Description

The present invention relates to sunflower plants ( Helianthus annuus) being resistant to the plant disease downy mildew and especially to sunflower plants ( Helianthus annuus ) being resistant to the downy mildew disease causing pathogen Plasmopara halstedii. The present invention further relates to seeds and plant part of the present plants and to methods for identifying downy mildew resistant sunflower plants ( Helianthus annuus).

Plants employ diverse constitutive and inducible defense strategies to counteract colonization by microbial pathogens and attacks of herbivorous insects. Plant responses induced upon pathogen encounter are triggered by non-self-recognition of common microbial structures and by highly pathogen- specific effectors.

Obligate biotrophic phytopathogens such as the causal pathogens of powdery and downy mildew diseases employ highly specialized infection strategies. Genetic studies on

Arabidopsis have identified several downy mildew resistance genes that appear to be involved in susceptibility to downy mildew oomycete pathogens. In Arabidopsis, knockdown mutations provide protection against downy mildew. Map-based cloning revealed that a downy mildew resistance gene designated as DMR1 encodes homoserine kinase (HSK) protein which catalyzes the conversion of homoserine to homoserine- 4-phosphate, a step in the Asp-derived Thr biosynthesis pathway. The homoserine kinase (HSK) protein substrate homoserine, which is present at low levels in the wild-type, accumulates in dmrl mutant lines.

However, considering that DMR1 based resistance directly interferes with the biosynthesis of essential amino acids, the development of viable and commercially interesting plants has been hampered. For example, it is very difficult to obtain plants in which expression of homoserine kinase has been completely abolished for example through introduction of a premature stop-codon in the coding sequence because, generally, introducing a premature stop-codon results in lethality or a severe hampering of plant development.

Accordingly, using DMR1 based resistance in plants requires a delicate balancing between maintaining a sufficiently high concentration, or activity, of homoserine kinase (HSK) protein in a plant cell to allow for adequate synthesis of essential amino acids and a sufficiently low concentration, or activity, of homoserine kinase (HSK) in a plant cell to provide downy mildew resistance.

Helianthus, or sunflower, is a genus of plants comprising about 70 species in the family Asteraceae. The common name, "sunflower", typically refers to the annual species Helianthus annuus whose round flower heads in combination with the ligules look like the sun. Helianthus annuus is cultivated in temperate regions and some tropical regions as food crops for humans, cattle, and poultry, and as ornamental plants.

Plasmopara halstedii is a plant pathogen infecting sunflowers. The species is one of many pathogens commonly referred to as downy mildew. Plasmopara halstedii is an obligate biotroph that attacks flowering plants of the Asteraceae family. The pathogen causes significant yield losses due to seedling collapse (damping off), plant stunting, chlorosis, root browning and alteration of secondary metabolism of infected plants.

Once the pathogen has been detected in an area, management is essential, as Plasmopara halstedii is nearly impossible to eradicate. Between long surviving resting spores and high levels of secondary inoculum, Plasmopara halstedii can affect anywhere from 50% to 95% of sunflowers in the field in a single season.

Seed treatment has been shown to be effective in controlling the disease, as the establishment of Plasmopara halstedii in an area of soil is nearly irreversible. The compounds metalaxyl and oxadixyl have been shown to protect seeds in the case of infection, and treatments containing these compounds are commercially available. However, some strains of Plasmopara halstedii show resistance to metalaxyl-based fungicides.

Considering the above, there is a need in the art for sunflowers, or Helianthus annuus, plants in which resistance is encoded by a genetic determinant or resistance gene. It is an object of the present invention, amongst other objects, to address this need in the art.

This object of the present invention, amongst other objects, is met by the provision of Helianthus annuus plants as outlined in the appended claims.

Specifically, this object of the present invention, amongst other objects, is met by Helianthus annuus plants, which plants are resistant to downy mildew disease due to one or more amino acid substitutions in the homoserine kinase protein shown in SEQ ID No. 1 or a reduced expression of the gene encoding SEQ ID No.l and of which the coding sequence, including 5’and 3’untranslated regions, is shown in SEQ ID No. 3.

