Host-induced RNA interference targeting the neuromotor gene FMRFamide-like peptide-14 (Mi-flp14) perturbs Meloidogyne incognita parasitic success in eggplant.

KEY MESSAGE: The study demonstrates the successful management of Meloidogyne incognita in eggplant using Mi-flp14 RNA interference, showing reduced nematode penetration and reproduction without off-target effects across multiple generations. Root-knot nematode, Meloidogyne incognita, causes huge yield losses worldwide. Neuromotor function in M. incognita governed by 19 neuropeptides is vital for parasitism and parasite biology. The present study establishes the utility of Mi-flp14 for managing M. incognita in eggplant in continuation of our earlier proof of concept in tobacco (US patent US2015/0361445A1). Mi-flp14 hairpin RNA construct was used for generating 19 independent transgenic eggplant events. PCR and Southern hybridization analysis confirmed transgene integration and its orientation, while RT-qPCR and Northern hybridization established the generation of dsRNA and siRNA of Mi-flp14. In vitro and in vivo bio-efficacy analysis of single-copy events against M. incognita showed reduced nematode penetration and development at various intervals that negatively impacted reproduction. Interestingly, M. incognita preferred wild-type plants over the transgenics even when unbiased equal opportunity was provided for the infection. A significant reduction in disease parameters was observed in transgenic plants viz., galls (40-48%), females (40-50%), egg masses (35-40%), eggs/egg mass (50-55%), and derived multiplication factor (60-65%) compared to wild type. A unique demonstration of perturbed expression of Mi-flp14 in partially penetrated juveniles and female nematodes established successful host-mediated RNAi both at the time of penetration even before the nematodes started withdrawing plant nutrients and later stage, respectively. The absence of off-target effects in transgenic plants was supported by the normal growth phenotype of the plants and T-DNA integration loci. Stability in the bio-efficacy against M. incognita across T1- to T4-generation transgenic plants established the utility of silencing Mi-flp14 for nematode management. This study demonstrates the significance of targeting Mi-flp14 in eggplant for nematode management, particularly to address global agricultural challenges posed by M. incognita.

Homologs of Caenorhabditis elegans Chemosensory Genes Have Roles in Behavior and Chemotaxis in the Root-Knot Nematode Meloidogyne incognita.

Nematode chemosensation is a vital component of their host-seeking behavior. The globally important phytonematode Meloidogyne incognita perceives and responds (via sensory organs such as amphids and phasmids) differentially to various chemical cues emanating from the rhizosphere during the course of host finding. However, compared with the free-living worm Caenorhabditis elegans, the molecular intricacies behind the plant nematode chemotaxis are a yet-unexploited territory. In the present study, four putative chemosensory genes of M. incognita, namely, Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 were molecularly characterized. Mi-odr-1 mRNA was found to be expressed in the cell bodies of amphidial neurons and phasmids of M. incognita. Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 transcripts were highly expressed in early life stages of M. incognita, consistent with a role of these genes in host recognition. Functional characterization of Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 via RNA interference revealed behavioral defects in M. incognita and perturbed attraction to host roots in Pluronic gel medium. Knockdown of Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 resulted in defective chemotaxis of M. incognita to various volatile compounds (alcohol, ketone, aromatic compound, ester, thiazole, pyrazine), nonvolatiles of plant origin (carbohydrate, phytohormone, organic acid, amino acid, phenolic), and host root exudates in an agar-Pluronic gel-based assay plate. In addition, ascaroside-mediated signaling was impeded by downregulation of chemosensory genes. This new information that behavioral response in M. incognita is modulated by specific olfactory genes can be extended to understand chemotaxis in other nematodes.

Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant.

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T1, T2 and T3 transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

A novel in vitro chemotaxis bioassay to assess the response of Meloidogyne incognita towards various test compounds.

Plant-parasitic, root-knot nematodes (Meloidogyne spp.) are a serious problem in agri- and horticultural crops worldwide. Understanding their complex host recognition process is essential for devising efficient and environmental-friendly management tactics. In this study, the authors report a new, simple, inexpensive, efficient, and quantitative method to analyze the chemotaxis of M. incognita second-stage juveniles (J2s) using a combination of pluronic gel and agar in a petri dish. The authors quantitatively defined the concentration gradient formation of acid fuchsin on the assay plate. Using this novel assay method, the authors have accurately measured the nematode response (attraction or repulsion) to various volatile (isoamyl alcohol, 1-butanol, benzaldehyde, 2-butanone, and 1-octanol) and non-volatile (root exudates of tomato, tobacco, and marigold) compounds. Isoamyl alcohol, 1-butanol, and 2-butanone were attractive to J2s through a broad range of concentrations. On the contrary, J2s were repelled when exposed to various concentrations of 1-octanol. Despite being attractive at lower concentrations, undiluted benzaldehyde was repulsive to J2s. Tomato and tobacco root exudates were attractive to J2s while marigold root exudates repelled J2s. The present quantitative assay method could be used as a reference to screen and identify new candidate molecules that attract or repel nematodes.Plant-parasitic, root-knot nematodes (Meloidogyne spp.) are a serious problem in agri- and horticultural crops worldwide. Understanding their complex host recognition process is essential for devising efficient and environmental-friendly management tactics. In this study, the authors report a new, simple, inexpensive, efficient, and quantitative method to analyze the chemotaxis of M. incognita second-stage juveniles (J2s) using a combination of pluronic gel and agar in a petri dish. The authors quantitatively defined the concentration gradient formation of acid fuchsin on the assay plate. Using this novel assay method, the authors have accurately measured the nematode response (attraction or repulsion) to various volatile (isoamyl alcohol, 1-butanol, benzaldehyde, 2-butanone, and 1-octanol) and non-volatile (root exudates of tomato, tobacco, and marigold) compounds. Isoamyl alcohol, 1-butanol, and 2-butanone were attractive to J2s through a broad range of concentrations. On the contrary, J2s were repelled when exposed to various concentrations of 1-octanol. Despite being attractive at lower concentrations, undiluted benzaldehyde was repulsive to J2s. Tomato and tobacco root exudates were attractive to J2s while marigold root exudates repelled J2s. The present quantitative assay method could be used as a reference to screen and identify new candidate molecules that attract or repel nematodes.

