Comparative genomics-based insights into Xanthomonas indica, a non-pathogenic species of healthy rice microbiome with bioprotection function.

Xanthomonas species are major pathogens of plants and have been studied extensively. There is increasing recognition of the importance of non-pathogenic species within the same genus. With this came the need to understand the genomic and functional diversity of non-pathogenic Xanthomonas (NPX) at the species and strain level. This study reports isolation and investigation into the genomic diversity and variation in NPX isolates, chiefly Xanthomonas indica, a newly discovered NPX species from rice. The study establishes the relationship of X. indica strains within clade I of Xanthomonads with another NPX species, X. sontii, also associated with rice seeds. Identification of highly diverse strains, open-pan genome, and systematic hyper-variation at the lipopolysaccharide biosynthetic locus when compared to pathogenic Xanthomonas indicates the acquisition of new functions for adaptation. Furthermore, comparative genomics studies established the absence of major virulence genes such as type III secretion system and effectors, which are present in the pathogens, and the presence of a known bacterial-killing type IV secretion system (X-T4SS). The diverse non-pathogenic strains of X. indica and X. sontii were found to protect rice from bacterial leaf blight pathogen, X. oryzae pv. oryzae (Xoo). The absence of phenotype of an X-T4SS mutant suggests redundancy in the genetic basis of the mechanisms involved in the bioprotection function, which may include multiple genetic loci, such as putative bacteriocin-encoding gene clusters and involvement of other factors such as nutrient and niche competition apart from induction of innate immunity through shared microbial-associated molecular patterns. The rice-NPX community and its pathogenic counterpart can be a promising model for understanding plant-microbe-microbiome interaction studies.IMPORTANCEThe Xanthomonas group of bacteria is known for its characteristic lifestyle as a phytopathogen. However, the discovery of non-pathogenic Xanthomonas (NPX) species is a major shift in understanding this group of bacteria. Multi-strain, in-depth genomic, evolutionary and functional studies on each of these NPX species are still lacking. This study on diverse non-pathogenic strains provides novel insights into genome diversity, dynamics, and evolutionary trends of NPX species from rice microbiome apart from its relationship with other relatives that form a sub-clade. Interestingly, we also uncovered that NPX species protect rice from pathogenic Xanthomonas species. The plant protection property shows their importance as a part of a healthy plant microbiome. Furthermore, finding an open pan-genome and large-scale variation at lipopolysaccharide biosynthetic locus indicates a significant role of the NPX community in host adaptation. The findings and high-quality genomic resources of NPX species and the strains will allow further systematic molecular and host-associated microbial community studies for plant health.

Co-overexpression of SWEET sucrose transporters modulates sucrose synthesis and defence responses to enhance immunity against bacterial blight in rice.

Enhancing carbohydrate export from source to sink tissues is considered to be a realistic approach for improving photosynthetic efficiency and crop yield. The rice sucrose transporters OsSUT1, OsSWEET11a and OsSWEET14 contribute to sucrose phloem loading and seed filling. Crucially, Xanthomonas oryzae pv. oryzae (Xoo) infection in rice enhances the expression of OsSWEET11a and OsSWEET14 genes, and causes leaf blight. Here we show that co-overexpression of OsSUT1, OsSWEET11a and OsSWEET14 in rice reduced sucrose synthesis and transport leading to lower growth and yield but reduced susceptibility to Xoo relative to controls. The immunity-related hypersensitive response (HR) was enhanced in the transformed lines as indicated by the increased expression of defence genes, higher salicylic acid content and presence of HR lesions on the leaves. The results suggest that the increased expression of OsSWEET11a and OsSWEET14 in rice is perceived as a pathogen (Xoo) attack that triggers HR and results in constitutive activation of plant defences that are related to the signalling pathways of pathogen starvation. These findings provide a mechanistic basis for the trade-off between plant growth and immunity because decreased susceptibility against Xoo compromised plant growth and yield.

Multiomics-assisted characterization of rice-Yellow Stem Borer interaction provides genomic and mechanistic insights into stem borer resistance in rice.