The present inventors have surprisingly found that through amino acid substitution or a reduced, but not absent, expression, a sufficient activity of homoserine kinase (HSK) can be maintained in Helianthus annuus to allow for synthesis of essential amino acids and,

simultaneously, a sufficient low activity of homoserine kinase (HSK) can be obtained to provide downy mildew resistance.

The present one or more mutations in SEQ ID No. 1 can be provided by generally known plant mutagenesis techniques such as ethyl methane sulfonate (EMS) or radiation or by genetic modification of the coding sequence as shown in SEQ ID No. 3. Reduced expression can be obtained through generally known techniques such as RNAi or manipulation of the promoter sequence directly 5’ upstream of the coding sequence of SEQ ID No. 3. According to a preferred embodiment, the downy mildew disease is caused by the pathogen Plasmopara halstedii.

According to another preferred embodiment, the present one or more amino acid substitutions in the homoserine kinase protein shown in SEQ ID No. 1 provide a homoserine concentration of 50 to 2000 ng/mg fresh weight in the leaves of said plant. As indicated above, DMR1 based resistance requires a delicate balancing between maintaining a sufficiently high concentration, or activity, of homoserine kinase (HSK) protein in a plant cell to allow for synthesis of essential amino acids and a sufficiently low concentration, or activity, of homoserine kinase (HSK) protein in a plant cell to provide downy mildew resistance.

The present inventors have surprisingly found that in planta homoserine concentrations of at least 50 ng/mg fresh weight in the leaves, either through amino acid substitutions or reduced expression, provide the present downy mildew resistance. It is noted that in susceptible plants homoserine concentrations are considerably lower than 50 ng/mg fresh weight.

According to yet another preferred embodiment, the present one or more amino acid substitutions in the homoserine kinase protein shown in SEQ ID No. 1 , or reduced expression of the gene encoding SEQ ID No. 1, provide an in planta homoserine concentration of 100 to 700 ng/mg fresh in the leaves.

According to a more preferred embodiment of the invention, the present

Helianthus annuus plants comprise an amino acid substitution at position 247, more preferably an amino acid substitution of methionine with a non-polar amino acid at position 247, most preferably an amino acid substitution methionine for isoleucine (M247I) at position 247. The amino acid sequence of the latter is shown in SEQ ID No. 2 and the coding sequence, including 5’and 3’untranslated regions, in SEQ ID No. 4.

According to another more preferred embodiment of the present invention, the present Helianthus annuus plants comprise one or more amino acid substitutions in the homoserine kinase protein shown in SEQ ID No. 1 such as SEQ ID No. 2, wherein the protein is

homozygously encoded in the genome of the Helianthus annuus plants.

Considering the benefits of the Helianthus annuus plants as outlined above, the present invention also relates to seeds or plant parts thereof and to a downy mildew resistance providing homoserine kinase protein comprising one or more amino acid substitutions in the homoserine kinase protein shown in SEQ ID No. 1 , preferably the homoserine kinase protein comprising the amino acid sequence of SEQ ID No. 2.

The present invention also allows for methods for identifying a downy mildew resistant Helianthus annuus plant, wherein the method comprises the step of establishing the presence of one or more amino acid substitutions in the homoserine kinase protein shown in SEQ ID No. 1 , preferably the step of establishing the presence of SEQ ID No. 2.

The present invention will be further outlined in the examples below. In the examples, reference is made to figures wherein:

Figure 1: shows that SEQ ID No. 1 can functionally complement the Arabidopsis dmrl mutant;

Figure 2: shows that SEQ ID No. 2 dmrl mutant M247I accumulates medium levels of homoserine but mutant A249T does not;

Figure 3: shows an impression of DM infection assay on whole seedlings. Pictures were taken 14 days after inoculation and the two lower panels are wild-type control lines;

Figure 4: shows Helianthus annuus dmrl mutant (M4 generation) DM test (seedling dip inoculation, 5*E4 spores/ml, DM-isolate 307. Sporulation was induced at 13 dpi and scored at 14dpi.

