Rhizoctonia solani AG1-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 investigated 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, and 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 the 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. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Phytopathogens pose a severe threat to agriculture and strengthening the 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), whereas AtRAV1 overexpressing lines exhibit disease resistance against them. Enhanced expression of various defense genes and activation of mitogen-activated protein kinases (AtMPK3 and AtMPK6) are observed in the R. solani infected overexpressing lines, but not in the atrav1 mutant plants. An in vitro phosphorylation assay suggests AtRAV1 to be a novel phosphorylation target of AtMPK3. Bimolecular fluorescence complementation and yeast two-hybrid assays support physical interactions between AtRAV1 and AtMPK3. Overexpression of the native as well as phospho-mimic but not the phospho-defective variant of AtRAV1 imparts 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 the 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, whereas silencing of the gene enhances disease susceptibility.

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.

Burkholderia gladioli strain NGJ1, isolated from healthy rice seedling, was earlier demonstrated to have mycophagous properties on a broad range of fungi, including Rhizoctonia solani , a causal agent of deadly sheath blight disease of rice. The purified Bg_9562 protein exerts broad-spectrum antifungal activity.

Many plant-associated bacteria produce plant- mimicking hormones which are involved in modulating host physiology. However, their function in modulating bacterial physiology has not been reported. Here we show that the XopQ protein, a type-III effector of the rice pathogen, Xanthomonas oryzae pv. oryzae ( Xoo ), is involved in cytokinin biosynthesis. Xoo produces and secretes an active form of cytokinin which enables the bacterium to maintain a planktonic lifestyle and promotes virulence. RNA-seq analysis indicates that the cytokinin produced by Xoo is required for the regulation of several genes which are involved in biofilm formation. We have also identified the Xoo isopentenyl transferase gene, which is involved in the cytokinin biosynthesis pathway and is required for maintaining planktonic behaviour and virulence. Furthermore, mutations in the predicted cytokinin receptor kinase (PcrK) and the downstream response regulator (PcrR) of Xoo phenocopy the cytokinin biosynthetic mutants, but are not complemented by supplementation with exogenous cytokinin. Cytokinin biosynthetic functions are encoded in a number of diverse bacterial genomes suggesting that cytokinin may be a widespread signalling molecule in the bacterial kingdom.

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 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 non-canonical 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.

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.

Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24-hour intervals from 0 days post-inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon-induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.

Plant diseases pose a serious threat to sustainable agriculture as controlling them in eco-friendly manner remains a challenge. In this study, we establish RAV1 as a master transcriptional regulator of defense genes in model plant Arabidopsis. The overexpression of AtRAV1 provided disease resistance against necrotrophic fungal pathogen ( Rhizoctonia solani) infection in A. thaliana . The transgenic lines exhibited enhanced expression of several defense genes including mitogen associated protein kinases (MAPKs) and the amplitude of their expression was further enhanced upon pathogen infection. Conversely, the atrav1 mutant plants were unable to induce the expression of these defense genes and were highly susceptible to infection. Our data suggests that upon pathogen attack, AtRAV1 transcriptionally upregulate the expression of MAPKs ( AtMPK3, AtMPK4 and AtMPK6 ) and AtMPK3 and AtMPK6 are essential for AtRAV1 mediated disease resistance. Further, we demonstrate that AtRAV1 is a phosphorylation target of AtMPK3 (but not AtMPK6) and the phospho-defective variants of AtRAV1 are unable to induce disease resistance in A. thaliana . Considering the presence of AtRAV1 orthologs in diverse plant species, we propose that they can be gainfully deployed to control economically important diseases. In deed we observe that overexpression of tomato ortholog of AtRAV1 ( SlRAV1 ) provides broad spectrum disease resistance against bacterial ( Ralstonia solanacearum) , fungal ( R. solani ) and viral (Tomato leaf curl virus) infections in tomato.

Bacteria use various kinds of toxins to either inhibit the growth of co-habiting bacteria or when needed control their own growth. Here we report that Burkholderia and certain other bacteria have altered the potential defensive function of Tox-REase-5 domain containing toxins into offensive function. The Burkholderia gladioli strain NGJ1 encodes such toxins as type VI secretion system (T6SS) effectors (Tse) and potentially deploys them to kill co-habiting rice endophytic bacteria. Notably, the immunity (Tsi) proteins associated with Tse effectors demonstrate functional similarity with the antitoxin of type II toxin-antitoxin (TA) system. Genome analysis of diverse bacteria revealed that various Tse orthologs are either encoded as TA or T6SS effectors. In addition, potential evolutionary events associated with conversion of TA into T6SS effectors have been delineated. Our results indicate that the transposition of IS3 elements has led to the operonic fusion of certain T6SS related genes with TA genes resulting in their conversion into T6SS effectors. Such a genetic change has enabled bacteria to utilize novel toxins to precisely target co-habiting bacteria.

Our study demonstrates that simultaneous overexpression of RGB1 and RGG1 genes provides multiple stress tolerance in rice by inducing stress responsive genes and better management of ROS scavenging/photosynthetic machineries. 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. Marker-free 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 significantly 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.

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.

In this study, we report Ralstonia solanacearum pathogenicity in the early stages of tomato seedlings by an innovative root inoculation method. Pathogenicity assays were performed under gnotobiotic conditions in microfuge tubes by employing only 6- to 7-day-old tomato seedlings for root inoculation. Tomato seedlings inoculated by this method exhibited the wilted symptom within 48 h and the virulence assay can be completed in 2 weeks. Colonization of the wilted seedlings by R. solanacearum was confirmed by using gus staining as well as fluorescence microscopy. Using this method, mutants in different virulence genes such as hrpB, phcA, and pilT could be clearly distinguished from wild-type R. solanacearum. The method described here is economic in terms of space, labor, and cost as well as the required quantity of bacterial inoculum. Thus, the newly developed assay is an easy and useful approach for investigating virulence functions of the pathogen at the seedling stage of hosts, and infection under these conditions appears to require pathogenicity mechanisms used by the pathogen for infection of adult plants.

Ralstonia solanacearum (F1C1) is a Gram-negative plant pathogenic bacterium that causes lethal wilt disease in a wide range of plant species. This pathogen is very well known for its unpredictable behavior during infection and wilting its host. Because of its mysterious infection behavior, virulence and pathogenicity standardization are still a big challenge in the case of R. solanacearum. Here, we report an innovative pathogenicity assay of R. solanacearum (F1C1) in the early stage of tomato seedlings by root dip inoculation. In this assay, we employed 6-7days old tomato seedlings for infection grown under nutrients free and gnotobiotic condition. After that, pathogenicity assay was performed by maintaining the inoculated seedlings in 1.5 or 2 ml sterile microfuge tubes. During infection, wilting symptom starts appearing from ~48 h post inoculation and the pathogenicity assay gets completed within seven days of post inoculation. This method is rapid, consistent as well as less resource dependent in terms of labor, space and cost to screen large numbers of plants. Hence, this newly developed assay is an easy and useful approach to study pathogen virulence functions and its interaction with the host plant during wilting and disease progression at the seedling stage.