KEY MESSAGE: By deploying a multi-omics approach, we unraveled the mechanisms that might help rice to combat Yellow Stem Borer infestation, thus providing insights and scope for developing YSB resistant rice varieties. Yellow Stem Borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), is a major pest of rice, that can lead to 20-60% loss in rice production. Effective management of YSB infestation is challenged by the non-availability of adequate sources of resistance and poor understanding of resistance mechanisms, thus necessitating studies for generating resources to breed YSB resistant rice and to understand rice-YSB interaction. In this study, by using bulk-segregant analysis in combination with next-generation sequencing, Quantitative Trait Loci (QTL) intervals in five rice chromosomes were mapped that could be associated with YSB resistance at the vegetative phase in a resistant rice line named SM92. Further, multiple SNP markers that showed significant association with YSB resistance in rice chromosomes 1, 5, 10, and 12 were developed. RNA-sequencing of the susceptible and resistant lines revealed several genes present in the candidate QTL intervals to be differentially regulated upon YSB infestation. Comparative transcriptome analysis revealed a putative candidate gene that was predicted to encode an alpha-amylase inhibitor. Analysis of the transcriptome and metabolite profiles further revealed a possible link between phenylpropanoid metabolism and YSB resistance. Taken together, our study provides deeper insights into rice-YSB interaction and enhances the understanding of YSB resistance mechanism. Importantly, a promising breeding line and markers for YSB resistance have been developed that can potentially aid in marker-assisted breeding of YSB resistance among elite rice cultivars.

Strong culm: a crucial trait for developing next-generation climate-resilient rice lines.

Lodging, a phenomenon characterized by the bending or breaking of rice plants, poses substantial constraints on productivity, particularly during the harvesting phase in regions susceptible to strong winds. The rice strong culm trait is influenced by the intricate interplay of genetic, physiological, epigenetic, and environmental factors. Stem architecture, encompassing morphological and anatomical attributes, alongside the composition of both structural and non-structural carbohydrates, emerges as a critical determinant of lodging resistance. The adaptive response of the rice culm to various biotic and abiotic environmental factors further modulates the propensity for lodging. Advancements in next-generation sequencing technologies have expedited the genetic dissection of lodging resistance, enabling the identification of pertinent genes, quantitative trait loci, and novel alleles. Concurrently, contemporary breeding strategies, ranging from biparental approaches to more sophisticated methods such as multi-parent-based breeding, gene pyramiding, genomic selection, genome-wide association studies, and haplotype-based breeding, offer perspectives on the genetic underpinnings of culm strength. This review comprehensively delves into physiological attributes, culm histology, epigenetic determinants, and gene expression profiles associated with lodging resistance, with a specialized focus on leveraging next-generation sequencing for candidate gene discovery.

Combined strategy employing MutMap and RNA-seq reveals genomic regions and genes associated with complete panicle exsertion in rice.

Complete panicle exsertion (CPE) in rice is an important determinant of yield and a desirable trait in breeding. However, the genetic basis of CPE in rice still remains to be completely characterized. An ethyl methane sulfonate (EMS) mutant line of an elite cultivar Samba Mahsuri (BPT 5204), displaying stable and consistent CPE, was identified and named as CPE-110. MutMap and RNA-seq were deployed for unraveling the genomic regions, genes, and markers associated with CPE. Two major genomic intervals, on chromosome 8 (25668481-25750456) and on chromosome 11 (20147154-20190400), were identified to be linked to CPE through MutMap. A non-synonymous SNP (G/A; Chr8:25683828) in the gene LOC_Os08g40570 encoding pyridoxamine 5'-phosphate oxidase with the SNP index 1 was converted to Kompetitive allele-specific PCR (KASP) marker. This SNP (KASP 8-1) exhibited significant association with CPE and further validated through assay in the F2 mapping population, released varieties and CPE exhibiting BPT 5204 mutant lines. RNA-seq of the flag leaves at the booting stage, 1100 genes were upregulated and 1305 downregulated differentially in CPE-110 and BPT 5204. Metabolic pathway analysis indicated an enrichment of genes involved in photosynthesis, glyoxylate, dicarboxylate, porphyrin, pyruvate, chlorophyll, carotenoid, and carbon metabolism. Further molecular and functional studies of the candidate genes could reveal the mechanistic aspects of CPE. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01412-1.

