Browsing by Author "Kumar, Rahul"

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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, Gopaljee
Bacteria 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.
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, Gopaljee
There 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.
Burkholderia gladioli strain NGJ1 deploys a prophage tail-like protein for mycophagy
(Shared Science Publishers OG, 2018) Kumar, Rahul; Yadav, Sunil Kumar; Swain, Durga Madhab; Jha, Gopaljee
Fungal pathogens are responsible for approximately two third of the infectious plant diseases. Historically they have been associated with several devastating famines, causing death and disabilities in humans. Mostly fungal diseases are being controlled by using fungicides which otherwise have adverse side effects on the health of consumers as well as environment. Due to extensive usages, pathogens have evolved resistance against most of the commonly used fungicides and rendered them ineffective. Controlling fungal disease in a sustainable and eco-friendly fashion remains a challenge. The antifungal biocontrol agents are being considered as potent, alternative and ecofriendly approach to manage fungal diseases. In our recent work, we have identified a rice associated bacterium; Burkholderia gladioli strain NGJ1 which demonstrates broad spectrum fungal eating (mycophagous) property. We determined that the bacterium utilizes its type III secretion system (Injectisome) machinery to deploy a prophage tail-like protein (Bg_9562) into fungal cells to devour them. The purified Bg_9562 protein from overexpressing recombinant E. coli strain demonstrates broad spectrum antifungal activity. Overall our study opens up a new opportunity to exploit prophage taillike protein as potent antifungal compound to control plant as well as animal fungal diseases.
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, Gopaljee
Background: 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
Concurrent overexpression of rice G-protein β and γ subunits provide enhanced tolerance to sheath blight disease and abiotic stress in rice
(Springer Nature Publishing AG, 2019) Swain, Durga Madhab; Sahoo, Ranjan Kumar; Chandan, Ravindra Kumar; Ghosh, Srayan; Kumar, Rahul; Jha, Gopaljee; Tuteja, Narendra
The heterotrimeric G-proteins act as signalling molecules and modulate various cellular responses including stress tolerance in eukaryotes. The gamma (γ) subunit of rice G-protein (RGG1) was earlier reported to promote salinity stress tolerance in rice. In the present study, we report that a rice gene-encoding beta (β) subunit of G-protein (RGB1) gets upregulated during both biotic (upon a necrotrophic fungal pathogen, Rhizoctonia solani infection) and drought stresses. Markerfree transgenic IR64 rice lines that simultaneously overexpress both RGB1 and RGG1 genes under CaMV35S promoter were raised. The overexpressing (OE) lines showed enhanced tolerance to R. solani infection and salinity/drought stresses. Several defense marker genes including OsMPK3 were signifcantly upregulated in the R. solani-infected OE lines. We also found the antioxidant machineries to be upregulated during salinity as well as drought stress in the OE lines. Overall, the present study provides evidence that concurrent overexpression of G-protein subunits (RGG1 and RGB1) impart multiple (both biotic and abiotic) stress tolerance in rice which could be due to the enhanced expression of stress-marker genes and better management of reactive oxygen species (ROS)-scavenging/photosynthetic machinery. The current study suggests an improved approach for simultaneous improvement of biotic and abiotic stress tolerance in rice which remains a major challenge for its sustainable cultivation.
Genome-wide analysis of auxin response factor (ARF) gene gamily from tomato and analysis of their role in flower and fruit development
(Springer Science, 2011) Kumar, Rahul; Tyagi, Akhilesh K.; Sharma, Arun K.
Auxin response transcription factors have been widely implicated in auxin-mediated responses during various developmental processes ranging from root and shoot development to flower and fruit development in plants. In order to use them for improvement of agronomic traits related to fruit, we need to have better understanding of their role during fruit development. In this study, 17 SlARF genes have been identified from tomato (Solanum lycopersicum), using various publically available tomato EST databases. Phylogenetic analysis of the 23 AtARF and 17 SlARF proteins results in formation of three major classes and a total of 14 sister pairs, including seven SlARF-AtARF, four SlARF-SlARF and three AtARF-AtARF sister pairs, providing insights into various orthologous relationships between AtARFs and SlARFs. Further, search for orthologs of these SlARFs resulted in identification of nine, ten, four and three ARF genes from potato, tobacco, N. benthemiana and pepper, respectively. A phylogenetic analysis of these genes, along with their orthologs from Solanaceae species, suggests the presence of a common set of the ARF genes in this family. Comparison of the expression of these SlARF genes in wild type and rin mutant provides an insight into their role during different stages of flower and fruit development. This study suggests that ARF genes may play diverse role during flower and fruit development. Comprehensive data generated here will provide a platform for identification of ARF genes and elucidation of their function during reproductive development stages in Solanaceae in general and fruit development in tomato, in particular.
Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive GH3 genes during development and response to different stimuli in tomato (Solanum lycopersicum)
(Springer Science, 2012) Kumar, Rahul; Agarwal, Priyanka; Tyagi, Akhilesh K.; Sharma, Arun K.
