LEVEL IN A PLANT

Plant-parasitic nematodes (PPN) are nearly microscopic, worm-shaped animals virtually invisible to the naked eye when in the soil. Although a few nematode species feed on above ground plant parts, such as leaves, stems, flowers and seeds, the majority of these parasites feed on underground parts of plants, including roots, bulbs and tubers. Most PPN feed on root tissue and damage their host mainly by inhibiting root growth resulting in reduced water uptake. Further, these nematodes favour microbial infections through wound sites or serve as vectors for pathogenic viruses. While some species exhibit a hit-and-run strategy, remaining migratory throughout their life cycle into the plant root, an increase in complexity of host- parasite interactions is associated in sedentary parasite species with their enhanced capacity to manipulate host plant genes in their favour. These endoparasitic nematodes settle down after an initial migratory phase and assume a sedentary life style while transforming plant cells into complex feeding structures. Nematodes of this category represent the most damaging species for crops. Some of these nematodes have a relatively specialized host range (e.g. , cyst nematodes Heterodera and Globodera genus) while others are able to successfully infest thousands of unrelated host plant species (e.g., root-knot nematodes Meloidogyne spp.).

Because root-knot nematodes represent the most economically-important PPN, they constitute the most explored group of species and can now be considered as one of the most advanced models for understanding mechanisms of plant parasitism in nematodes. As other PPN, sedentary endoparasites have a stylet that is used to pierce and penetrate plant cell walls, to release esophageal secretions into the host tissue and to take up nutrients. During their infective life-cycle, root-knot nematode larvae penetrate plant root tissue and migrate along the vascular cylinder. By injecting secretions into plant cells, they induce the formation of a feeding site indispensable for their development. As a consequence of the formation of these feeding structures, root-knots or galls are observed as symptoms of the infestation. Plant nutrient and water uptake are substantially reduced by the resulting damage to the root system, and infested plants are therefore weak and give low yields. Once the feeding structure is established, female nematodes continue their development and eventually become pear- shaped and produce hundreds to thousands of eggs. These eggs are then extruded as an egg- mass, protected within a gelatinous matrix, at the outer surface of the root.

Plant-parasitic nematodes (PPN) cause significant damage to agriculture throughout the world. A global survey in 1987 evaluated crop losses at $78-125 billion per year (Sasser and Freckman, 1987). More recent direct global estimates are not available, but when the increase in agricultural productivity is taken into account, the extrapolated 2001 loss for crops totalled $ 1 18 billion (1 1 % of production) (McCarter, 2009). The current figure is thus probably much higher. Measures such as growing resistant crop varieties and the use of nematicides are extensively employed to control PPN infections. Billions of euros have been spent annually on soil fumigants and other nematicides. Chemical controls against nematodes are not only costly but they are highly toxic and hazardous, and involve application of environmentally unacceptable compounds. Such toxicological problems and environmental damage caused by nematicides have led to banning of the most efficient chemicals that were commonly used so far in Europe (EC directive 2007/619/EC). In the absence of alternative control methods or development of specific and environmentally safe molecules, it is to be feared severe crop losses within major sectors of the agricultural industry. Indeed, nematode problems recently re-emerged in some areas where the use of traditional nematicides has been abandoned for a short while (Djian-Caporalino, 2012; Wesemael et al. , 2011 ).

It appears from the foregoing that there is a need for methods for limiting the damages caused by the plant-parasitic nematodes.

Mining the genomes of root-knot nematodes Meloidogyne incognita and Meloidogyne hapla through an evolutionary and comparative genomics approach, the inventors have searched genes conserved in various plant parasites while otherwise absent from the genomes of non target species such as those of chordates, plants, annelids, insect pollinators and mollusks. The inventors have identified a set of root-knot nematode genes absent from non- target species but present in several plant parasites. Further bioinformatics pruning of this set of genes yielded 16 candidate genes (15 genes encoding effector like proteins and 1 gene encoding a transcription factor) present both in M. incognita and M. hapla. These 16 candidate genes were respectively silenced using siRNA technique in M. incognita. As much as 12 of the candidate genes turned out to show a significant and reproducible diminution of M. incognita infestation on tomato plants when silenced with siRNAs (namely the candidate genes Minc00801, Minc01632, Minc02483, Minc03313, Minc03866, MincOSOOl, Minc08013, Minc08335, Minc09526, Mincl2224, Mind 7713 and Minc07817). The remaining 4 candidate genes turned out to show a significant but not reproducible reduction of M. incognita infestation when silenced with siRNAs (namely the candidate genes Minc08014, Mincl0706, Mind 4652 and Mind 7987). The nucleotide sequence of the coding region (CDS) of the genes Minc01632, Minc02483, Minc05001, Minc03866, Minc09526, Mind 7713, Minc03313, Minc08335, Minc08013, Minc00801, Mind 2224, Mind 4652, Mind 0706, Minc08014, Mind 7987 and Minc07817 of M. incognita are respectively represented as SEQ ID NO: 235 to 250. The nucleotide sequence of the CDS of the orthologous genes Mh01632, MH02483, MhOSOOl, Mh03866, MH09526, MM7713, Mh03313, Mh08335, Mh08013, Mh00801, MM2224, MM4652, Mhl0706, MH08014, MM7987 and Mh07817 of M. hapla are respectively represented as SEQ ID NO: 251 to 266. In particular, during infestation tests on tomato plants, the inventors have shown that:

- siRNA silencing of candidate genes Minc01632, Minc02483, Minc05001, Minc03866 or Minc09526 induced a significant and reproducible reduction of both the number of galls and egg masses on tomato (the candidate genes are classified by decreasing order of the amplitude of diminution of the number of egg masses),

- siRNA silencing of candidate genes Mind 7713, Minc03313, Minc07817, Minc08335, Minc08013, Minc00801, Mincl2224 induced a significant and reproducible reduction of galls or egg masses on tomato (the candidate genes are classified by decreasing order of the amplitude of diminution of the number of egg masses ), and

- siRNA silencing of candidate genes Mincl4652, Mincl0706, Minc08014 and Mincl 7987 induced a significant but not reproducible reduction of galls or egg masses on tomato (the candidate genes are classified by decreasing order of the effect on the egg mass).

The inventors have also shown in vivo in the model plant A. thaliana that transgenic A. thaliana plants expressing an amiRNA directed against the gene Minc01632 had a reduced plant-parasitic nematode infestation level compared to control plants.

Overall, out of the 34,780 predicted protein-coding genes from the M. incognita and M. hapla whole genomes, the inventors have selected these 1 candidate genes.

The inventors have found that the 15 effector-like proteins comprise a signal peptide sequence including a MERCI effector-specific motif, and no transmembrane region.

As used herein, an effector-like protein refers to a protein that presents characteristics similar to those of known effectors. Effectors are proteins secreted by the nematodes and that support the process of plant parasitism. Approximately 100 confirmed effectors are known from root-knot nematodes (Vens et al., 201 1). Effectors usually bear a signal peptide for secretion and have no transmembrane region. A MERCI peptide motif is frequently found inside the signal peptide of effectors (Vens et al., 2011).

As used herein, a signal peptide refers to a short polypeptide sequence (typically 5-35 amino acid residues), mainly composed of hydrophobic amino acids that have a tendency to form a single alpha-helix. This signal peptide is found at the N-terminal part of the protein and is responsible for directing the protein towards a secretion pathway. In the effector-like proteins, the signal peptide usually corresponds to the first 31 amino acids of the proteins. Methods for identifying a signal peptide in proteins are well-known to a person skilled in the art. By way of example, signal peptides can be detected in root-knot nematode proteins, by using the software SignalP 3.0 (Nielsen et al, 1997) or SignalP 4.0 (Petersen et al., 201 1). In SignalP 3.0 software, predictions of signal peptides are made according to two approaches, Neural Networks (NN) and Hidden Markov Models (HMM): a signal peptide is considered present when its detection is significant according to at least one of the two approaches.

As used herein, a MERCI effector-specific motif refers to a short polypeptide sequence identified as frequently present in the signal peptides of known root-knot nematode effector proteins but absent from those of housekeeping proteins. The MERCI effector-specific motifs are identified using the MERCI (Motif - EmeRging and with Classes-Identification) software (Vens et al, 2011). The MERCI software first searches for conserved sequences of amino acids in the proteins of interest. If no exact conservation of the amino acid itself is found, the MERCI software searches for conservation of the physico-chemical properties of an amino acid at a given position. Using the MERCI software, Vens et al. (2011) have identified a total of 4 MERCI peptide motifs of 12 or 15 amino acid residues in root-knot nematode effectors. MOTIF 1 consists of 15 amino acid residues classified according to the RasMol's classification (Sayle et al, 1995) from the N-terminal to the C-terminal as follows: large, hydrophobic, neutral, buried, neutral, neutral, buried, buried, neutral, acyclic, acyclic, hydrophobic, neutral, acyclic, acyclic. MOTIF 2 consists of 15 amino acid residues classified according to the RasMol's classification from the N-terminal to the C-terminal as follows: neutral, buried, neutral, large, buried, neutral, neutral, neutral, hydrophobic, hydrophobic, neutral, acyclic, acyclic, acyclic, buried. MOTIF 3 consists of 15 amino acid residues classified according to the RasMol's classification from the N-terminal to the C-terminal as follows: hydrophobic, neutral, buried, acyclic, neutral, neutral, neutral, buried, neutral, large, neutral, acyclic, neutral, polar, acyclic. MOTIF 4 consists of 12 amino acid residues classified according to the RasMol's classification from the N-terminal to the C-terminal as follows: neutral, neutral, L (leucine), buried, hydrophobic, buried, neutral, hydrophobic, neutral, neutral, acyclic, neutral. The amino acid sequence of the 15 effector-like proteins identified by the inventors contains at least one of these four MERCI motifs in their signal peptide sequence.

