| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 103 === The hrp (hypersensitive response and pathogenicity) genes of genus Xanthomonas encode factors that are required for pathogenicity in host plants and hypersensitive response in non-host plants. These genes are expressed in planta or when cultured in minimal medium (XVM2). HrpX and HrpG are the main transcriptional regulators for hrp genes, while HrpX is regulated by HrpG; mutation in which causes the loss of pathogenicity. X. campestris pv. campestris strain 11A (Xc11A), a spontaneous avirulent mutant derived from Xc11, has an extremely low level of hrpX mRNA. Microarray analysis performed here showed that expression of most Xc17 hrp genes was enhanced in XVM2, which was generally higher than those of Xc11A, except that the level of hrpG was 1.8 times higher than that in Xc17. Since it has previously been shown that phosphorylation of HrpG is required for regulation of the downstream genes, it is possible that the loss of pathogenicity in Xc11A is correlated to defect in HrpG. With this in mind, a HrpXhis expression plasmid, that could express active HrpXhis, was constructed. Then subcloning was performed by placing the hrpXhis gene downstream of the lac promoter for constructive expression of HrpXhis, which was then introduced into Xc11 and Xc11A, which were used to infect the host plant. Transformants with this construct integrated into the chromosome were also made. Data showed that none of these constructs confers pathogenicity. The above results suggest that low levels of hrpX expression may not be the main reason for Xc11 and Xc11A to lose virulence.
The lytic phage phiL7, specifically infecting Xc17, has four lytic genes: p27 (holin), p28 (lysozyme), p29 (Rz) and p29.1 (Rz1). It has previously been shown that co-expression of p27 and p28 has the strongest antibacterial effect on Xc17. This study showed that expressing p28 alone did not affect the growth of DH5α. However, co-expression of p27 and p28 readily killed DH5α and Xag, although no cell lysis was caused. Furthermore, recombinant plasmids which could express GST-p27 and GST-p28 proteins, both with antibacterial effects, were also constructed; however, the expression levels were too low for further studies.
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