Combinatorial in vitro RNAi of two neuropeptide genes and a pharyngeal gland gene on Meloidogyne incognita

Abstract

Root-knot nematodes are the most economically important group of plant-parasitic nematodes. In the present study, functional validation using in vitro RNAi was carried out on Meloidogyne incognita with two FMRFamide-like peptide genes, flp-14 and flp-18, and a subventral pharyngeal gland specific gene, 16D10. It was found that RNAi silencing of each gene reduced the attraction of M. incognita at different time intervals both in combination and individually. Silencing of the genes reduced nematode infection by 23-30% and development as indicated by a reduction in the number of females by 26-62%. Reproduction was decreased by 27-73% and fecundity was decreased by 19-51%. In situ hybridisation revealed the expression of flp-18 in cells associated with the ventral and retro vesicular ganglia of the central nervous system. qRT-PCR supported the correlation between phenotypic effects of silencing with that of transcript quantification.

Simultaneous RNAi Knockdown of Three FMRFamide-Like Peptide Genes, Mi-flp1, Mi-flp12, and Mi-flp18 Provides Resistance to Root-Knot Nematode, Meloidogyne incognita

“…Hence, it is possible that Mi-flp1 could have similar functional role in M. incognita. Previously, we had reported the expression site of Mi-flp12 and Mi-flp18 in the nerve cells, involved in the various biological and physiological process of M. incognita J2s (Banakar et al, 2015;Banakar and Rao, 2016). These findings support the involvement of the three FLPs in locomotion, host finding, reproduction and other physiological roles in the nematodes (White et al, 1986;Kimber et al, 2002Kimber et al, , 2007Rogers et al, 2003;Johnston et al, 2010;Kumari et al, 2017).…”

“…RNAi has been the mainstay tool for functional validation of genes in phytonematodes (Lilley et al, 2007;Dutta et al, 2015a,b). However, reports of gene silencing to explore the function of FLPergic molecules in plant nematodes are finite (Kimber et al, 2007;Dalzell et al, 2010;Papolu et al, 2013;Dong et al, 2014;Banakar et al, 2015). Here, we disrupted three individual FLPs and their combinatorial fusion gene through in vitro RNAi (dsRNA soaking) in M. incognita J2s which resulted in perturbed the expression of target transcripts of each of the FLPs.…”

“…Subsequently, PCR product was used for preparing the DIG-labeled sense and antisense DNA probes, separately (Roche, Switzerland). J2s fixation, permeabilization, hybridization of probes and signal detection and staining procedures were followed as described by Banakar et al (2015). Processed nematodes were placed on microscope slides, covered with 0.28 mm glass coverslips and sealed with nail varnish.…”

“…Silencing effect of Mi-flp1, Mi-flp12, Mi-flp18 and their synthetic fusion gene on M. incognita development and reproduction was carried out by inoculating dsRNA-soaked nematodes on 7-days old adzuki bean (Vigna angularis var. angularis) roots in CYG seed growth pouches (Banakar et al, 2015 andShivakumara et al, 2016). Six root tips of each plant were inoculated with about 15-20 J2s, and incubated in a growth chamber at 27 • C with 70% humidity and a light intensity of 300 lux (Jeio Tech Company Ltd., South Korea).…”

“…Investigation on the FLPs genes function through RNAi has shown reduced migration, penetration and aberrant behavioral phenotypes in M. incognita, Meloidogyne graminicola, and Globodera pallida (Urwin et al, 2002;Kimber et al, 2007;Dalzell et al, 2010;Atkinson L. E. et al, 2013;Papolu et al, 2013;Dong et al, 2014;Banakar et al, 2015;Kumari et al, 2017). Further, effectiveness of in vitro RNAi of FLP genes (Gp-flp1, Gp-flp12, and Gp-flp18) in G. pallida (Kimber et al, 2007) and host-delivered RNAi of Mi-flp14, Mi-flp18 in M. incognita (Papolu et al, 2013) indicate that it would be intriguing to investigate the synergistic or additive impacts, if any, of concomitant knockdown of more than one flp gene.…”