Strain PXO99 Research Articles

XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

Pathogen-induced methylglyoxal negatively regulates rice bacterial blight resistance by inhibiting OsCDR1 protease activity

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), a globally devastating disease of rice (Oryza sativa) that is responsible for significant crop loss. Sugars and sugar metabolites are important for pathogen infection, providing energy and regulating events associated with defense responses; however, the mechanisms by which they regulate such events in BB are unclear. As an inevitable sugar metabolite, methylglyoxal (MG) is involved in plant growth and responses to various abiotic stresses, but the underlying mechanisms remain enigmatic. Whether and how MG functions in plant biotic stress responses is almost completely unknown. Here, we report that the Xoo strain PXO99 induces OsWRKY62.1 to repress transcription of OsGLY II genes by directly binding to their promoters, resulting in overaccumulation of MG. MG negatively regulates rice resistance against PXO99: osglyII2 mutants with higher MG levels are more susceptible to the pathogen, whereas OsGLYII2-overexpressing plants with lower MG content show greater resistance than the wild type. Overexpression of OsGLYII2 to prevent excessive MG accumulation confers broad-spectrum resistance against the biotrophic bacterial pathogens Xoo and Xanthomonas oryzae pv. oryzicola and the necrotrophic fungal pathogen Rhizoctonia solani, which causes rice sheath blight. Further evidence shows that MG reduces rice resistance against PXO99 through CONSTITUTIVE DISEASE RESISTANCE 1 (OsCDR1). MG modifies the Arg97 residue of OsCDR1 to inhibit its aspartic protease activity, which is essential for OsCDR1-enhanced immunity. Taken together, these findings illustrate how Xoo promotes infection by hijacking a sugar metabolite in the host plant.

A Novel Glycerol Kinase Gene OsNHO1 Regulates Resistance to Bacterial Blight and Blast Diseases in Rice.

Glycerol-induced resistance to various pathogens has been reported in different plants. Glycerol kinase (GK), a vital rate-limiting enzyme that catalyzes glycerol conversion to glycerol-3-phosphate (G3P), participates in responses to both abiotic and biotic stresses. However, its physiological importance in rice defenses against pathogens remains unclear. In this research, quantification analysis revealed that GK levels were significantly induced in rice leaves infected by Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99. A typical GK-encoding gene OsNHO1 was cloned in rice. The transcriptional levels of OsNHO1 were significantly induced by salicylic acid, jasmonic acid, and Xoo-PXO99. Ectopic expression of OsNHO1 partially rescued the resistance to P. s. pv. phaseolicola in the Arabidopsis nho1 mutant. In the overexpressing transgenic rice lines (OsNHO1-OE), the content of GK and the transcriptional level of OsNHO1 were increased and the resistance to bacterial blight and blast was improved, while reduced OsNHO1 expression impaired the resistance in OsNHO1-RNAi lines. The wax contents and expression of the wax synthesis regulatory genes were significantly increased in the overexpression lines but decreased in the OsNHO1-RNAi lines. We then confirmed the interaction partner of OsNHO1 using yeast two-hybrid and bimolecular fluorescence complementation assays. The transcription of the interaction partner-encoding genes OsSRC2 and OsPRs in OsNHO1-RNAi lines was downregulated but upregulated in OsNHO1-OE lines. Thus, we concluded that OsNHO1 provided disease resistance by affecting the wax content and modulating the transcription levels of PR genes.

Identification of bacterial blight resistance genes in rice landraces from Yunnan Province, China

