U.S.-Japan Joint Research on Molecular Biology of Host-specific Toxins as Pathogenicity

1.每种病原体参与AM-和HC-毒素生物合成的非核糖体多功能酶系统。炭旋孢霉TOX 2基因座HTS 1基因（22 kb）编码570 kD蛋白，该蛋白由4个结构域组成：结构域B在重组杆状病毒/昆虫细胞中表达时具有L-脯氨酸激活和异构化作用。编码聚酮合酶的两个必需TOX 1基因座位于T小种菌株的不同染色体上。由于在T小种和O小种的常规遗传杂交中，它们与相互易位的断点连锁，因此，没有分子技术是无法解决这些问题的。3.利用插入突变技术克隆了梨链格孢菌致病型中的AK毒素合成基因。通过质粒拯救的方法，从一个Tox^-β的基因组DNA中分离出了位于该突变DNA侧翼的序列。通过用拯救质粒的转化DNA侧翼的序列筛选野生型菌株的基因组DNA文库来克隆野生型基因组序列。4.发现了一个抗病基因Hm 1，它编码一种酶，该酶能解除引起玉米叶斑病的Chocliobolus carbonum 1号小种释放的HC-毒素。3-6.本研究的部分结果发表在鸟取大学学报上宿主特异性毒素国际专题讨论会（1994年）。

1.Non-ribozomal, multi-function enzyme systems involved in biosynthesis of AM-and HC-toxins by each pathogen. TOX2 locus in H.carbonum has HTS1 gene (22 kb) encoding 570 kD protein which have 4 domains : Domain B had L-proline-activation and isomerization when expressed in recombinant baculovirus/insect cell culture.2.Function and chromosomal location of the Cochliobolus heterostrophus TOX1 locus were investigated by tagging by insertional mutagenesis & REMI procedure. Two essential TOX1 loci that encode a polyketide synthase are on different chromosomes of race T strains. They could not be resolved without molecular technology because in conventional genetic crosses between race T and race O they are linked to the break point of a reciprocal translocation.3.Attempts to clone a gene for AK-toxin biosynthesis in Alternaria alternata Japanese pear pathotype was made by using insertional mutagenesis. The sequences flanking the transfoming DNA were isolated from genomic DNA of a Tox^- transformant by means of plasmid rescue. Wild-type genomic sequences were cloned by screening a genomic DNA library of the wild-type strain with the sequences flanking the transforming DNA of the rescued plasmid. The selected genomic clones have been characterizing.4.A disease resistance gene Hm1 was found to code the enzyme which detoxify HC-toxin released from Chocliobolus carbonum race 1, cause of leaf spot of maize.5.Pathological roles of fungal melanin, beta-1,3-glucanase and cutinase were examined by technology of molecular genetics.6.Part of the results obtained in the project was published as a Proceedings of Tottori University International Symposium on Host-Specific toxin (1994).