As used herein, a transmembrane region refers to a portion of a protein, which, given the physico-chemical properties of its constituting amino-acids is thermodynamically stable in a cell membrane. In a protein, transmembrane regions reside in the cell membrane. Secreted proteins are usually devoid of transmembrane region.

Transgenic plants expressing siRNAs, and more generally RNAs {e.g., dsRNAs, amiRNAs), capable of driving post-transcriptional inhibition {e.g., post-transcriptional silencing) of one or several of these 16 candidate genes can be produced to obtain plants with a reduced plant-parasitic nematode infestation level.

Accordingly, the present invention provides a method for reducing the plant-parasitic nematode infestation level in a plant, comprising expressing in said plant at least one RNA capable of driving post-transcriptional inhibition of at least one target gene from said nematode,

wherein said gene is selected from the group consisting of a gene encoding an effectorlike protein and a gene encoding a transcription factor,

wherein said effector-like protein:

- comprises a signal peptide including 1, 2, 3 or 4 different MERCI effector-specific motifs selected from the group consisting of:

MOTIF 1: large, hydrophobic, neutral, buried, neutral, neutral, buried, buried, neutral, acyclic, acyclic, hydrophobic, neutral, acyclic, acyclic,

MOTIF 2: neutral, buried, neutral, large, buried, neutral, neutral, neutral, hydrophobic, hydrophobic, neutral, acyclic, acyclic, acyclic, buried,

MOTIF 3: hydrophobic, neutral, buried, acyclic, neutral, neutral, neutral, buried, neutral, large, neutral, acyclic, neutral, polar, acyclic, and

MOTIF 4: neutral, neutral, L, buried, hydrophobic, buried, neutral, hydrophobic, neutral, neutral, acyclic, neutral,

- comprises no transmembrane region, and - consists of a polypeptide sequence having at least 35% identity, or by order of increasing preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and preferably having 100% identity, with a polypeptide selected from the group consisting of SEQ ID NO. 1 (Minc01632), SEQ ID NO. 2 (Minc02483), SEQ ID NO. 3 (Minc05001), SEQ ID NO. 4 (Minc03866), SEQ ID NO. 5 (Minc09526), SEQ ID NO. 6 (Mincl7713), SEQ ID NO. 7 (Minc03313), SEQ ID NO. 8 (Minc08335), SEQ ID NO. 9 (Minc08013), SEQ ID NO. 10 (Minc00801), SEQ ID NO. 11 (Mincl2224), SEQ ID NO. 12 (Mincl4652), SEQ ID NO. 13 (Mincl0706), SEQ ID NO. 14 (Minc08014) and SEQ ID NO. 15 (Mincl7987), preferably with a polypeptide selected from the group consisting of SEQ ID NO. 1 (Minc01632), SEQ ID NO. 2 (Minc02483), SEQ ID NO. 3 (Minc05001), SEQ ID NO. 4 (Minc03866), SEQ ID NO. 5 (Minc09526), SEQ ID NO. 6 (Mincl7713), SEQ ID NO. 7 (Minc03313), SEQ ID NO. 8 (Minc08335), SEQ ID NO. 9 (Minc08013), SEQ ID NO. 10 (Minc00801), SEQ ID NO. 11 (Mincl2224), more preferably with a polypeptide selected from the group consisting of SEQ ID NO. 1 (Minc01632), SEQ ID NO. 2 (Minc02483), SEQ ID NO. 3 (Minc05001), SEQ ID NO. 4 (Minc03866), SEQ ID NO. 5 (Minc09526), most preferably with the polypeptide of sequence SEQ ID NO. 1 (Minc01632), and

wherein said transcription factor consists of a polypeptide sequence having at least 35% identity, or by order of increasing preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and preferably having 100% identity, with the polypeptide of sequence SEQ ID NO. 16 (Minc07817).

Unless otherwise specified, the percents of identity between two sequences which are mentioned herein are calculated from an alignment of the two sequences over the whole length of any of the two sequences, preferably over the whole length of the two sequences. By way of example, one can use the « needle » program (Needleman et al., 1970).

The proteins of SEQ ID NO. 1 to 16 of M. incognita are respectively encoded by the CDS of SEQ ID NO. 235 to 250.

As used herein, at least one target gene means 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or 16 target genes, preferably 1, 2, 3, 4, 5 or 6 target genes, more preferably 1, 3 or 6 target genes.

In an advantageous embodiment, the method for reducing the plant-parasitic nematode infestation level in a plant, comprises expressing in said plant two RNAs capable of driving post-transcriptional inhibition of two target genes respectively from said nematode, wherein said two target genes respectively encode two effector-like proteins as defined above selected from the group consisting of a polypeptide sequence having at least 35% identity, or by order of increasing preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and preferably having 100% identity, with a polypeptide selected from the group consisting of SEQ ID NO. 1 (Minc01632), SEQ ID NO. 2 (Minc02483), SEQ ID NO. 4 (Minc03866), SEQ ID NO. 5 (Minc09526), SEQ ID NO. 8 (Minc08335) and SEQ ID NO. 10 (Minc00801). Preferably, said two target genes encode two effector-like proteins as defined above consisting of a polypeptide sequence having at least 35% identity, or by order of increasing preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and preferably having 100% identity, with the polypeptide of SEQ ID NO. 1 (Minc01632) and SEQ ID NO. 2 (Minc02483) respectively.

In another advantageous embodiment, the method for reducing the plant-parasitic nematode infestation level in a plant, comprises expressing in said plant three RNAs capable of driving post-transcriptional inhibition of three target genes respectively from said nematode, wherein said genes respectively encode three effector-like proteins as defined above selected from the group consisting of a polypeptide sequence having at least 35% identity, or by order of increasing preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and preferably having 100% identity, with a polypeptide selected from the group consisting of SEQ ID NO. 1 (Minc01632), SEQ ID NO. 2 (Minc02483), SEQ ID NO. 4 (Minc03866), SEQ ID NO. 5 (Minc09526), SEQ ID NO. 8 (Minc08335) and SEQ ID NO. 10 (Minc00801). Preferably, said three target genes encode three effector-like proteins as defined above consisting of a polypeptide sequence having at least 35% identity, or by order of increasing preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and preferably having 100% identity, with the polypeptide of SEQ ID NO. 2 (Minc02483), SEQ ID NO. 4 (Minc03866) and SEQ ID NO. 10 (Minc00801) respectively.

Advantageously, the amino acid sequence of the signal peptide including a MERCI effector-specific motif of an effector-like protein according to the present invention is selected from the group consisting of SEQ ID NO. 38 to 52 (corresponding to the signal peptide present in the amino acid sequence of the proteins Minc01632, Minc02483, Minc05001, Minc03866, Minc09526, Mincl7713, Minc03313, Minc08335, Minc08013, Minc00801, Mincl2224, Mincl4652, Mincl0706, Minc08014 and Mincl7987 respectively.

Advantageously, the amino acid sequence of the MERCI effector-specific motif is selected from the group consisting of SEQ ID NO. 54 (MOTIF 4 present in the amino acid sequence of the protein Minc01632), 55 (MOTIF 1 present in the amino acid sequence of the protein Minc01632), 56 (MOTIF 1 present in the amino acid sequence of the protein Minc02483), 57 (MOTIF 3 present in the amino acid sequence of the protein Minc05001), 58 (MOTIF 3 present in the amino acid sequence of the protein Minc03866), 59 (MOTIF 1 present in the amino acid sequence of the protein Minc03866), 60 (MOTIF 1 present in the amino acid sequence of the protein Minc09526), 61 (MOTIF 1 present in the amino acid sequence of the protein Minc09526), 62 (MOTIF 1 present in the amino acid sequence of the protein Mincl7713), 63 (MOTIF 3 present in the amino acid sequence of the protein Mincl7713), 64 (MOTIF 3 present in the amino acid sequence of the protein Minc03313), 65 (MOTIF 2 present in the amino acid sequence of the protein Minc08335), 66 (MOTIF 4 present in the amino acid sequence of the protein Minc08335), 67 (MOTIF 1 present in the amino acid sequence of the protein Minc08013), 68 (MOTIF 1 present in the amino acid sequence of the protein Minc08013), 69 (MOTIF 2 present in the amino acid sequence of the protein Minc00801), 70 (MOTIF 1 present in the amino acid sequence of the protein Mine 12224), 71 (MOTIF 2 present in the amino acid sequence of the protein Mine 12224), 72 (MOTIF 2 present in the amino acid sequence of the protein Mincl2224), 73 (MOTIF 4 present in the amino acid sequence of the protein Mincl4652), 74 (MOTIF 1 present in the amino acid sequence of the protein Mincl 0706), 75 (MOTIF 2 present in the amino acid sequence of the protein Minc08014), 76 (MOTIF 2 present in the amino acid sequence of the protein Minc08014), 77 (MOTIF 4 present in the amino acid sequence of the protein Minc08014) and 78 (MOTIF 1 present in the amino acid sequence of the protein Mine 17987).

