The Interactions of Pseudomonas Syringae Harpins, HRP Secretion Proteins, and AVR Proteins in Eliciting Either theHypersensitive Response or Pathogenesis in Higher Plants

9305178柯尔默高等植物的过敏性反应(HR)的特征是植物细胞在“不亲和”病原体(一种只在另一种植物中引起疾病的微生物)入侵的地方迅速、局部地死亡，并与对许多线虫、真菌病毒和细菌的抗性有关。紫丁香假单胞菌和其他植物病原菌对HR的诱导是由hrp基因控制的，hrp基因也是这些细菌在各自寄主植物中增殖和致病所必需的。该研究的总体目标是利用两个最新的进展来剖析HRP基因的功能，并确定AVR基因对HRP基因的表型依赖性的一般基础。我们预计：(I)紫丁香叶霉菌株需要Harpins来启动与植物的代谢活跃的相互作用，(Ii)Harpin的分泌需要HrpH和其他几种HRP蛋白，(Iii)Harpin的产生必须受到仔细的调控才能成功致病，(Iv)AVR蛋白依赖于HRP分泌系统和/或Harpin来产生其表型。这项工作可能涉及三种丁香疫霉菌株：(I)P.S.Sringae 61，其克隆的HRP基因簇已被广泛鉴定，其独特之处在于，它在非病原菌如大肠杆菌中发挥作用。(Ii)P.S.获得克隆的avrB基因后，与Rpg1大豆品种发生不亲和性的大豆小种4和(Iii)P.S.番茄DC3000，其与拟南芥的亲和性因获得avrB而逆转。该项目的早期工作将决定P.S.甘草小种4或P.S.番茄DC3000将是AVR蛋白功能研究的重点。我们打算(1)刻画P.S.Syringae 61 hrpZ基因和编码的Harpin基因的初级序列、激发子活性结构域和亚细胞位置。(2)确定hrpH和hrpZ突变对P.S.初始相互作用的相对影响。紫丁香61与豆类和烟草(或P.S.番茄DC3000与A.thaliana或P.S.大豆小种4)。(3)克隆和鉴定P.S.甘草小种4号(或P.S.番茄DC3000)hrpZ基因，构建Harpin缺失的衍生物，过量产生Harpin而不是Harpin，或含有hrpZ启动子的反式融合；测定这些衍生物(有或没有avrB+质粒)在烟草和差异寄主品种上的表型。%由于HRP分泌蛋白的同源物存在于一些动物病原体中，这些假说的成功检验应该会对解决细菌致病性的研究产生广泛的影响。***

9305178 Collmer The hypersensitive response (HR) of higher plants is characterized by the rapid, localized death of plant cells at the site of invasion by an "incompatible" pathogen (a microorganism that causes disease only in another plant) and is associated with resistance against many nematodes, fungi viruses and bacteria. Elicitation of the HR by Pseudomonas syringae and other plant pathogenic bacteria is controlled by hrp genes, which are also required for the multiplication and pathogenicity of these bacteria in their respective host plants. The overall goal of the research is to use two recent advances to dissect the functions of the hrp genes and to determine the general basis for the phenotypic dependence of avr genes on hrp genes. We expect that (i) harpins are needed for P. syringae strains to initiate a metabolically active interaction with the plant, (ii) HrpH and several other Hrp proteins are needed for the secretion of harpin, (iii) harpin production must be carefully regulated for successful pathogenesis, and (iv) Avr proteins depend on the Hrp secretion system and/or harpin to produce their phenotypes. The work will involve potentially three strains of P. syringae: (i) P.s. syringae 61, whose cloned cluster of hrp genes has been extensively characterized and is unique in that, it functions in nonpathogens like E. coli. (ii) P.s. glycinea race 4, which becomes incompatible with Rpg1 soybean cultivars upon acquisition of the cloned avrB gene, and (iii) P.s. tomato DC3000, whose compatibility with Arabidopsis thaliana is reversed by acquisition of avrB. Early work on the project will determine whether P.s. glycinea race 4 or P.s. tomato DC3000 will be the focus of work on Avr protein function. We intend to (1) Characterize the P.s. syringae 61 hrpZ gene and the encoded harpin with respect to primary sequence, elicitor-active domains, and subcellular location. (2) Determine the relative effects of hrpH and hrpZ mutations on the initial interac tion of P.s. syringae 61 with bean and tobacco (or of P.s. tomato DC3000 with A. thaliana or of P.s. glycinea race 4 with soybean). (3) Clone and characterize the P.s. glycinea race 4 (or P.s. tomato DC3000) hrpZ gene, construct derivatives that are deficient in harpin overproduce harpin produce harpin instead of harpin or contain a hrpZ promoter- uida fusion in trans; determine the phenotype of these derivatives (with or without an avrB+ plasmid) on tobacco and differential host cultivars. %%% Because homologs of Hrp secretion proteins are present in some animal pathogens, successful testing of these hypothesis should have a broad impact on research addressing bacterial pathogenicity. ***