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ABSTRACT: Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of bacterial leaf streak, a devastating disease in rice. Xoc uses a type III secretion (T3S) system, which is encoded by the hrp-hrc-hpa (hypersensitive response and pathogenicity, hrp-conserved and hrp-associated) genes, to inject repertoires of T3S effectors (T3Es) into plant cells. Many of the hrp-hrc-hpa genes have roles in pathogenesis, but the role of hrpE3, which shows homology to hpaE in X. campestris pv. vesicatoria (Xcv), is poorly understood. In this study, hrpE3 was shown to be transcribed independent of the hrpD operon, and its expression was dependent on a promoter within hpaB. The expression of hrpE3 was positively regulated by HrpG and HrpX, a finding probably caused by an imperfect plant-inducible promoter (PIP) box (TTCGT-N16 -TTCGA) in the hrpE3 promoter. The secretion of HrpE3 was dependent on T3S, and subcellular localization of HrpE3 was cytoplasmic and nuclear in plant cells. A mutation in hrpE3 reduced the virulence of Xoc by decreasing disease lesion length and bacterial growth in planta. Full virulence was restored to the mutant when Xoc hrpE3, but not Xcv hpaE, was expressed in trans. The differences in transcription, secretion via the T3S system and bacterial virulence in plants were attributed to N-terminal amino acid differences between Xoc HrpE3 and Xcv HpaE. Collectively, the results demonstrate that hrpE3 encodes a T3E protein which is delivered into the plant cell through the T3S system, localizes to the cytoplasm and nucleus, and is required for full virulence in rice.
Molecular Plant Pathology 05/2013; · 3.90 Impact Factor
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ABSTRACT: The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.
Applied and environmental microbiology 01/2012; 78(8):2888-95. · 3.69 Impact Factor
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ABSTRACT: Xanthomonas oryzae pv. oryzicola is the causal agent of bacterial leaf streak in rice and injects repertoires of T3S effectors (T3SEs), which are normally regulated by a global regulator HrpX, into plant cells to suppress plant innate immunity for disease development. To establish a visualization technique to identify HrpX regulon genes in this pathogen, we chose six known or unknown T3SE genes of X. oryzae pv. oryzicola (strain RS105) as the targets in this report. The promoters of these candidates, whether or not containing a PIP-box, were fused with the gfp (green fluorescent protein) reporter gene, and the gfp reporters were introduced into the wild-type RS105 and the hrpX mutant RΔhrpX. GFP expression in the wild-type strain could be observed under fluorescence microscopy, but fluorescence was not observed in the hrpX mutant. Consequently, transcription and secretion detection demonstrated that these HrpX regulon members were repressed when hrpX was mutated and were not secreted into the hrp-inducing medium XOM3 because of a mutation in hrcV (T3SS deficient). These data suggest that the gfp reporter system is a feasible visualization tool for the identification of HrpX regulon genes and discovery of new T3SEs in the X. oryzae pv. oryzicola-rice pathosystem.
Archives of Microbiology 10/2011; 194(4):281-91. · 1.43 Impact Factor
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ABSTRACT: Previously, seventeen extracellular polysaccharide-associated mutants of Xanthomonas oryzae pv. oryzicola (Xoc) were acquired from our randomly Tn5-inserted mutant library. To know the Tn5-inserted genes of these mutants and their contribution to EPS production and virulence in rice,
in this study, we first identified and characterized the Tn5-targeted genes in these mutants and then inoculated them in susceptible rice for virulence assessment.
Tn5 transposon was inserted in genes of the gum, xan and wxoc clusters in the majority of EPS-reduced mutants. Of the EPS-reduced mutants, three were due to the Tn5 insertion in Xoryp_4217, Xoryp 2488 and Xoryp_0918 genes, respectively. In six EPS-increased mutants, three were in mutagenesis in fimO, pilY and xopQ genes, respectively, resulting in higher EPS production than the wild-type strain RS105. Other three were because of the mutation in Xoryp2392, Xoryp_4221 and Xoryp_3511 genes. Interestingly, XocORF-3511 only exists in X. oryzae but not in other Xanthomomas species. Virulence assays in rice showed that the less EPS production by the mutant, the weaker the virulence in rice. However, those mutants in higher EPS production did not increase virulence significantly in rice compared to that by the wild-type strain.
Our findings will help further understand the metabolic pathways for EPS synthesis in Xoc and the specific roles of EPS-associated genes in Xoc-rice interactions.
ACTA MICROBIOLOGICA SINICA 10/2011; 51(10):1334-41.
