Tomato transgenic plants expressing hairpin construct of a nematode protease gene conferred enhanced resistance to root-knot nematodes.

Tushar K Dutta,Pradeep K Papolu,Divya Choudhary,Anil Sirohi,Uma Rao,Prakash Banakar

doi:10.3389/fmicb.2015.00260

Tushar K Dutta, Pradeep K Papolu + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2015.00260

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Abstract

Root-knot nematodes (Meloidogyne incognita) cause substantial yield losses in vegetables worldwide, and are difficult to manage. Continuous withdrawal of environmentally-harmful nematicides from the global market warrants the need for novel nematode management strategies. Utility of host-delivered RNAi has been demonstrated in several plants (Arabidopsis, tobacco, and soybean) that exhibited resistance against root-knot and cyst nematodes. Herein, a M. incognita-specific protease gene, cathepsin L cysteine proteinase (Mi-cpl-1), was targeted to generate tomato transgenic lines to evaluate the genetically modified nematode resistance. In vitro knockdown of Mi-cpl-1 gene led to the reduced attraction and penetration of M. incognita in tomato, suggesting the involvement of Mi-cpl-1 in nematode parasitism. Transgenic expression of the RNAi construct of Mi-cpl-1 gene resulted in 60–80% reduction in infection and multiplication of M. incognita in tomato. Evidence for in vitro and in vivo silencing of Mi-cpl-1 was confirmed by expression analysis using quantitative PCR. Our study demonstrates that Mi-cpl-1 plays crucial role during plant-nematode interaction and plant-mediated downregulation of this gene elicits detrimental effect on M. incognita development, reinforcing the potential of RNAi technology for management of phytonematodes in crop plants.

Highlights

Root-knot nematodes (Meloidogyne spp.) are one of the potential constraints for cultivation of vegetables in the tropical and subtropical countries
According to quantitative real-time PCR (qRT-PCR) results, approximately 76% reduction in mRNA abundance was observed in Micpl-1 double-stranded RNA (dsRNA) fed J2s compared to J2s treated with soaking buffer alone or gfp dsRNA
Cathepsin L-like cysteine proteinases are an attractive group of candidate genes for RNA interference (RNAi)-induced downregulation, because they are crucial for parasitic aspects of plant-nematode interaction, but they lack significant homology to genes in other organisms

Summary

Introduction

Root-knot nematodes (Meloidogyne spp.) are one of the potential constraints for cultivation of vegetables in the tropical and subtropical countries. Nematodes inject a cascade of effector proteins of esophageal gland origin into plant cells via its stylet (Hassan et al, 2010). These effectors are thought to be involved in host pathogen interaction starting from the host recognition process to degradation of plant cell walls in order to facilitate the migration of nematode, culminating in the establishment of hypermetabolic, multinucleate feeding cell (giant cell, GC) which serves as the permanent food source for nematode development and reproduction (Davis et al, 2008).

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