCACCACCAAAAACCATGAC TGTCAAG

ATGTTCAAGTCTATCATGGCAGTCGATAAGAAAGTGGCTGATGGGAGGCTTAGACTC ATCCTTT

TGAAAGGTCCACTCGGCAACTGTGTATTCACTGGCGACTATGACAAAAAGGCCCTCG ACGAAAC TCTTCACGCATTCTGCAAATCCTAA

SEQ ID NO. 3 (N73-SPWR1 protein)

MDSAYNGEYKALLNELIQGMECAKQLRVHLNSAASSETQYFFLQRILSSYEKALLIL KWRLVGQ

SHPVAPPLPGAPEPSISLDRSPDINNNGFKEQQDYNVSKKRKAMPTWTEQVRVGAEN GLEGPTE

DGYSWRKYGQKDILGAKYPRSYYRCTFRLMHNCWATKQVQRSDDDPTVFDITYKGAH TCTLAPT

TSVPPLRSPEKQELKQIHHQNENFQAMQSNQMLMNLRASLRVNTDGLDTKETAFPFS FPPTFSG

LTDENQHFQSSQVDDNAVALGTYSPSFVSPTTPESNYFSASQQHTNAFKGVHISSAN TSSTNSP

IGGLDLDYSLHPATLDPNFPFDTLEFLT

SEQ ID NO. 4 (N73-SPWR2 protein)

MASSVCSPRHSLFLSSSTKSPLRDCPVRFPAFNAFHSRRRLVSATRLKVSASSPAPA VDQSPSR

AASRAPTVVEVDLGNRSYPIYIGSGLLDQPELLQRHVHGKRVLVVTNSTVAPLYLDK TISALTD

GNPNVSVESVILPDGEQFKNMENLMKIFDKSIESRLDRRCTFVALGGGVIGDMCGYA AASYLRG

VNFIQIPTTVMAQVDSSVGGKTGINHPLGKNMIGAFYQPQCVLVDTDTLNTLPDREL ASGLAEV

IKYGLIRDAEFFEWQEKNMPSLLARDPSAFAYAIKRSCENKAEVVSLDEKESGLRAT LNLGHTF

GHAIETGVGYGQWLHGEAVAAGTVMAVDMSHHLGWIDDLLAQRVGNILKEAKLPNAP PKTMTVK

MFKSIMAVDKKVADGRLRLILLKGPLGNCVFTGDYDKKALDETLHAFCKS

SEQ ID NO. 5 (N73-SPWR2-promoter sequence):

AGAGAGAGAAAGAAACGTCGCGATTAAGGTGGAAATGAGGATTAAGCATTGTGGGAG TTTTAAG

CTTTTAGGTGGGTAGGAGTGGAAGGGTGGTAGGTGGAATGAGACCTGAGAAGGATTT GGGATTT

GCGAAAATGGTTTCTGGCACAAGTTCTTCAAGGCCCTACCCTACTACTACTTTATTT CATTTTT

GCAGCCTCAAACACGAGCTCAATTCAATTATTGAAATTGATTAAGGATTACTACAGG TGTTTGG

TTATATGGTTTTGAGGGATTTAAATCCTAATTTTTGTCTGCTTGTTTGTGTTTTTTA ATAAGTC

AGAATAATATATTTTTTAAGTTGTTGGATCTAAATTTGTCCACCTCTCTAAGATTTT GTTAGTA

TTTGTCTTCAATTAATACAAATTTAAAAGGGAAAAGAGTCAAAGAAATTTTGGCTGA AAAGTCA

ATTAGTCCCTTAACATTTTAAATGTGCGATTACACCTATCAACAATTCATTTTGATG CAATAAA

ACTCAAAAACCGATTAATGACCTGGTATCATAGGTCATTAGGGATTCAGCCACCTTC CGATGAA TTTTCAGGCAAAAAAAACGATCGACAATCGGTTGCCAGTCACAGAGAAGGAAGGAAGGAG ACCA

TTGTGGTCGCCTAACAAACCCACCACCCATTTTAAAATAAATCTCATTGATTTCAAA TGGTAAA

ATAGTTAATATACCCATTAATCCCAACCGGCTGATAAAAACGCTACATTCATTAGCT TTTCAAT

ACCAAACACTTCAAAATAACTTATTAACATGTCAAACAGCTAAACTTGGTCAAATAA GCTAAAA

TTTGACTGATCAGCTAATAACCAACCACCCCGCATGTGTCCGTATCCATAACGAGCA TAACCAT

CTTCCTGCTACGTAGCGATTTCGGCAGCATAACTACTATTTGGAGGTCAACCAGACA AGCAGAA

AAATAAAATTGTTTGAAGTGATAAATTTAACTAAAGTCTACTTTTGCAAAACAGTGA GCATTGA

AACAAACTGGACAGCTATCTCAGAAACATCTAAGATGAACTACAAATGCTTAAAAAA TGAAATC

GCACATTCGCACCAATTACACCGGATCATTTTTACCAACAGATGATGGACACTAATA TATCCCT T TAG AT T GAG AAC TTTTATGTCCTGC T AC AAAGT GGG

SEQ ID NO. 6 (G87-SPWR1, wild-type):

