Xoo Virulence Research Articles

Alternative sigma factor RpoN2 is required for flagellar motility and full virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, harbors a single polar flagellum for motility. How the flagellar system is regulated and how it is related to bacterial pathogenesis are not well understood. The genomic sequence of Xoo strain PXO99A revealed a flagellar regulon containing over 60 contiguous genes. A gene encoding alternative sigma factor 54 (σ54), named as rpoN2, is located in the central region of the regulon. RT-PCR analysis demonstrated rpoN2 was transcribed in an operon with flgRR, and fleQ. Single gene deletion mutants of the rpoN2 operon were generated. The rpoN2 and fleQ mutant lost swimming motility, whereas the flgRR mutant remained motile. Quantitative RT-PCR analysis further demonstrated that expression of regulatory genes fliA and flgM, and structural genes flgG, flhB, and fliC were significantly down-regulated in the rpoN2 and fleQ mutants. These results indicated that RpoN2 and FleQ synergistically controlled flagellar motility by regulating gene expression. Interestingly, the rpoN2 mutant, but not the fleQ mutant was impaired in its virulence on rice. In addition, we showed that the flagellin gene fliC mutant, which was non-motile, was not defective in virulence. Thus, we concluded that flagellar motility might not be essential for Xoo virulence in rice, and RpoN2 probably regulated bacterial virulence through a manner independent of its role in controlling flagellar gene expression.

Biochemical Approach for Virulence Factors’ Identification in Xanthomonas Oryzae Pv. Oryzae

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) leads to a substantial yield reduction of up to 50% in most rice-growing regions. Host plant resistance is an effective control method, and more than 30 resistance genes have been identified in rice genotypes. To understand the interaction of the pathogen in a susceptible reaction of the host plant, Xoo culture filtrate and treated culture filtrates were used to treat two rice genotypes using four strains Mai1, PXO88, Dak1 and Dak16. The study revealed that Xoo culture filtrate, heated culture filtrate and proteinase K treated culture filtrate induced typical bacterial blight symptoms on rice genotypes IRBB4 and FKR14 with a maximum lesion length of about 23.1 cm for culture filtrate. Heated culture filtrate phytotoxicity effects on both rice genotypes was with highest lesion length of about 6.9 cm, while 13.4 cm was the maximum length induced by a proteinase K treated fraction. After ethyl acetate treatment of the culture filtrate, a considerable reduction of the phytotoxicity was observed. Therefore we suggest that a low molecular-weight toxin may be present in the ethyl acetate extract should not play a major role in Xoo virulence and speculate that EPS, Xylanase, polygalacturonase, proteinaceouse contribute to Xoo virulence.

Genetic Analysis and Molecular Identification of Virulence in Xanthomonas oryzae pv. oryzae Isolates.

Bacterial leaf blight (BLB) of rice is a very destructive disease worldwide and is caused by Xanthomonas oryzae pv. oryzae (Xoo). The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr, and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence genotypes (Mta and Mtb) with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Xoo virulence genotypes were known to exist within country and there was evidence of Xoo pathogen migration between countries. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa.

Xanthomonas oryzae pv. oryzae Type III Effector XopN Targets OsVOZ2 and a Putative Thiamine Synthase as a Virulence Factor in Rice

Xanthomonas oryzae pv. oryzae (Xoo) is spread systemically through the xylem tissue and causes bacterial blight in rice. We evaluated the roles of Xanthomonas outer proteins (Xop) in the Xoo strain KXO85 in a Japonica-type rice cultivar, Dongjin. Five xop gene knockout mutants (xopQ KXO85, xopX KXO85, xopP1 KXO85, xopP2 KXO85, and xopN KXO85) were generated by EZ-Tn5 mutagenesis, and their virulence was assessed in 3-month-old rice leaves. Among these mutants, the xopN KXO85 mutant appeared to be less virulent than the wild-type KXO85; however, the difference was not statistically significant. In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85. These observations indicate that the roles of Xop in Xoo virulence are dependent on leaf stage. We chose the xopN gene for further characterization because the xopN KXO85 mutant showed the greatest influence on virulence. We confirmed that XopNKXO85 is translocated into rice cells, and its gene expression is positively regulated by HrpX. Two rice proteins, OsVOZ2 and a putative thiamine synthase (OsXNP), were identified as targets of XopNKXO85 by yeast two-hybrid screening. Interactions between XopNKXO85 and OsVOZ2 and OsXNP were further confirmed in planta by bimolecular fluorescence complementation and in vivo pull-down assays. To investigate the roles of OsVOZ2 in interactions between rice and Xoo, we evaluated the virulence of the wild-type KXO85 and xopN KXO85 mutant in the OsVOZ2 mutant line PFG_3A-07565 of Dongjin. The wild-type KXO85 and xopN KXO85 mutant were significantly less virulent in the mutant rice line. These results indicate that XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.

English

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo), is a very destructive rice disease worldwide. The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence molecular type (Mt) which were Mta and Mtb with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa.   Key words: Bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo), Xoo virulence pathotype, molecular typing, genomic DNA, Operon primer, Xoo virulence genotype, Xoo pathogen migration, West Africa.

Five phylogenetically close rice SWEET genes confer TAL effector‐mediated susceptibility to Xanthomonas oryzae pv. oryzae

Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo). We predict and validate that expression of OsSWEET14 is induced by a novel TAL effector, Tal5, from an African Xoo strain. Artificial TAL effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence. The Tal5 target box differs from those of the already known TAL effectors TalC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo talC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TalC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence. Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence.

Comparative Transcriptome Profiling Reveals Different Expression Patterns in Xanthomonas oryzae pv. oryzae Strains with Putative Virulence-Relevant Genes

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight, which is a major rice disease in tropical Asian countries. An attempt has been made to investigate gene expression patterns of three Xoo strains on the minimal medium XOM2, PXO99 (P6) and PXO86 (P2) from the Philippines, and GD1358 (C5) from China, which exhibited different virulence in 30 rice varieties, with putative virulence factors using deep sequencing. In total, 4,781 transcripts were identified in this study, and 1,151 and 3,076 genes were differentially expressed when P6 was compared with P2 and with C5, respectively. Our results indicated that Xoo strains from different regions exhibited distinctly different expression patterns of putative virulence-relevant genes. Interestingly, 40 and 44 genes involved in chemotaxis and motility exhibited higher transcript alterations in C5 compared with P6 and P2, respectively. Most other genes associated with virulence, including exopolysaccharide (EPS) synthesis, Hrp genes and type III effectors, including Xanthomonas outer protein (Xop) effectors and transcription activator-like (TAL) effectors, were down-regulated in C5 compared with P6 and P2. The data were confirmed by real-time quantitative RT-PCR, tests of bacterial motility, and enzyme activity analysis of EPS and xylanase. These results highlight the complexity of Xoo and offer new avenues for improving our understanding of Xoo-rice interactions and the evolution of Xoo virulence.