ARS | Publication request: Understanding the molecular mechanism of instability of the avirulence gene AVR-Pita1 in field isolates of Maganporthe oryzae

Submitted to: Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: August 5, 2010
Publication Date: August 12, 2010
Citation: Dai, Y., Correll, J.C., Jia, Y. 2010. Understanding the molecular mechanism of instability of the avirulence gene AVR-Pita1 in field isolates of Maganporthe oryzae [abstract]. 5th International Rice Blast Conference, August 12-14, 2010, Little Rock, Arkansas. Poster Abstract I-CP-12.

Technical Abstract: The avirulence gene AVR-Pita1 in Magnaporthe oryzae triggers a resistance response in rice plants that contain the resistance gene Pi-ta. Understanding the evolution of the AVR-Pita1 gene in field isolates should benefit the deployment of Pi-ta for the control of rice blast disease. A total of 187 field isolates of M. oryzae collected from U.S., China, Colombia, Egypt, India, and the Philippines over a 40 year period were used for this analysis. We first determined the pathogenicity of isolates on rice cultivars with and without Pi-ta. It was found that 151 isolates were avirulent toward Pi-ta containing rice cultivars while 36 were virulent. Using AVR-Pita1 specific primers, AVR-Pita1 was amplified by PCR, and the resulting PCR products were sequenced from the DNA of all 151 avirulent isolates as well as 4 virulent isolates collected from China. Alignment of sequence assemblies revealed 38 highly similar AVR-Pita1 haplotypes. It was found that most DNA sequence variation occurs in the exon regions, and the majority of this variation resulted in amino acid substitutions. A total of 27 highly variable haplotypes excluding AVR-Pita1 was predicted based on the 38 different DNA sequence assemblies. For the 4 virulent isolates from which the AVR-Pita1 allele was amplified, two additional nucleotides were found in the first exon of AVR-Pita1 of these virulent isolates. The insertion of these two nucleotides resulted in a frame-shift that produced a predicted truncated AVR-Pita1 metalloprotease; in the 32 virulent isolates where AVR-Pita1 was undetectable by PCR, the 5’ portion of the AVR-Pita1 allele was predicted to be deleted from the genomes of 29 isolates while the entire AVR-Pita1 was predicted to be deleted in the remaining 3 virulent isolates using Southern blot analysis. These findings suggest that frame-shift, partial and complete deletions of AVR-Pita1 are three mechanisms that the fungus uses to overcome Pi-ta mediated resistance. To determine the function of AVR-Pita1, field isolates from Arkansas were transformed with one to many copies of AVR-Pita1 from isolate O-137 by homologous based recombination. The presence of AVR-Pita1 in virulent isolates was confirmed by PCR using AVR-Pita1 specific primers and the results were verified by DNA sequencing and Southern blot analysis using the AVR-Pita1 coding region as the probe. Results of pathogenicity assays demonstrated that isolates transformed with AVR-Pita1 were re-stored to avirulence on Pi-ta-containing rice cultivars. In summary, we demonstrated the molecular diversity and instability of AVR-Pita1 among field isolates of M. oryzae. The impact of these findings on the understanding of the molecular mechanisms of disease resistance in rice will be discussed. References: 1. Dai. Y. 2010. Structural and Functional Analysis of the Avirulence Gene AVR-Pita1 of Magnaporthe oryzae. University of Arkansas MS thesis. 2. Zhou, E., Jia, Y., Singh, P., Correll, J.C., Lee, F.N., 2007. Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence. Fung. Genet. Biol. 44:1024-1034.