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Item OsNUOR enhances disease susceptibility by interfering with reactive oxygen species homeostasis and ferroptosis-like cell death(Oxford University Press, 2026)Necrotrophic fungal pathogens such as Rhizoctonia solani, the causal agent of rice (Oryza sativa) sheath blight disease, enhance reactive oxygen species (ROS) production to induce necrosis in infected tissues. Here, we present evidence that the host alternative NADH:ubiquinone oxidoreductase (OsNUOR) facilitates R. solani infection by promoting an oxidative-stress-enriched environment and inducing iron-dependent ferroptosis-like cell death. OsNUOR overexpression (OE) lines exhibit enhanced disease susceptibility, whereas knock-out (KO) lines developed through genome editing demonstrate increased resistance. Infected OE lines have enhanced accumulation of ROS, lipid peroxides, and ferric ions (Fe3+); a significant reduction in antioxidative enzyme (including glutathione peroxidase) activity; and depletion of glutathione levels. In KO lines, the redox status of infected tissues is maintained, and the antioxidative defense is activated. Our data suggest that upregulation of OsNUOR induces mitochondrial ROS accumulation and modulates redox signalling, leading to Fe3+ accumulation and lipid peroxidation that promote necrosis in rice. KO lines are compromised in these processes and therefore exhibit disease resistance. We demonstrate that treatment with ferroptosis inhibitors prevents necrotic lesions, whereas ferroptosis inducers enhance disease severity. Overall, our study reveals the importance of ferroptosis-like cell death in promoting necrosis during R. solani infection in rice.Item Methionine biosynthetic genes and methionine sulfoxide reductase A are required for Rhizoctonia solani AG1-IA to cause sheath blight disease in rice(John Wiley & Sons, 2024) Das, Joyati; Ghosh, Srayan; Tyagi, Kriti; Sahoo, Debashis; Jha, GopaljeeRhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate-active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative-stress-enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double-stranded RNA-mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease-causing ability of Rs_MET13-silenced R. solani and facilitates its growth on 10 mM H2O2-containing minimal-media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA-silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray-induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice.Item Evolution of pathogenicity-associated genes in Rhizoctonia solani AG1-IA by genome duplication and transposon-mediated gene function alterations(BioMed Central Ltd, 2023) Francis, Aleena; Ghosh, Srayan; Tyagi, Kriti; Prakasam, V.; Rani, Mamta; Singh, Nagendra Pratap; Pradhan, Amrita; Sundaram, R. M.; Priyanka, C.; Laha, G. S.; Kannan, C.; Prasad, M. S.; Chattopadhyay, Debasis; Jha, GopaljeeBackground: Rhizoctonia solani is a polyphagous fungal pathogen that causes diseases in crops. The fungal strains are classified into anastomosis groups (AGs); however, genomic complexity, diversification into the AGs and the evolution of pathogenicity-associated genes remain poorly understood. Results: We report a recent whole-genome duplication and sequential segmental duplications in AG1-IA strains of R. solani. Transposable element (TE) clusters have caused loss of synteny in the duplicated blocks and introduced differential structural alterations in the functional domains of several pathogenicity-associated paralogous gene pairs. We demonstrate that the TE-mediated structural variations in a glycosyl hydrolase domain and a GMC oxidoreductase domain in two paralogous pairs affect the pathogenicity of R. solani. Furthermore, to investigate the association of TEs with the natural selection and evolution of pathogenicity, we sequenced the genomes of forty-two rice field isolates of R. solani AG1-IA. The genomic regions with high population mutation rates and with the lowest nucleotide diversity are enriched with TEs. Genetic diversity analysis predicted the genes that are most likely under diversifying and purifying selections. We present evidence that a smaller variant of a glucosamine phosphate N-acetyltransferase (GNAT) protein, predicted to be under purifying selection, and an LPMP_AA9 domain-containing protein, predicted to be under diversifying selection, are important for the successful pathogenesis of R. solani in rice as well as tomato. Conclusions: Our study has unravelled whole-genome duplication, TE-mediated neofunctionalization