Insights into the Diversity of Transcription Activator-Like Effectors in Indian Pathotype Strains of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a major rice pathogen, and its genome harbors extensive inter-strain and inter-lineage variations. The emergence of highly virulent pathotypes of Xoo that can overcome major resistance (R) genes deployed in rice breeding programs is a grave threat to rice cultivation. The present study reports on a long-read Oxford nanopore-based complete genomic investigation of Xoo isolates from 11 pathotypes that are reported based on their reaction toward 10 R genes. The investigation revealed remarkable variation in the genome structure in the strains belonging to different pathotypes. Furthermore, transcription activator-like effector (TALE) proteins secreted by the type III secretion system display marked variation in content, genomic location, classes, and DNA-binding domain. We also found the association of tal genes in the vicinity of regions with genome structural variations. Furthermore, in silico analysis of the genome-wide rice targets of TALEs allowed us to understand the emergence of pathotypes compatible with major R genes. Long-read, cost-effective sequencing technologies such as nanopore can be a game changer in the surveillance of major and emerging pathotypes. The resource and findings will be invaluable in the management of Xoo and in appropriate deployment of R genes in rice breeding programs.

Xanthomonas oryzae pv. oryzae Exoribonuclease R Is Required for Complete Virulence in Rice, Optimal Motility, and Growth Under Stress.

Exoribonuclease R (RNase R) is a 3' hydrolytic exoribonuclease that can degrade structured RNA. Mutation in RNase R affects virulence of certain human pathogenic bacteria. The aim of this study was to determine whether RNase R is necessary for virulence of the phytopathogen that causes bacterial blight in rice, Xanthomonas oryzae pv. oryzae (Xoo). In silico analysis has indicated that RNase R is highly conserved among various xanthomonads. Amino acid sequence alignment of Xoo RNase R with RNase R from various taxa indicated that Xoo RNase R clustered with RNase R of order Xanthomonadales. To study its role in virulence, we generated a gene disruption mutant of Xoo RNase R. The Xoo rnr- mutant is moderately virulence deficient, and the complementing strain (rnr-/pHM1::rnr) rescued the virulence deficiency of the mutant. We investigated swimming and swarming motilities in both nutrient-deficient minimal media and nutrient-optimal media. We observed that RNase R mutation has adversely affected the swimming and swarming motilities of Xoo in optimal media. However, in nutrient-deficient media only swimming motility was noticeably affected. Growth curves in optimal media at suboptimal temperature (15°C cold stress) indicate that the Xoo rnr- mutant grows more slowly than the Xoo wild type and complementing strain (rnr-/pHM1::rnr). Given these findings, we report for the first time that RNase R function is necessary for complete virulence of Xoo in rice. It is also important for motility of Xoo in media and for growth of Xoo at suboptimal temperature.

Xanthomonas indica sp. nov., a Novel Member of Non-Pathogenic Xanthomonas Community from Healthy Rice Seeds.

Xanthomonas is a major group of pathogenic bacteria infecting staple food crops like rice. Increasingly it is being recognized that non-pathogenic Xanthomonas (NPX) are also important members of a healthy plant microbiome. However, the vast majority of the species described in this genus are of pathogenic nature, and only a few NPX species have been reported till now. Genomic and taxonogenomic analysis of NPX is needed for the management of this important group of bacteria. In this study, two yellow-pigmented bacterial isolates were obtained from healthy rice seeds in Punjab, India. The isolates designated PPL560T and PPL568 were identified as members of the genus Xanthomonas based on biochemical tests and 16S rRNA gene sequence analysis retrieved from the whole-genome sequences. Isolates formed a distinct monophyletic lineage with Xanthomonas sontii and Xanthomonas sacchari as the closest relatives in the phylogenetic tree based on core gene content shared by the representative species of the genus Xanthomonas. Pairwise ortho Average Nucleotide Identity and digital DNA-DNA hybridization values calculated against other species of Xanthomonas were below their respective cut-offs. In planta studies revealed that PPL560T and PPL568 are non-pathogenic to rice plants upon leaf clip inoculation. The absence of type III secretion system-related genes and effectors further supported their non-pathogenic status. Herein, we propose Xanthomonas indica sp. nov. as novel species of the genus Xanthomonas with PPL560T = MTCC 13185 = CFBP 9039 = ICMP 24394 as its type strain and PPL568 as another constituent member.

Interaction of the Xanthomonas effectors XopQ and XopX results in induction of rice immune responses.

