| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 93 === Abstract
Bacterial spot is a new disease of carambola in Taiwan. Based on physiological characteristics, the pathogen was identified as a strain of Pseudomonoas syringae and named as a new pathovar: P. syringae pv. averrhoi (Pav). Many phytopathogenic bacteria use a type III secretion system (TTSS) to transfer virulence proteins into their hosts. TTSS encoded by a 25 Kb cluster of hrp/hrc gene in P. syringae is required to cause diseases on their hosts and to elicit the hypersensitive response (HR). HrcC is an outer membrane protein of TTSS involved in macromolecular traffic across the bacterial outer membrane. Since the biological functions of HrcC in Pav involved in pathogenesis is still unknown, the aim of this study is to clone hrpC operon from PavHL1. The primers corresponding to the conserved regions of genes in other P. syringae pathovars were designed, the chromosome DNA of PavHL1 strain was used as a template, then polymerase chain reaction (PCR) was performed to gain 3.3 Kb hrpC operon fragment. Based on the DNA sequencing and derived amino acid sequence analysis using SDSC Biology Workbench, five putative open reading frames were found in the hrpC operon, designated as hrpF, hrpG, hrcC. hrpT and hrpV genes. To further investigate the role of HrcC in host-bacteria interactions, a non-polar mutation of PavHL1 in which hrcC was deleted and replaced with an nptII cassette lacking a rho-independent transcription terminator was constructed by using marker-exchanged mutagensis. According to the HR test and western blot with HrpZ serum, results show that HrcC is required for the HR eliciting and the secretion of HrpZ (a harpin protein). Recently it was reported that the hrpZ from P. syringae pv. tabaci is defective. However, P. syringae pv. tabaci induced typical HR in its nonhost tomato plant, indicating the existence of other HR-elicitors besides harpin. It was also reported flagellin, encoded by fliC gene, is a potent elicitor of hypersensitive cell death in plant cell. Thus, PavHL1 and PavHL1 hrcC mutant carrying fliC gene from P. syringae pv. tabaci were constructed. Motility and the HR eliciting in tobacco leaves assay of PavHL1 and PavHL1 hrcC mutant revealed that both fliC containing PavHL1 and PavHL1 hrcC mutant still have no mobility and hrcC mutant carrying fliC can not cause the HR neither. It was also reported the upstream region of hrcC gene from P. syringae pv. syringae 61 was found similar to the conserved sequence of a harp box. Then, a 0.5 Kb DNA fragment that contains the upstream region of PavHL1 hrcC gene was subcloned into pNCHU413 and used lacZ gene as a reporter. The results suggest that the upstream region of hrcC gene from PavHL1 has a promoter activity but does not contain a very typical harp box sequence. On the other hand, hrpV is a negative regulator gene in Psy61 and overexpressing hrpV represses the expression of hrp/hrc genes. To study relation of hrpV and hrp/hrc genes, hrpV mutants of PavHL1 and PavPA5 were constructed by marker-exchanged mutagenesis. PavHL1 or PavPA5 hrpV mutant and their complemented strains do not show the obviously earlier or delayed HR phenotypes in tobacco leaves. These obtained results in this study were obviously different from those in other pathovars of P. syringae. Therefore, it is very worthy to study further on genetics of P.syringae pv. averrhoi in terms of pathogenesis.
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