RESPONSE OF DIVERSE RICE GERMPLASM TO BIOTIC AND ABIOTIC STRESSES
Location: Dale Bumpers National Rice Research Center
Title: 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.
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.
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.