Microarray/Mutation Studies to Identify New Virulence Genes in Erwinia Chrysanthemi

大量的经验证据表明，只有当病原菌感染宿主时，它们才会高水平地表达毒力基因。事实上，可以存在于各种替代生态位中的病原体很可能在所有这些生态位中都表达独特的基因集。利用基因芯片和体内表达技术(IVET)，研究了植物病原菌欧文氏菊花欧文氏菌在叶寄主和室内培养基中的基因表达谱。这些结果为新的致病基因提供了强有力的见解，这些基因对毒力很重要。特别是，相当数量的III类毒力基因在植物中上调。与I类和II类毒力基因不同，III类基因似乎不直接参与破坏宿主，但可能在适应病原体在宿主环境中生存和生长方面起重要作用。构建了几个植物上调的菊花基因的突变。一个编码先前未知的多肽合成酶的I类毒力基因的突变，以及两个新的III类毒力基因的突变，都大大降低了寄主非洲紫罗兰叶片上细菌的毒力。本项目的目标是：(I)继续通过IVET筛选寄主上调基因；(Ii)对从IVET和以前的微阵列检测获得的寄主上调基因进行系统突变和毒力分析；以及(Iii)通过在携带所克隆的菊花HRP基因簇的大肠杆菌中进行功能克隆，鉴定受菊花致病基因簇HRP调控的毒力基因。这些交叉互补的方法有望识别对植物寄主上的细菌毒力至关重要的新基因。

Substantial empirical evidence indicates that pathogenic bacteria express virulence genes at high levels only when they infect a host. Indeed, it is likely that pathogens that can exist in various alternative ecological niches express unique gene sets in all of them. Using microarray and in vivo expression technique (IVET), the gene expression pattern of a plant pathogenic bacterium, Erwinia chrysanthemi, grown in its leaf host as compared to growth in laboratory culture medium was studied. These results have offered powerful insights into new pathogen genes that are important for virulence. In particular, a substantial number of class III virulence genes were upregulated in planta. The class III genes do not appear to be directly involved in damaging the host, as is the case with class I and II virulence genes, but are probably important in adapting the pathogen to survive and grow in the host environment. Mutations in several of the plant upregulated E. chrysanthemi genes were constructed. Mutations in a putative class I virulence gene encoding a previously unidentified peptide synthase, and two novel class III virulence genes, all greatly reduced virulence of the bacteria on host African violet leaves. The objectives of this project are to: (i) continue screening of host upregulated genes by IVET; (ii) perform systematic mutation and virulence assays of host upregulated genes obtained from IVET and previous microarray assays; and (iii) identify virulence genes regulated by the pathogenicity gene cluster hrp of E. chrysanthemi by functional cloning in E. coli cells carrying the cloned E. chrysanthemi hrp gene cluster. These cross-complementary approaches are expected to identify new genes important for bacterial virulence on plant hosts.