Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae
Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae
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DOI：
10.1111/j.1364-3703.2009.00534.x
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发表时间：
2009-05-01
影响因子：
4.9
通讯作者：
Sone, Teruo
中科院分区：
文献类型：
--
作者：
Miki, Shinsuke;Matsui, Kotaro;Sone, Teruo
为了克隆和分析日本稻瘟菌田间分离物的无毒基因AVR-Pia，对稻瘟菌的突变体进行了研究。分离到一株产芽孢杆菌Ina 168。该突变体被命名为Ina 168 m95 -1，对含有Pia抗性基因的水稻品种Aichi-asahi具有致病性。分离到一个在突变体中缺失的DNA片段（命名为PM 01），该片段与对爱知朝日的无毒力共分离。从基因组DNA文库中分离出三个包含PM 01侧翼区域的粘粒克隆。其中一个克隆（46 F3）与突变表型互补，这清楚地表明该克隆含有无毒基因AVR-Pia。克隆46 F3含有转座因子的插入。将46 F3插入片段分成片段I-VI，并将这些片段分别克隆到潮霉素抗性载体中，用于转化突变体Ina 168 m95 -1。转化体的接种测定显示片段V（3.5kb）含有AVR-Pia。通过片段V的缺失分析，AVR-Pia定位于1199-bp的DNA片段，其包括与细菌细胞色素c样蛋白具有弱同源性的255-bp开放阅读框架。该区域的限制性片段长度多态性分析表明，该DNA序列与AVR-Pia基因座共分离，该基因座是使用中国分离株构建的遗传图谱。
In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.