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Item Rs_MEP1 is required for the pathogenesis of Rhizoctonia solani AG1-IA in plants(The American Phytopathological Society (APS), 2026) Pradhan, Amrita; Sahoo, Debashis; Bhati, Vikrant; Kumar, Rahul; Yadav, Rajni; Ghosh, Srayan; Pawar, Prashant Anupama-Mohan; Jha, GopaljeeRhizoctonia solani AGI-IA is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. Efforts are being made to identify pathogenicity-associated genes in R. solani and modulate them to develop a disease control strategy. Here, we investigate the roles of some predicted pathogenicity-associated genes of R. solani that have previously been reported to be upregulated during infection in rice. The tobacco rattle virus-based host-induced gene silencing of the selected pathogenicity-associated genes revealed that silencing of Rs_MEP1, a zinc-containing Peptidase_M43 domain-metalloprotease, severely compromises R. solani infection in tomato. Moreover, double-stranded RNA-mediated silencing of Rs_MEP1 prevented R. solani infection in rice. The signal sequence trap assay indicated the secretory nature of Rs_MEP1, while the reporter assay suggested its localization in the plant apoplast. Notably, agrobacterium-mediated transient overexpression of Rs_MEP1 induces necrotic cell death responses in plants. We provide evidence that Rs_MEP1 interacts with GH19 family of rice chitinases and potentially modulates their functions. Overall, our study emphasizes that Rs_MEP1 facilitates R. solani in promoting necrotic responses and targets rice GH19 chitinases to impart disease susceptibility in plants.Item A prophage tail-like protein facilitates the endophytic growth of Burkholderia gladioli and mounting immunity in tomato(John Wiley & Sons, 2023) Chandan, Ravindra Kumar; Kumar, Rahul; Kabyashree, Kristi; Yadav, Sunil Kumar; Roy, Mandira; Swain, Durga Madhab; Jha, GopaljeeA prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.Item Nicotinic acid catabolism modulates bacterial mycophagy in Burkholderia gladioli strain NGJ1(American Society for Microbiology, 2023) Das, Joyati; Kumar, Rahul; Yadav, Sunil Kumar; Jha, GopaljeeBurkholderia gladioli strain NGJ1 exhibits mycophagous activity on a broad range of fungi, including Rhizoctonia solani, a devastating plant pathogen. Here, we demonstrate that the nicotinic acid (NA) catabolic pathway in NGJ1 is required for mycophagy. NGJ1 is auxotrophic to NA and it potentially senses R. solani as a NA source. Mutation in the nicC and nicX genes involved in NA catabolism renders defects in mycophagy and the mutant bacteria are unable to utilize R. solani extract as the sole nutrient source. As supplementation of NA, but not FA (fumaric acid, the end product of NA catabolism) restores the mycophagous ability of ΔnicC/ΔnicX mutants, we anticipate that NA is not required as a carbon source for the bacterium during mycophagy. Notably, nicR, a MarR-type of transcriptional regulator that functions as a negative regulator of the NA catabolic pathway is upregulated in ΔnicC/ΔnicX mutant and upon NA supplementation the nicR expression is reduced to the basal level in both the mutants. The ΔnicR mutant produces excessive biofilm and is completely defective in swimming motility. On the other hand, ΔnicC/ΔnicX mutants are compromised in swimming motility as well as biofilm formation, potentially due to the upregulation of nicR. Our data suggest that a defect in NA catabolism alters the NA pool in the bacterium and upregulates nicR which in turn suppresses bacterial motility as well as biofilm formation, leading to mycophagy defects. IMPORTANCE Mycophagy is an important trait through which certain bacteria forage over fungal mycelia and utilize fungal biomass as a nutrient source to thrive in hostile environments. The present study emphasizes that nicotinic acid (NA) is important for bacterial motility and biofilm formation during mycophagy by Burkholderia gladioli strain NGJ1. Defects in NA catabolism potentially alter the cellular NA pool, upregulate the expression of nicR, a negative regulator of biofilm, and therefore suppress bacterial motility as well as biofilm formation, leading to mycophagy defects.Item RAV1 family members function as transcriptional regulators and play a positive role in plant disease resistance(John Wiley & Sons, 2023) Chandan, Ravindra Kumar; Kumar, Rahul; Swain, Durga