Bacterial blight (BB), a serious bacterial disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo) affects rice growth and yield. Yunnan Province is regarded as a center of rice diversity in China and indeed the world, and has abundant rice landrace resources, which may offer prospective candidate donors in rice improvement and breeding. In this study, a set of 200 rice landraces were evaluated to determine their resistance to 10 pathogenic Xoo strains resistance by the leaf-clipping method. The results indicated that the tested rice landraces had different resistance levels against different Xoo strains. Multiple comparisons showed that the Xoo strain PXO99 was virulent to the tested rice landraces. Sixty-six rice landraces conferred resistance against at least one Xoo strain. These resistant rice landraces screened were then performed the presence of 14 cloned BB resistance genes by closely linked molecular markers and designed specific primers. The results showed that none of these resistant accessions contained xa13, Xa21, Xa27, and Xa45(t) homologous fragments, while 9, 24, 4, 7, 9, 15, 1, 5, 4 and 27 accessions contained Xa1, Xa2/Xa31(t), Xa14, Xa3/Xa26, Xa4, xa5, Xa7, Xa10, Xa23 and xa25 homologous fragments, respectively. Sequence analysis further revealed that nucleotide variations around functional nucleotide polymorphisms region were observed within these accessions containing the Xa1, Xa2/Xa31(t), Xa14, Xa3/Xa26, Xa4, xa5, Xa10, Xa23 and xa25 homologous fragments. These results along with phenotypic resistance spectrum supported that these accessions carried nine resistance homologous genes. Only one accession (Qishanggu_Wenshan) carried the Xa7 resistance gene. We also found that some resistant rice landraces, especially Xilandigu_Baoshan, and Laoyaling_Lincang without the above resistance genes, which mediated broad spectrum resistance to multiple Xoo strains, were identified as potential sources for breeding rice lines resistance to BB.

Melatonin Treatment Inhibits the Growth of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes rice bacterial blight (BB), one of the most widespread and destructive diseases in rice-growing regions worldwide. Melatonin enhances pathogen resistance by inducing plant innate immunity, but the direct effect of melatonin on plant pathogenic bacteria is poorly understood. In this study, we investigated the direct effects of melatonin on Xoo. Exogenous melatonin at 200 μg/mL significantly inhibited the proliferation of Xoo and reduced the mRNA expression of five genes involved in cell division. This concentration of melatonin also inhibited the motility and biofilm formation of Xoo. Notably, melatonin was observed to alter the length of Xoo cells. To provide deeper insights into the mechanisms underlying this antibacterial activity, we examined global gene expression changes in Xoo strain PXO99 in response to the application of 200 μg/mL melatonin using RNA sequencing (RNA-Seq). A wide range of differentially expressed genes (DEGs) related to catalytic activity and metal-binding activity were downregulated in Xoo cells in response to the melatonin treatment. In addition, DEGs responsible for carbohydrate and amino acid metabolism were also downregulated. These results suggest that the inhibitory mechanism of melatonin on Xoo proliferation may involve the regulation of cell division in combination with a reduction in the concentration or activity of enzymes involved in metabolism.

The GGDEF-domain protein GdpX1 attenuates motility, exopolysaccharide production and virulence in Xanthomonas oryzae pv.oryzae.

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that is synthesized by diguanylate cyclase (DGC) with the GGDEF-domain, regulates diverse virulence phenotypes in pathogenic bacteria. Although 11 genes encoding GGDEF-domain proteins have been shown in the genome of Xanthomonas oryzae pv.oryzae (Xoo) strain PXO99(A) , the causal pathogen of bacterial blight of rice, however, little is known about their roles in the c-di-GMP regulation of virulence in the pathogen. GdpX1, one of the GGDEF-domain proteins in Xoo was investigated in this study to reveal its regulatory function of bacterial virulence expression through genetic analysis. GdpX1 was functionally characterized in virulence expression through deletion and overexpression analysis. Bioinformatics analysis revealed the GGDEF-domain in GdpX1 was well conserved, indicating it is a putative DGC. Deletion of gdpX1 resulted in significant increases in virulence, exopolysaccharide (EPS) production and flagellar motility. In contrast, overexpression of gdpX1 dramatically reduced these virulence phenotypes. qRT-PCR analysis showed genes related to the type III secretion system (T3SS), EPS synthesis, and flagellar motility, were up-regulated in ∆gdpX1 and down-regulated in the gdpX1-overexpressed strains. In addition, overexpression of gdpX1 promoted biofilm formation and xylanase activity. GdpX1 is the first GGDEF-domain protein functionally characterized in Xoo, which functions as a negative regulator of bacterial virulence via suppression of virulence-related gene transcription. Identification and functional characterization of GdpX1 provided additional insights into molecular mechanisms of c-di-GMP regulation of bacterial virulence expression.

