十字花科黑腐病菌聚半乳糖醛酸酶基因 pglA 之調控

Abstract

Abstract The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) causes black rot in crucifers. This bacterium produces large mounts of an exopolysaccharide (EPS), xanthan gum, and an array of extracellular enzymes, including proteases, pectinases and cellulases. These substances are collectively required for pathogenicity. In addition to these substances, Avr (avirulence) proteins and the hrp genes encoded Type III secretory pathway are also essential for virulence. Regulation of the virulence factors in Xcc is not fully understood, but it is known that rpf (regulation of pathogenicity factors) gene cluster and the global transcription factor Clp（cAMP receptor protein-like protein）are involved in regulation of EPS and the extracellular enzymes. In Xcc, pglA is a pectinase gene encoding one of the polygalacturonases. Sequence analysis performed previously indicated that the upstream region of this gene has a region conserved to a Clp-binding site and a putative HrpX-binding site (plant-inducible promoter, also called PIP box). The purposes of this study were to investigate the regulation of the pglA gene by Clp, rpf system and HrpX protein. My sequence analysis revealed that PglA belongs to family 28 of glycosyl hydrolases, which typically have four conserved domains (NTD, DD, HG, and RIK). In plate assays, the pglA mutant constructed by marker exchange exhibited extracellular polygalacturonase at a level similar that of the wild-type, indicating that pglA is not the major pectinase in Xcc. In pathogenicity tests, the symptoms caused by the pglA mutant appeared slightly slower than Xc17. Using 5’RACE technique, nucleotide A at 102 nt upstream of the pglA translation initiation codon GTG was mapped as the 5’ end of pglA mRNA, the transcription star site. Gel retardation assays revealed that Clp can directly bind to the pglA promoter. Nested fragments of the pglA upstream region were separately cloned into the broad host range promoter-probing vector pFY13-9 to form transcriptional fusion constructs, each of which was then introduced by electroporation into the wild-type Xc17, clp mutant Xc17(clp::Gm), rpfF mutant Xc17(rpfF::Gm), and hrp mutant Xc17(hrpX::Gm). Results of reporter assays indicated that when the cells were grown in the modified XVM2 medium supplemented with 0.5% polygalacturonic acid, the pglA promoter activity was greatly reduced in hrpX mutant compared to that in than Xc17, but increased by 2.27 and 1.76 times in clp and rpfF mutant, respectively.