Madhab; Ghosh, Srayan; Bhagat, Prakash Kumar; Patel, Sunita; Bagler, Ganesh; Sinha, Alok Krishna; Jha, GopaljeePhytopathogens pose a severe threat to agriculture and strengthening plant defense response is an important strategy for disease control. Here, we report that AtRAV1, an AP2 and B3 domain-containing transcription factor is required for basal plant defense in Arabidopsis thaliana. The atrav1 mutant lines demonstrate hyper-susceptibility against fungal pathogens (Rhizoctonia solani and Botrytis cinerea) while AtRAV1 overexpressing (OE) lines exhibit disease resistance against them. Enhanced expression of various defense genes and activation of MAP kinases (AtMPK3 and AtMPK6) are observed in the R. solani infected OE lines, but not in the atrav1 mutant plants. In-vitro phosphorylation assay suggests AtRAV1 to be a novel phosphorylation target of AtMPK3. The bimolecular fluorescence complementation and yeast two-hybrid assay support physical interactions between AtRAV1 and AtMPK3. Overexpression of the native as well as phospho-mimic but not the phospho-defective variant of AtRAV1 impart disease resistance in the atrav1 mutant A. thaliana lines. On the other hand, overexpression of AtRAV1 fails to impart disease resistance in the atmpk3 mutant. These analyses emphasize that AtMPK3-mediated phosphorylation of AtRAV1 is important for the elaboration of defense response in A. thaliana. Considering that RAV1 homologs are conserved in diverse plant species, we propose that they can be gainfully deployed to impart disease resistance in agriculturally important crop plants. Indeed, overexpression of SlRAV1 (a member of the RAV1 family) imparts disease tolerance against not only fungal (R. solani and B. cinerea) but also against bacterial (Ralstonia solanacearum) pathogens in tomato, while silencing of the gene enhances disease susceptibility.Item The alternative sigma factors, rpoN1 and rpoN2 are required for mycophagous activity of Burkholderia gladioli strain NGJ1(John Wiley & Sons, 2022) Das, Joyati; Kumar, Rahul; Yadav, Sunil Kumar; Jha, GopaljeeBacteria utilize RpoN, an alternative sigma factor (σ54) to grow in diverse habitats, including nitrogen-limiting conditions. Here, we report that a rice-associated mycophagous bacterium Burkholderia gladioli strain NGJ1 encodes two paralogues of rpoN viz. rpoN1 and rpoN2. Both of them are upregulated during 24 h of mycophagous interaction with Rhizoctonia solani, a polyphagous fungal pathogen. Disruption of either one of rpoNs renders the mutant NGJ1 bacterium defective in mycophagy, whereas ectopic expression of respective rpoN genes restores mycophagy in the complementing strains. NGJ1 requires rpoN1 and rpoN2 for efficient biocontrol to prevent R. solani to establish disease in rice and tomato. Further, we have identified 17 genes having RpoN regulatory motif in NGJ1, majority of them encode potential type III secretion system (T3SS) effectors, nitrogen assimilation, and cellular transport-related functions. Several of these RpoN regulated genes as well as certain previously reported T3SS apparatus (hrcC and hrcN) and effector (Bg_9562 and endo-β-1,3-glucanase) encoding genes are upregulated in NGJ1 but not in ΔrpoN1 or ΔrpoN2 mutant bacterium, during mycophagous interaction with R. solani. This highlights that RpoN1 and RpoN2 modulate T3SS, nitrogen assimilation as well as cellular transport systems in NGJ1 and thereby promote bacterial mycophagy.Item Plant viral vectors: expanding the possibilities of precise gene editing in plant genomes(Springer Nature Publishing AG, 2021) Kujur, Stuti; Senthil-Kumar, Muthappa; Kumar, RahulThe lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.Item Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors(EMBO Press, 2021) Yadav, Sunil Kumar; Magotra, Ankita; Ghosh, Srayan; Krishnan, Aiswarya; Pradhan, Amrita; Kumar, Rahul; Das, Joyati; Sharma, Mamta; Jha, GopaljeeBacteria utilize type VI secretion system (T6SS) to deliver antibacterial toxins to target co-habiting bacteria. Here, we report that Burkholderia gladioli strain NGJ1 deploys certain T6SS effectors (TseTBg), having both DNase and RNase activities to kill target bacteria. RNase activity is prominent on NGJ1 as well as other bacterial RNA while DNase activity is pertinent to only other bacteria. The associated immunity (TsiTBg) proteins harbor noncanonical