CRISPR/Cas9-induced knockout of an amino acid permease gene (AAP6) reduced Arabidopsis thaliana susceptibility to Meloidogyne incognita.

BACKGROUND: Plant-parasitic root-knot nematode (Meloidogyne incognita) causes global yield loss in agri- and horticultural crops. Nematode management options rely on chemical method. However, only a handful of nematicides are commercially available. Resistance breeding efforts are not sustainable because R gene sources are limited and nematodes have developed resistance-breaking populations against the commercially available Mi-1.2 gene-expressing tomatoes. RNAi crops that manage nematode infection are yet to be commercialized because of the regulatory hurdles associated with transgenic crops. The deployment of the CRISPR/Cas9 system to improve nematode tolerance (by knocking out the susceptibility factors) in plants has emerged as a feasible alternative lately. RESULTS: In the present study, a M. incognita-responsive susceptibility (S) gene, amino acid permease (AAP6), was characterized from the model plant Arabidodpsis thaliana by generating the AtAAP6 overexpression line, followed by performing the GUS reporter assay by fusing the promoter of AtAAP6 with the ß-glucuronidase (GUS) gene. Upon challenge inoculation with M. incognita, overexpression lines supported greater nematode multiplication, and AtAAP6 expression was inducible to the early stage of nematode infection. Next, using CRISPR/Cas9, AtAAP6 was selectively knocked out without incurring any growth penalty in the host plant. The 'Cas9-free' homozygous T3 line was challenge inoculated with M. incognita, and CRISPR-edited A. thaliana plants exhibited considerably reduced susceptibility to nematode infection compared to the non-edited plants. Additionally, host defense response genes were unaltered between edited and non-edited plants, implicating the direct role of AtAAP6 towards nematode susceptibility. CONCLUSION: The present findings enrich the existing literature on CRISPR/Cas9 research in plant-nematode interactions, which is quite limited currently while compared with the other plant-pathogen interaction systems.

Induced knockdown of Mg-odr-1 and Mg-odr-3 perturbed the host seeking behavior of Meloidogyne graminicola in rice.

Root-knot nematode Meloidogyne graminicola is one of the most destructive plant parasites in upland as well as direct seeded rice. As an integral part of nematode biology, host finding behavior involves perceiving and responding to different chemical cues originating from the rhizosphere. A sustainable management tactic may include retardation of nematode chemoreception that would impair them to detect and discriminate the host stimuli. Deciphering the molecular basis of nematode chemoreception is vital to identify chokepoints for chemical or genetic interventions. However, compared to the well-characterized chemoreception mechanism in model nematode Caenorhabditis elegans, plant nematode chemoreception is yet underexplored. Herein, the full-length cDNA sequences of two chemotaxis-related genes (Mg-odr-1 and Mg-odr-3) were cloned from M. graminicola. Both the genes were markedly upregulated in the early developmental stages of M. graminicola suggesting their involvement in host finding processes. RNAi-induced independent knockdown of Mg-odr-1 and Mg-odr-3 caused behavioral aberration in second-stage juveniles of M. graminicola which in turn perturbed the nematodes' host finding ability and parasitic success inside rice roots. Additionally, nematodes' chemotactic response to different host root exudates, volatile and nonvolatile compounds was affected. Our results demonstrating the role of specific chemosensory genes in modulating M. graminicola host seeking behavior can enrich the existing knowledge of plant nematode chemoreception mechanism, and these genes can be targeted for novel nematicide development or in planta RNAi screens.

Molecular and functional characterization of chemosensory genes from the root-knot nematode Meloidogyne graminicola.

BACKGROUND: Root-knot nematode Meloidogyne graminicola has emerged as a major threat in rice agroecosystems owing to climate change-induced changes in cultivation practices. Synthetic nematicides are continually being withdrawn from the nematode management toolbox because of their ill effects on the environment. A sustainable strategy would be to develop novel nematicides or resistant plants that would target nematode sensory perception, which is a key step in the host finding biology of plant-parasitic nematodes (PPNs). However, compared to the extensive literature on the free-living nematode Caenorhabditis elegans, negligible research has been performed on PPN chemosensory biology. RESULTS: The present study characterizes the five chemosensory genes (Mg-odr-7, Mg-tax-4, Mg-tax-4.1, Mg-osm-9, and Mg-ocr-2) from M. graminicola that are putatively associated with nematode host-finding biology. All the genes were highly transcribed in the early life stages, and RNA interference (RNAi)-induced downregulation of each candidate gene perturbed the normal behavioural phenotypes of M. graminicola, as determined by examining the tracking pattern of juveniles on Pluronic gel medium, attraction to and penetration in rice root tip, and developmental progression in rice root. In addition, a detrimental effect on nematode chemotaxis towards different volatile and nonvolatile organic compounds and host root exudates was documented. CONCLUSION: Our findings enrich the existing literature on PPN chemosensory biology and can supplement future research aimed at identifying a comprehensive chemosensory signal transduction pathway in PPNs.