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Evidence for HrpXo-dependent expression of type II secretory proteins in Xanthomonas oryzae pv. oryzae.

ABSTRACT: Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

SUBMITTER: Furutani A

PROVIDER: S-EPMC344398 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Evidence for HrpXo-dependent expression of type II secretory proteins in Xanthomonas oryzae pv. oryzae.

Furutani Ayako A Tsuge Seiji S Ohnishi Kouhei K Hikichi Yasufumi Y Oku Takashi T Tsuno Kazunori K Inoue Yasuhiro Y Ochiai Hirokazu H Kaku Hisatoshi H Kubo Yasuyuki Y

Journal of bacteriology 20040301 5

Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type I ...[more]

PMID: 14973015

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Project description:BackgroundPathogenic Gram-negative bacteria interact with their eukaryotic hosts by deploying the type III translocon to inject effector proteins into the cytosol of eukaryotic cells. The translocon compositions, the number and biochemical characteristics of type III translocators in animal-pathogenic bacteria have been well elucidated, but information is lacking for plant-pathogenic bacteria. With extensive studies on biological functions of the Hpa1 protein secreted by the type III secretion system in Xanthomonas oryzae pv. oryzae (Xoo), we show here that Hpa1 is a type III translocator based on measurements of two proteins categorized as transcription activator-like (TAL) effector.ResultsHpa1 was functionally associated with the TAL effector PthXo1 or AvrXa10 by genetic analysis of the wild-type Xoo strain and related mutants or recombinant strains. Inoculation experiments suggested that Hpa1 is required not only for the virulent role of PthXo1 in the susceptible rice variety Nipponbare, but also for the avirulent function of AvrXa10 on the resistant rice variety IRBB10. Hpa1 is unrelated to the secretion of PthXo1 and AvrXa10 out of bacterial cells. However, Hpa1 is critical for both TAL effectors to be translocated from bacterial cells into the cytosol of rice cells based on replicate experiments performed on the susceptible and resistant varieties, respectively. Hpa1-mediated translocation of PthXo1 is coincident with induced expression of rice SWEET11 gene, which is the regulatory target of PthXo1, resulting in the occurrence of the bacterial blight disease in the susceptible rice variety. By contrast, the immune hypersensitive response is induced in agreement with induced expression of rice Xa10 gene, which is the target of AvrXa10, only when AvrXa10 is translocated from bacteria into cells of the resistant rice variety. All the virulent or avirulent performances of the TAL effectors are nullified by directed mutation that removes the α-helix motif from the Hpa1 sequence.ConclusionsThe genetic and biochemical data demonstrate that Hap1 is a type III translocator at least for TAL effectors PthXo1 and AvrXa10. The effect of the directed mutation suggests that Hpa1 depends on its α-helical motif to fulfil the translocator function.

Project description:BackgroundPlant-pathogen interactions at early stages of infection are important to the fate of interaction. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is a devastating disease in rice. Although in vivo and in vitro systems have been developed to study rice-Xoo interactions, both systems have limitations. The resistance mechanisms in rice can be better studied by the in vivo approach, whereas the in vitro systems are suitable for pathogenicity studies on Xoo. The current in vitro system uses minimal medium to activate the pathogenic signal (expression of pathogenicity-related genes) of Xoo, but lacks rice-derived factors needed for Xoo activation. This fact emphasizes the need of developing a new in vitro system that allow for an easy control of both pathogenic activation and for the experiment itself.ResultsWe employed an in vitro system that can activate pathogenicity-related genes in Xoo using rice leaf extract (RLX) and combined the in vitro assay with RNA-Seq to analyze the time-resolved genome-wide gene expression of Xoo. RNA-Seq was performed with samples from seven different time points within 1 h post-RLX treatment and the expression of up- or downregulated genes in RNA-Seq was validated by qRT-PCR. Global analysis of gene expression and regulation revealed the most dramatic changes in functional categories of genes related to inorganic ion transport and metabolism, and cell motility. Expression of many pathogenicity-related genes was induced within 15 min upon contact with RLX. hrpG and hrpX expression reached the maximum level within 10 and 15 min, respectively. Chemotaxis and flagella biosynthesis-related genes and cyclic-di-GMP controlling genes were downregulated for 10 min and were then upregulated. Genes related to inorganic ion uptake were upregulated within 5 min. We introduced a non-linear regression fit to generate continuous time-resolved gene expression levels and tested the essentiality of the transcriptionally upregulated genes by a pathogenicity assay of lesion length using single-gene knock-out Xoo strains.ConclusionsThe in vitro system combined with RNA-Seq generated a genome-wide time-resolved pathogenic gene expression profile within 1 h of initial rice-Xoo interactions, demonstrating the expression order and interaction dependency of pathogenic genes. This combined system can be used as a novel tool to study the initial interactions between rice and Xoo during bacterial blight progression.

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Evidence for HrpXo-dependent expression of type II secretory proteins in Xanthomonas oryzae pv. oryzae.

Publications

Evidence for HrpXo-dependent expression of type II secretory proteins in Xanthomonas oryzae pv. oryzae.

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