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ABSTRACT: Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight in rice, interacts with rice plants in a gene-for-gene manner. The specificity of the interaction is dictated by avirulence (avr) genes in the pathogen and resistance (R) genes in the host. To date, no avr genes that correspond to recessive R genes have been isolated. We isolated an avrBs3/pthA family gene, avrxa5, from our previously isolated clone p58, which was originally from strain JXOIII. The avrxa5 gene converted the PXO99(A) strain from compatible to incompatible in rice cultivars containing the recessive xa5 gene, but not in those containing the dominant Xa5 gene. Sequencing indicated that avrxa5, which is highly similar to members of the avrBs3/pthA family, encodes a protein of 1238 amino acid residues with a conserved carboxy-terminal region containing three nuclear localization signals and a transcription activation domain. It has 19.5 34-amino-acid direct repeats, but the 13th amino acid is missing in the fifth and ninth repetitive units. Domain swapping of the repetitive regions between avrxa5 and avrXa7 changed the avirulence specificity of the genes in xa5 and Xa7 rice lines, respectively. This indicates that avrxa5 is distinct from previously characterized avrBs3/pthA members. The specificity of avrxa5 toward recessive xa5 in rice could help us better understand the molecular mechanisms of plant-pathogen specific interactions.
Science China. Life sciences 12/2010; 53(12):1440-9. · 2.02 Impact Factor
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ABSTRACT: Xanthomonas oryzae pv. oryzae (Xoo) possesses a type III secretion system (T3 S), encoded by a hpa-hrp-hrc cluster, including hrpD6, to inject T3S effectors into plant cells to trigger hypersensitive response (HR) in nonhost tobacco and pathogenicity in susceptible host rice. However, it is unclear what roles of Xoo hrpD6 gene plays in HR in tobacco and in pathogenicity in rice.
In this study, we constructed a deletion mutant of hrpD6 gene by using marker-exchange method. PCR and Southern blot analysis demonstrated that the hrpD6 gene was knocked out successfully.
in planta assays indicated the hrpD6 mutant, delta PhrpD6, lost the ability to induce HR in tobacco, to trigger water-soaked symptoms in seedlings rice and to cause bacterial blight in adult rice. Importantly, the bacterial growth in rice tissues was tremendously reduced. Complementation assays confirmed that hrpD6 gene could restore HR induction in tobacco, pathogenicity and bacterial growth in rice to the mutant delta PhrpD6. Reverse transcriptional polymerase chain reaction (RT-PCR) revealed that the expression of hrpD6 was not only induced by rice cells, but also controlled by hrpG and hrpX. Intriguyingly, the expression of hpal, encoding a harpin protein, was found to be dependent on hrpD6, implying that hrpD6 regulates the expression of hpal. Immunobloting assay confirmed that the mutation of hrpD6 affect the secretion of Hpal through T3S.
The mutant lost the ability of triggering hypersensitive response in nonhost tobacco and pathogenicity in host rice is due to that hrpD6 regulates the expression of hpal gene and the mutation in hrpD6 affects the secretion of T3S effectors, like Hpa1, through T3SS. Our results provide molecular clues to understand whether hrpD6 is involved in the formation of T3S apparatus and in regulation of other hpa-hrp-hrc gene expression or not for HR induction in tobacco and pathogenicity in rice.
ACTA MICROBIOLOGICA SINICA 09/2010; 50(9):1155-63.
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ABSTRACT: The type III secretion system (T3SS) is considered one of the major virulence factors in many bacterial pathogens. This report demonstrates that RssB, ClpXP, and RpoS play a role in T3SS regulation of Dickeya dadantii 3937. ClpP is a serine-type protease which associates with the ClpX chaperone to form a functional Clp proteolytic complex for degradation of proteins. With the assistance of recognition factor RssB, ClpXP degrades the RpoS sigma factor. RpoS positively regulates the expression of the rsmA gene encoding an RNA-binding regulatory protein. By interacting with the hrpL mRNA, RsmA reduces HrpL production and downregulates the T3SS genes in the HrpL regulon. In addition, ClpXP, RssB, and RpoS affect pectinolytic enzyme production in D. dadantii 3937, probably through RsmA. The ClpXP and RssB proteins are essential for bacterial virulence.
Molecular Plant-Microbe Interactions 07/2010; 23(7):871-8. · 4.43 Impact Factor
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Shihui Yang,
Quan Peng,
Qiu Zhang, Lifang Zou,
Yan Li,
Christelle Robert,
Leighton Pritchard,
Hui Liu,
Raymond Hovey,
Qi Wang,
Paul Birch,
Ian K Toth,
Ching-Hong Yang
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ABSTRACT: BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.
PLoS ONE 01/2010; 5(10):e13472. · 4.09 Impact Factor
