MOLECULAR PATHOLOGY OF PLANT INFECTION MEDIATED BY HOST-SPECIFIC TOXINS

开展了一项为期3年的跨学科研究，旨在阐明寄主特异性毒素(HST)致病真菌的分子基础。1)从病原菌的萌发液中分离出ACT-、BZR-、AT-和稻瘟病菌毒素，并对前两种毒素的结构进行了鉴定。ACT-毒素与AK-和AF-毒素在结构上与十碳五烯酸相同，而BZR-毒素由环状脱肽的同系物组成，只有当它们至少存在两个成员时才具有生物活性(共毒素)。另一方面，结构-活性相关性研究表明，…的寄主范围之间存在一致性互隔交链孢菌的日本梨、草莓和柑橘致病类型及其产生的AK-、AF-和ACT-毒素的敏感范围，表明该真菌的寄主专一性是在分子水平上精确确定的。2)根据中间代谢物的结构分析和示踪实验，提出了AK-、AF-和ACT-毒素的生物合成途径。3)从生理生化和超微结构方面剖析了HSTs作用的级联信号转导过程。4)建立了互隔交链孢霉高效的整合转化体系：将一个高度重复的核糖体RNA基因簇(RDNA)序列插入到pDH25的Xbal位点，构建了载体pDH25rla。在此基础上，分离了3个真菌黑素生物合成基因(ALM、BRM1、BRM2)，作为克隆致病基因的模型。5)用于HST受体研究，制备了抗HST抗体。在感病梨愈伤组织的质膜部分中检测到可能的受体蛋白，而在来自同一感病愈伤组织的抗性细胞系中检测到可能的受体蛋白。较少

An interdisiplinary project for 3 years was conducted to elucidate the molecular basis of plant infection by toxigenic fungal pathogens with host-specific toxins (HST). A proposed scheme for disease establishment that an HST released during spore germination binds a putative toxin-binding site (receptor) in potential host plant, where the HST-signal induces the state of accessibility to the invader, and then the producing fungus is eventually able to colonize the plant, was developed as follows:1) ACT-, BZR-, AT- and Pyricularia oryzae-toxins were isolated from germination fluids of the pathogens, and structures of the former two toxins were elucidated. ACT-toxin shared in structure a decatrtienoic acid with AK- and AF-toxins, while BZR-toxin composed of homologs of cyclic depsipeptides that were bioactive only when they co-exisited at least two members (co-toxin). On the other hand, a structure-activity corelation study revealed that there is coincidence between the host ranges of the … More Japanese pear, strawberry and tangerine pathotypes of Alternaria alternata and the plant ranges sensitive to AK-, AF- and ACT-toxins they produced, suggesting that host-specificity of the fungus is precisely determined at the molecular level.2) Biosynthetic pathways of AK-, AF- and ACT-toxins were proposed, depending on structural analysis of intermediate metabolites and tracer experiments. Enzymatic basis for AM-toxin synthesis also examined.3) Cascade-like signal transduction processes of HSTs actions were dissected by means of physiological, biochemical and ultrastructural approaches.4) A highly efficient integrative transformation system was estalished for Alternaria alternata: the vector pDH25rla was constructed by inserting a highly repetitive ribosomal RNA gene cluster (rDNA) sequence into a unique Xbal site of pDH25. Using this, 3 genes for fungal melanin biosynthesis (ALM, BRM1, BRM2) were isolated as a model for cloning pathogenicity genes.5) For HST-receptor studies, anti-HST anibodies were generated. A possible receptor protein was detected in the plasma membrane fraction of susceptible pear calli but not of resistant cell line derived from the same susceptible calli. Less