of genes and evolution of pathogenicity traits in R. solani AG1-IA. The pathogenicity-associated genes identified during the study can serve as novel targets for disease control.Item Heterologous overexpression of PDH45 gene of pea provides tolerance against sheath blight disease and drought stress in rice(Elsevier B.V., 2022) Sahoo, Ranjan Kumar; Chandan, Ravindra Kumar; Swain, Durga Madhab; Tuteja, Narendra; Jha, GopaljeeBiotic and abiotic stress tolerant crops are required for sustainable agriculture as well as ensuring global food security. In a previous study, we have reported that heterologous overexpression of pea DNA helicase (PDH45), a DEAD-box family member protein, provides salinity stress tolerance in rice. The improved management of photosynthetic machinery and scavenging of reactive oxygen species (ROS) are associated with PDH45 mediated salinity stress tolerance. However, the role of PDH45 in biotic and other abiotic stress (drought) tolerance remains unexplored. In the present study, we have generated marker-free transgenic IR64 rice lines that overexpress PDH45 under the CaMV35S promoter. The transgenic rice lines exhibited a significant level of tolerance against sheath blight disease, caused by Rhizoctonia solani, a polyphagous necrotrophic fungal pathogen. The defense as well as antioxidant responsive marker genes were significantly upregulated in the PDH45 overexpressing (OE) rice lines, upon pathogen infection. Moreover, the OE lines exhibited tolerance to drought stress and various antioxidant as well as drought responsive marker genes were significantly upregulated in them, upon drought stress. Overall, the current study emphasizes that heterologous overexpression of PDH45 provides abiotic as well as biotic stress tolerance in rice. Tolerance against drought as well as sheath blight disease by overexpression of a single gene (PDH45) signifies the practical implication of the present study. Moreover, considering the conserved nature of the gene in different plant species, we anticipate that PDH45 can be gainfully deployed to impart tolerance against multiple stresses in agriculturally important crops.Item Foliar application or seed priming of cholic acid-glycine conjugates can mitigate/prevent the rice bacterial leaf blight disease via activating plant defense genes(Frontiers Media S.A., 2021) Pal, Garima; Mehta, Devashish; Singh, Saurabh; Magal, Kalai; Gupta, Siddhi; Jha, Gopaljee; Bajaj, Avinash; Ramu, Vemanna S.Xanthomonas Oryzae pv. oryzae (Xoo) causes bacterial blight and Rhizoctonia solani (R. solani) causes sheath blight in rice accounting for >75% of crop losses. Therefore, there is an urgent need to develop strategies for the mitigation of these pathogen infections. In this study, we report the antimicrobial efficacy of Cholic Acid-Glycine Conjugates (CAGCs) against Xoo and R. solani. We show that CAGC C6 is a broad-spectrum antimicrobial and is also able to degrade biofilms. The application of C6 did not hamper plant growth and showed minimal effect on the plant cell membranes. Exogenous application of C6 on pre-infection or post-infection of Xoo on rice susceptible genotype Taichung native (TN1) can mitigate the bacterial load and improve resistance through upregulation of plant defense genes. We further demonstrate that C6 can induce plant defense responses when seeds were primed with C6 CAGC. Therefore, this study demonstrates the potential of CAGCs as effective antimicrobials for crop protection that can be further explored for field applications.Item Understanding the molecular intricacies of rice-Rhizoctonia solani interactions(American Phytopathological Society, 2019) Ghosh, S.; Kanwar, P.; Jha, GopaljeeSheath blight disease is a devastating disease in rice that causes huge crop losses worldwide. It is caused by a necrotrophic fungus Rhizoctonia solani. In spite of global efforts till now there are no reports of complete resistance against this pathogen. We observed that R. solani has a brief biotrophic phase wherein mycelial grows parallel to rice veins without showing any morphological and anatomical changes. However, at later stage, i.e. necrotrophic phase, infection cushions along with anatomical changes are observed at the site of disease symptoms. Through transcriptome and metabolome studies we have identified several candidate host susceptibility factors and pathogenicity determinants that might play an important role during pathogenesis of R. solani in rice. Furthermore, using whole genome studies, we have identified several gene/gene families