Identification of plant-parasitism genes in nematodes in silico screening and in vivo validation in Meloidogyne incognita.

Abstract : Root-knot nematodes, Meloidogyne spp., are root parasites that annually cause ca. 100 billion Euros losses in crop production worldwide. An initial analysis of the genome of Meloidogyne incognita has allowed the identification of thousands of genes that apparently did not have homolog in model eukaryotes. A proportion of these M. incognita-restricted gene models may be involved in functions linked to plant-parasitism. Our objective was to identify drug target genes to control primarily root-knot nematodes with potential applications to other plant pathogenic and parasitic species. Our hypothesis was, if a gene is involved in a key parasitism process, it might be conserved in plant-parasitic or plant-pathogenic species and absent from non-parasitic species. Our strategy to identify druggable parasitism target genes was to perform a comparative analysis of M. incognita and M. hapla predicted protein coding genes against a selection of fully sequenced genomes and transcriptomes of various eukaryotes with different lifestyles. Our pipeline conserves all sequences shared with parasitic or plant-associated species and eliminates those conserved in species that could be negatively affected by newly developed drugs or control means. In parallel, we performed automatic functional annotation of the identified genes. Those annotations include detection of signal peptide, Pfam domains, assignment of gene ontology (G.O.) terms, and identification of specific motifs. All these features and annotations were stored in a relational database and it is thus possible to produce any combination of feature to filter out genes of interest. Comparison of the relative abundance of G.O. terms assigned to genes restricted to Meloidogyne species versus those assigned to the whole gene sets of M. incognita and M. hapla revealed a number of differences. A second objective was to develop a new RNA interference (RNAi) procedure using siRNA to artificially silence target genes and analyze their role in parasitism. SiRNA are smaller than long dsRNA usually used for silencing in M. incognita. SiRNA might be easier to introduce by soaking in M. incognita. SiRNA are also more specific to a target sequence as they are 21 base-long and their specificity towards target mRNAs can be assessed by BLAST searches in M. incognita genome. SiRNA targeted to various positions on the mRNA secondary structure were tested to optimize siRNA design. Several transfection conditions were tested and validated by quantitative PCR to optimize reproducibility and target mRNA expression inhibition efficiency. To test the effect of gene silencing on nematode infectivity, nematodes treated with optimized siRNA were used for inoculation assays on tomato plants (Solanum esculentum cv. St Pierre). Infectivity was measured by counting gall and egg masse numbers. With this new method we will test the effect of gene silencing for a series of the most promising targets identified during bioinformatics screens.