Xanthomonas oryzae pv. oryzae uses several type III secretion system (T3SS) secreted effectors, namely XopN, XopQ, XopX and XopZ, to suppress rice immune responses that are induced following treatment with cell wall degrading enzymes. Here we show that a T3SS secreted effector XopX interacts with two of the eight rice 14-3-3 proteins. Mutants of XopX that are defective in 14-3-3 binding are also defective in suppression of immune responses, suggesting that interaction with 14-3-3 proteins is required for suppression of host innate immunity. However, Agrobacterium-mediated delivery of both XopQ and XopX into rice cells results in induction of rice immune responses. These immune responses are not observed when either protein is individually delivered into rice cells. XopQ-XopX-induced rice immune responses are not observed with a XopX mutant that is defective in 14-3-3 binding. Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays indicate that XopQ and XopX interact with each other. A screen for Xanthomonas effectors that can suppress XopQ-XopX-induced rice immune responses led to the identification of five effectors, namely XopU, XopV, XopP, XopG and AvrBs2, that could individually suppress these immune responses. These results suggest a complex interplay of Xanthomonas T3SS effectors in suppression of both pathogen-triggered immunity and effector-triggered immunity to promote virulence on rice.

Dual Activities of Receptor-Like Kinase OsWAKL21.2 Induce Immune Responses.

Plant pathogens secrete cell wall-degrading enzymes that degrade various components of the plant cell wall. Plants sense this cell wall damage as a mark of infection and induce immune responses. However, the plant functions that are involved in the elaboration of cell wall damage-induced immune responses remain poorly understood. Transcriptome analysis revealed that a rice (Oryza sativa) receptor-like kinase, WALL-ASSOCIATED KINASE-LIKE21 (OsWAKL21.2), is up-regulated following treatment with either Xanthomonas oryzae pv oryzae (a bacterial pathogen) or lipaseA/esterase (LipA; a cell wall-degrading enzyme of X. oryzae pv oryzae). Overexpression of OsWAKL21.2 in rice induces immune responses similar to those activated by LipA treatment. Down-regulation of OsWAKL21.2 attenuates LipA-mediated immune responses. Heterologous expression of OsWAKL21.2 in Arabidopsis (Arabidopsis thaliana) also activates plant immune responses. OsWAKL21.2 is a dual-activity kinase that has in vitro kinase and guanylate cyclase activities. Interestingly, kinase activity of OsWAKL21.2 is necessary to activate rice immune responses, whereas in Arabidopsis, OsWAKL21.2 guanylate cyclase activity activates these responses. Our study reveals a rice receptor kinase that activates immune responses in two different species via two different mechanisms.

Ectopic Expression of a Cell-Wall-Degrading Enzyme-Induced OsAP2/ERF152 Leads to Resistance against Bacterial and Fungal Infection in Arabidopsis.

Pathogen secreted cell-wall-degrading enzymes (CWDEs) induce plant innate immune responses. The expression of rice transcription factor APETALA2/ethylene response factor-152 (OsAP2/ERF152) is enhanced in leaves upon treatment with different CWDEs and upon wounding. Ectopic expression of OsAP2/ERF152 in Arabidopsis leads to induction of immune responses such as callose deposition and upregulation of both salicylic acid- and jasmonic acid/ethylene-responsive defense genes. Arabidopsis transgenics expressing OsAP2/ERF152 exhibited resistance to infections caused by both bacterial and fungal pathogens (Pseudomonas syringae pv. tomato DC3000 and Rhizoctonia solani AG1-IA, respectively). Ectopic expression of OsAP2/ERF152 results in transient activation of mitogen-activated protein kinases 3/6 (MPK3/6), which could be leading to the induction of a broad range immunity in Arabidopsis.

Overexpression of OsPUB41, a Rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in Rice and Arabidopsis.

BACKGROUND: Cell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses. RESULTS: Expression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses. CONCLUSION: The results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice.

Overexpression of a cell wall damage induced transcription factor, OsWRKY42, leads to enhanced callose deposition and tolerance to salt stress but does not enhance tolerance to bacterial infection.

BACKGROUND: Members of the WRKY gene family play important roles in regulating plant responses to abiotic and biotic stresses. Treatment with either one of the two different cell wall degrading enzymes (CWDEs), LipaseA and CellulaseA, induces immune responses and enhances the expression of OsWRKY42 in rice. However, the role of OsWRKY42 in CWDE induced immune responses is not known. RESULTS: Expression of the rice transcription factor OsWRKY42 is induced upon treatment of rice leaves with CWDEs, wounding and salt. Overexpression of OsWRKY42 leads to enhanced callose deposition in rice and Arabidopsis but this does not enhance tolerance to bacterial infection. Upon treatment with NaCl, Arabidopsis transgenic plants expressing OsWRKY42 exhibited high levels of anthocyanin and displayed enhanced tolerance to salt stress. Treatment with either cellulase or salt induced the expression of several genes involved in JA biosynthesis and response in Arabidopsis. Ectopic expression of OsWRKY42 results in reduced expression of cell wall damage and salt stress induced jasmonic acid biosynthesis and response genes. OsWRKY42 expressing Arabidopsis lines exhibited enhanced tolerance to methyl jasmonate mediated growth inhibition. CONCLUSION: The results presented here suggest that OsWRKY42 regulates plant responses to either cell wall damage or salinity stress by acting as a negative regulator of jasmonic acid mediated responses.