MDSAYNGEYKALLNELIQGMECAKQLRVHLNSAASSETQYFFLQRILSSYEKALLIL KWRLVGQ

SHPVAPPLPGAPEPSISLDGSLDINNNGFKEQQDYNVSKKRKAMPTWTEQVRVGAEN GLEGPTE

DGYSWRKYGQKDILGAKYPRSYYRCTFRLMHNCWATKQVQRSDDDPTVFDITYKGAH TCNLAPT

TSVPPLRSPENQELKQIHHQNENFQAMQSNQMLMNLRASLRVNTDGLDTKETAFPFS FPPTFSG

LTDENQHFQSSQVDDNAVALGTYSPSFVSPPLPNRTTSLLHSNTTQMPSREFIYPQP NI INKLS NWGLGLGLLTSPCNF

SEQ ID NO. 7 (G87-SPWR2, wild-type):

MASSVCSPRHSLFLSSSTKSPLRDCPVRFPACNAFHSRRRLVSATRLKVSASSPAPA VDHSPSR

AASRAPTVVEVDLGNRSYPIYIGSGLLDQPELLQRHIHGKRVIVVTNTTVAPLYLDK TVSALTD

GNPNVSVESVILPDGEQFKNMENLMRIFDKSIESRLDRRCTFVALGGGVIGDMCGYA AASYLRG

VNFIQIPTTVMAQVDSSVGGKTGINHPLGKNMIGAFYQPQCVLVDTDTLNTLPDREL ASGLAEV

IKYGLIRDAEFFEWQEKNMPSLLARDPSAFAYAIKRSCENKAEVVSLDEKESGLRAT LNLGHTF

GHAIETGVGYGQWLHGEAVAAGTVMAVDMSHHLGWIDDLLAQRVGNILKEAKLPNAP PKTMTVK

MFKSIMAVDKKVADGRLRLILLKGPLGNCVFTGDYDKKALDETLNAFCKS

SEQ ID NO. 8 (G87-SPWR1-CDS, wild-type):

ATGGACAGCGCTTATAATGGGGAATACAAGGCTCTACTCAATGAGTTAATCCAAGGG ATGGAAT

GTGCAAAGCAGCTAAGAGTTCATCTCAACTCTGCAGCTTCTTCTGAAACACAATACT TTTTTCT

GCAGAGGATACTCTCTTCTTATGAGAAAGCCCTGTTGATTCTCAAATGGAGGTTGGT AGGGCAA

TCCCACCCGGTGGCACCACCTCTGCCGGGTGCCCCTGAACCTTCCATCTCTCTCGAT GGGAGCC

T CGAC AT T AAC AACAACGGTT TT AAGGAGC AGCAGGACT AT AAT GT AT CAAAAAAGAGAAAGGC

GATGCCAACATGGACCGAACAAGTAAGAGTTGGCGCTGAGAATGGCCTCGAAGGCCC TACAGAA

GATGGATATAGTTGGCGAAAATACGGGCAGAAAGACATTCTTGGAGCTAAATATCCC AGGAGCT ACTACAGATGCACGTTTCGCCTAATGCACAACTGCTGGGCAACTAAGCAAGTGCAGAGAT CAGA

TGATGATCCAACGGTATTTGACATCACATACAAGGGGGCACATACATGCAACTTAGC TCCCACC

ACCTCAGTTCCCCCGCTGAGATCGCCTGAAAATCAAGAACTCAAGCAAATCCATCAC CAGAACG

AGAATTTCCAGGCAATGCAATCAAACCAGATGCTAATGAACCTGAGAGCAAGCCTGA GAGTGAA

CACAGATGGATTGGACACAAAGGAAACCGCATTCCCCTTCTCCTTCCCTCCAACATT CTCGGGA

CTTACAGACGAGAATCAACACTTCCAGAGCTCACAGGTTGATGACAATGCAGTGGCA TTGGGCA

CATACTCACCCTCCTTTGTTTCCCCACCACTCCCGAATCGAACTACTTCTCTGCTTC ACAGCAA

CACCACACAAATGCCTTCAAGGGAGTTCATATATCCTCAGCCAAACATCATCAACAA ACTCTCC

AATTGGGGGCTTGGACTTGGACTACTCACTTCACCCTGCAACTTTAG

SEQ ID NO. 9 (G87-SPWR2-CDS, wild-type):

