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Journal of Bacteriology, March 2004, p. 1374-1380, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1374-1380.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in Xanthomonas oryzae pv. oryzae

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522,¹ Research Institute of Molecular Genetics,² Laboratory of Plant Pathology and Biotechnology, Kochi University, Nankoku, Kochi 783-8502,³ Laboratory of Molecular Plant Pathology, School of Bioresources, Hiroshima Prefectural University, Shobara 727-0023,⁴ Faculty of Agriculture, Miyazaki University, Miyazaki 889-2155,⁵ National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan⁶

Received 17 June 2003/ Accepted 10 November 2003

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.

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