In plants, auxin-mediated responses are regulated by diverse proteins. One such class of proteins, i.e. GH3, is involved in the conjugation of IAA to amino acids and provides a negative feedback loop to control auxin homoeostasis. In order to have a better understanding of the mechanism of the auxin action, 15 genes encoding GH3 members were identified using existing EST databases of tomato. Their orthologs were identified from tobacco, potato, N. benthemiana, pepper, and petunia. Phylogenetic analysis of AtGH3, SlGH3, and their Solanaceae orthologs provided insights into various orthologous relationships among these proteins. These genes were found to be responsive to a variety of signals including, phytohormones and environmental stresses. Analysis of AuxRE elements in their promoters showed variability in the sequence as well as number of this element. Up-regulation of only 11 SlGH3 genes, in response to exogenous auxin, suggested possible relationship between the diversity in the sequence and number of AuxRE element with the auxin inducibility. Expression analysis of SlGH3 genes in different vegetative and reproductive tissues/stages suggested limited or no role for most of the SlGH3 genes at the initiation of fruit ripening. However, up-regulation of SlGH3-1 and -2 at the onset of fruit ripening indicates that these genes could have a role in fruit ripening. The present study characterizes GH3 gene family of tomato and its evolutionary relationship with members of this family from other Solanaceae species and Arabidopsis. It could help in the identification of GH3 genes and revelation of their function during vegetative/reproductive development stages from other Solanaceae members.
Genomic survey, gene expression, and interaction analysis suggest diverse roles of ARF and Aux/IAA proteins in Solanaceae
(Springer, 2015) Kumar, Rahul; Agarwal, Priyanka; Pareek, Amit; Tyagi, Akhilesh K.; Sharma, Arun K.
Auxin response factor (ARF) and Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are the foremost regulators of auxin action and play an essential role in the coordination of many aspects of plant growth and development. Though many members of both ARF and Aux/IAA gene families have been identified and characterized in tomato, they are less studied in other Solanaceae species. In the present study, we focused on gaining insights into their functional conservation as well as diversification during auxin-mediated responses in Solanaceae. First, we identified their full complement in tomato, potato, pepper, Nicotiana benthamiana, eggplant, and petunia and found that both the gene families have expanded in N. benthamiana. We also looked into the structural variations associated with all the members of these two classes of genes in tomato and showed that huge natural variation exists in their sequence in wild relatives. The comprehensive gene expression analysis provided evidence of high conservation in the expression of orthologous ARFs and Aux/IAAs during fruit development and ripening in tomato and pepper. Furthermore, the molecular changes caused by exogenous plant hormones and abiotic stress conditions on their transcript levels were investigated which showed that many members of both the gene families may participate in various hormone- and stress-mediated responses in tomato and potato. Some of these genes may play a role in linking the hormone-controlled plant growth and stress-related signaling pathways. Finally, we demonstrate that single tomato ARF can interact with multiple Aux/IAA proteins and vice versa. Overall, our study will be very helpful in establishing both conserved as well as non-conserved functions of these genes in Solanaceae.
Identification and establishment of genomic identity of Ralstonia solanacearum isolated from a wilted chilli plant at Tezpur, North East India
(Indian Academy of Sciences, 2013) Kumar, Rahul; Barman, Anjan; Jha, Gopaljee; Ray, Suvendra Kumar
The bacterial wilt disease caused by Ralstonia solanacearum is an ever-increasing threat to tropical as well as temperate regions of the world. Though the disease has been reported from different parts of India, appropriate identification of the pathogen at molecular level is still incomplete. In this study we report the isolation and molecular characterization of a R. solana- cearum strain F1C1 from wilted chilli plant collected from a field near Tezpur University, Assam, India, using techniques such as multiplex PCR, 16SrDNA sequencing, multilocus typing, pathogenicity test, twitching motility and natural transformation. Our results suggest that F1C1 is a phylotype-I strain of R. solanacearum species complex. Additionally, we also report presence of other bacterial species in the ooze collected from wilted plants.
Identification and validation of sex-linked SCAR markers in dioecious Hippophae rhamnoides L. (Elaeagnaceae)
(Springer Science, 2012) Korekar, Girish; Sharma, Ram Kumar; Kumar, Rahul; Bisht, Naveen C.; Srivastava, Ravi B.; Ahuja, Paramvir Singh; Stobdan, Tsering
The actinorhizal plant seabuckthorn (Hippophae rhamnoides L., Elaeagnaceae) is a wind pollinated dioecious crop. To distinguish male genotypes from female genotypes early in the vegetative growth phase, we have developed robust PCR-based marker(s). DNA bulk samples from 20 male and 20 female plants each were screened with 60 RAPD primers. Two primers, OPA-04 and OPT-06 consistently amplified female-specific (FS) polymorphic fragments of 1,164 and 868 bp, respectively, that were absent in the male samples. DNA sequence of the two markers did not exhibit significant similarity to previously characterized sequences. A sequence-characterized amplified region marker HrX1 (JQ284019) and HrX2 (JQ284020) designed for the two fragments, continued to amplify the FS allele in 120 female plants but not in 100 male plants tested in the current study. Thus, HrX1 and HrX2 are FS markers that can determine the sex of seabuckthorn plants in an early stage and expedite cultivations for industrial applications.