EXAMPLES

Example 1: Complementation assay

The coding sequence of the Sunflower DMR1 gene was cloned into a plant expression vector driven by the Cauliflower mosaic virus 35S promoter. The resulting construct was transformed into the Arabidopsis dmrl mutant (background is Landsberg edsl) using the Agrobacterium mediated floral dipping method. Transgenic seedlings were infected with downy mildew ( Hyaloperonospora Arabidopsidis) and sporulation levels (disease severity) were compared to susceptible control (“edsl”) and resistant control (“edsl.dmrl”). As shown in Figure 1, transgenic expression of sunflower DMR1 restored susceptibility of the Arabidopsis dmrl mutant. Hence, sunflower DMR1 is functional.

Example 2: Measurement of homoserine concentrations

Leaves from DMR1 mutants and wild- type control plants were sampled and weighed. Samples were homogenized and extracted with water. Samples were re-extracted with chloroform for further cleanup. Half volume of acetonitrile was added to the upper phase, samples were spun and the supernatant was used for metabolite analysis using HPLC-MS/MS. External standards were used for quantitation of homoserine and other amino-acids. Reference samples were included, which represent dmrl mutants from Arabidopsis and other species, with previously confirmed homoserine-accumulation. As shown in Figure 2, analysis of two different sunflower dmrl mutants, M247I and A249T, revealed that M247I is a homoserine-accumulator, whereas A249T is not.

Example 3: Plasmopara halstedii infection assay

Seeds from mutant and wild-type sunflowers were germinated on wet filter-paper for 4 days. Meanwhile, DM-infected sunflower plantlets (DM-maintenance/culture) were put in 100% RH to induce DM-sporulation. DM spores were harvested by shaking infected leaves in water. Spores were counted on a haemocytometer and the spore-suspension was adjusted to 50,000 spores per ml. The 4d-old seedlings were incubated in the spore-suspension for a few hours, and then potted. After 13 days, sporulation was induced by overnight incubation of the pots with seedlings at 100%RH. Sporulation was scored by counting the number of cotyledons and first true leaves that show sporulation (Figures 3 and 4). While 40-80% of wild-type leaves show sporulation, sporulation on leaves of dmrl M247I mutants is less than 5%.

The sequences of the corresponding SEQ ID Nos 1 to 4 referred herein are presented below in a sequence listing. In SEQ ID No. 1 , the wild-type amino acid methionine at position 247 is indicated in bold. In SEQ ID No. 2, the present resistance providing isoleucine at position 247 is indicated in bold. In SEQ ID No. 3 encoding SEQ ID No. 1 , the coding sequence is indicated in uppercase while 5’ upstream and 3’ downstream (regulatory) sequences are indicated in lowercase. In SEQ ID No. 4 encoding SEQ ID No. 2, the coding sequence is indicated in uppercase while 5’ upstream and 3’ downstream (untranslated) sequences are indicated in lowercase. The mutation G to A responsible for the present amino acid substitution is indicated in bold in SEQ ID Nos 3 and 4.

SEQUENCE LISTING

<110> SciENZA Biotechnologies 5 B.V.

