Ramesh V. Sonti

Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99A

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice (Oryza sativa L.), a major disease that constrains production of this staple crop in many parts of the world. We report here on the complete genome sequence of strain PXO99A and its comparison to two previously sequenced strains, KACC10331 and MAFF311018, which are highly similar to one another. The PXO99A genome is a single circular chromosome of 5,240,075 bp, considerably longer than the genomes of the other strains (4,941,439 bp and 4,940,217 bp, respectively), and it contains 5083 protein-coding genes, including 87 not found in KACC10331 or MAFF311018. PXO99A contains a greater number of virulence-associated transcription activator-like effector genes and has at least ten major chromosomal rearrangements relative to KACC10331 and MAFF311018. PXO99A contains numerous copies of diverse insertion sequence elements, members of which are associated with 7 out of 10 of the major rearrangements. A rapidly-evolving CRISPR (clustered regularly interspersed short palindromic repeats) region contains evidence of dozens of phage infections unique to the PXO99A lineage. PXO99A also contains a unique, near-perfect tandem repeat of 212 kilobases close to the replication terminus. Our results provide striking evidence of genome plasticity and rapid evolution within Xanthomonas oryzae pv. oryzae. The comparisons point to sources of genomic variation and candidates for strain-specific adaptations of this pathogen that help to explain the extraordinary diversity of Xanthomonas oryzae pv. oryzae genotypes and races that have been isolated from around the world.

Marker assisted introgression of bacterial blight resistance in Samba Mahsuri, an elite indica rice variety

Samba Mahsuri (BPT5204) is a medium slender grain indica rice variety that is very popular with farmers and consumers across India because of its high yield and excellent cooking quality. However, the variety is susceptible to several diseases and pests, including bacterial blight (BB). We have used PCR based molecular markers in a backcross-breeding program to introgress three major BB resistance genes (Xa21, xa13 and xa5) into Samba Mahsuri from a donor line (SS1113) in which all the three genes are present in a homozygous condition. At each backcross generation, markers closely linked to the three genes were used to select plants possessing these resistance genes (foreground selection) and microsatellite markers polymorphic between donor and recurrent parent were used to select plants that have maximum contribution from the recurrent parent genome (background selection). A selected BC4F1 plant was selfed to generate homozygous BC4F2 plants with different combinations of BB resistance genes. The three-gene pyramid and two-gene pyramid lines exhibited high levels of resistance against the BB pathogen. Under conditions of BB infection, the three-gene pyramid lines exhibited a significant yield advantage over Samba Mahsuri. Most importantly, these lines retain the excellent grain and cooking qualities of Samba Mahsuri without compromising the yield as determined in multi-location trials. This work demonstrates the successful application of marker-assisted selection for targeted introgression of multiple resistance genes into a premium quality rice variety.

Two New Complete Genome Sequences Offer Insight into Host and Tissue Specificity of Plant Pathogenic Xanthomonas spp.

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.

Mutants of Xanthomonas oryzae pv. oryzae deficient in general secretory pathway are virulence deficient and unable to secrete xylanase

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a serious disease of rice. A virulence- and xylanase-deficient mutant of Xoo was isolated following ethyl methane sulfonate (EMS) mutagenesis. A cosmid clone that restored virulence and xylanase secretion was obtained from a genomic library by functional complementation. Transposon mutagenesis and marker exchange studies revealed genes on the cloned DNA that were required for xylanase production and virulence. Sequence analysis with transposon-specific primers revealed that these genes were homologues of xps F and xps D, which encode components of a protein secretion system in Xanthomonas campestris pv. campestris. Enzyme assays showed xylanase accumulation in the periplasmic space and cytoplasm of the xps F mutant and the complementing clone restored transport to the extracellular space.

rpfF mutants of Xanthomonas oryzae pv. oryzae are deficient for virulence and growth under low iron conditions.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. In the related bacterium Xanthomonas campestris pv. campestris, the rpfF gene is involved in production of a diffusible extracellular factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes. Transposon insertions in the rpfF homolog of X. oryzae pv. oryzae are deficient for virulence and production of a DSF but are proficient for EPS and extracellular enzyme production. The rpfF X. oryzae pv. oryzae mutants exhibit an unusual tetracycline susceptibility phenotype in which exogenous iron supplementation is required for phenotypic expression of a tetracycline resistance determinant that is encoded on an introduced plasmid. The rpfF X. oryzae pv. oryzae mutants also overproduce one or more siderophores and exhibit a growth deficiency under low iron conditions as well as in the presence of reducing agents that are expected to promote the conversion of Fe+3 to Fe+2. Exogenous iron supplementation promotes migration of rpfF X. oryzae pv. oryzae mutants in rice leaves. The results suggest that rpfF may be involved in controlling an iron-uptake system of X. oryzae pv. oryzae and that an inability to cope with the conditions of low iron availability in the host may be the reason for the virulence deficiency of the rpfF X. oryzae pv. oryzae mutants.