that might contribute to the aggressiveness of the R. solani strains. Although gene manipulation studies in R. solani is difficult we have devised ways wherein we can functionally characterize these pathogenicity determinants. Overall the present study will help in better understanding of the rice-R. solani pathosystem which would eventually be helpful in developing strategies for durable sheath blight disease tolerance in rice.Item Host alternative NADH:ubiquinone oxidoreductase serves as susceptibility factor to promote pathogenesis of Rhizoctonia solani in plants(American Phytopathological Society, 2019) Kant, Ravi; Tyagi, Kriti; Ghosh, Srayan; Jha, GopaljeePhytopathogens have evolved mechanisms to utilize host genes (commonly known as susceptibility factors) to promote its pathogenesis. Rhizoctonia solani is one of the highly destructive fungal pathogens of various plants, including rice. Previously we had reported differentially regulated rice genes during pathogenesis of R. solani. In this study, we analyzed the role of tomato homologs of two of the rice genes i.e Isoflavone reductase (IFR) and alternative NADH:ubiquinone oxidoreductase (NUOR) as potential susceptibility factors for R. solani. Virus induced gene silencing (VIGS) of NUOR gene in tomato resulted in compromised susceptibility against R. solani, while IFR-silenced plants demonstrated susceptibility similar to that of control plants. NUOR silencing in tomato led to homogenous accumulation of reactive oxygen species (ROS) (optimum range) upon R. solani infection. In addition, expression and enzyme activities of some of the host defense and anti-oxidant genes were enhanced, while H2O2 content, lipid peroxidation and electrolyte leakage were reduced in NUOR-silenced plants. Similarly, transient silencing of OsNUOR gene provided tolerance against R. solani infection in rice. Overall, the data presented in this study suggests that NUOR serves as a host susceptibility factor to promote pathogenesis of R. solani.Item Bacteria-fungal confrontation and fungal growth prevention assay(Bio-protocol LLC., 2018) Kumar, Rahul; Swain, Durga Madhab; Yadav, Sunil Kumar; Tyagi, Isha; Kumar, Rajeev; Das, Joyati; Ghosh, Srayan; Jha, GopaljeeThere are some bacteria which can grow and multiply at the cost of living fungal biomass. They can potentially utilize fungi as a source of nutrients to forage over them. Such phenomenon is known as bacterial mycophagy, however, its mechanistic insights need to be explored to identify the molecules involved in mycophagy for potential utilization in controlling various fungal diseases. Recently we have demonstrated that a rice-associated bacteria Burkholderia gladioli strain NGJ1 exhibits mycophagous ability on several fungi, including Rhizoctonia solani, the necrotrophic fungal pathogen causing sheath blight disease in rice. We hereby describe our validated and efficient methods used to study B. gladioli strain NGJ1-R. solani interactions. These methodologies would be useful for designing assays to study the confrontation between bacteria and fungi which in turn enable discovery of novel antifungal molecules from such bacteria.Item Alterations in rice chloroplast integrity, photosynthesis and metabolome associated with pathogenesis of Rhizoctonia solani(Nature Publishing Group, 2017) Ghosh, S; Kanwar, P; Jha, GopaljeeSheath blight disease is caused by a necrotrophic fungal pathogen Rhizoctonia solani and it continues to be a challenge for sustainable rice cultivation. In this study, we adopted a multi-pronged approach to understand the intricacies of rice undergoing susceptible interactions with R. solani. Extensive anatomical alteration, chloroplast localized ROS, deformed chloroplast ultrastructure along with decreased photosynthetic efficiency were observed in infected tissue. GC-MS based metabolite profiling revealed accumulation of glycolysis and TCA cycle intermediates, suggesting enhanced respiration. Several aromatic and aliphatic amino acids along with phenylpropanoid intermediates were also accumulated, suggesting induction of secondary metabolism during pathogenesis. Furthermore, alterations in carbon metabolism along with perturbation of hormonal signalling were highlighted in this study. The gene expression analysis including RNAseq profiling reinforced observed metabolic alterations in the infected tissues. In conclusion, the present study unravels key events associated during susceptible rice-R. solani interactions and identifies metabolites and transcripts that are accumulated in infected tissues.
