Exploration of the sequential fundamental processes in the early infection stage of Ralstonra solanacearum

青枯雷尔氏菌首先附着在寄主植物的根表面并侵入根皮层的细胞间隙。R.青枯菌在细胞间隙和维管薄壁组织内迅速繁殖和定殖，最终侵入原生木质部的维管。植物细胞信号被细菌外膜上的PrhA识别并转移到hrpB表达。采用共培养的方法，对红毛菊进行了研究。在液体培养基中，我们研究了在感染过程的这些早期阶段，致病性基因表达的调节，包括hrp基因。已知负责EPS产生的eps基因在感染的晚期以细胞密度依赖性方式被PhcA诱导。虽然prhA的表达是组成型的，但参与植物信号级联的其他基因和hrp调节子中的基因的表达取决于细胞密度。这些基因的最大表达的细胞密度小于eps基因的细胞密度，表明这些基因的表达在达到最大表达后被抑制。通过转座子突变，PhcA被证明是一个负调控基因的hrp调节子。已知phcA本身的表达通过群体感应以细胞密度依赖性方式被激活。综上所述，我们提出了在感染的早期阶段涉及HrpB和PhcA的全球监管计划。在R.青枯菌侵入细胞间隙，hrpB被植物信号诱导并激活hrp调节子，从而构建III型分泌系统。R.青枯菌细胞可以通过III型系统在分泌蛋白的帮助下发育，并且细胞密度达到phcA开始表达的阈值。表达的PhcA抑制参与植物信号级联和hrp调节子的基因，同时激活eps基因和其他降解酶基因。

Ralstonia solanacearum first attaches to host plant root surfaces and invades the intercellular spaces of the root cortex. R. solanacearum rapidly multiplies and colonizes within intercellular spaces and vascular parenchyma to finally break into the protoxylem vessels. Plant cell signals are recognized by PrhA on the bacterial outer membrane and transferred to hrpB expression. By using the co-cultivation method of R. solanacearum with Arabidopsis thaliana in the liquid medium, we have investigated the regulation of pathogenicity gene expression, including hrp genes, at these early stages of the infection process. The eps genes responsible for EPS production are known to be induced at the late stage of infection in a cell density-dependent manner by PhcA. While the expression of prhA was constitutive, other genes involved in plant signal cascade and genes in hrp regulon were expressed depending on cell densities. Cell densities of maximal expression for these genes were less than those for eps genes, indicating expression of these genes are repressed after they reach to the maximum. By transposon mutagenesis, PhcA was demonstrated to be a negative regulator of genes in hrp regulon. The expression of phcA itself is known to be activated in a cell density-dependent manner through quorum sensing. Taken all together, we propose the global regulation scheme at early stages of infection involving HrpB and PhcA. After R. solanacearum invades the intercellular spaces, hrpB is induced in response to plant signal and activates hrp regulon, which constructs type III secretion system. R. solanacearum cells can develop with the aid of secreted proteins through the type III system, and cell densities reach to the threshold, in which phcA starts to express. The expressed PhcA represses genes involved in plant signal cascade and hrp regulon, and at the same time activates eps genes and other genes for degradation enzymes.