helix–turn–helix motifs and demonstrate transcriptional repression activity, similar to the antitoxins of type II toxin– antitoxin (TA) systems. Genome analysis reveals that homologs of TseTBg are either encoded as TA or T6SS effectors in diverse bacteria. Our results indicate that a new ORF (encoding a hypothetical protein) has evolved as a result of operonic fusion of TA type TseTBg homolog with certain T6SS-related genes by the action of IS3 transposable elements. This has potentially led to the conversion of a TA into T6SS effector in Burkholderia. Our study exemplifies that bacteria can recruit toxins of TA systems as T6SS weapons to diversify its arsenal to dominate during inter-bacterial competitions.Item Identification, evolutionary profiling, and expression analysis of F-box superfamily genes under phosphate deficiency in tomato(Elsevier B.V., 2021) Akash; Parida, Adwaita Prasad; Srivastava, Alok; Mathur, Saloni; Sharma, Arun Kumar; Kumar, RahulF-box genes are an integral component of the Skp1-cullin-F-box (SCF) complex in eukaryotes. These genes are primarily involved in determining substrate specificities during cellular proteolysis. Here we report that 410 members constitute the F-box superfamily in tomato. Based on the incidence of C-terminal domains, these genes fell into ten subfamilies, leucine-rich repeat domain-containing F-box members constituting the largest subfamily. The F-box genes are present on all 12 chromosomes with varying gene densities. Both segmental and tandem duplication events contribute significantly to their expansion in the tomato genome. The syntenic analysis revealed close relationships among F-box homologs within Solanaceae species genomes. Transcript profiling of F-box members identified several ripening-associated genes with altered expression in the ripening mutants. RNA-sequencing data analysis showed that phosphate (Pi) deficiency affected 55 F-box transcripts in the Pi-deficient seedlings compared to their control seedlings. The persistent up-regulation of eight members, including two phloem protein 2B (PP2–B) genes, PP2–B15, and MATERNAL EFFECT EMBRYO ARREST 66 (MEE66) homologs, at multiple time-points in the roots, shoot, and seedling, point towards their pivotal roles in Pi starvation response in tomato. The attenuation of such upregulation in sucrose absence revealed the necessity of this metabolite for robust activation of these genes in the Pi-deficient seedlings. Altogether, this study identifies novel F-box genes with potential roles in fruit ripening and Pi starvation response and unlocks new avenues for functional characterization of candidate genes in tomato and other related species.Item Calcium regulates the mycophagous ability of Burkholderia gladioli strain NGJ1 in a type III secretion system-dependent manner(BioMed Central Ltd, 2020) Yadav, Sunil Kumar; Das, Joyati; Kumar, Rahul; Jha, GopaljeeBackground: A rice associated bacterium Burkholderia gladioli strain NGJ1 demonstrates mycophagy, a phenomenon wherein bacteria feed on fungi. Previously, we have reported that NGJ1 utilizes type III secretion system (T3SS) to deliver a prophage tail-like protein (Bg_9562) into fungal cells to establish mycophagy. Results: In this study, we report that calcium ion concentration influences the mycophagous ability of NGJ1 on Rhizoctonia solani, an important fungal pathogen. The calcium limiting condition promotes mycophagy while high calcium environment prevents it. The expression of various T3SS apparatus encoding genes of NGJ1 was induced and secretion of several potential T3SS effector proteins (including Bg_9562) into extracellular milieu was triggered under calcium limiting condition. Using LC-MS/MS proteome analysis, we identified several calcium regulated T3SS effector proteins of NGJ1. The expression of genes encoding some of these effector proteins was upregulated during mycophagous interaction of NGJ1 with R. solani. Further, mutation of one of these genes (endo-β-1, 3- glucanase) rendered the mutant NGJ1 bacterium defective in mycophagy while complementation with full length copy of the gene restored its mycophagous activity. Conclusion: Our study provides evidence that low calcium environment triggers secretion of various T3SS effectors proteins into the extracellular milieu and suggests the importance of cocktail of these proteins in promoting mycophagy