<120> DOWNY MILDEW RESISTANT SUNFLOWER PLANTS

<130> 4/2WL15/61

<160> 4

<170> BiSSAP 1.3.6

<210> 1

<211> 376

<212> PRT

<213> Helianthus annuus

<400> 1

Met Ala lie Cys His His His Pro Thr Phe His Thr His His Ala Phe 1 5 10 15

Pro Pro Thr Thr Thr Pro Thr Pro His Leu His Leu His Leu Arg Leu

20 25 30

Pro Ser Ser Phe Arg Cys Asn Leu Ser Val Thr Pro Pro Leu

35 40 45

Glu Pro Glu Pro Val Tyr Thr Ser Val Lys Ser Phe Ala Pro Ala Thr 50 55 60

Val Ala Asn Leu Gly Pro Gly Phe Asp Phe Leu Gly Cys Ala Val Asp 65 70 75 80

Gly lie Gly Asp Phe Val Thr Leu Lys Val Asp Pro Gin Thr Pro Pro

85 90 95

Gly Glu lie Ser lie Ser Asp lie Thr Gly Thr Gly Asn Ser Ala Lys

100 105 110

Lys Leu Ser Lys Asn Pro Asn Trp Asn Cys Ala Gly He Ala Ala lie

115 120 125

Ser Val Met Lys Met Leu Asn lie Arg Ser Val Gly Leu Ser Leu Glu 130 135 140

Leu Glu Lys Gly Leu Pro Leu Gly Ser Gly Leu Gly Ser Ser Ala Ala 145 150 155 160

Ser Ala Ala Ala Ala Ala Val Ala Val Asn Glu Leu Phe Gly Gly Lys 165 170 175

Leu Pro Ala Ser Glu Leu Val Leu Ala Gly Leu Glu Ser Glu Ala Lys

180 185 190

Val Ser Gly Tyr His Ala Asp Asn lie Ala Pro Ala lie Met Gly Gly

195 200 205

Phe Val Leu Val Arg Ser Tyr Asp Pro Leu Glu Leu lie Ser Leu Arg 210 215 220

Phe Pro Ser Asp Lys Asn Leu Cys Phe Val Leu Val Asn Pro Glu Phe 225 230 235 240

Glu Ala Pro Thr Lys Lys Met Arg Ala Ala Leu Pro Thr Glu lie Ser

245 250 255

Met Ser Gin His lie Trp Asn Ser Ser Gin Ala Gly Ala Leu Val Ala

260 265 270

Ala Val Leu Gin Gly Asp Leu Val Gly Leu Gly Lys Ala Leu Ser Asn

275 280 285

Asp Lys lie Val Glu Pro Lys Arg Ala Pro Leu lie Pro Gly Met Glu

290 295 300

Glu Val Lys Arg Ala Ala Leu Glu Ala Gly Ala Phe Gly Cys Thr He

305 310 315 320

Ser Gly Ala Gly Pro Thr Ala Val Ala lie Val Asp Asp Glu Glu Lys

325 330 335

Gly Arg Val lie Gly Glu Lys Met Val Glu Ala Phe Met Val Gly Gly

340 345 350

Asn Leu Lys Ala Gin Ala Met Val Lys Lys Leu Asp Arg Val Gly Ala

355 360 365

Arg Leu Val Ser Ser Ser Ser Arg

370 375

<210> 2

<211> 376

<212> PRT

<213> Helianthus annuus

<400> 2

Met Ala lie Cys His His His Pro Thr Phe His Thr His His Ala Phe 1 5 10 15

Pro Pro Thr Thr Thr Pro Thr Pro His Leu His Leu His Leu Arg Leu

20 25 30

Pro Ser Ser Phe Arg Cys Asn Leu Ser Val Thr Ser Pro Pro Lys Leu 35 40 45

Glu Pro Glu Pro Val Tyr Thr Ser Val Lys Ser Phe Ala Pro Ala Thr 50 55 60

Val Ala Asn Leu Gly Pro Gly Phe Asp Phe Leu Gly Cys Ala Val Asp 65 70 75 80

Gly lie Gly Asp Phe Val Thr Leu Lys Val Asp Pro Gin Thr Pro Pro

85 90 95

Gly Glu lie Ser lie Ser Asp lie Thr Gly Thr Gly Asn Ser Ala Lys