ATGGCTTCCTCTGTCTGTTCACCCAGACATTCCCTCTTTCTTTCTAGCTCCACCAAA TCCCCCC

TGCGTGATTGCCCCGTGCGCTTTCCTGCCTGCAATGCTTTTCATTCAAGGCGGCGTT TAGTCTC

TGCCACACGACTGAAAGTTAGTGCCAGCTCCCCAGCTCCGGCGGTGGATCATTCGCC TAGTAGA

GCTGCGTCCCGGGCACCCACAGTCGTTGAGGTTGATTTGGGTAACCGGAGCTACCCG ATTTACA

TCGGGTCTGGACTCCTTGATCAACCTGAACTCCTTCAAAGGCATATTCATGGAAAGA GAGTTAT

TGTTGTGACAAACACAACCGTTGCTCCATTGTATTTGGATAAAACTGTTAGTGCTTT AACAGAT

GGGAATCCTAATGTTAGTGTTGAAAGTGTGATTTTGCCAGATGGAGAGCAGTTTAAG AACATGG

AAAATCTCATGAGAATCTTTGATAAATCGATTGAATCACGATTGGATAGGCGTTGTA CTTTTGT

TGCCCTTGGCGGTGGAGTAATTGGTGATATGTGTGGGTATGCTGCTGCTTCATACCT TCGAGGA

GTCAATTTCATACAGATTCCTACAACTGTTATGGCACAGGTAGATTCTTCAGTTGGT GGGAAAA

CTGGCATTAACCACCCACTTGGTAAAAATATGATCGGTGCATTCTACCAACCCCAAT GTGTGCT

TGTAGACACGGATACTTTGAATACCCTACCAGACAGAGAATTAGCTTCTGGTCTTGC TGAGGTT

ATAAAATATGGACTTATTAGAGATGCTGAATTTTTTGAGTGGCAAGAGAAGAATATG CCATCTT

TGCTAGCAAGGGACCCAAGTGCATTTGCTTATGCTATCAAACGTTCCTGTGAAAACA AGGCTGA

GGTGGTGTCCTTGGATGAAAAGGAAAGTGGATTAAGGGCAACACTGAACTTGGGCCA CACGTTT

GGCCATGCAATAGAAACTGGTGTTGGCTATGGTCAATGGCTACATGGAGAAGCTGTT GCAGCTG

GAACGGTTATGGCTGTTGATATGTCACATCACTTAGGATGGATTGACGACTTACTAG CACAGCG

TGTTGGTAATATCCTTAAAGAGGCAAAGTTGCCCAATGCACCACCAAAAACCATGAC TGTCAAG

ATGTTCAAGTCTATCATGGCAGTCGATAAAAAAGTGGCTGATGGGAGGCTTAGACTC ATCCTTT

TGAAAGGTCCACTCGGCAACTGTGTATTCACTGGCGACTATGACAAAAAGGCCCTCG ACGAAAC TCTTAACGCATTCTGCAAATCCTAA

SEQ ID NO. 10 (G87-SPWR2-promoter, wild-type):

AGAGAGAGAAAGAAACGTCGCGATTAAGGTGGAAATGAGGATTAAGCATTGTGGGAG TTTTAAG

CTTTTAGGTGGGTAGGAGTGGAAGGGTGGTAGGTGGAATGAGACCTGAGAAGGATTT GGGATTT GGGAAAATGGTTTCTGGCACAACTTCTTCAAGGCCCTACCTTACAACTACTTTATTTCAT TTTT

GCAGCCTCACACACGAGCTCAATTCAATTATTGAAATTGATTAAGGATTACTACTGG TGTTGGT

TTATATGGTTTTGAGGGATTTAAATCCTAATTTTTGTCGGCTTGTTTGTGTTTTTTA ATAGCTC

AGAATAACATTTTTTAAAGTTGTTGGATCTAAATTTGTCTACATTTTTTGAGGGATC TAAATCC TAGTGTGGTTTCAAAAAAAAAAGAAGAAGATTTTGTTAGTGTTTTTCTTCATCACAAGAA AAGT

GACAAACAAATTCCTAGTTCCAACAGTACTCTATCAAATCCAAAAAAAGCACAATCA AAAGGGG

GTGCCAGCATTCGTATGTTTTTGTAATGCTTTTAATTCCTCAGCTTTAGATTATTCA TATGTGG

GTTTGAGCTTTAGATTATTCATATGTGGGTTTGGTTATCAAGCAATGCAGGTTGATT GCTAGAG

GCATGGGTGACTGTGTTGTTCGCCATGTCAGAAGGTCAGCGAATCATATTGCTCACG TACTTGC TCGGGCAACTGGTTCTTCTACTGTCCTTAGTGTGTGGGATTATATTCCTCCAGTTTGTAT TTCG

GACTTGGTTTTATATTAATATATGATGTTACTTTGTTTTTCAAAAAAAAGGGGGTGC CGGCTTG

GGATTCGACACTTCTTTTCTAAGAATAGATTTGCACTGTGCCAAACAAGCTCTAACA CAGTAGA

AAACAGTTGCAAAAAAGTGCGCATTGAAAGAAACTGGACAGCTATCTCAGAAACATA TAAGATG

AACTACAAATGCTTAAAAAATGAAATCGCACCAATTACACCGGATCATTTTTACCAA CAGATGA TGACACTAATATAATATATCCCTTTACATTGACAACTTTTATGTCCTGCTACAAAGTGGG