The inventors have determined that the effector-like proteins Minc01632, Minc02483,

Minc05001, Minc03866, Minc09526, Mincl7713, Minc03313, Minc08335, Minc08013, Minc00801 , Mincl2224, Mincl4652, Mincl0706, Minc08014 and Mincl7987 of M. incognita as described above show respectively 58%, 80%, 74%, 60%, 47%, 81%, 95%, 79%, 60%, 87%, 79%, 68%, 68%, 63% and 73% identity with the heterologous effector-like proteins in M, hapla (Mhap01632, Mhap02483, Mhap05001 , Mhap03866, Mhap09526, Mhapl7713, Mhap03313, Mhap08335, Mhap08013, Mhap00801, Mhapl2224, Mhapl4652, Mhapl0706, Mhap08014 and Mhapl7987 respectively). Said heterologous effector-like proteins in M. hapla are referred to as SEQ ID NO. 17 to 31 respectively. They are respectively encoded by the CDS of SEQ ID NO. 251 to 266.

The inventors have determined that the transcription factor Minc07817 of M. incognita shows 81 ) identity with the heterologous transcription factor in M. hapla (Mhap07817). Said heterologous transcription factor in M. hapla of Minc07817 is referred to as SEQ ID NO. 32.

The inventors have determined that the effector-like proteins Minc01632, Minc02483, Minc05001 and Minc03313 of M. incognita as described above show respectively 44%, 44%, 35% and 66% identity with the orthologous effector-like proteins in the cyst nematode Globodera pallida (Gpal01632, Gpal02483, Gpal05001 and Gpal03313 respectively). Said heterologous proteins in G. pallida are referred to as SEQ ID NO. 33 to 36 respectively.

The nucleotide sequence of the CDS of the genes Gpal01632, Gpal02483, Gpal05001, Gpal03313 and Gpal07817 of Globodera pallida are respectively represented as SEQ ID NO: 267 to 271.

The inventors have determined that the transcription factor Minc07817 of M. incognita shows 38% identity with the orthologous transcription factor in G. pallida (Gpal07817). Said heterologous transcription factor in G. pallida of Minc07817 is referred to as SEQ ID NO. 37.

Advantageously, the RNA capable of driving post- transcriptional inhibition of at least one target gene from a nematode is selected from the group consisting of a sense RNA (for co-suppression), an antisense RNA, a dsRNA and an amiRNA, preferably an amiRNA.

Preferred RNAs capable of driving post-transcriptional inhibition of at least one target gene are dsRNAs capable of driving the post-transcriptional silencing of the targeted gene with homologous sequences. Post transcriptional gene silencing is induced by means of the cellular mechanism called RNA interference (RNAi). Various methods and DNA constructs for RNAi are available in the art (for review, Watson et al, 2005). Typically, DNA constructs for delivering a dsRNA in a plant include at least a fragment of at least 15 contiguous nucleotides of the cDNA of the target gene, under transcriptional control of a promoter active in said plant. Currently, the more widely used DNA constructs are those encoding a dsRNA that produces - through cleavage by RNases into the plant cell - a small interfering RNA (siRNA), a hairpin RNA (hpRNA) or an artificial microRNA (amiRNA). In these constructs, the fragment of the target gene is inversely repeated, with generally a linker region between the repeats.

Transgenic plants expressing a dsRNA targeting a gene from a plant-parasitic nematode are known in the art (see for review Rosso et al, 2009; Atkinson et al, 2012; Kozub et al, 2012; Schwab et al, 2006). In particular, transgenic plants expressing a dsRNA targeting a gene from a root-knot nematode have been obtained in Arabidopsis (Huang et al, 2006), tomato (Charlton et al, 2010), tobacco (Yadav B.C. et al, 2006) or soybean (Ibrahim et al, 2011). Transgenic plants expressing a dsRNA targeting a gene from cyst nematode have been obtained in Arabidopsis (Patel et al. , 2008 and 2010; Sindhu et al. , 2009).

Advantageously, the dsRNA targeting a gene as defined above comprises a first RNA sequence of 18 to 25 nucleotides, preferably of 19 to 24 nucleotides, more preferably of 19, 20 or 21 nucleotides, wherein said first RNA sequence is encoded by a 18 to 25 nucleotide sequence, preferably of 19 to 24 nucleotide sequence, more preferably by a 19, 20 or 21 nucleotide sequence, having at least 90% identity, and by order of increasing preference, at least 95%, 96%, 97%, 98%, or 99 % identity, and preferably 100% identity, with a 18 to 25 contiguous nucleotide fragment, preferably of 19 to 24 contiguous nucleotide fragment, more preferably by a 19, 20 or 21 contiguous nucleotide fragment of said target gene, and a second RNA sequence that is the complementary of said first RNA sequence, said first and second sequences being in opposite orientations, and optionally linked with a spacer sequence (a linker region).

According to another preferred embodiment of the invention the dsRNA targeting a gene as defined above produces (through cleavage by the DICER RNase into a plant cell) a siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises a 15 to 30 nucleotide sequence having at least 90% identity, or by order of increasing preference at least 95%, 96%, 97%, 98% or 99% identity, or having 100% identity, with a 15 to 30 contiguous nucleotide fragment of said gene, and the antisense strand comprises a 15 to 30 nucleotide sequence that is complementary to the sense strand.

Advantageously, the sense strand of the siRNA comprises a 19 to 24 nucleotide sequence, preferably a 19, 20 or 21 nucleotide sequence, that is complementary to a 19 to 24 nucleotide sequence, preferably a 19, 20 or 21 nucleotide sequence, respectively of the antisense strand of said siRNA.

The siRNA can comprise blunt end(s) or 1 to 10 overhanging nucleotides at the 5' and/or 3' end of each strand. Advantageously, the siRNA comprises two overhanging thymine nucleotides (TT) at the 3' end of each strand.

The sense strand and the antisense strand of the siRNA can be connected together via a 1 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) nucleotide linker.

According to another preferred embodiment of the invention, the dsRNA targeting a gene as defined above produces (through cleavage by the Dicer-Likel RNase inside the nucleus plant cell) an amiRNA consisting of a 19 to 24 nucleotide, preferably a 21 nucleotide single stranded RNA having 0 to 5 mismatch, preferably 0 mismatch (i.e., 100% identity) with a 19 to 24 contiguous nucleotide fragment, preferably a 21 contiguous nucleotide fragment, of the mRNA encoded by said gene.

In a particular embodiment of dsRNAs capable of producing a siRNA in the plant cell it can be mentioned:

- the dsRNA targeting the gene encoding SEQ ID NO. 1 (Minc01632) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 79 (GGTTCAGAGATTCCTTGTC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 80 (GACAAGGAATCTCTGAACC),

- the dsRNA targeting the gene encoding SEQ ID NO. 2 (Minc02483) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 81 (TTGACAACCAATTGCCGTT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 82 (AACGGCAATTGGTTGTCAA),

- the dsRNA targeting the gene encoding SEQ ID NO. 3 (Minc05001) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 83 (ATGACGGCATTATCCGTTC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 84 (GAACGGATAATGCCGTCAT),

- the dsRNA targeting the gene encoding SEQ ID NO. 4 (Minc03866) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 85 (GCAGGAGTTGTATGCAAGC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 86 (GCTTGCATACAACTCCTGC),

- the dsRNA targeting the gene encoding SEQ ID NO. 5 (Minc09526) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 87

(GAGATAGAAGTGAGTGTAC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 88 (GTACACTCACTTCTATCTC),

- the dsRNA targeting the gene encoding SEQ ID NO. 6 (Mincl7713) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 89 (TCCACATTATCGCGCAGGA) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 90 (TCCTGCGCGATAATGTGGA),

- the dsRNA targeting the gene encoding SEQ ID NO. 7 (Minc03313) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 91 (CGCTCCATGCATTAGGTTC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 92 (GAACCTAATGCATGGAGCG), - the dsRNA targeting the gene encoding SEQ ID NO. 8 (Minc08335) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 93 (CGACAGCGAAGAAGACTAC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 94 (GTAGTCTTCTTCGCTGTCG),

- the dsRNA targeting the gene encoding SEQ ID NO. 9 (Minc08013) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 95 (AGAACGGATCAATCGAGCA) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 96 (TGCTCGATTGATCCGTTCT),

- the dsRNA targeting the gene encoding SEQ ID NO. 10 (Minc00801) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 97

(ACGTTCACAGAACTCTTAC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 98 (GTAAGAGTTCTGTGAACGT),

- the dsRNA targeting the gene encoding SEQ ID NO. 11 (Mine 12224) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 99 (GACGAAGCTGGTCAATGCT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 100 (AGCATTGACCAGCTTCGTC),

- the dsRNA targeting the gene encoding SEQ ID NO. 12 (Mine 14652) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 101 (CAAGCAATGCTGTTCATGC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 102 (GCATGAACAGCATTGCTTG),

- the dsRNA targeting the gene encoding SEQ ID NO. 13 (Mincl0706) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 103 (TGGCATTCAAGTTGGTCCT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 104 (AGGACCAACTTGAATGCCA),

- the dsRNA targeting the gene encoding SEQ ID NO. 14 (Minc08014) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 105 (ATCCGGCAAGTCTGCTTGT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 106 (ACAAGCAGACTTGCCGGAT),

- the dsRNA targeting the gene encoding SEQ ID NO. 15 (Mincl7987) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 107

(AGGACAACAAGGAGGCATT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 108 (AATGCCTCCTTGTTGTCCT),

- the dsRNA targeting the gene encoding SEQ ID NO. 16 (Minc07817) comprising a sense strand encoded by the nucleotide sequence SEQ ID NO. 109 (GGATGAACCGTTGGAACAA) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 110 (TTGTTCC AACGGTTC ATCC) .

