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標題: 楊桃細菌性斑點病菌的hrpC 基因串之選殖及分析
Cloning and characterization of hrpC operon from Pseudomonas syringae pv. averrhoi
作者: 侯寶勝
關鍵字: Pseudomonas syringae pv. averrhoi;楊桃細菌性斑點病;hrp/hrc;HrcC;hrpC operon;hrpV;第三型蛋白分泌系統
出版社: 生物科技學研究所
楊桃細菌性斑點病為台灣發生之新病害,經生理生化特性分析,顯示此病原菌屬於Pseudomonas syringae 的一種新病原型 (pathovar),定名為 Pseudomonas syringae pv. averrhoi (Pav)。P. syringae有一個25 Kb大小序列的hrp/hrc基因組,主要的轉譯蛋白質可組成的第三型蛋白分泌系統,此系統與其在寄主植物產生致病性,和在非寄主植物或具抗性的寄主植物中誘發過敏性反應上,為一不可或缺的主要因子,依據前人研究,HrcC蛋白為構成第三型分泌系統的膜外蛋白。本文從楊桃細菌性斑點病原細菌PavHL1中選殖出hrpC operon 的基因以進一步分析其生物活性。首先利用PCR方式,以P. s. pv. syringae 61 hrcC基因的引子對,PavHL1菌株染色體DNA為模版,增幅出2.1 Kb大小的DNA片段,經定序後確定為PavHL1 hrcC基因,並以此序列及其上下游的保留性序列設計引子對,利用PCR方式增幅出hrcC基因上下游的基因序列,經分析後含有3.3 Kb大小完整的hrpC operon,包含有hrpF、hrpG、hrcC、hrpT、hrpV等五個基因序列,與hrpU operon下游977 bp大小的hrcU基因。進一步利用不具有terminator的nptII基因,藉由三親本接合作用及標記致變術,構築hrcCPav基因之非極性突變株,探討其生物活性。最近研究發現P. s. pv. tabaci hrpZ基因不完整,但仍能引起非寄主蕃茄的過敏性反應,而其hrcC突變株亦能引起非寄主番茄輕微的過敏性反應,顯示有非第三型分泌系統的過敏原存在。另有研究也顯示由fliC基因所轉譯的鞭毛次單元蛋白 (flagellin),也能引起非寄主植物的過敏性反應,故選殖P. s. pv. tabaci的fliC基因送入PavHL1菌株及PavHL1 hrcC突變株,發現含有fliC基因的PavHL1及其hrcC突變株並未能增加其游動性,同時將含有fliC基因的PavHL1 hrcC突變株接種於菸草上也未觀察到flagellin所誘導的過敏性反應。另有研究指出P. s. pv. syringae 61的hrcC基因上游區域有類似的harp box序列存在,故構築一段0.5 Kb大小的hrcC基因上游區域DNA片段,以lacZ為報導基因分析啟動子的活性,發現PavHL1 hrcC基因上游區域確有啟動子的活性,但在受到HrpL調控時,啟動子活性較不如hrpC operon上游區域的harp box高。P. s. pv. syringae 61的hrpV基因為一負調控因子,其大量表現時會抑制hrp/hrc基因的表現,故在楊桃細菌性斑點病菌上構築hrpVPav突變株,探討是否會影響hrp/hrc基因的表現。將PavHL1與PavPA5 hrpV基因突變株及其突變互補菌株分別接種於菸草,發現並沒有出現顯著的過敏性反應延遲或提早的現象。這些結果顯示楊桃細菌性斑點病菌與P. syringae其他不同病原型的菌株可能有部分的差異,值得日後深入的研究。

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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