Methionine biosynthetic genes and methionine sulfoxide reductase A are required for Rhizoctonia solani AG1-IA to cause sheath blight disease in rice.

Rhizoctonia 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.

Evolution of pathogenicity-associated genes in Rhizoctonia solani AG1-IA by genome duplication and transposon-mediated gene function alterations.

BACKGROUND: 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.

RAV1 family members function as transcriptional regulators and play a positive role in plant disease resistance.

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.

Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors.

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.

Enzymatic and non-enzymatic functional attributes of plant microbiome.

Microbiome plays an important role in plant growth and adaptation to various environmental conditions. The cross-talk between host plant and microbes (including microbe-microbe interactions) plays a crucial role in shaping the microbiome. Recent studies have highlighted that plant microbiome is enriched in genes encoding enzymes and natural products. Several novel antimicrobial compounds, bioactive natural products and lytic/degrading enzymes with industrial implications are being identified from the microbiome. Moreover, advancements in metagenomics and culture techniques are facilitating the development of synthetic microbial communities to promote sustainable agriculture. We discuss the recent advancements, opportunities and challenges in harnessing the full potential of plant microbiome.

RS_CRZ1, a C2H2-Type Transcription Factor Is Required for Pathogenesis of Rhizoctonia solani AG1-IA in Tomato.

Rhizoctonia solani is a necrotrophic fungal pathogen that causes disease in diverse plant species. In recent years, the genomic and transcriptomic studies have identified several candidate pathogenicity determinants of R. solani; however, most of them remain to be validated. In this study, we report a viral vector-based host-induced gene silencing (HIGS) as well as a dsRNA (double-stranded RNA)-based approach to effectively downregulate genes of R. solani AG1-IA (BRS1 strain) during pathogenesis in tomato. We tested a few of the in-planta upregulated R. solani genes and observed that silencing of one of them, i.e., RS_CRZ1 (a C2H2 type zinc finger transcription factor) significantly compromises the pathogenesis of R. solani in tomato. The RS_CRZ1-silenced plants not only exhibited significant reduction in disease symptoms, but the depth of pathogen colonization was also compromised. Furthermore, we identified the R. solani genes that were coregulated with RS_CRZ1 during the pathogenicity process. The HIGS-mediated silencing of a few of them [CL1756Contig1; subtilisin-like protease and CL1817Contig2; 2OG-Fe(II) oxygenase] compromised the pathogenesis of R. solani in tomato. The ectopic expression of RS_CRZ1 complemented the crz1 mutant of yeast and restored tolerance against various metal ion stress. Overall, our study reveals the importance of RS_CRZ1 in managing the hostile environment encountered during host colonization. Also, it emphasizes the relevance of the HIGS and dsRNA-based gene silencing approach toward functional characterization of pathogenicity determinants of R. solani.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Fungal effectors, the double edge sword of phytopathogens.

Phyto-pathogenic fungi can cause huge damage to crop production. During millions of years of coexistence, fungi have evolved diverse life-style to obtain nutrients from the host and to colonize upon them. They deploy various proteinaceous as well as non-proteinaceous secreted molecules commonly referred as effectors to sabotage host machinery during the infection process. The effectors are important virulence determinants of pathogenic fungi and play important role in successful pathogenesis, predominantly by avoiding host-surveillance system. However, besides being important for pathogenesis, the fungal effectors end-up being recognized by the resistant cultivars of the host, which mount a strong immune response to ward-off pathogens. Various recent studies involving different pathosystem have revealed the virulence/avirulence functions of fungal effectors and their involvement in governing the outcome of host-pathogen interactions. However, the effectors and their cognate resistance gene in the host remain elusive for several economically important fungal pathogens. In this review, using examples from some of the biotrophic, hemi-biotrophic and necrotrophic pathogens, we elaborate the double-edged functions of fungal effectors. We emphasize that knowledge of effector functions can be helpful in effective management of fungal diseases in crop plants.

Concurrent overexpression of rice G-protein ß and γ subunits provide enhanced tolerance to sheath blight disease and abiotic stress in rice.

MAIN CONCLUSION: 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 (ß) 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.

Host Alternative NADH:Ubiquinone Oxidoreductase Serves as a Susceptibility Factor to Promote Pathogenesis of Rhizoctonia solani in Plants.

Phytopathogens have evolved mechanisms to utilize host genes (commonly known as susceptibility factors) to promote their pathogenesis. Rhizoctonia solani is a highly destructive fungal pathogen of various plants, including rice. We previously reported rice genes that were differentially regulated during R. solani pathogenesis. In this study, we analyzed the role of tomato homologs of two rice genes, isoflavone reductase (IFR) and alternative NADH:ubiquinone oxidoreductase (NUOR), as potential susceptibility factors for R. solani. Virus-induced gene silencing of NUOR in tomato resulted in compromised susceptibility against R. solani, whereas IFR-silenced plants demonstrated susceptibility similar to that of control plants. NUOR silencing in tomato led to homogenous accumulation of reactive oxygen species (optimum range) upon R. solani infection. In addition, the expression and enzyme activity of some host defense and antioxidant genes was enhanced, whereas H2O2 content, lipid peroxidation, and electrolyte leakage were reduced in NUOR-silenced plants. Similarly, transient silencing of OsNUOR provided tolerance against R. solani infection in rice. Overall, the data presented in this study suggest that NUOR serves as a host susceptibility factor to promote R. solani pathogenesis.