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, Rahul
F-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.
Identification, phylogeny and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato
(Springer, 2010) Sharma, Manoj K.; Kumar, Rahul; Solanke, Amolkumar U.; Sharma, Rita; Tyagi, Akhilesh K.; Sharma, Arun K.
Ethylene responsive transcription factors have been shown to be intimately connected to plant development, defense responses and stress signaling pathways and in order to use them for plant improvement, we need to have better understanding of these proteins. In this study, 85 ERF genes have been identified from tomato using raw EST data in various public repositories. Phylogenetic analysis with tomato ERF domains revealed their distribution in all the groups, previously identified in model systems. MEME motif analysis resulted in identification of conserved domains, characteristic to member of each clade, in addition to ERF domain. Expression analysis during vegetative and reproductive stages of development using QPCR and tomato GeneChip arrays, revealed their tissue-specific/preferential accumulation. In total, 57 genes were found to be differentially expressed during temporal stages of tomato fruit development. The expression analysis of 23 ERF family genes representing each clade in response to seven abiotic stress treatments revealed their differential expression in response to more than one abiotic stress treatments. Results suggest that ERF genes play diverse roles in plant's life and comprehensive data generated will be helpful in conducting functional genomics studies to understand their precise role during plant development and stress response.
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, Gopaljee
Bacteria 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.
Nicotinic acid catabolism modulates bacterial mycophagy in Burkholderia gladioli strain NGJ1
(American Society for Microbiology, 2023) Das, Joyati; Kumar, Rahul; Yadav, Sunil Kumar; Jha, Gopaljee
Burkholderia 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.
Plant viral vectors: expanding the possibilities of precise gene editing in plant genomes
(Springer Nature Publishing AG, 2021) Kujur, Stuti; Senthil-Kumar, Muthappa; Kumar, Rahul
The 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.
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, Gopaljee
A 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.
A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi
(Nature Publishing Group, 2017) Swain, Durga Madhab; Yadav, Sunil Kumar; Tyagi, Isha; Kumar, Rahul; Kumar, Rajeev; Ghosh, Srayan; Das, Joyati; Jha, Gopaljee
Some bacteria can feed on fungi, a phenomenon known as mycophagy. Here we show that a prophage tail-like protein (Bg_9562) is essential for mycophagy in Burkholderia gladioli strain NGJ1. The purified protein causes hyphal disintegration and inhibits growth of several fungal species. Disruption of the Bg_9562 gene abolishes mycophagy. Bg_9562 is a potential effector secreted by a type III secretion system (T3SS) and is translocated into fungal mycelia during confrontation. Heterologous expression of Bg_9562 in another bacterial species, Ralstonia solanacearum, confers mycophagous ability in a T3SS-dependent manner. We propose that the ability to feed on fungi conferred by Bg_9562 may help the bacteria to survive in certain ecological niches. Furthermore, considering its broad-spectrum antifungal activity, the protein may be potentially useful in biotechnological applications to control fungal diseases.
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, Gopaljee
Phytopathogens 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.
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, Gopaljee
Rhizoctonia 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.
Transcriptome analysis of rin mutant fruit and in silico analysis of promoters of differentially regulated genes provides insight into LeMADS-RIN-regulated ethylene-dependent as well as ethylene-independent aspects of ripening in tomato
(Springer Science, 2012) Kumar, Rahul; Sharma, Manoj K.; Kapoor, Sanjay; Tyagi, Akhilesh K.; Sharma, Arun K.
A thorough understanding of molecular mechanisms underlying ripening is the prerequisite for genetic manipulation of fruits for better shelf-life and nutritional quality. Mutation in LeMADS-RIN, a MADS-box gene, leads to non-ripening phenotype of rin fruits in tomato. Characterization of ripening-inhibitor (rin) mutant has elucidated important role of ethylene in the regulation of climacteric fruit ripening. A complete understanding of this mutation will unravel novel genetic regulatory mechanisms involved in fruit ripening. In this study, fruit transcriptomes of two genotypes, including a cultivated Indian cultivar Solanum lycopersicum cv. Pusa Ruby and a homozygous line harboring the rin mutation (LA1795) were compared to get better insight into RIN-regulated ethylene-dependent and ethylene-independent events during ripening. Cluster analysis of ripening-related genes indicated a major shift in their expression profiles in rin mutant fruit. A total of 112 genes, exhibiting expression patterns similar to that of LeMADS-RIN in wild-type fruits, showed down regulation of expression in the rin mutant. In silico analysis of putative promoters of these genes for the presence of CArG box along with ERE and ethylene inducibility of these genes revealed that genes lacking CArG box in their regulatory regions could be indirectly regulated by LeMADS-RIN. New regulators of ethylene-dependent aspect of ripening were also identified. In this study, we have made an attempt to distinguish between ethylene-dependent and ethylene-independent aspects of ripening, which will be useful for developing strategies to improve fruit-related agronomic traits in tomato and other crops.