Functional Analysis Of Seven Putative Secrete Proteins In BAC Clone 805L15 Of Magnaporthe Oryzae Isolate 81278ZB15

The isolate 81278ZB15 collected in Fujian province contains several avirulence genes including Avr-Pi1, Avr-Pi2 and Avr-Pi4a. By map-based cloning, the Avr gene cluster was positioned in the BAC clone 805L15. The sequences of clone 805L15 were obtained but stil with 2 gaps unfilled.In this study, basing on the analysis results through bioinformatics software, 48 genes were annotated from the sequence of BAC clone 805L15, and among which there were eight genes encoding extracellular secreted protein, and one gene encoding GPI- signal-anchor protein. Among those genes, overexpression vectors of No. 1, 8, 16, 23, 32, 37, 43 genes were constructed by using yeast homologous recombination approach. At the same time, the knock-out vector of gene No. 23 was constructed. The gene overexpression and knock-out mutants were inoculated on isogenic lines C101LAC Pi-1 (t), C101A51 Pi-2 (t), C104PKT Pi-3 (t), C101PKT Pi-4a, C105TTP-4L-23 Pi -4b, CO39 by spraying spore suspension on rice young leaves. The results showed that the pathogenicity of all transformants were the same to GUY11 wild type strain, suggesting that these transformants may not contain Avr-Pi1 , Avr-Pi2 and Avr-Pi4a genes. However, sporulation of No. 8 gene overexpression mutant was increased, and onion epidermal infection capacity of No.43 gene overexpression mutant was decreased, but the pathogenicity of these two mutants on rice plants was not attenuated.So according to all the results above, in order to clone the avirulence gene cluster of Avr-Pi1, Avr-Pi2 and Avr-Pi4a, the sequence of BAC clone 805L15 should be re-splicied, and meanwhile, the genes which 81278ZB15 contained should be re-predicted. Or the avirulence gene should be re-positioned,a new BAC clone be obtained, and be predicted gene. Further study is necessary.