RNAi-mediated disruption of neuropeptide genes, nlp-3 and nlp-12, cause multiple behavioral defects in Meloidogyne incognita.

Owing to the current deficiencies in chemical control options and unavailability of novel management strategies, root-knot nematode (M. incognita) infections remain widespread with significant socio-economic impacts. Helminth nervous systems are peptide-rich and appear to be putative drug targets that could be exploited by antihelmintic chemotherapy. Herein, to characterize the novel peptidergic neurotransmitters, in silico mining of M. incognita genomic and transciptomic datasets revealed the presence of 16 neuropeptide-like protein (nlp) genes with structural hallmarks of neuropeptide preproproteins; among which 13 nlps were PCR-amplified and sequenced. Two key nlp genes (Mi-nlp-3 and Mi-nlp-12) were localized to the basal bulb and tail region of nematode body via in situ hybridization assay. Mi-nlp-3 and Mi-nlp-12 were greatly expressed (in qRT-PCR assay) in the pre-parasitic juveniles and adult females, suggesting the association of these genes in host recognition, development and reproduction of M. incognita. In vitro knockdown of Mi-nlp-3 and Mi-nlp-12 via RNAi demonstrated the significant reduction in attraction and penetration of M. incognita in tomato root in Pluronic gel medium. A pronounced perturbation in development and reproduction of NLP-silenced worms was also documented in adzuki beans in CYG growth pouches. The deleterious phenotypes obtained due to NLP knockdown suggests that transgenic plants engineered to express RNA constructs targeting nlp genes may emerge as an environmentally viable option to manage nematode problems in crop plants.

Insights on Novel Effectors and Characterization of Metacaspase (RS107_6) as a Potential Cell Death-Inducing Protein in Rhizoctonia solani.

Effectors play an important role in host-pathogen interactions. Though an economically significant disease in rice, knowledge regarding the infection strategy of Rhizoctonia solani is obscure. In this study, we performed a genome-wide identification of the effectors in R. solani based on the characteristics of previously reported effector proteins. A total of seven novel effectors (designated as RS107_1 to RS107_7) in the disease mechanism of R. solani were identified and were predicted to be non-classically secreted proteins with functionally conserved domains. The function, reactivity, and stability of these proteins were evaluated through physiochemical characterization. The target proteins involved in the regulation of rice defense mechanisms were identified. Furthermore, the effector genes were cloned and RS107_6 (metacaspase) was heterologously expressed in Escherichia coli to obtain a purified protein of ~36.5 kDa. The MALD-TOF characterization confirmed that the protein belonged to a metacaspase of the Peptidase_C14 protein family, 906 bp in size, and encoded a polypeptide of 301 amino acids. These findings suggest that the identified effectors can potentially serve as a virulence factor and can be targeted for the management of sheath blight in rice.

Meloidogyne incognita (Nematoda: Meloidogynidae) sterol-binding protein Mi-SBP-1 as a target for its management.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes.

Knockdown of a mucin-like gene in Meloidogyne incognita (Nematoda) decreases attachment of endospores of Pasteuria penetrans to the infective juveniles and reduces nematode fecundity.

Mucins are highly glycosylated polypeptides involved in many host-parasite interactions, but their function in plant-parasitic nematodes is still unknown. In this study, a mucin-like gene was cloned from Meloidogyne incognita (Mi-muc-1, 1125 bp) and characterized. The protein was found to be rich in serine and threonine with numerous O-glycosylation sites in the sequence. Quantitative real-time polymerase chain reaction (qRT-PCR) showed the highest expression in the adult female and in situ hybridization revealed the localization of Mi-muc-1 mRNA expression in the tail area in the region of the phasmid. Knockdown of Mi-muc-1 revealed a dual role: (1) immunologically, there was a significant decrease in attachment of Pasteuria penetrans endospores and a reduction in binding assays with human red blood cells (RBCs), suggesting that Mi-MUC-1 is a glycoprotein present on the surface coat of infective second-stage juveniles (J2s) and is involved in cellular adhesion to the cuticle of infective J2s; pretreatment of J2s with different carbohydrates indicated that the RBCs bind to J2 cuticle receptors different from those involved in the interaction of Pasteuria endospores with Mi-MUC-1; (2) the long-term effect of RNA interference (RNAi)-mediated knockdown of Mi-muc-1 led to a significant reduction in nematode fecundity, suggesting a possible function for this mucin as a mediator in the interaction between the nematode and the host plant.