In another particular embodiment of dsRNAs capable of producing an amiRNA in the plant cell it can be mentioned: - the dsRNA targeting the gene encoding SEQ ID NO. 1 (Minc01632) comprising a strand encoded by the nucleotide sequence SEQ ID NO. 229 (AAGACAAGGAATCTCTGAACC),

- the dsRNA targeting the gene encoding SEQ ID NO. 2 (Minc02483) comprising a strand encoded by the nucleotide sequence SEQ ID NO. 230

(AAAACGGCAATTGGTTGTCAA),

- the dsRNA targeting the gene encoding SEQ ID NO. 4 (Minc03866) comprising a strand encoded by the nucleotide sequence SEQ ID NO. 231 (AAGCTTGCATACAACTCCTGC),

- the dsRNA targeting the gene encoding SEQ ID NO. 5 (Minc09526) comprising a strand encoded by the nucleotide sequence SEQ ID NO. 233 (AAGTACACTCACTTCTATCTC),

- the dsRNA targeting the gene encoding SEQ ID NO. 8 (Minc08335) comprising a strand encoded by the nucleotide sequence SEQ ID NO. 232 (AAGTAGTCTTCTTCGCTGTCG),

- the dsRNA targeting the gene encoding SEQ ID NO. 10 (Minc00801) comprising a strand encoded by the nucleotide sequence SEQ ID NO. 228 (AAGTAAGAGTTCTGTGAACGT).

The plant-parasitic nematode is preferably a root-knot nematode (genus Meloidogyne) or a cyst nematode such as a nematode of the genus Globodera (in particular G. pallid), preferably a root-knot nematode.

Advantageously, the nematode is selected from the group consisting of M. javanica, M. arenaria, M. incognita and M. hapla, preferably M. incognita.

The present invention applies to monocot- or dicotyledon plants of agronomical or ornamental interest, such as alfalfa, banana, barley, beans, cotton, cucurbits, flax, fruit trees, maize, oats, peas, potato, rape, rice, rye, sorghum, soybean, sugar cane, sugarbeet, sunflower, tobacco, tomato, triticale, wheat.

The present invention also provides an isolated RNA for silencing a gene from a nematode encoding an effector-like protein or a transcription factor as defined above, comprising or consisting of a 15 to 30 nucleotide, preferably 18 to 25 nucleotide, more preferably 19 to 21 nucleotide RNA having at least 90% identity, or by order of increasing preference at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and preferably having 100% identity, with respectively a 15 to 30 contiguous nucleotide fragment, preferably 18 to 25 contiguous nucleotide fragment, more preferably 19 to 21 contiguous nucleotide fragment of said target gene.

Preferably, said target gene encodes a protein selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 16.

In particular, said isolated RNA consists of a dsRNA as described above, preferably an isolated siRNA as described above. By way of example, said isolated dsRNA targeting a gene from a nematode encoding an effector-like protein or a transcription factor, selected from the group consisting of:

- a dsRNA targeting the gene encoding SEQ ID NO. 1 (Minc01632) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 79 (GGTTCAGAGATTCCTTGTC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 80 (GACAAGGAATCTCTGAACC),

- a dsRNA targeting the gene encoding SEQ ID NO. 2 (Minc02483) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 81 (TTGACAACCAATTGCCGTT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 82 (AACGGCAATTGGTTGTCAA),

- a dsRNA targeting the gene encoding SEQ ID NO. 3 (Minc05001) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 83 (ATGACGGCATTATCCGTTC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 84 (GAACGGATAATGCCGTCAT),

- a dsRNA targeting the gene encoding SEQ ID NO. 4 (Minc03866) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 85 (GCAGGAGTTGTATGCAAGC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 86 (GCTTGCATACAACTCCTGC),

- a dsRNA targeting the gene encoding SEQ ID NO. 5 (Minc09526) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 87

(GAGATAGAAGTGAGTGTAC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 88 (GTACACTCACTTCTATCTC),

- a dsRNA targeting the gene encoding SEQ ID NO. 6 (Mincl7713) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 89 (TCCACATTATCGCGCAGGA) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 90 (TCCTGCGCGATAATGTGGA),

- a dsRNA targeting the gene encoding SEQ ID NO. 7 (Minc03313) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 91 (CGCTCCATGCATTAGGTTC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 92 (GAACCTAATGCATGGAGCG),

- a dsRNA targeting the gene encoding SEQ ID NO. 8 (Minc08335) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 93 (CGACAGCGAAGAAGACTAC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 94 (GTAGTCTTCTTCGCTGTCG),

- a dsRNA targeting the gene encoding SEQ ID NO. 9 (Minc08013) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 95 (AGAACGGATCAATCGAGCA) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 96 (TGCTCGATTGATCCGTTCT),

- a dsRNA targeting the gene encoding SEQ ID NO. 10 (Minc00801) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 97 (ACGTTCACAGAACTCTTAC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 98 (GTAAGAGTTCTGTGAACGT),

- a dsRNA targeting the gene encoding SEQ ID NO. 11 (Mincl2224) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 99 (GACGAAGCTGGTCAATGCT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 100 (AGCATTGACCAGCTTCGTC),

- a dsRNA targeting the gene encoding SEQ ID NO. 12 (Mincl4652) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 101 (CAAGCAATGCTGTTCATGC) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 102 (GC ATGAAC AGCATTGCTTG),

- a dsRNA targeting the gene encoding SEQ ID NO. 13 (Mincl0706) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 103 (TGGCATTCAAGTTGGTCCT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 104 (AGGACCAACTTGAATGCCA),

- a dsRNA targeting the gene encoding SEQ ID NO. 14 (Minc08014) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 105 (ATCCGGCAAGTCTGCTTGT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 106 (ACAAGCAGACTTGCCGGAT),

- a dsRNA targeting the gene encoding SEQ ID NO. 15 (Mincl7987) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 107

(AGGACAACAAGGAGGCATT) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 108 (AATGCCTCCTTGTTGTCCT),

- a dsRNA targeting the gene encoding SEQ ID NO. 16 (Minc07817) comprises a sense strand encoded by the nucleotide sequence SEQ ID NO. 109 (GGATGAACCGTTGGAACAA) and an antisense strand encoded by the nucleotide sequence SEQ ID NO. 110 (TTGTTCCAACGGTTCATCC).

The present invention also provides an isolated amiRNA for silencing a gene from a nematode encoding an effector-like protein as defined above selected from the group consisting of:

- the amiRNA targeting the gene encoding SEQ ID NO. 1 (Minc01632) of sequence

SEQ ID NO. 229 (AAGACAAGGAATCTCTGAACC),

- the amiRNA targeting the gene encoding SEQ ID NO. 2 (Minc02483) of sequence SEQ ID NO. 230 (AAAACGGCAATTGGTTGTCAA),

- the amiRNA targeting the gene encoding SEQ ID NO. 4 (Minc03866) of sequence SEQ ID NO. 231 (AAGCTTGCATACAACTCCTGC),

- the amiRNA targeting the gene encoding SEQ ID NO. 5 (Minc09526) of sequence SEQ ID NO. 233 (AAGTACACTCACTTCTATCTC),

- the dsRNA targeting the gene encoding SEQ ID NO. 8 (Minc08335) of sequence SEQ ID NO. 232 (AAGTAGTCTTCTTCGCTGTCG), - the amiRNA targeting the gene encoding SEQ ID NO. 10 (Minc00801) of sequence SEQ ID NO. 228 (A AGTA AG AGTTCTGTG A ACGT) .

The present invention also provides a composition comprising an isolated RNA for silencing a gene from a nematode encoding an effector-like protein or a transcription factor as defined above and at least one suitable carrier, excipient or diluents.

This composition can be in the form suitable for ingestion by a plant-parasitic nematode, e.g., in solid, liquid or gel form.

This composition can further comprise a pesticide, herbicide or insecticide agent.

Advantageously, this composition is suitable for reducing the plant-parasitic nematode infestation level in a plant.

The present invention also provides the use of an isolated RNA or a composition according to the present invention for reducing the plant-parasitic nematode infestation level in a plant.

The present invention also provides a method for reducing the plant-parasitic nematode infestation level in a plant comprising contacting said plant-parasitic nematode with an effective amount of an isolated RNA or a composition according to the present invention.

Said isolated RNA can be a small interfering RNA (siRNA), a hairpin RNA (hpRNA) or an artificial microRNA (amiRNA), as described above.

Said isolated RNA can be present in irrigation water of said plant.

The present invention also provides a chimeric DNA construct encoding a dsRNA for silencing a target gene as defined above, comprising:

- a promoter functional in a plant cell;

- a first DNA sequence of 18 to 25 bp, preferably of 19 to 24 bp, more preferably of 19, 20 or 21 bp, consisting of a fragment of a cDNA encoded by said target gene, or having at least 90% identity, and by order of increasing preference, at least 95%, 96%, 97%, 98%, or 99% identity, and preferably 100% identity, with said fragment, said DNA sequence being placed under transcriptional control of said promoter;

- a second DNA sequence that is the complementary of said first DNA, said first and second sequences being in opposite orientations;

- a spacer sequence separating said first and second sequence, such that these first and second DNA sequences are capable, when transcribed, of forming a single double-stranded RNA molecule.

The spacer can be a random fragment of DNA.

A large choice of promoters suitable for expression of heterologous genes in plants is available in the art.