100 105 110

Lys Leu Ser Lys Asn Pro Asn Trp Asn Cys Ala Gly He Ala Ala lie

115 120 125

Ser Val Met Lys Met Leu Asn lie Arg Ser Val Gly Leu Ser Leu Glu 130 135 140

Leu Glu Lys Gly Leu Pro Leu Gly Ser Gly Leu Gly Ser Ser Ala Ala 145 150 155 160

Ser Ala Ala Ala Ala Ala Val Ala Val Asn Glu Leu Phe Gly Gly Lys

165 170 175

Leu Pro Ala Ser Glu Leu Val Leu Ala Gly Leu Glu Ser Glu Ala Lys

180 185 190

Val Ser Gly Tyr His Ala Asp Asn lie Ala Pro Ala lie Met Gly Gly

195 200 205

Phe Val Leu Val Arg Ser Tyr Asp Pro Leu Glu Leu He Ser Leu Arg 210 215 220

Phe Pro Ser Asp Lys Asn Leu Cys Phe Val Leu Val Asn Pro Glu Phe 225 230 235 240

Glu Ala Pro Thr Lys Lys lie Arg Ala Ala Leu Pro Thr Glu lie Ser

245 250 255

Met Ser Gin His lie Trp Asn Ser Ser Gin Ala Gly Ala Leu Val Ala

260 265 270

Ala Val Leu Gin Gly Asp Leu Val Gly Leu Gly Lys Ala Leu Ser Asn

275 280 285

Asp Lys lie Val Glu Pro Lys Arg Ala Pro Leu lie Pro Gly Met Glu 290 295 300

Glu Val Lys Arg Ala Ala Leu Glu Ala Gly Ala Phe Gly Cys Thr lie 305 310 315 320

Ser Gly Ala Gly Pro Thr Ala Val Ala lie Val Asp Asp Glu Glu Lys

325 330 335

Gly Arg Val lie Gly Glu Lys Met Val Glu Ala Phe Met Val Gly Gly

340 345 350

Asn Leu Lys Ala Gin Ala Met Val Lys Lys Leu Asp Arg Val Gly Ala 355 360 365

Arg Leu Val Ser Ser Ser Ser Arg

370 375

<210> 3

<211> 1254

<212> DNA

<213> Helianthus annuus

<400> 3

tacgcctcca cttccacttc cacctgcaac actctctctc tctctctcta caactcaaca 60

ATGGCAATCT GCCACCACCA CCCCACATTC CACACCCACC ACGCCTTCCC ACCCACCACC 120

ACCCCCACCC CCCACCTCCA CCTCCACCTC CGTCTACCCT CATCTTTCCG CTGCAACCTC 180

TCGGTCACTT CACCCCCGAA ACTTGAACCC GAACCCGTTT ACACCTCTGT CAAATCCTTC 240

GCCCCAGCCA CCGTAGCCAA CCTGGGTCCC GGCTTCGACT TTCTAGGCTG CGCCGTTGAC 300

GGAATCGGAG ACTTCGTCAC CCTCAAAGTC GACCCGCAAA CCCCACCCGG CGAAATCTCA 360

ATCTCCGACA TCACCGGAAC AGGTAACTCC GCTAAAAAAC TAAGCAAAAA CCCTAACTGG 420

AATTGTGCCG GAATAGCCGC CATTTCTGTT ATGAAGATGC TCAATATTAG ATCTGTGGGT 480

CTGTCTTTAG AACTAGAAAA AGGGTTACCG TTGGGTAGTG GTTTAGGGTC TAGCGCCGCC 540

AGTGCAGCCG CTGCAGCCGT AGCTGTTAAT GAACTGTTTG GTGGGAAGTT GCCGGCATCG 600

GAGTTGGTGC TCGCCGGACT TGAATCCGAG GCGAAGGTGT CCGGTTATCA TGCTGATAAT 660

ATTGCTCCGG CTATTATGGG TGGGTTTGTG TTGGTACGGA GTTATGATCC TTTAGAGTTG 720

ATTTCTTTAA GGTTTCCAAG TGATAAGAAT TTGTGTTTCG TGTTGGTGAA TCCCGAATTC 780

GAAGCGCCCA CGAAGAAGAT GCGGGCGGCG TTGCCAACGG AGATATCGAT GTCGCAACAT 840

ATATGGAACA GTAGTCAGGC GGGTGCGTTG GTGGCGGCTG TGTTGCAGGG GGATTTGGTT 900 GGGTTGGGAA AGGCGTTGTC GAACGATAAG ATTGTGGAGC CGAAGCGGGC GCCTTTGATT 960