Rice Leaf Transcriptional Profiling Suggests a Functional Interplay Between Xanthomonas oryzae pv. oryzae Lipopolysaccharide and Extracellular Polysaccharide in Modulation of Defense Responses During Infection.

Treatment of rice leaves with isolated Xanthomonas oryzae pv. oryzae lipopolysaccharide (LPS) induces the production of callose deposits, reactive oxygen species, and enhanced resistance against subsequent bacterial infection. Expression profiling of X. oryzae pv. oryzae LPS-treated rice (Oryza sativa subsp. indica) leaves showed that genes involved in the biosynthetic pathways for lignins, phenylpropanoids, chorismate, phenylalanine, salicylic acid, and ethylene, as well as a number of pathogenesis-related proteins are up-regulated. Gene ontology categories like cell-wall organization, defense response, stress response, and protein phosphorylation/kinases were found to be upregulated, while genes involved in photosynthesis were down-regulated. Coinfiltration with xanthan gum, the xanthomonas extracellular polysaccharide (EPS), suppressed LPS-induced callose deposition. Gene expression analysis of rice leaves that are treated with an EPS-deficient mutant of X. oryzae pv. oryzae indicated that a number of defense-regulated functions are up-regulated during infection. These transcriptional responses are attenuated in rice leaves treated with an EPS-deficient mutant that is also deficient in the O-antigen component of LPS. Overall, these results suggest that the O-antigen component of X. oryzae pv. oryzae LPS induces rice defense responses during infection and that these are suppressed by bacterial EPS.

Action of Multiple Cell Wall-Degrading Enzymes Is Required for Elicitation of Innate Immune Responses During Xanthomonas oryzae pv. oryzae Infection in Rice.

Xanthomonas oryzae pv. oryzae secretes a number of plant cell wall-degrading enzymes (CWDEs) whose purified preparations induce defense responses in rice. These defense responses are suppressed by X. oryzae pv. oryzae using type 3 secretion system (T3SS) effectors and a type 3 secretion system mutant (T3SS(-)) of X. oryzae pv. oryzae is an inducer of rice defense responses. We assessed the role of individual CWDEs in induction of rice defense responses during infection, by mutating them in the genetic background of a T3SS(-). We mutated the genes for five different plant CWDEs secreted by X. oryzae pv. oryzae, including two cellulases (clsA and cbsA), one xylanase (xyn), one pectinase (pglA), and an esterase (lipA), singly in a T3SS(-) background. We have demonstrated that, as compared with a T3SS(-) of X. oryzae pv. oryzae, a cbsA(-)T3SS(-), a clsA(-)T3SS(-), and a xyn(-)T3SS(-) are deficient in induction of rice immune responses such as callose deposits and programmed cell death. In comparison, a lipA(-) T3SS(-) and a pglA(-)T3SS(-) is as efficient in induction of host defense responses as a T3SS(-). Overall, these results indicate that the collective action of X. oryzae pv. oryzae-secreted ClsA, CbsA, and Xyn proteins is required for induction of rice defense responses during infection.

Mutations in the Predicted Active Site of Xanthomonas oryzae pv. oryzae XopQ Differentially Affect Virulence, Suppression of Host Innate Immunity, and Induction of the HR in a Nonhost Plant.

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ-/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl ß-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.

Upregulation of jasmonate biosynthesis and jasmonate-responsive genes in rice leaves in response to a bacterial pathogen mimic.