Genome analysis provides insight about pathogenesis of Indian strains of Rhizoctonia solani in rice.

The Rhizoctonia solani species complex is comprised of strains belonging to different anastomosis groups and causes diseases in several economically important crops, including rice. However, individuals within same anastomosis group exhibit distinct morphological and pathological differences on the same host. In this study, we have sequenced the genome of two aggressive Indian strains (BRS11 and BRS13) belonging to AG1-IA anastomosis group and compared them with the available genome of R. solani AG1-IA. We identified several SNPs and Indels in both of these genomes, in comparison to the AG1-IA genome. Furthermore, we observed expansion and emergence of orthogroups in these Indian strains and identified those potentially associated with pathogenesis. Amongst them, transposable elements, cell wall degrading enzymes, transcription factors, and oxalate decarboxylase were noteworthy. The current study unravels genetic variations and identifies genes that might account for pathogenicity variations amongst R. solani strains.

Bacteria-fungal Confrontation and Fungal Growth Prevention Assay.

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.

Identification of candidate pathogenicity determinants of Rhizoctonia solani AG1-IA, which causes sheath blight disease in rice.

Sheath blight disease is one of the predominant diseases of rice and it is caused by the necrotrophic fungal pathogen Rhizoctonia solani. The mechanistic insight about its widespread success as a broad host range pathogen is limited. In this study, we endeavor to identify pathogenicity determinants of R. solani during infection process in rice. Through RNAseq analysis, we identified a total of 65 and 232 R. solani (strain BRS1) genes to be commonly upregulated in three different rice genotypes (PB1, Tetep, and TP309) at establishment and necrotrophic phase, respectively. The induction of genes encoding extracellular protease, ABC transporter, and transcription factors were notable during establishment phase. While during necrotrophic phase, several CAZymes, sugar transporters, cellular metabolism, and protein degradation-related genes were prominently induced. We have also identified few putative secreted effector encoding genes that were upregulated during pathogenesis. The qPCR analysis further validated the phase-specific expression dynamics of some selected putative effectors and pathogenicity-associated genes. Overall, the present study reports identification of key genes and processes that might be crucial for R. solani pathogenesis. The ability to effectively damage host cell wall and survive in hostile plant environment by managing oxidative stress, cytotoxic compounds, etc. is being proposed to be important for pathogenesis of R. solani in rice. The functional characterization of these genes would provide key insights about this important pathosystem and facilitate development of strategies to control this devastating disease.

A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi.

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.Some bacteria can feed on live fungi through unclear mechanisms. Here, the authors show that a T3SS-secreted protein, which is homologous to phage tail proteins, allows a Burkholderia gladioli strain to kill and feed on various fungal species.

Alterations in rice chloroplast integrity, photosynthesis and metabolome associated with pathogenesis of Rhizoctonia solani.

Sheath 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.

Draft Genome Sequence of Broad-Spectrum Antifungal Bacterium Burkholderia gladioli Strain NGJ1, Isolated from Healthy Rice Seeds.

We report here the draft genome sequence of Burkholderia gladioli strain NGJ1. The strain was isolated from healthy rice seeds and exhibits broad-spectrum antifungal activity against several agriculturally important pathogens, including Rhizoctonia solani, Magnaporthe oryzae, Venturia inaequalis, and Fusarium oxysporum.

Identification and functional analysis of AG1-IA specific genes of Rhizoctonia solani.

Rhizoctonia solani is an important necrotrophic fungal pathogen which causes disease on diverse plant species. It has been classified into 14 genetically distinct anastomosis groups (AGs), however, very little is known about their genomic diversity. AG1-IA causes sheath blight disease in rice and controlling this disease remains a challenge for sustainable rice cultivation. Recently the draft genome sequences of AG1-IA (rice isolate) and AG1-IB (lettuce isolate) had become publicly available. In this study, using comparative genomics, we report identification of 3,942 R. solani genes that are uniquely present in AG1-IA. Many of these genes encode important biological, molecular functions and exhibit dynamic expression during in-planta growth of the pathogen in rice. Based upon sequence similarity with genes that are required for plant and human/zoonotic diseases, we identified several putative virulence/pathogenicity determinants amongst AG1-IA specific genes. While studying the expression of 19 randomly selected genes, we identified three genes highly up-regulated during in-planta growth. The detailed in silico characterization of these genes and extent of their up-regulation in different rice genotypes, having variable degree of disease susceptibility, suggests their importance in rice-Rhizoctonia interactions. In summary, the present study reports identification, functional characterization of AG1-IA specific genes and predicts important virulence determinants that might enable the pathogen to grow inside hostile plant environment. Further characterization of these genes would shed useful insights about the pathogenicity mechanism of AG1-IA on rice.