CCGGGGATGG AGGAGGTGAA GAGGGCTGCG TTGGAGGCGG GGGCGTTTGG GTGTACGATT 1020

AGTGGGGCGG GGCCCACGGC AGTGGCGATT GTGGATGATG AGGAGAAGGG GAGGGTGATT 1080

GGGGAGAAGA TGGTGGAGGC GTTTATGGTC GGTGGGAATT TGAAAGCTCA GGCTATGGTT 1140

AAGAAGTTGG ATAGAGTTGG TGCTAGGCTT GTTAGCAGCA GTTCAAGATG Attatgaata 1200 tgattgtgtt tggatcttga agatgtttgt gatggtgggg taaggttttt tttt 1254

<210> 4

<211> 1254

<212> DNA

<213> Helianthus annuus

<400> 4

tacgcctcca cttccacttc cacctgcaac actctctctc tctctctcta caactcaaca 60

ATGGCAATCT GCCACCACCA CCCCACATTC CACACCCACC ACGCCTTCCC ACCCACCACC 120

ACCCCCACCC CCCACCTCCA CCTCCACCTC CGTCTACCCT CATCTTTCCG CTGCAACCTC 180

TCGGTCACTT CACCCCCGAA ACTTGAACCC GAACCCGTTT ACACCTCTGT CAAATCCTTC 240

GCCCCAGCCA CCGTAGCCAA CCTGGGTCCC GGCTTCGACT TTCTAGGCTG CGCCGTTGAC 300

GGAATCGGAG ACTTCGTCAC CCTCAAAGTC GACCCGCAAA CCCCACCCGG CGAAATCTCA 360

ATCTCCGACA TCACCGGAAC AGGTAACTCC GCTAAAAAAC TAAGCAAAAA CCCTAACTGG 420

AATTGTGCCG GAATAGCCGC CATTTCTGTT ATGAAGATGC TCAATATTAG ATCTGTGGGT 480

CTGTCTTTAG AACTAGAAAA AGGGTTACCG TTGGGTAGTG GTTTAGGGTC TAGCGCCGCC 540

AGTGCAGCCG CTGCAGCCGT AGCTGTTAAT GAACTGTTTG GTGGGAAGTT GCCGGCATCG 600 GAGTTGGTGC TCGCCGGACT TGAATCCGAG GCGAAGGTGT CCGGTTATCA TGCTGATAAT 660

ATTGCTCCGG CTATTATGGG TGGGTTTGTG TTGGTACGGA GTTATGATCC TTTAGAGTTG 720

ATTTCTTTAA GGTTTCCAAG TGATAAGAAT TTGTGTTTCG TGTTGGTGAA TCCCGAATTC 780

GAAGCGCCCA CGAAGAAGAT ACGGGCGGCG TTGCCAACGG AGATATCGAT GTCGCAACAT 840

ATATGGAACA GTAGTCAGGC GGGTGCGTTG GTGGCGGCTG TGTTGCAGGG GGATTTGGTT 900

GGGTTGGGAA AGGCGTTGTC GAACGATAAG ATTGTGGAGC CGAAGCGGGC GCCTTTGATT 960

CCGGGGATGG AGGAGGTGAA GAGGGCTGCG TTGGAGGCGG GGGCGTTTGG GTGTACGATT 1020

AGTGGGGCGG GGCCCACGGC AGTGGCGATT GTGGATGATG AGGAGAAGGG GAGGGTGATT 1080

GGGGAGAAGA TGGTGGAGGC GTTTATGGTC GGTGGGAATT TGAAAGCTCA GGCTATGGTT 1140

AAGAAGTTGG ATAGAGTTGG TGCTAGGCTT GTTAGCAGCA GTTCAAGATG Attatgaata 1200 tgattgtgtt tggatcttga agatgtttgt gatggtgggg taaggttttt tttt 1254