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

大量的实验证据表明，病原菌只有在感染宿主时才会高水平表达毒力基因。 事实上，可能存在于各种替代生态位中的病原体在所有这些生态位中都表达独特的基因组。 利用基因芯片技术和体内表达技术（IVET），研究了植物病原细菌欧文氏菌（Erwiniachenii）在其叶片宿主中生长和在实验室培养基中生长的基因表达模式。 这些结果为了解对毒力重要的新病原体基因提供了有力的见解。 特别是，大量的III类毒力基因在植物中上调。 III类基因似乎不直接参与破坏宿主，如I类和II类毒力基因的情况，但可能是重要的适应病原体在宿主环境中生存和生长。几种植物中的突变上调了E.构建了cheapi基因。 在一个推定的I类毒力基因编码一个以前未鉴定的肽合酶，和两个新的III类毒力基因的突变，都大大降低了宿主非洲紫罗兰叶上的细菌的毒力。 本项目的目标是：（i）继续通过IVET筛选宿主上调基因;（ii）对从IVET和先前的微阵列检测中获得的宿主上调基因进行系统的突变和毒力检测;和（iii）鉴定由E. chalcii的功能性克隆。大肠杆菌细胞，chelii hrp基因簇。 这些交叉互补的方法，预计将确定新的基因重要的细菌对植物宿主的毒力。

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.