Dataset Information

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

ABSTRACT: 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.

SUBMITTER: Miki S

PROVIDER: S-EPMC6640357 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

Miki Shinsuke S 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

Molecular plant pathology 20090501 3

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 we ...[more]

PMID: 19400839

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Project description:BackgroundRice blast disease is one of the most destructive fungal disease of rice worldwide. The avirulence (AVR) genes of Magnaporthe oryzae are recognized by the cognate resistance (R) genes of rice and trigger race-specific resistance. The variation in AVR is one of the major drivers of new races. Detecting the variation in the AVR gene in isolates from a population of Magnaporthe oryzae collected from rice production fields will aid in evaluating the effectiveness of R genes in rice production areas. The Pik gene contains 5 R alleles (Pik, Pikh, Pikp, Pikm and Piks) corresponding to the AVR alleles (AVR-Pik/kh/kp/km/ks) of M. oryzae. The Pik gene specifically recognizes and prevents infections by isolates of M. oryzae that contain AVR-Pik. The molecular variation in AVR-Pik alleles of M. oryzae and Pik alleles of rice remains unclear.ResultsWe studied the possible evolutionary pathways of AVR-Pik alleles by analyzing their DNA sequence variation and assaying their avirulence to the cognate Pik alleles of resistance genes under field conditions in China. The results of PCR products from genomic DNA showed that 278 of the 366 isolates of M. oryzae collected from Yunnan Province, China, carried AVR-Pik alleles. Among the isolates from six regions of Yunnan, 66.7-90.3% carried AVR-Pik alleles. Moreover, 10 AVR-Pik haplotypes encoding five novel AVR-Pik variants were identified among 201 isolates. The AVR-Pik alleles evolved to virulent from avirulent forms via stepwise base substitution. These findings demonstrate that AVR-Pik alleles are under positive selection and that mutations are responsible for defeating race-specific resistant Pik alleles in nature.ConclusionsWe demonstrated the polymorphism and distribution of AVR-Pik alleles in Yunnan Province, China. By pathogenicity assays used to detect the function of the different haplotypes of AVR-Pik, for the first time, we showed the avoidance and stepwise evolution of AVR-Pik alleles in rice production areas of Yunnan. The functional AVR-Pik possesses diversified sequence structures and is under positive selection in nature.

Project description:The product of the avirulence (Avr) gene of Magnaporthe oryzae can be detected by the product of the corresponding resistance (R) gene of rice and activates immunity to rice mediated by the R gene. The high degree of variability of M. oryzae isolates in pathogenicity makes the control of rice blast difficult. That resistance of the R gene in rice has been lost has been ascribed to the instability of the Avr gene in M. oryzae. Further study on the variation of the Avr genes in M. oryze field isolates may yield valuable information on the durable and effective deployment of R genes in rice production areas. AvrPiz-t and Piz-t are a pair of valuable genes in the Rice-Magnaporthe pathosystem. AvrPiz-t is detectable by Piz-t and determines the effectiveness of Piz-t. To effectively deploy the R gene Piz-t, the distribution, variation, and evolution of the corresponding Avr gene AvrPiz-t were found among 312 M. oryzae isolates collected from Yunnan rice production areas of China. PCR amplification and pathogenicity assays of AvrPiz-t showed that 202 isolates (64.7%) held AvrPiz-t alleles and were avirulent to IRBLzt-T (holding Piz-t). There were 42.3-83.3% avirulent isolates containing AvrPiz-t among seven regions in Yunnan Province. Meanwhile, 11 haplotypes of AvrPiz-t encoding three novel AvrPiz-t variants were identified among 100 isolates. A 198 bps insertion homologous to solo-LTR of the retrotransposon inago2 in the promoter region of AvrPiz-t in one isolate and a frameshift mutation of CDS in another isolate were identified among 100 isolates, and those two isolates had evolved to virulent from avirulent. Synonymous mutation and non-AUG-initiated N-terminal extensions keeps the AvrPiz-t gene avirulence function in M. oryzae field isolates in Yunnan. A haplotype network showed that H3 was an ancestral haplotype. Structure variance for absence (28.2%) or partial fragment loss (71.8%) of AvrPiz-t was found among 39 virulent isolates and may cause the AvrPiz-t avirulence function to be lost. Overall, AvrPiz-t evolved to virulent from avirulent forms via point mutation, retrotransposon, shift mutation, and structure variance under field conditions.

Dataset Information

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

Publications

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

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