Gopaljee Jha

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.

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.

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.

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.

Bacterial type two secretion system secreted proteins : Double-edged swords for plant pathogens

The type two secretion system (T2S) is important for virulence of a number of gram-negative bacterial plant pathogens. Most of the T2S-secreted proteins that have been characterized to date are involved in degrading different components of plant cell walls. Functional redundancy appears to exist among T2S-secreted proteins because significant effects on virulence are observed only in strains in which multiple secreted proteins are mutated. Several T2S-secreted proteins have been shown to induce plant defense responses, including hypersensitive response-like reactions. Bacterial pathogens can suppress these defense responses, and recent results indicate that suppression is mediated through the type three secretion system.

The Venturia Apple Pathosystem: Pathogenicity Mechanisms and Plant Defense Responses

Venturia inaequalis is the causal agent of apple scab, a devastating disease of apple. We outline several unique features of this pathogen which are useful for molecular genetics studies intended to understand plant-pathogen interactions. The pathogenicity mechanisms of the pathogen and overview of apple defense responses, monogenic and polygenic resistance, and their utilization in scab resistance breeding programs are also reviewed.

De Novo Transcriptome Sequencing and Analysis for Venturia inaequalis, the Devastating Apple Scab Pathogen

Venturia inaequalis is the causal agent of apple scab, one of the most devastating diseases of apple. Due to several distinct features, it has emerged as a model fungal pathogen to study various aspects of hemibiotrophic plant pathogen interactions. The present study reports de novo assembling, annotation and characterization of the transcriptome of V. inaequalis. Venturia transcripts expressed during its growth on laboratory medium and that expressed during its biotrophic stage of infection on apple were sequenced using Illumina RNAseq technology. A total of 94,350,055 reads (50 bp read length) specific to Venturia were obtained after filtering. The reads were assembled into 62,061 contigs representing 24,571 unique genes. GO analysis suggested prevalence of genes associated with biological process categories like metabolism, transport and response to stimulus. Genes associated with molecular function like binding, catalytic activities and transferase activities were found in majority. EC and KEGG pathway analyses suggested prevalence of genes encoding kinases, proteases, glycoside hydrolases, cutinases, cytochrome P450 and transcription factors. The study has identified several putative pathogenicity determinants and candidate effectors in V. inaequalis. A large number of transcripts encoding membrane transporters were identified and comparative analysis revealed that the number of transporters encoded by Venturia is significantly more as compared to that encoded by several other important plant fungal pathogens. Phylogenomics analysis indicated that V. inaequalis is closely related to Pyrenophora tritici-repentis (the causal organism of tan spot of wheat). In conclusion, the findings from this study provide a better understanding of the biology of the apple scab pathogen and have identified candidate genes/functions required for its pathogenesis. This work lays the foundation for facilitating further research towards understanding this host-pathogen interaction.

Current trends and future prospects of biotechnological interventions through tissue culture in apple.

Apple (Malus domestica Borkh.), which is a widely cultivated, important economic fruit crop with nutritive and medicinal importance, has emerged as a model horticultural crop in this post-genomic era. Apple cultivation is heavily dependent on climatic condition and is susceptible to several diseases caused by fungi, bacteria, viruses, insects, etc. Extensive research work has been carried out to standardize tissue culture protocols and utilize them in apple improvement. We review the in vitro shoot multiplication, rooting, transformation and regeneration methodologies in apple and tabulate various such protocols for easy reference. The utility and limitation of transgenesis in apple improvement have also been summarized. The concepts of marker-free plants, use of non-antibiotic resistance selectable markers, and cisgenic and intragenic approaches are highlighted. Furthermore, the limitations, current trends and future prospects of tissue culture-mediated biotechnological interventions in apple improvement are discussed.

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.

Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant-microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 A and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 A, beta = 90.8 degrees . Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 A, beta = 92.6 degrees and diffract to 1.86 A. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.