Xanthomonas oryzae pv. oryzae requires H-NS-family protein XrvC to regulate virulence during rice infection.

Histone-like nucleoid-structuring (H-NS) proteins, which are conserved in Gram-negative bacteria, bind DNA and act as the global transcriptional repressors. In this study, we identified and characterized the xrvC gene encoding a H-NS protein in Xathomonas oryzae pv. oryzae (Xoo) Philippines strain PXO99(A) Compared with the wild type, the xrvC-deficient mutant of PXO99(A) (named PXO99ΔxrvC) showed a reduced growth rate in both nutrient-rich and nutrient-limited media. Interestingly, PXO99ΔxrvC exhibited significantly reduced virulence on rice cultivar IRBB214, but its virulence on 31 other rice cultivars was not affected. Transcriptional analysis revealed that the expression of hrpG, hrpX and hpa1 and of 15 out of 18 tested non-TAL (transcription activator-like) effector genes was decreased significantly in the xrvC mutant compared with that in the wild type. In addition, loss of xrvC also impaired the induction of the rice susceptibility gene Os8N3 in IRBB214 by PXO99(A) Our results suggest that the xrvC gene is involved in bacterial growth, and it plays a vital role in virulence by positively regulating the expression of hrp genes and non-TAL effector genes in PXO99(A) and the susceptibility gene Os8N3 in rice.

Dynamic and Coordinated Expression Changes of Rice Small RNAs in Response to Xanthomonas oryzae pv. oryzae

Endogenous small RNAs are newly identified players in plant immune responses, yet their roles in rice (Oryza sativa) responding to pathogens are still less understood, especially for pathogens that can cause severe yield losses. We examined the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae (Xoo) virulent strain PXO99, the causal agent of rice bacterial blight disease. Dynamic expression changes of some miRNAs and trans-acting siRNAs were identified, together with a few novel miRNA targets, including an RLK gene targeted by osa-miR159a.1. Coordinated expression changes were observed among some small RNAs in response to Xoo infection, with small RNAs exhibiting the same expression pattern tended to regulate genes in the same or related signaling pathways, including auxin and GA signaling pathways, nutrition and defense-related pathways. These findings reveal the dynamic and complex roles of small RNAs in rice-Xoo interactions, and identify new targets for regulating plant responses to Xoo.

Rice responds to endophytic colonization which is independent of the common symbiotic signaling pathway.

As molecular interactions of plants with N2 -fixing endophytes are largely uncharacterized, we investigated whether the common signaling pathway (CSP) shared by root nodule symbioses (RNS) and arbuscular mycorrhizal (AM) symbioses may have been recruited for the endophytic Azoarcus sp.-rice (Oryza sativa) interaction, and combined this investigation with global approaches to characterize rice root responses to endophytic colonization. Putative homologs of genes required for the CSP were analyzed for their putative role in endophytic colonization. Proteomic and suppressive subtractive hybridization (SSH) approaches were also applied, and a comparison of defense-related processes was carried out by setting up a pathosystem for flooded roots with Xanthomonas oryzae pv. oryzae strain PXO99 (Xoo). All tested genes were expressed in rice roots seedlings but not induced upon Azoarcus sp. inoculation, and the oscyclops and oscastor mutants were not impaired in endophytic colonization. Global approaches highlighted changes in rice metabolic activity and Ca(2+) -dependent signaling in roots colonized by endophytes, including some stress proteins. Marker genes for defense responses were induced to a lesser extent by the endophytes than by the pathogen, indicating a more compatible interaction. Our results thus suggest that rice roots respond to endophytic colonization by inducing metabolic shifts and signaling events, for which the CSP is not essential.

The broad bacterial blight resistance of rice line CBB23 is triggered by a novel transcription activator-like (TAL) effector of Xanthomonas oryzae pv. oryzae.

