Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.
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.
Miki
Shinsuke
S
Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
Matsui
Kotaro
K
Kito
Hideki
H
Otsuka
Keisuke
K
Ashizawa
Taketo
T
Yasuda
Nobuko
N
Fukiya
Satoru
S
Sato
Junko
J
Hirayae
Kazuyuki
K
Fujita
Yoshikatsu
Y
Nakajima
Toshihiko
T
Tomita
Fusao
F
Sone
Teruo
T
D000383:Agriculture; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D020090:Chromosome Segregation; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003360:Cosmids; D003433:Crosses, Genetic; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007564:Japan; D020082:Magnaporthe / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D012275:Oryza / Q000382:microbiology*; D010641:Phenotype; D019105:Random Amplified Polymorphic DNA Technique; D017384:Sequence Deletion; D014170:Transformation, Genetic; D014774:Virulence
D004271:DNA, Fungal