They can be obtained for instance from plants, plant viruses, or bacteria such as those of the Agrobacterium lineage. They include constitutive promoters, i.e. promoters which are active in most tissues and cells and under most environmental conditions, tissue or cell specific promoters which are active only or mainly in certain tissues {e.g. root cells) or certain cell types (e.g. giant cells), and inducible promoters that are activated by physical or chemical stimuli, such as those resulting from nematode infection.

One can use constitutive promoters that are commonly used in plant cells such as the cauliflower mosaic virus (CaMV) 35S promoter, the Nos promoter and the rubisco promoter.

However, the most suitable promoters for silencing a target gene as defined above in a plant in order to reduce the plant-parasitic nematode infestation level are promoters that are tissue-specific, having a preferential or exclusive expression in the tissues infected by the nematodes, in particular the giant cells, and/or promoters that are inducible by nematode infection, i.e. promoters that drive an increased level of expression in cells infected by nematodes, when compared to non-infected cells. Said promoters may be naturally occurring promoter, may comprise a nematode responsive element isolated from a naturally occurring promoter, or may be a synthetic promoter.

Non-limitative examples of preferred promoters for carrying out the invention include:

- the elongation specific endo-l,4-p-glucanase (cell) promoter of Arabidopsis thaliana, which is described in U.S. Patent No. 7,1 19,254 as being up-regulated in root-knot nematode feeding cells;

- the promoter of the POX gene of A. thaliana (Vercauteren et al, 2001);

- the promoter of the Trypsin inhibitor gene of A. thaliana (Vercauteren et al., 2001);

- the promoter of the endomembrane protein gene of A. thaliana (Vercauteren et al., 2001);

- the promoter of the DAP decarboxylase gene of A. thaliana (Vercauteren et al, 2001);

- the promoter of the WRKY23 (AttOOOl) gene of A. thaliana (Barthels et al., 1997);

- the promoter of the AtFH6 gene (Favery et al., 2004), also disclosed in International Application WO 2005/063989 (SEQ ID NO: 1 of WO 2005/063989).

The DNA constructs of the invention generally also include a transcriptional terminator (for instance the 35S transcriptional terminator, or the nopaline synthase (Nos) transcriptional terminator).

These DNA constructs can be obtained and introduced in a host cell or organism by the well-known techniques of recombinant DNA and genetic engineering. The choice of the recombinant vector depends on the intended host and on the intended method of transformation of said host. A variety of methods for genetic transformation of plant cells or plants are available in the art for many plant species, dicotyledons or monocotyledons. By way of non-limitative examples, one can mention virus mediated transformation, transformation by microinjection, by electroporation, microprojectile mediated transformation, Agrobacterium mediated transformation, and the like.

The invention also comprises a plant cell or a plant genetically modified by a construct of the invention. The polynucleotide may be transiently expressed; it can also be incorporated in a stable extrachromosomal replicon, or integrated in the chromosome. In particular, the invention relates to a transgenic plant containing a transgene comprising a chimeric DNA construct of the invention. The expression of said chimeric DNA construct, resulting in decreased level of expression of a target gene as defined above, provides to said transgenic plant a reduction of the plant-parasitic nematode infestation level when compared with a plant devoid of said transgene.

The invention also encompasses isolated organs or tissues of said plants (such as seeds, leafs, flowers, roots).

The present invention also provides a method for screening a candidate compound that can be used for reducing the plant-parasitic nematode infestation level in a plant comprising the steps of:

- contacting a candidate compound with a plant-parasitic nematode expressing an effector-like protein or a transcription factor as defined above;

- determining whether the candidate compound modulates the expression of the gene encoding said effector-like protein or said transcription factor;

wherein said candidate compound that decreases the expression of the gene encoding said effector-like protein or said transcription factor is identified as a candidate compound capable of reducing the plant-parasitic nematode infestation level in a plant.

The step of contacting a candidate compound with a plant-parasitic nematode can be carried out by incubating the nematode in a solution containing candidate compound using the soaking method.

The plant-parasitic nematode is preferably a root-knot nematode (genus Meloidogyne) or a cyst nematode such as a nematode of the genus Globodera (in particular G. pallida), preferably a root-knot nematode. Advantageously, it is selected from the group consisting of M.javanica, M. arenaria, M. incognita and hapla, preferably M. incognita.

Methods for determining the expression of a gene in a nematode are well known to a person skilled in the art. For instance, expression of a gene coding for an effector-like protein or a transcription factor as defined above can be determined by quantifying the effector-like protein or the transcription factor as defined above in a cell lysate, by means of antibodies specific for said effector-like protein or transcription factor, chromatography techniques, mass spectrometry techniques. Expression of a gene encoding an effector-like protein or a transcription factor as defined above can also be determined by quantifying mRNA encoded by said effector-like gene or transcription factor gene, including quantitative RT-PCR using primers specifically hybridizing to said mRNA.

The candidate compound can be a dsRNA, a siRNA, a hpRNA, an amiRNA or organic chemicals.

Foregoing and other objects and advantages of the invention will become more apparent from the following detailed description and accompanying drawings. It is to be understood however that this foregoing detailed description is exemplary only and is not restrictive of the invention.

Figure 1: Transcript abundance percentage change in M. incognita infective juveniles soaked in a siRNA solution relative to control. siRNAs induced significant change in the targeted transcripts expression level. Transcript level for each of the targeted gene was measured by qPCR 24h after soaking treatment and compared to their transcript level in control sample treated with siRNA targeting no sequence in the M. incognita genome.

Figure 2: Transcript level measured 16h after soaking to test bounce effect. qPCR analysis was performed 16h after soaking on Minc07817, Minc03866 and Minc05001 treated with siRNA.

Figure 3: Effect of siRNA on nematode mobility / viability. Controls are J2 larvae soaked with siRNA targeting no sequence in the M. incognita genome, accession numbers indicate M. incognita genes targeted by siRNAs. Viability was assessed lh (A) and 16h (B) after soaking by counting the number of dead nematodes (1 ^st bars = far left bars), the number of individuals with movements restricted to extremities (2 ^nd bars), movements on the whole length of the body (3 ^rd bars) and fast undulations (4 ^th bars = far right bars) under microscope observation. Three independent replicates were analyzed. Error bars represent standard error of the mean.

Figure 4: Effect of siRNA on nematode infestation. Controls are J2 larvae soaked with siRNA targeting no sequence in the M. incognita genome, accession numbers indicate M. incognita genes targeted by siRNAs. Infection tests were performed in duplicate represented as left bars for the first replicate and right bars for the second replicate. Variations of number of galls and of numbers of egg masses, measured six weeks after inoculation, are represented in the top panels and in the bottom panel, respectively. Error bars were calculated by standard error of the mean (SEM). P-Value above each bar indicates measures statistically different from controls. A significant change between the two duplicates is represented by p-values above a line spanning duplicates. P-value signification codes are as follows: 0.0001 '***', 0.001 '**', 0.01 '*', 0.05

Figure 5: Table showing the percent reduction in number of egg masses and galls after siRNA treatment for the 16 target genes.

Figure 6: Organization of six Mine genes studied and position of the target sequences for siRNA and amiRNA. The untranslated 5 'and 3' regions are represented by white boxes, exons by black boxes, introns by broken lines and the sequence targeted by siRNA / amiRNA by a grey line.

EXAMPLE I: EXPERIMENTAL VALIDATION OF THE 16 TARGET GENES BY M. INCOGNITA INFESTATION ON TOMATO PLANTS AFTER GENE SILENCING

Materials and Methods

siRNA design and siRNA treatment for viability tests and infection assays

siRNAs (see Table 1 below) were prepared using the Silencer® siRNA Construction Kit (AMI 620, Ambion®, Austin, TX) and siRNA yields were determined using a NanoDrop 2000 spectrophotometer (NanoDrop® Products, Wilmington, DE). Table 1. Small interfering RNAs used to silence M. incognita target genes

F = Forward oligomer, R= Reverse oligomer.

For control it was used a siRNA designed to have no sequence similarity in the M. incognita genome (Dalzell et al, 2010a) as confirmed by blastn searches. siRNAs were conserved at -80°C in 2 μg aliquots until use. Eggs of M. incognita were collected from tomato plants (Solanum esculentum cv. St Pierre) cultured in greenhouse. Eggs were collected as described by Rosso et al. (1999) and J2s were hatched in water. About 10,000 J2s were soaked in 40 μΐ final volume of spring water in the presence of 0.05 mg/ml siRNA for 1 hour. Worms were washed twice with water by centrifugation at 10,000 g for 1 min and suspended in 100 μΐ of water. For viability assay, the number of dead J2s was counted under microscope observation on 100 individuals. It was also reported movement quality and rapidity for 25 individuals. For movement quality it was observed extremity and whole length movement and the quality of movement was noticed by rapidity of undulation or absence of undulation. For infection assays, roots of 24 tomato plants aged of four weeks were each inoculated with 250 M. incognita infective J 2 larvae, previously incubated over night in a water solution containing siRNAs as described by Arguel et al. (2012). Two replicates of the infection assays were performed at three weeks intervals. Galls and egg masses were counted six weeks after inoculation. Statistical analyses were performed by an ANOVA test using the R software.

Quantitative-PCR

RNA was isolated from approximately 500 J2s soaked for lhour in 0.05 mg ml siRNA and incubated for 16h or 24h in water, using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA). Purity and concentration of the RNA was determined on a NanoDrop_2000 spectrophotometer (NanoDrop Products, Wilmington, DE). Reverse transcription was carried out using the iScript cDNA Synthesis Kit (Bio-Rad laboratories, Marnes la Coquette, France). The primers for qPCR were determined using primer3 software (Rozen et al, 2000) and synthesised by Eurogentec (Seraing, Belgium) (see Table 2 below).