Meloidogyne incognita Fatty Acid- and Retinol- Binding Protein (Mi-FAR-1) Affects Nematode Infection of Plant Roots and the Attachment of Pasteuria penetrans Endospores.

Root-knot nematode (RKN) Meloidogyne incognita is an economically important pest of crops. Pasteuria penetrans, is a nematode hyperparasitic bacterium capable of suppressing the reproduction of RKN and thereby useful for its management. Secreted fatty acid and retinol-binding proteins are unique in nematodes and are engaged in nutrient acquisition, development and reproduction; they are also a component of the nematode cuticle and thought to be involved in the interface between hosts and parasites. Attachment of endospores to the cuticle of second stage juveniles of RKN is the primary step of infection and several factors have been identified to facilitate attachment. In this study, the full length of Mi-far-1 (573 bp) was cloned from M. incognita and characterized. Analysis revealed that the Mi-far-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a glycosylation site. Quantitative PCR showed the highest expression in the fourth stage juveniles and in situ hybridization revealed the presence of Mi-far-1 mRNA in the hypodermis below the cuticle. Single copy insertion pattern of Mi-far-1 in M. incognita genome was detected by Southern blotting. Knockdown of Mi-far-1 showed significantly increased attachment of P. penetrans' endospores on juvenile cuticle surface and also affected host finding, root infection and nematode fecundity.

Overexpression of Pea DNA Helicase 45 (PDH45) imparts tolerance to multiple abiotic stresses in chili (Capsicum annuum L.).

Imparting tolerance to abiotic stresses is of global importance as they inflict significant yield losses in field as well as in vegetable crops. Transcriptional activators, including helicases are identified to play a pivotal role in stress mitigation. Helicases, also known as molecular motors, are involved in myriad cellular processes that impart intrinsic tolerance to abiotic stresses in plants. Our study demonstrates the potential of a Pea DNA Helicase 45 (PDH45), in combating multiple abiotic stresses in chili. We harnessed Agrobacterium-mediated in planta transformation strategy for the generation of stable, single copy transgenic events. Precise molecular detection of the transgenes by sqRT-PCR coupled with genomic Southern analysis revealed variation in the integration of PDH45 at distinct loci in independent transgenic events. Characterization of five promising transgenic events showed both improved response to an array of simulated abiotic stresses and enhanced expression of several stress-responsive genes. While survival and recovery of transgenic events were significantly higher under gradual moisture stress conditions, under imposition of moderate stress, the transgenic events exhibited invigorated growth and productivity with concomitant improvement in water use efficiency (WUE). Thus, our study, unequivocally demonstrated the cardinal role of PDH45 in alleviating multiple abiotic stresses in chili.

Host-Induced Silencing of Two Pharyngeal Gland Genes Conferred Transcriptional Alteration of Cell Wall-Modifying Enzymes of Meloidogyne incognita vis-à-vis Perturbed Nematode Infectivity in Eggplant.

The complex parasitic strategy of Meloidogyne incognita appears to involve simultaneous expression of its pharyngeal gland-specific effector genes in order to colonize the host plants. Research reports related to effector crosstalk in phytonematodes for successful parasitism of the host tissue is yet underexplored. In view of this, we have used in planta effector screening approach to understand the possible interaction of pioneer genes (msp-18 and msp-20, putatively involved in late and early stage of M. incognita parasitism, respectively) with other unrelated effectors such as cell-wall modifying enzymes (CWMEs) in M. incognita. Host-induced gene silencing (HIGS) strategy was used to generate the transgenic eggplants expressing msp-18 and msp-20, independently. Putative transformants were characterized via qRT-PCR and Southern hybridization assay. SiRNAs specific to msp-18 and msp-20 were also detected in the transformants via Northern hybridization assay. Transgenic expression of the RNAi constructs of msp-18 and msp-20 genes resulted in 43.64-69.68% and 41.74-67.30% reduction in M. incognita multiplication encompassing 6 and 10 events, respectively. Additionally, transcriptional oscillation of CWMEs documented in the penetrating and developing nematodes suggested the possible interaction among CWMEs and pioneer genes. The rapid assimilation of plant-derived carbon by invading nematodes was also demonstrated using 14C isotope probing approach. Our data suggests that HIGS of msp-18 and msp-20, improves nematode resistance in eggplant by affecting the steady-state transcription level of CWME genes in invading nematodes, and safeguard the plant against nematode invasion at very early stage because nematodes may become the recipient of bioactive RNA species during the process of penetration into the plant root.