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is not only a disease devastating rice production worldwide, but also an ideal model system for the study of the interaction between plants and their bacterial pathogens. The rice near-isogenic line (NIL) CBB23, derived from a cross between a wild rice Oryza rufipogon accession (RBB16) and a susceptible indica rice variety (Jingang 30), is highly resistant to all field Xoo strains tested so far. Although the BB resistance of CBB23 has been widely used in rice breeding programmes, the mechanism of its extremely broad-spectrum resistance remains unknown. Here, we report the molecular cloning of an avirulence gene, designated as avrXa23, from Xoo strain PXO99(A) . We validate that AvrXa23, a novel transcription activator-like effector, specifically triggers the broad-spectrum BB resistance in CBB23. The prevalence of avrXa23 in all 38 Xoo strains surveyed may explain the broad-spectrum feature of BB resistance in CBB23. The results will significantly facilitate the molecular cloning of the corresponding resistance (R) gene in the host, and provide new insights into our understanding of the molecular mechanism for broad-spectrum disease resistance in plants.

Addition of transcription activator‐like effector binding sites to a pathogen strain‐specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak

Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

Oryza rufipogon, a valuable source for resistance to bacterial blight of rice

With 2 tables Abstract Six hundred and sixty-two accessions of Oryza rufipogon were screened against two Philippine strains (PXO61 and PXO99) of Xanthomonas oryzae pv. oryzae (Xoo) for their resistance to bacterial blight (BB). Of the 662 accessions, 13.6% were resistant and 7.9% were moderately resistant to PXO61 strain, while 5.7% and 6.8% accessions were found resistant and moderately resistant, respectively, against strain PXO99. The accessions found resistant to one or both the strains were further evaluated with five Philippine isolates and four Indian isolates having different virulence profile. Fourteen accessions were highly resistant to all the five strains of the Philippines, while six accessions were found to have broad-spectrum resistance to all the four Indian strains of Xoo. Of the 662 accessions screened, only two accessions designated as Acc # 104423 and 80671 were found resistant to all the Philippines and Indian strains making them highly valuable donors for durable broad- spectrum resistance to BB.

Characterization of Xanthomonas oryzae-Responsive cis-Acting Element in the Promoter of Rice Race-Specific Susceptibility Gene Xa13

The rice Xa13 gene, whose promoter harbors a UPT (up-regulated by transcription activator-like [TAL] effector) box, UPTPthXo1, plays a pivotal role in the race-specific pathogenicity caused by Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99. PXO99 causes rice disease by inducing Xa13. It is unknown, however, whether the UPTPthXo1 box is the only PXO99-responsive cis-regulating elements in the activation of Xa13 expression. We analyzed the expression of a series of end- and site-truncated and site-mutated Xa13 promoters in rice and the binding of PXO99 protein to the intact, partial, or site-mutated UPTPthXo1 boxes. In the Xa13 promoter, UPTPthXo1 box is the only Xoo-responsive cis-acting element that results in PXO99-induced Xa13 expression. The 5'-terminal second, third, and fourth nucleotides of the box are important for bacterial protein binding and gene activation; mutation of any one of these sites abolished PXO99-induced gene expression. Furthermore, the 3'-half of the UPTPthXo1 box is also required for protein binding and gene activation. These findings will enhance our understanding of the molecular mechanism of the interaction of rice and Xoo via UPT boxes and TAL effectors.

Identification and functional characterization of small non-coding RNAs in Xanthomonas oryzae pathovar oryzae