Functional interplay between two Xanthomonas oryzae pv,. oryzae secretion systems in modulating virulence on rice.

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S-) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S- T3S- double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA- mutant or a ClsA- LipA- double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.

A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence

Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1265 amino acids that exhibits significant similarity to non‐fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti‐XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a β‐helix conformation‐like pertactin, a non‐fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non‐fimbrial adhesin‐like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.

Role of an In Planta-Expressed Xylanase of Xanthomonas oryzae pv. oryzae in Promoting Virulence on Rice

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. We demonstrated earlier that the type II secretion system (T2S) is important for virulence of X. oryzae pv. oryzae and that several proteins, including a xylanase, are secreted through this system. In this study, the xynB gene encoding for the secreted xylanase was cloned as a 6.9-kb EcoRI fragment (pRR7) that also included a paralog called xynA. As in X. oryzae pv. oryzae, xynA and xynB are adjacent to each other in X. axonopodis pv. citri, whereas only the xynA homolog is present in X. campestris pv. campestris. Mutations in xynB but not xynA affect secreted xylanase activity. Western blot analysis using anti-XynB antibodies on exudates from infected rice leaves indicated that this xylanase is expressed during in planta growth. Another T2S-secreted protein was identified to be a lipase/esterase (LipA) based on the sequence tags obtained by tandem mass spectrometry analysis and biochemical assays. Mutations in either xynB or lipA partially affected virulence. However, a lipA-xynB double mutant was significantly reduced for virulence, and the pRR7 clone containing an intact xynB gene could complement the virulence-deficient phenotype of the lipA-xynB mutant. Our results suggest that there is functional redundancy among the T2S secreted proteins of X. oryzae pv. oryzae in promoting virulence on rice.

Acquisition and Evolution of Plant Pathogenesis–Associated Gene Clusters and Candidate Determinants of Tissue-Specificity in Xanthomonas

Background Xanthomonas is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown. Methodology/Principal Findings To assess potential contributions to host- and tissue-specificity, pathogenesis-associated gene clusters were compared across genomes of eight Xanthomonas strains representing vascular or non-vascular pathogens of rice, brassicas, pepper and tomato, and citrus. The gum cluster for extracellular polysaccharide is conserved except for gumN and sequences downstream. The xcs and xps clusters for type II secretion are conserved, except in the rice pathogens, in which xcs is missing. In the otherwise conserved hrp cluster, sequences flanking the core genes for type III secretion vary with respect to insertion sequence element and putative effector gene content. Variation at the rpf (regulation of pathogenicity factors) cluster is more pronounced, though genes with established functional relevance are conserved. A cluster for synthesis of lipopolysaccharide varies highly, suggesting multiple horizontal gene transfers and reassortments, but this variation does not correlate with host- or tissue-specificity. Phylogenetic trees based on amino acid alignments of gum, xps, xcs, hrp, and rpf cluster products generally reflect strain phylogeny. However, amino acid residues at four positions correlate with tissue specificity, revealing hpaA and xpsD as candidate determinants. Examination of genome sequences of xanthomonads Xylella fastidiosa and Stenotrophomonas maltophilia revealed that the hrp, gum, and xcs clusters are recent acquisitions in the Xanthomonas lineage. Conclusions/Significance Our results provide insight into the ancestral Xanthomonas genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates.

Introduction of bacterial blight resistance into Triguna, a high yielding, mid-early duration rice variety.

Bacterial blight (BB) is a serious disease of rice in India. We have used molecular marker-assisted selection in a backcross breeding program to introgress three genes (Xa21, xa13, and xa5) for BB resistance into Triguna, a mid-early duration, high yielding rice variety that is susceptible to BB. At each generation in the backcross program, molecular markers were used to select plants possessing these resistance genes and to select plants that have maximum contribution from the Triguna genome. A selected BC3F1 plant was selfed to generate homozygous BC(3)F(2) plants with different combinations of BB resistance genes. Plants containing the two-gene combination, Xa21 and xa13, were found to exhibit excellent resistance against BB. Single plant selections for superior agronomic characteristics were performed on the progeny of these plants, from BC(3)F(3) generation onwards. The selected plants were subjected to yield trials at the BC(3)F(8) generation and were found to have a significant yield advantage over Triguna. The newly developed lines are being entered into national multi-location field trials. This work represents a successful example of the application of molecular marker-assisted selection for BB resistance breeding in rice.