Novel Pathogenicity Genes of Soft-rot Erwinias

我们分离出了4个胡萝卜Erwina cartovora亚种的原生营养毒性突变体。胡萝卜菌（Ecc）似乎在克服宿主抗性方面存在缺陷。这些突变体在植物生长初期（10至17个对数代）与Ecc的致病菌株相似，但未能达到显示软腐病表型所需的细菌阈值（1 × 108 cfu）。我们有独立的克隆来补充这些突变。从其中一个互补位点获得的两个亚克隆似乎抑制了Ecc和E. carotovora亚sp中果胶裂解酶、聚半乳糖醛酸酶、纤维素酶和蛋白酶的产生。并因此赋予这些植物致病细菌一种无毒的表型。我们提出的研究目标是：(1)Ecc致病性基因的分子特征及其在培养物和植物发病过程中的表达调控；(2)通过克隆DNA序列确定抑制细胞外酶产生的分子基础。使用无毒溶胸突变体分离的致病性基因的性质和机制将通过一系列综合实验来证明，包括(a)通过饱和转座子诱变、S1定位和DNA测序分析这些操纵子的结构和调控成分；(b)定位基因产物，利用抗体和报告基因融合确定基因激活的时间和调控，以及(c) DNA迁移迟缓和DNaseI足迹分析来表征反式作用的调控因子。实验还概述了关闭任何反式作用因子(s)，这可能是负责抑制细胞外酶的生产。这项研究将为植物与微生物相互作用的分子基础提供见解，并将增加我们对植物致病菌胞外酶表达调控的理解。将进行实验以确定这些影响是否由克隆载体引起。将对克隆的细菌基因进行测序，并进行必要的实验，以确定补充AH2552突变的基因与抑制植物细胞降解酶产生的DNA序列之间的物理关系。这项研究将为植物与微生物相互作用的分子基础提供见解，并将增加我们对植物致病菌胞外酶表达调控的理解。

We have isolated four prototrophic pectolytic avirulent mutants of Erwina carotovora subsp. carotovora (Ecc) which seem to be defective in overcoming host resistant. These mutants show initial in plants growth (10 to 17 logarithmic generations) similar to pathogenic strains of Ecc but fail to reach the threshold of bacteria (1 x 108 cfu) required to demonstrate the soft-rot disease phenotype. We have isolated clones which complement these mutations. Two subclones obtained from one of these complementing loci seem to inhibit production of pectate lyases, polygalacturonase, cellulase and protease in for Ecc and E. carotovora subsp. atroseptica and thereby impart an avirulent phenotype to these phytopathogenic bacteria. The goals of our proposed research are (1) the molecular characterization of pathogenicity genes of Ecc and their regulation of expression in cultures and during pathogenesis in planta, and (2) to determine the molecular basis of inhibition of production of extracellular enzymes by cloned DNA sequences. The nature and mechanisms of pathogenicity genes, isolated using avirulent pectolytic mutants, will be demonstrated by integrated series of experiments including (a) analysis of the structural and regulatory components of these operons by saturation transposon mutagenesis, S1 mapping and DNA sequencing, (b) localization of gene product and determination of the timing and regulation of gene activation using antibodies and reporter gene fusions, and (c) DNA mobility retardation and DNaseI foot-printing analysis to characterize the trans-acting regulatory factors. Experiments also are outlined to close any trans-acting factor(s) which may be responsible for the inhibition of the extracellular enzyme production. This research will provide insight into the molecular basis of plant-microbe interactions and will add to our understanding of the regulation of expression of extracellular enzymes in phytopathogenic bacteria. Experiments will be conducted to determine whether or not these effects are caused by the cloning vector. Cloned bacterial genes will be sequenced and experiments which are required to establish the physical relationships between the gene which complements the AH2552 mutation and the DNA sequence which inhibits production of plant cell-degrading enzymes will be carried out. This research will provide insight into the molecular basis of plant-microbe interactions and will add to our understanding of the regulation of expression of extracellular enzymes in phytopathogenic bacteria.