IDENTIFICATION OF KEY GENES INVOLVED IN ROOT KNOT NEMATODE (M. INCOGNITE) DEVELOPMENT FOR EFFECTIVE RESISTANCE IN PLANTS USING RNAi

Abstract

The southern root-knot nematode, Meloidogyne incognita, is sedentary endoparasite with an adverse social and economic impact worldwide. The cuticle and pharynx of plant-parasitic nematode is a less studied area of research with only few developmental genes identified till date. In this study, key genes involved in cuticle and pharynx development of M. incognita were identified and characterized. An orthologue of Caenorhabditis elegansdpy-10 (dumpy), dpy-31 and glp-1 (abnormal germline proliferation) genes were identified in M. incognita. The phylogenetic analysis revealed evolutionary conserved nature of these genes throughout the phylum Nematoda. Successful partial mRNA cloning of these three genes in pGEMT-Easy vector confirms their presence in M. incognita. Temporal regulated expression of dumpy genes was revealed by qRT-PCR expression analyses. Mi-dpy-10 showed higher expression at the second stage juveniles (J2s) of the nematode development in comparison to eggs and adult females. However, Mi-dpy-31 gene revealed higher expression in adult females in M. incognita suggesting different roles of dumpy genes during moulting process in nematode development. While Mi-glp-1 showed differential expression throughout the lifecycle of M. incognita, with relatively higher expression in the egg stage. The functional analysis of Mi-dpy-31 and Mi-glp-1 was conducted in a host-mediated gene silencing strategy by developing gene-specific dsRNA expressing RNAi lines. There was no significant reduction in terms of nematode infection in Mi-dpy-31-RNAi lines as equal number of galls, females and eggs were observed in RNAi and wild-type plants. The Mi-glp-1-RNAi lines on the other hand showed 47–50% reduction in the numbers of galls, females and egg viii masses. Intriguingly, an adverse effect on the phenotype of second-generation J2s, which were descendants of the infected females from the glp-1-RNAi lines, was noticed. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and disruption around the metacorporeal bulb of the pharynx. This indicates a role for this gene in organ (pharynx) development during embryogenesis in M. incognita. In Toto, these findings suggest that Mi-dpy-10 and Mi-dpy-31 play role in cuticle and Mi-glp-1 in pharynx development in M. incognita and their potential use as a target in controlling plant-parasitic nematode infection in a host-mediated gene silencing approach.