BackgroundSmall non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. Xanthomonas oryzae pathovar oryzae (Xoo) is an important plant pathogenic bacterium which causes serious bacterial blight of rice. However, little is known about the number, genomic distribution and biological functions of sRNAs in Xoo.ResultsHere, we performed a systematic screen to identify sRNAs in the Xoo strain PXO99. A total of 850 putative non-coding RNA sequences originated from intergenic and gene antisense regions were identified by cloning, of which 63 were also identified as sRNA candidates by computational prediction, thus were considered as Xoo sRNA candidates. Northern blot hybridization confirmed the size and expression of 6 sRNA candidates and other 2 cloned small RNA sequences, which were then added to the sRNA candidate list. We further examined the expression profiles of the eight sRNAs in an hfq deletion mutant and found that two of them showed drastically decreased expression levels, and another exhibited an Hfq-dependent transcript processing pattern. Deletion mutants were obtained for seven of the Northern confirmed sRNAs, but none of them exhibited obvious phenotypes. Comparison of the proteomic differences between three of the ΔsRNA mutants and the wild-type strain by two-dimensional gel electrophoresis (2-DE) analysis showed that these sRNAs are involved in multiple physiological and biochemical processes.ConclusionsWe experimentally verified eight sRNAs in a genome-wide screen and uncovered three Hfq-dependent sRNAs in Xoo. Proteomics analysis revealed Xoo sRNAs may take part in various metabolic processes. Taken together, this work represents the first comprehensive screen and functional analysis of sRNAs in rice pathogenic bacteria and facilitates future studies on sRNA-mediated regulatory networks in this important phytopathogen.

The bacterial pathogen Xanthomonas oryzae overcomes rice defenses by regulating host copper redistribution.

Pathogen effectors are virulence factors causing plant diseases. How the host targets of these effectors facilitate pathogen infection is largely unknown. An effector of Xanthomonas oryzae pv oryzae (Xoo) transcriptionally activates rice (Oryza sativa) susceptibility gene Xa13 to cause bacterial blight disease. Xa13 encodes an indispensable plasma membrane protein of the MtN3/saliva family, which is prevalent in eukaryotes with unknown biochemical function. We show that the XA13 protein cooperates with two other proteins, COPT1 and COPT5, to promote removal of copper from xylem vessels, where Xoo multiplies and spreads to cause disease. Copper, an essential micronutrient of plants and an important element for a number of pesticides in agriculture, suppresses Xoo growth. Xoo strain PXO99 is more sensitive to copper than other strains; its infection of rice is associated with activation of XA13, COPT1, and COPT5, which modulate copper redistribution in rice. The involvement of XA13 in copper redistribution has led us to propose a mechanism of bacterial virulence.

Metabolomic and transcriptomic analysis of the rice response to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae

Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo), gives rise to devastating crop losses in rice. Disease resistant rice cultivars are the most economical way to combat the disease. The TP309 cultivar is susceptible to infection by Xoo strain PXO99. A transgenic variety, TP309_Xa21, expresses the pattern recognition receptor Xa21, and is resistant. PXO99△raxST, a strain lacking the raxST gene, is able to overcome Xa21-mediated immunity. We used a single extraction solvent to demonstrate comprehensive metabolomics and transcriptomics profiling under sample limited conditions, and analyze the molecular responses of two rice lines challenged with either PXO99 or PXO99△raxST. LC–TOF raw data file filtering resulted in better within group reproducibility of replicate samples for statistical analyses. Accurate mass match compound identification with molecular formula generation (MFG) ranking of 355 masses was achieved with the METLIN database. GC–TOF analysis yielded an additional 441 compounds after BinBase database processing, of which 154 were structurally identified by retention index/MS library matching. Multivariate statistics revealed that the susceptible and resistant genotypes possess distinct profiles. Although few mRNA and metabolite differences were detected in PXO99 challenged TP309 compared to mock, many differential changes occurred in the Xa21-mediated response to PXO99 and PXO99△raxST. Acetophenone, xanthophylls, fatty acids, alkaloids, glutathione, carbohydrate and lipid biosynthetic pathways were affected. Significant transcriptional induction of several pathogenesis related genes in Xa21 challenged strains, as well as differential changes to GAD, PAL, ICL1 and Glutathione-S-transferase transcripts indicated limited correlation with metabolite changes under single time point global profiling conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-010-0218-7) contains supplementary material, which is available to authorized users.