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, secretes several cell wall degrading enzymes including cellulase (ClsA) and lipase/esterase (LipA). Prior treatment of rice leaves with purified cell wall degrading enzymes such as LipA can confer enhanced resistance against subsequent X. oryzae pv. oryzae infection. To understand LipA-induced rice defense responses, microarray analysis was performed 12 h after enzyme treatment of rice leaves. This reveals that 867 (720 upregulated and 147 downregulated) genes are differentially regulated (≥2-fold). A number of genes involved in defense, stress, signal transduction, and catabolic processes were upregulated while a number of genes involved in photosynthesis and anabolic processes were downregulated. The microarray data also suggested upregulation of jasmonic acid (JA) biosynthetic and JA-responsive genes. Estimation of various phytohormones in LipA-treated rice leaves demonstrated a significant increase in the level of JA-Ile (a known active form of JA) while the levels of other phytohormones were not changed significantly with respect to buffer-treated control. This suggests a role for JA-Ile in cell wall damage induced innate immunity. Furthermore, a comparative analysis of ClsA- and LipA-induced rice genes has identified key rice functions that might be involved in elaboration of damage-associated molecular pattern (DAMP)-induced innate immunity.

gltB/D mutants of Xanthomonas oryzae pv. oryzae are virulence deficient.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Upon clip inoculation of rice leaves, Xoo causes typical V-shaped lesions whose leading edge moves through the mid-veinal region. We have isolated a virulence deficient mutant of Xoo, referred to as BXO808 that causes limited lesions which primarily extend through the side-veinal regions of rice leaves. Functional complementation studies identified a clone, pSR19, from a cosmid genomic library that restored wild-type virulence and lesion phenotype to BXO808. Transposon mutagenesis of the pSR19 clone, marker exchange experiments, and targeted mutagenesis, revealed that the BXO808 phenotype is due to mutation in the gltB/D genes of Xoo, which encode glutamate synthase subunits α and ß, respectively. The gltB/D mutants that were generated in this study also exhibited virulence deficiency, an altered lesion phenotype and growth deficiency on minimal medium with low levels of ammonium as a sole nitrogen source. This is the first report that mutations in the gltB/D genes of Xoo cause virulence deficiency.

Mutational analysis of predicted active site residues of an exoglucanase secreted by Xanthomonas oryzae pv. oryzae to determine their role in catalysis and in virulence on rice.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice. As a part of its virulence repertoire, Xoo secretes a cell wall degrading enzyme Cellobiosidase (CbsA), which is a critical virulence factor and also a determinant of tissue specificity. CbsA protein is made up of an N-terminal catalytic domain and a C-terminal fibronectin type III domain. According to the CAZy classification, the catalytic domain of CbsA protein belongs to the glycosyl hydrolase-6 (GH6) family that performs acid-base catalysis. However, the identity of the catalytic acid and the catalytic base of CbsA is not known. Based on the available structural and biochemical data, we identified putative catalytic residues and probed them by site-directed mutagenesis. Intriguingly, the biochemical analysis showed that none of the mutations abolishes the catalytic activity of CbsA, an observation that is contrary to other GH6 family members. All the mutants exhibited altered enzymatic activity and caused significant virulence deficiency in Xoo emphasising the requirement of specific exoglucanase activity of wild-type CbsA for virulence on rice. Our study highlights the need for further studies and the detailed characterisation of bacterial exoglucanases.

Xanthomonas protegens sp. nov., a novel rice seed-associated bacterium, provides in vivo protection against X. oryzae pv. oryzae, the bacterial leaf blight pathogen.

Historically, Xanthomonas species are primarily known for their pathogenicity against plants, but recently, there have been more findings of non-pathogenic xanthomonads. In the present study, we report isolates from healthy rice seeds that belong to a new species, X. protegens, a protector of the rice plants against a serious pathogenic counterpart, i.e. X. oryzae pv. oryzae upon leaf clip co-inoculation. The new member species is non-pathogenic to rice and lacks a type III secretion system. The pangenome investigation revealed a large number of unique genes, including a novel lipopolysaccharide biosynthetic gene cluster, that might be important in its adaptation. The phylo-taxonogenomic analysis revealed that X. protegens is a taxonomic outlier species of X. sontii, a core, vertically transmitted rice seed endophyte with numerous probiotic properties. Interestingly, X. sontii is also reported as a keystone species of healthy rice seed microbiome. The findings and resources will help in the development of unique gene markers and evolutionary studies of X. sontii as a successful symbiont and X. oryzae as a serious pathogen. Here, we propose X. protegens sp. nov. as a novel species of the genus Xanthomonas with PPL118 = MTCC 13396 = CFBP 9164 = ICMP 25181 as the type strain. PPL117, PPL124, PPL125 and PPL126 are other strains of the species.