Table 2. Sequences of oligonucleotides used for qPCRs for M. incognita target and control genes

target gene primer primer sequence (5'- 3') SEQ ID amplicon name NO. length (bp)

Minc07817 N48b ATGGCTACAATACCTCCAAATAAT 143 124

N49b TAATTGTCTGTTCTGTCTGCCAAG 144

Minc00801 NF64 CTATGGAGATGAGGCAGGACAT 145 248

NR65 GTGAACGTTTCTTGCGGTATC 146

Minc01632 NF66 ATTGCTCTTTTGTGTTTAGAAATGG 147 155

NR67 CAAGGAATCTCTGAACCTTTGAAAT 148

Minc02483 F68 CGAAATTTAATGCGGATTGG 149 167

R69 TGTGGGCGATATGGCTTAAT 150

Minc03313 NF70 TGCCCACAAAAATATAGAAGAAAAT 151 133

NR71 AGATAACCACTGCTTTCCTCCATT 152

Minc03866 F72 AGCTGATCGTGCTGTCATTG 153 127

R73 ACAGAAAGACGCGCAATCAC 154

MincOSOOl NF74 TGTTACTGTCCAATCAGAAAATGAA 155 106

NR75 ACATATCCCATGTTGTGGACTAACT 156

Minc08013 F76b AGATCCACCCTTAGAAAGCCATAG 157 99

R77b TTGTCGTAAAGTCTTGTGGTGATAG 158 Minc08014 F78 TCCAGCGTTCTATGATTGTAATTG 159 108 R79 CACTGCTTAGGAATATCAATGCTC 160

Minc08335 NF80 TTATTATTAATTTCCCAAAGCCAATC 161 159

FR81 TATATTTTGTTCCCCTTCCTCCTTA 162

Minc09526 NF82 AGTGAGTGTACAGGATCAACATCTG 163 168

NR83 GCTGACATTTGCTGTCAGAGTATTA 164

Mincl0706 F84 CAGGTCTAACAGCCTCCTCAA 165 135

R85 CAGTTCCTTGTGGCATGAAT 166

Mincl2224 F86 TGGAGCTGTTACACCAGCAG 167 225

R87 AAGAATGACCGTTGGAGTGC 168

Mincl4652 F88 TATCAACCAGCAGCCACTCA 169 230

R89 CGTTGTCTGTGCCTTGAAGA 170

Mind 7713 F92 TACACCGGTCAGCAACATTC 171 89

R93 AGATTCCTTTGCCCTTCTGG 172

Mincl 7987 F90 CTCCAGGTGAGGATGACGAG 173 238

R91 ATGCCTGGTTGTCCAGTACC 174

GAPDH* GAP 17 GGTTATCTCAGCTCCGTCTGC 175 138

GAP 18 TAACCTTCGCAAGAGGAGCA 176

ACT* ACT23 AAGACGAAGCAGCTGTAGCC 177 230

ACT24 GGTGTTACGCACACAGTTCC 178

Mi-pg-1 * PolyF4 ATGAGTCAAACAATAACACCCCC 179 244

PolyR2 AATATATAAGGGCCAGACAAAG 180

*Accession numbers of control genes are as follow, ACT: actin (Minc06773), GAPDH: glyceraldehyde 3-phosphate dehydrogenase (Mincl0963) Mi-pg-1: polygalacturonase (Mincl 8543). T°C: annealing temperature used in PCR and quantitative-PCR run. Amplicon size in base pairs (bp) attempted while amplification with specific primer couple.

The cDNA was diluted 10 times, and 5μΙ_, was used per PCR reaction. Seven and a half microliters of 2X SYBR® Green Master Mix (Eurogentec, Liege, Belgium), 0.2 each of 100 μΜ of forward and reverse primer, and 4.6 iL of water were added to the cDNA. Thermocycling was carried out with one cycle at 95°C for 15 min, followed by 40 cycles of 95°C for 15 sec and 56°C for 1 min and 72°C for 30 sec. The dissociation curve of the final products was checked to ascertain the presence of a single amplification product. qPCR was performed on triplicate samples of each cDNA. Among the three tested candidate reference genes, i.e., M. incognita polygalacturonase (Mi-pg-1, Mincl 8543), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Mincl0963) and Actin a (Minc06773), GAPDH was determined as the most stable reference using Genorm algorithm (Vandesompele J. et al., 2002) and was selected as reference gene. For normalization, the threshold cycle values of GAPDH amplifications (CTG _A PD _H ) were subtracted from the threshold cycle values of the analyzed genes (CTexp). Transcript levels in arbitrary units (AU) were calculated with the formula: AU= 100 x 2 ^{(L lexp ~ WUH)} . Figure 1 shows results from two independent replicates.

Transcript analyses by in situ hybridization

Sense and antisense probes were synthesized from each target gene with specific primers designed with Primer3 software (Rozen et al, 2000) and synthesised by Eurogentec (Seraing, Belgium) (see Table 1 above). As controls, it was used the polygalacturonase Mi- pg-1 (Mincl 8543) gene for esophageal gland-specific labeling and the GAPDH Mincl0963 gene for ubiquitous labeling. In situ hybridizations were conducted as described previously (Jaubert et al, 2002). 10,000 J2s were hybridized with DIG-labeled specific probes at 40°C over night for each target transcript.

Results

Effect of siRNA on the 16 target genes

To test whether the designed siRNA interfere with the expression of each of the 16 target genes (15 effector-like proteins, namely Minc00801 , Minc01632, Minc02483, Minc03313, Minc03866, Minc05001 , Minc08013, Minc08014, Minc08335, Minc09526, Mincl0706, Mincl2224, Mincl4652, Mincl7713 and Mincl7987, and one transcription factor, namely Minc07817), it was performed Real-Time quantitative PCR (qPCR) experiments on soaked J2s.

Six siRNAs induced significant reduction in the corresponding targeted transcripts (Minc00801, Minc01632, Minc02483, Minc08335, Minc09526, Mincl7987) 24h after soaking treatment, compared to their expression level in control sample (Figure 1). On the other hand, Minc08013, Minc08014 and Mincl2224 siRNAs induced a silencing that was not reproducible between independent qPCR replicate experiments. Surprisingly, 7 siRNAs (Minc03313, Minc03866, Minc05001 , Mincl0706, Mincl4652, Mincl7713, Minc07817) induced significant and reproducible increase in transcript abundance when qPCR was performed 24h after soaking. A similar "bounce" effect, that could be due to a response of the nematode RNAi machinery to increased siRNA quantities within the cells, had already been reported in PPN after gene silencing (Dubreuil et al, 2007; Bakhetia et al, 2008; Dalzell et al, 2010b). Therefore, it was hypothesized that the transcripts may be present in higher quantity after an initial early silencing. To test this "bounce" hypothesis, it was performed qPCR analysis 16h after soaking on Minc07817, Minc03866 and Minc05001. Confirming siRNA knockdown and bounce effect, Minc03866 was significantly silenced 16h after soaking. However, Minc05001 expression was reduced but not significantly and Minc07817 expression was still higher than control (Figure 2). Overall, the qPCR results indicate that the designed siRNAs, were efficient to knockdown 7 out of the 16 target genes at the tested time points.

Effect of siRNA soaking on nematode viability and mobility

It was tested whether treatment with siRNAs had effect on nematode viability or mobility, by counting the number of dead nematodes and the number of individuals with altered movements 1 hour and 16 hours after soaking with anti-effector siRNAs. These measures were compared to those observed for control nematodes soaked with a siRNA having no similarity in the genome of M. incognita.

In terms of viability, 1 hour after soaking, it was started to observe a few dead nematodes (up to 10%) in 10 out of 16 tested genes, while no dead nematodes were observed in controls (Figure 3). 16 hours after soaking, the number of dead nematodes was substantially higher than in controls for a total of 9 targeted genes (Minc03313, Minc05001, Minc08014, Minc08335, Mincl0706, Mincl2224, Mincl4652, Mincl7987, Mincl7713).

It was observed no substantial change in terms of nematode mobility, 1 hour after soaking with siRNA compared to control nematodes. However, 16 hours after siRNA soaking, an effect on mobility was observed for some targeted genes. The number of nematodes with movement on the whole body length was reduced by 40% or more for 5 out of the 15 targeted genes (Minc02483, Minc03313, Minc08014, Mincl4652, Mincl7987). Similarly, it was observed reductions of 40% or more in the number of individuals presenting fast undulations for 4 out of the 15 siRNA-targeted genes (Minc02483, Minc08014, Mincl4652 and Mincl7987) (Figure 3).

Overall, it was noticed no systematic common effect of siRNA treatment on nematode viability and mobility.

Effect of siRNA soaking on nematode infestation

For each of the 16 identified targets, it was tested whether nematodes soaked with matching siRNAs showed a significant reduction in the numbers of galls and/or egg masses on infected plants as compared to control nematodes. Reduction in the number of galls implies that fewer nematodes have managed to induce a feeding structure. A reduction in the number of egg masses signifies that fewer female nematodes have managed to complete their development until the production of egg masses, a necessary step to propagate the infection at the next generation.

Overall, 12 out of the 16 siRNA-treated samples (Minc00801, Minc01632, Minc02483, Minc03313, Minc03866, Minc05001, Minc08013, Minc08335, Minc09526, Mincl2224, Mincl7713 and Minc07817) showed a significant and reproducible reduction in the number of galls or egg masses after nematode infestation compared to control nematodes (Figure 4).