Transcription activator‐like type III effector AvrXa27 depends on OsTFIIAγ5 for the activation of Xa27 transcription in rice that triggers disease resistance to Xanthomonas oryzae pv. oryzae

The transcription activator-like (TAL) type III effector AvrXa27 from Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99(A) activates the transcription of the host resistance gene Xa27, which results in disease resistance to bacterial blight (BB) in rice. In this study, we show that AvrXa27-activated Xa27 transcription requires host general transcription factor OsTFIIAgamma5. The V39E substitution in OsTFIIAgamma5, encoded by the recessive resistance gene xa5 in rice, greatly attenuates this activation in xa5 and Xa27 double homozygotes on inoculation with Xa27-incompatible strains. The xa5 gene also causes attenuation in the induction of Xa27 by AvrXa27 expressed in rice. The xa5-mediated attenuation of Xa27-mediated resistance to PXO99(A) is recessive. Intriguingly, xa5-mediated resistance to xa5-incompatible strains is also down-regulated in the xa5 and Xa27 double homozygotes. In addition, AvrXa27 expressed in planta shows weak virulence activity in the xa5 genetic background and causes enhanced susceptibility of the plants to BB inoculation. The results suggest that TAL effectors target host general transcription factors to directly manipulate the host transcriptional machinery for virulence and/or avirulence. The identification of xa5-mediated attenuation of Xa27-mediated resistance to Xoo provides a guideline for breeding resistance to BB when pyramiding xa5 with other resistance genes.

Identification and molecular characterization of twin-arginine translocation system (Tat) in Xanthomonas oryzae pv. oryzae strain PXO99

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. This study identified and characterized the contribution of the twin-arginine translocation (Tat) pathway to motility, chemotaxis, extracellular polysaccharide (EPS) production and virulence in X. oryzae pv. oryzae strain PXO99. The tatC disruption mutant (strain TCM) of strain PXO99 were generated, and confirmed both by PCR and Southern blotting. Strain PXO99 cells were highly motile in NYGB 0.3% soft agar plate. In contrast, the tatC mutation impaired motility. Furthermore, strain TCM cells lacked detectable flagella and exhibited almost no chemotaxis toward glucose under aerobic conditions, indicating that the Tat secretion pathway contributed to flagellar biogenesis and chemotactic responses. It was also observed that strain TCM exhibited a reductive production of extracellular polysaccharide (EPS) and a significant reduction of virulence on rice plants when compared with the wild type PXO99. However, the tatC mutation in strain PXO99 did not affect growth rate and the ability to induce hypersensitive response (HR) in nonhost tobacco (Nicotiana tabacum L. cv. Samsun). Our findings indicated that the Tat system of X. oryzae pv. oryzae played an important role in the pathogen's virulence.

Generation and characterisation of Tn5-tagged Xanthomonas oryzae pv. oryzae mutants that overcome Xa23-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23.

AvrXa3: A novel member of avrBs3 gene family from Xanthomonas oryzae pv. oryzae has a dual function

Abstract Two positive clones pUAV45 and pUAV47 were identified from the cDNA library of Jxo III, a race 3 strain of Xanthomonas oryzae pv. oryzae Dye (Xoo) in Japanese system, using Tn5 based technology. pUAV45 clone contained a 25.4 kb Xoo genomic DNA insert. Southern blot analysis with avrXa10 as the probe showed that DNA insert in pUAV45 shares homology with avrX10. Furthermore within the Xoo insert, a smaller 5.7 kb KpnI fragment (pUAV5k) was identified through hybridization with avrXa10. The transformation of pUAV45 and pUAV5k into the strain Pxo99 (race 6 in Philippine system) led to the decrease of Pxo99 pathogenicity on rice cultivar Wase Aikoku 3 (Xa3) and the increase of the pathogen pathogenicity on Cas209 (Xa10). The result of sequence analysis showed that there is a 2598 bp open reading frame (ORF) within the 5.7 kb Kpn1 fragment (pUAV5k). The ORF shared high identity (97%) with avrXa10. The deduced sequence of the ORF contained 8.5 tandem repeat units of 34-amino-acids, one leucine zipper (L...