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 Synthetic nematicides are continually being withdrawn from the nematode management toolbox because of their ill effects on the environment.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.

Stringent in-silico identification of putative G-protein-coupled receptors (GPCRs) of the entomopathogenic nematode Heterorhabditis bacteriophora.

The infective juveniles (IJs) of entomopathogenic nematode (EPN) Heterorhabditis bacteriophora find and infect their host insects in heterogeneous soil ecosystems by sensing a universal host cue (CO2) or insect/plant-derived odorants, which bind to various sensory receptors, including G protein-coupled receptors (GPCRs). Nematode chemosensory GPCRs (NemChRs) bind to a diverse set of ligands, including odor molecules. However, there is a lack of information on the NemChRs in EPNs. Here we identified 21 GPCRs in the H. bacteriophora genome sequence in a triphasic manner, combining various transmembrane detectors and GPCR predictors based on different algorithms, and considering inherent properties of GPCRs. The pipeline was validated by reciprocal BLAST, InterProscan, GPCR-CA, and NCBI CDD search. Functional classification of predicted GPCRs using Pfam revealed the presence of four NemChRs. Additionally, GPCRs were classified into various families based on the reciprocal BLAST approach into a frizzled type, a secretin type, and 19 rhodopsin types of GPCRs. Gi/o is the most abundant kind of G-protein, having a coupling specificity to all the fetched GPCRs. As the 21 GPCRs identified are expected to play a crucial role in the host-seeking behavior, these might be targeted to develop novel insect-pest management strategies by tweaking EPN IJ behavior, or to design novel anthelminthic drugs. Our new and stringent GPCR detection pipeline may also be used to identify GPCRs from the genome sequence of other organisms.

RNAi-mediated knockdown of gut receptor-like genes prohibitin and α-amylase altered the susceptibility of Galleria mellonella to Cry1AcF toxin.

BACKGROUND: Due to the prolonged usage of Bt-based biopesticides and Bt-transgenic crops worldwide, insects are continually developing resistance against Cry toxins. This resistance may occur if any mechanistic step in the insecticidal process is disrupted possibly because of the alteration in Cry-receptor binding affinity due to mutation in receptor genes. Compared to other lepidopteran insects, Cry receptor-related research has made asymmetric progress in the model insect Galleria mellonella. RESULTS: Present study describes the molecular characterization and functional analysis of five Cry toxin receptor-related genes (prohibitin, GLTP, α-amylase, ADAM and UDP-GT) and a gut repair gene (arylphorin) from the gut tissues of G. mellonella. Protein-protein docking analysis revealed that Cry1AcF putatively binds with all the five candidate proteins, suggesting their receptor-like function. These receptor-like genes were significantly overexpressed in the gut tissues of fourth-instar G. mellonella larvae upon early exposure to a sub-lethal dose of Cry1AcF toxin. However, targeted knockdown (by using bacterially-expressed dsRNAs) of these genes led to variable effect on insect susceptibility to Cry1AcF toxin. Insects pre-treated with prohibitin and α-amylase dsRNA exhibited significant reduction in Cry1AcF-induced mortality, suggesting their probable role as Cry receptor. By contrast, insects pre-treated with GLTP, ADAM and UDP-GT dsRNA exhibited no significant decline in mortality. This maybe explained by the possibility of RNAi feedback regulation (as few of the receptors belong to multigene family) or redundant role of GLTP, ADAM and UDP-GT in Cry intoxication process. CONCLUSION: Since the laboratory culture of G. mellonella develop Bt resistance quite rapidly, findings of the current investigation may provide some useful information for future Cry receptor-related research in the model insect.

Molecular characterization and functional analysis of Cry toxin receptor-like genes from the model insect Galleria mellonella.

Crystal (Cry) toxins produced from the soil bacterium, Bacillus thuringiensis (Bt), have gained worldwide attention for long due to their insecticidal potential. A number of receptor proteins located on the epithelial cells of the larval midgut were shown to be crucial for Cry intoxication in different insect pests belonging to order Lepidoptera, Diptera and Coleoptera. A beehive pest, Galleria mellonella, serves as an excellent insect model for biochemical research. However, information on the Cry receptor-like genes in G. mellonella is limited. In the present study, the full-length sequences of four putative Cry receptor genes (ABC transporter, alkaline phosphatase, aminopeptidase N and cadherin) were cloned from G. mellonella. All these receptor genes were substantially upregulated in the midgut tissue of fourth-instar G. mellonella larvae upon early exposure (6 h) to a sub-lethal dose of Cry1AcF toxin. Oral and independent delivery of bacterially-expressed dsRNAs corresponding to four receptor genes in G. mellonella suppressed the transcription of target receptors which in turn significantly reduced the larval sensitivity to Cry1AcF toxin. As the laboratory populations of G. mellonella develop Bt resistance in a relatively short time, molecular characterization of Cry receptor genes in G. mellonella performed in the present study may provide some useful information for future research related to the genetic basis of Bt resistance in the model insect.

Rapid and sensitive detection of potato cyst nematode Globodera rostochiensis by loop-mediated isothermal amplification assay.

Cyst nematodes of the species Globodera rostochiensis and G. pallida are devastating parasites of the potato crop. Early detection of cyst nematodes in the field is critical for adopting an appropriate management strategy. A specific and sensitive loop-mediated isothermal amplification (LAMP) assay using four oligonucleotide primers has been developed to amplify the internal transcribed spacer region (ITS) of ribosomal DNA of potato cyst nematode G. rostochiensis. The PCN-LAMP reaction could be completed within 75 min at 68 °C followed by termination at 85 °C for 7 min. The primers exhibited specificity for G. rostochiensis and did not detect any other tested genera of plant parasitic or entomopathogenic nematodes. LAMP reaction was highly sensitive, suitable for crude genomic DNA and could successfully detect G. rostochiensis DNA up to femtogram quantity. This assay is rapid, cost effective and requires minimal instrumentation. It will facilitate the detection of G. rostochiensis at field and point-of-care labs and help in the interception of infested plant material/soil samples at quarantine stations independent of a professional nematologist. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02830-8.