Functional Analysis Of Eleven Predicted Unique Genes Of Magnaporthe Oryzae Isolate FJ81278

To clone avirulence genes Avr-Pi1, Avr-Pi2 and Avr-Pi4a ,which are in the Magnaporthe oryzae isolate FJ81278, our previous study positioned the Avr gene cluster in the BAC clone 805L15. Then the BAC clone was sequenced and predicted genes which were possibly encoded by it. Throuth the comparison of these genes with isolate 70-15,we got some unque genes of isolate FJ81278, We chose four unque genes from them.At the same time, through bioinformatics approach seven genes encoding extracellular secreted protein from FJ81278 sequence. This study did functional analysis of these genes.We constructed 11 genes ( MoUng01, MoUng02, MoUng03, MoUng04, MoUng06, MoUng07, MoUng05, MoUng08, MoUng09, MoUng10, MoUng11)overexpression vectors, and transferred these clones into GUY11 protoplasts. Positive transformants were then inoculated on rice CO-39 isogenic lines, which are C101LAC Pi-1(t), C101A51 Pi-2, C104PKT Pi-3(t), C101PKT Pi-4a and C105TTP-4L-23 Pi-4b through spraying suspension of spores on rice leaves. The transformants did not alter virulence of their parent isolate GUY11, suggesting these genes may not be candidate avirulence genes or could not express normal function in these transformants. However, over-expression of unique genes MoUng03, MoUng05, MoUng07 increased the sporulation, but MoUng04, MoUng06 was opposite. Over-expression of unique genes MoUng01, MoUng09 decreased their onion epidermal infection capacity. But the pathogenicity of these over-expression transformants on rice plants was not changed.The virulence of 4 predicted special genes on BAC clone 805L15 toward CO39 NILs showed that they are not avirulence genes Avr-Pi1,Avr-Pi2å’ŒAvr-Pi4a. Then, we knew 13 predicted genes on 805L15 are not avirulence genes Avr-Pi1,Avr-Pi2å’ŒAvr-Pi4a at the moment. In order to check the assembly of 805L15 sequence, we did southern blot with the 9 cosmid clones DNA. The probes were the 5 molecular markers, which linked to avirulence genes Avr-Pi1, Avr-Pi2 and Avr-Pi4a.The results showed that the assembly of BAC clone sequence are incurect. So, the sequence of 805L15 needs to be reassembly, or several cosmid clones need to be sequenced. Thus, we can obtain the correct sequence of 805L15 and analyse the genes on it again. Hopefully, we can clone the avirulence genes Avr-Pi1,Avr-Pi2å’ŒAvr-Pi4a.