Among all silencing experiments and replicates, significant reductions in terms of gall numbers ranged from 21.08 % (Minc08014, replicate 1) to 60.84 % (Minc08335, replicate 1), (see Figure 5). Significant reductions in terms of number of egg masses ranged from 24.89% (Minc08014, replicate 2) to 71.12% (Minc05001, replicate 2). Considering observations that were reproducible and significant in both replicates, genes that presented the highest reproducible reduction in number of galls were Minc01632 (57.48% and 43.67% reduction in replicates 1 and 2, respectively), Minc03866 (38.36% and 41.20% reduction in replicates 1 and 2, respectively), Minc05001 (33.03% and 51.54% reductions in replicates 1 and 2, respectively) and Minc08013 (33.03% and 51.54% reductions in replicates 1 and 2, respectively). In terms of egg mass numbers, genes presenting the most important reproducible effects were Minc01632 (70.09% and 54.17% reduction in replicates 1 and 2, respectively), Minc02483 (39.54% and 60.85% in replicates 1 and 2, respectively), and Minc09526 (37.08% and 39.74% in replicates 1 and 2, respectively).

Interestingly, siRNAs targeted against gene Minc01632 are those responsible for both the most important reproducible diminution of the number of galls and of the number of egg masses.

In situ hybridizations of the 16 novel candidate targets

Among the 16 siRNA experiments (15 on effector-like genes and one on the transcription factor), 12 yielded significant and reproducible reduction of the number of galls or egg masses. Because all these genes are novel candidates and nothing is known about their possible functions, it was investigated whether information could be gained from their expression localization using in-situ hybridization (methods). Results of in-situ hybridizations could be grouped in 5 different categories, (i) Genes with ubiquitous expression detected (Minc01632, Minc08335, Mincl2224). (ii) Genes with expression detected specifically in secretory subventral gland cells (Minc03866). (iii) Genes with expression detected specifically in the intestinal tract (Minc00801 , Mincl7713). (iv) Genes with expression detected specifically in the nerve ring, a structure surrounding the metacorpus (Minc02483). (v) Genes that returned no detectable signal (Minc03313, Minc05001, Minc08013, Minc09526, Minc07817). No further indication could be deduced from genes expressed ubiquitously or from those that returned no detectable signal. However, the gene expressed in the subventral gland cell (Minc03866) could well encode an effector protein eventually secreted in plant tissue during infestation. Minc02483, expressed in the nerve ring is also interesting in itself. Indeed, the nerve ring is a structure surrounding the metacorpus that acts as a pump to inject secretion or take-up nutrients from the plant. These two genes with interesting expression localizations are part of a group of 5 genes (Minc01632, Minc02483, Minc03866, Minc05001 and Minc09526) that yielded significant and reproducible reductions of both the number of egg masses and galls in siRNA assays and thus are among the most promising novel targets. Besides Minc03866 (subventral gland cell) and Minc02483 (nerve ring), Minc01632 showed an ubiquitous expression and the two remaining genes (Minc05001 and Minc09526) returned no detectable signal.

EXAMPLE II: PROOF OF CONCEPT STUDY ON THE EFFICACY OF SMALL RNA TO INHIBIT THE EXPRESSION OF 6 TARGET GENES FROM M. INCOGNITA BY THE STRATEGY OF "FEEDING"

Materials and Methods

Bioinformatic analysis

The nucleotide sequences of six genes of interest (Minc00801, Minc01632, Minc02483,

Minc03866, Minc08335 and Minc09526) were aligned against the Arabidopsis genome sequence using BLAST program (TAIR website; https://www.arabidopsis.org/blast) to verify the absence of homologs in the plant. In order to identify the number of potential targets for designed amiRNAs in Arabidopsis, the nucleotide sequences of these amiRNAs were aligned against the Arabidopsis transcripts using the BLAST program. Cloning of amiRNA

Cloning of amiRNAs was performed according to the protocol of Schwab et al, 2006 and Ossowski et al. 2008, as amended. Using the website http://wmd3.weigelworld.org/, the sequences of 4 primers (denoted I to IV) for each amiRNA to clone were generated, as well as the primers "A" and "B" which have been modified to allow the cloning into the donor vector of type pDON (Gateway system). PCR amplifications were carried out in four steps. PCR (a), (b), and (c) were performed on 1 ng of plasmid pRS300 (Ossowski et al. 2008) in 50 μΐ final volume containing 0.2 μΜ of each primer, respectively (A+IV), (III+II) and (I+B), 0.2 miM dNTPs, 1 mM MgS0 ₄ , IX Platinum® Pfx buffer and 1U Platinum® Pfx Taq DNA polymerase High Fidelity, according to the following conditions: (a) and (c) 95°C 3 min; 30 cycles 95°C 30 sec 53°C 30 sec 68°C 40 sec; 68°C 7 min; (b) 95°C 3 min; 30 cycles 95°C 30 sec 50°C 30 sec 68°C 40 sec; 68°C 7 min. The PCR products were loaded onto a gel and purified using the QIAquick gel extraction kit (Qiagen), eluted in 20 μΐ. PCR (d) was performed in 50 μΐ final volume containing 0.5 μΐ of PCR (a) (b) and (c), 0.2 μΜ of primers "A" and "B", 0.2 mM dNTPs, 1 mM MgS0 ₄ , IX Platinum® Pfx buffer and 1U Platinum® Pfx Taq DNA polymerase High Fidelity, according to the program 95°C 3 min; 30 cycles 95°C 30 sec 55°C 30 sec 68°C 1 min 30 sec; 68°C 7 min. The PCR product was loaded and purified onto a gel and about 50 ng were used for recombination into the donor vector (BP reaction), according to the manufacturer's protocol (Invitrogen). The fragments of interest were then introduced into the vectors of destination pK2GW7, pK7m34GW2-8m21GW3- 9m56GW4 (= pK7m34GW234) or pK7m34GW2-8m21GW3 (= pK7m34GW23) by LR reaction following the manufacturer's protocol (Invitrogen). The constructs were sequenced {by GATC), and then introduced into Agrobacterium tumefaciens strain GV3101. The list of primers used for this study is found in Table 3 below.

Table 3. Sequences of primers used

Purpose amiRNA Name of the Sequence SEQ ID NO primer

I miR-s control gaAAAGGCTGGCGTTAAATGTACtctctcttttgtattcc 181

Il miR-a control gaGTACATTTAACGCCAGCCTTTtcaaagagaatcaatga 182

Cloning pDON 207; III_miR*s_control gaGTCCATTTAACGCGAGCCTTTtcacaggtcgtgatatg 183 pDONR P3P4 ;

pDONR P5P6 amiCTRL IV_miR*a_control gaAAAGGCTCGCGTTAAATGGACtctacatatatattcct 184

I_ miR-s_03866 gaAAGCTTGCATACAACTCCTGCtctctcttttgtattcc 185

U_miR-a 03866 gaGCAGGAGTTGTATGCAAGCTTtcaaagagaatcaatga 186

Ill_miR*s_03866 gaGCCGGAGTTGTATCCAAGCTTtcacaggtcgtgatatg 187

Cloning pDONR 207 ami03866 [V_miR*a_03866 gaAAGCTTGGATACAACTCCGGCtctacatatatattcct 188

Cloning pDONR 207 ami01632 I_miR-s_01632 gaAAGACAAGGAATCTCTGAACCtctctcttttgtattcc 1 89 H_miR-a_01632 gaGGTTCAGAGATTCCTTGTCTTtcaaagagaatcaatga 190

III_miR*s_01632 gaGGCTCAGAGATTCGTTGTCTTtcacaggtcgtgatatg 191

IV_miR*a_01632 gaAAGACAACGAATCTCTGAGCCtctacatatatattcct 1 2

I_miR-s_00801 gaAAGTAAGAGTTCTGTGAACGTtctctcttttgtattcc 193

II_miR-a_00801 gaACGTTCACAGAACTCTTACTTtcaaagagaatcaatga 194

III_miR*s_00801 gaACATTCACAGAACACTTACTTtcacaggtcgtgatatg 195

Cloning pDONR P3P4 ami00801 IV_miR*a_00801 gaAAGTAAGTGTTCTGTGAATGTtctacatatatattcct 196

I_miR-s_09526 gaAAGTACACTCACTTCTATCTCtctctcttttgtattcc 197

H_miR-a_09526 gaGAGATAGAAGTGAGTGTACTTtcaaagagaatcaatga 198 lIl_miR*s_09526 gaGAAATAGAAGTGACTGTACTTtcacaggtcgtgatatg 199

Cloning pDONR P3P4 ami09526 IV_miR*a_09526 gaAAGTACAGTCACTTCTATTTCtctacatatatattcct 200

I_miR-s_02483 gaAAAACGGCAATTGGTTGTCAAtctctcttttgtattcc 201 ll_miR-a_02483 gaTTGACAACCAATTGCCGTTTTtcaaagagaatcaatga 202

III_miR*s_02483 gaTTAACAACCAATTCCCGTTTTtcacaggtcgtgatatg 203

Cloning pDONR P5P6 ami02483 IV_miR*a_02483 gaAAAACGGGAATTGGTTGTTAAtctacatatatattcct 204

I_miR-s_08335 gaAAGTAGTCTTCTTCGCTGTCGtctctcttttgtattcc 205

[I_miR-a_08335 gaCGACAGCGAAGAAGACTACTTtcaaagagaatcaatga 206 ni_miR*s_08335 gaCGCCAGCGAAGAACACTACTTtcacaggtcgtgatatg 207

Cloning pDONR P5P6 ami08335 [V_miR*a_08335 gaAAGTAGTGTTCTTCGCTGGCGtctacatatatattcct 208

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGA 209

ATTB 1-A ATTCCTGCAGCCCCAAA

Cloning in pDONR GGGGACCACTTTGTACAAGAAAGCTGGGTGGA 210 207 ATTB2-B TCCCCCCATGGCGATG

GGGGACAACTTTGTATAGAAAAGTTGCGAATTC 21 1

ATTB4f-A CTGCAGCCCCAAA

Cloning in pDONR GGGGACAACTTTGTATAATAAAGTTGGATCCCC 212 P3P4 ATTB3r-B CCATGGCGATG

GGGGCAACTTTGTATTAAAAAGTTGCGAATTCC 213

ATTB6f_A TGCAGCCCCAAA

Cloning in pDONR GGGGACAACTTTGTATACAAAAGTTGGATCCCC 214 P5P6 ATTB5r_B CCATGGCGATG Ql-PREmir319-rev TTTGGCGACTCGGTATTTGG 215

Ql-PREamiCTRL- 217 for GTCCATTTAACGCGAGCCTTT

amiCTRL Ql-amiCTRL-for AAAGGCTGGCGTTAAATGTACa 218

Ql-PREami3866- 219 ami03866 for CCGGAGTTGTATCCAAGCTT

Ami01632 Q1 -amil632-for AGACAAGGAATCTCTGAACC 220

Validation of the

ami00801 Ql-PREami801 -for ACATTCACAGAACACTTACTT 221 expression in planta

Ami09526 Ql-ami9526-for AAGTACACTCACTTCTATCTCaa 222

Ql-PREami2483- 223 for TTAACAACCAATTCCCGTTT

ami02483 Ql-ami2483-for AACGGCAATTGGTTGTCAAa 224

Ql-PREami8335- 225 ami0833S for CGCCAGCGAAGAACACTACTTtc

pre-Mir-159a_L TGGATCCCATAAGCCCTAATCC 226 rairl59a pre-Mir-159a_R AGAGTAAAAGCCATTAAAGGGCAA 227

Genetic transformation of Arabidopsis

Ws Arabidopsis plants were transformed by floral dip (Clough and Bent, 1998). The transformed lines were selected on MS medium supplemented with 1 % agar 1 % sucrose pH 6.4, containing 50 μ^πιΐ kanamycin. About 10 primary transformants (Tl) were obtained and multiplied in the ground. The segregating of T2 plants was determined by seeding on selective medium (kanamycin). The lines with a single insertion were individualized and multiplied. Homozygous T3 lines for the insertion were selected and used for analysis in interaction with nematodes.

Validation of expression of amiRNA in planta

The total RNA from a pool of T2 plants were extracted using the TriZol Reagent following the manufacturer's protocol (Life Technologies). 1 μg RNA was reverse-transcribed using the kit miScript II RT kit (Qiagen), which allows in particular the reverse transcription of small RNAs. Semi-quantitative PCR allowing the detection of the precursor of mature amiRNA were performed on Ιμΐ cDNA diluted to the 10 ^th in 25 μΐ final volume containing

2 μΜ of each primer, IX MyTaq Buffer, 1U MyTaq (Bioline) according to the program: 94°C

3 min; 33 cycles 94°C 20 sec 54°C 30 sec 72°C 30 sec; 72°C 7 min. Depending on the amiRNA, the primer pairs Ql -PREami-for or Ql-ami-for/Q2-PREmir319-rev were used. The precursor of the microRNA mirl59a was amplified using primers pre-Mir-159a_L and pre- Mir-159a_R and used as reference gene. 15 μΐ were then loaded onto a 2% agarose gel. After revelation with BET, the gels were scanned with a FLA-3000 PhosphoTmager (Fujifilm) in FluoroStage IP mode with excitation at 473 nm and a 580 nm emission filter. Using the software Image Gauge, the band intensities of amiRNA and mirl59a were quantified and the ratio "Intensity amiRNA" / "Intensity reference microRNA" was calculated. The plant lines with the highest expression level of amiRNA were selected for nematode resistance testing. The pairs of primers used for detecting amiRNAs are described in Table 3 above.

Nematode resistance testing

The seeds were sown on MS medium supplemented with 1% agar 1% sucrose pH 6.4, vernalized 48 hours at 4°C and then transferred to 20°C with a photoperiod of 16h day / 8h night. 7 days later, 6 plants were transplanted to fresh medium contained in square Petri dishes (4-5 dishes per plant line, i.e. 24 to 30 individuals). 14 days after transplanting, the seedlings were inoculated with sterilized J2 nematode larvae (200 larvae per plant). The number of galls was recorded 15 days after infection, and the egg masses produced was assessed 5-6 weeks after infection.

Results

Bioinformatic analysis

The gene structure for the six targets (Minc00801, Minc01632, Minc02483, Minc03866, Minc08335 and Minc09526) was confirmed: exon-intron boundaries, initiation and termination codons, and reading frames. The results are shown in Figure 6. The alignment against the Arabidopsis genome sequence using BLAST program has confirmed the absence of homologous genes in the plant.

Table 4 below shows the sequences of amiRNA directed against the corresponding target genes, and the number of potential targets in Arabidopsis.

Table 4. Target genes and sequences of amiRNA (corresponding to the siRNA used for soaking) and number of potential off-targets in Arabidopsis. The reduction in the numbers of galls and egg masses in the soaking experiment with the siRNA (see Example I above) is reminded.

Cloning of amiRNA

Table 5 below shows the genetic constructions (vectors in Agrobacteria) performed. Construction for combined targeting of 3 target genes (3866x801x2483) or of 2 target genes (1632x9526) were also obtained.

Table 5. Genetic constructs and Arabidopsis transformations performed in the present study

Plant lines expressing an amiRNA directed against Minc01632 and (validation of the) expression of the amiRNA in planta

Among all the lines having a single T-DNA integration, those with the highest amiRNA expression were selected. The nematode resistance tests were performed on these lines. Resistance tests showed a significant 21.5% reduction of the gall number in plants expressing the single ami01632 construct compared to control plants, as shown in Table 6 below.

Table 6. Nematode resistance test of ami01632-expressing lines.

REFERENCES

Arguel M.J. et al, 2012, Genes, 3:391-408

Atkinson H.J. et al, 2012, Curr Opin Biotechnol., 23:251-256

Bakhetia M. et al., 2008, Int J Parasitol., 38: 1589-1597 Barthels N. et al, 1997, Plant Cell, 9:21 19-2134

Charlton W.C. et al, 2010, Int J Parasitol., 40:855-864

Clough S.J. and Bent A.F., 1998, Plant J., 16:735-743

Dalzell J.J. et al, 2010a, Int J Parasitol., 40:91 -100

Dalzell J.J., et al, 2010b, Int J Parasitol., 40: 1303-1310.

Djian-Caporalino C, 2012, EPPO Bulletin, 42: 127-137

Dubreuil G. et al, 2007, New Phytol., 176:426-436

Favery B. et al, 1998, Embo J. 17:6799-6811

Huang G. et al, 2006, Proc Natl Acad Sci U S A.,103: 14302-14306

Ibrahim H.M.M. et al, 2011, Exp Parasitol., 127:90-99

Jaubert S. et al, 2002, FEBS Letters, 522:109-112

link V.P. et al, 2009, Planta, 230:53-71

Kozub N.O. et al, 2012, Cytology and Genetics, 46:251-262

McCarter J.P., 2009, Molecular Approaches Toward Resistance to Plant-Parasitic Nematodes. In: Berg RH, Taylor C, editors. Cell Biology of Plant Nematode Parasitism: Springer Berlin Heidelberg, pp. 239-267

Needleman S.B. and Wunsch CD., 1970, J. Mol. Biol., 48:443-453

Nielsen H. et al, 1997, Protein Engineering, 10:1-6

Ossowski et al. 2008 Plant J., 53: 674-690.

Patel N. et al, 2008, J Nematol., 40:299-310

Patel N. et al, 2010, J Exp Bot, 61 :235-248

Petersen T.N. et al, 201 1, Nature Methods, 8:785-786

Rosso M.N. et al, 1999, Mol Plant Microbe Interact., 12:585-591

Rosso M.N. et al, 2009, Annu Rev PhytopathoL, 47:207-232

Rozen S. et al, 2000, Methods Mol Biol., 132: 365-386

Sasser J.N. and Freckman D.W., 1987, A world perspective on nematology; the role of society. In: Veech JA, Dickerson DW, editors. Vistas on nematology. Hyattsville, USA: Society of Nematologists. pp. 7-14

Sayle, R. and Milner- White, E., 1995, Trends Biochem. Sci., 20:374

Schwab R. et al, 2006, Plant Cell, 18:1121-1133

Sindhu A.S. et al, 2009, J Exp Bot., 60:315-324

Vandesompele J. et al, 2002, Genome Biol. 3:RESEARCH0034

Vens C. et al, 201 1, Bioinformatics, 27:1231-1238

Vercauteren I. et al, 2001, Mol Plant Microbe Interact., 14:288-299

Watson J.M. et al, 2005, FEBS Letters, 579:5982-5987

Wesemael W.M.L. et al, 2011 , Nematology, 13: 3-16

Yadav B.C. et al, 2006, Mol Biochem Parasitol., 148:219-222