Genetic Diversification Of The Avirulence Gene Avrpii In Magnaporthe Oryzae Populations

Rice(Oryza sativa)is a staple food for more than half of the world population.Rice blast caused by Magnaporthe oryzae is one of the most destructive rice diseases.It threatens the stability of rice production worldwide.Utilization of host resistance(R)genes is the most efficient environment-friendly and economically sustainable approach to control rice blast.The “gene-for-gene” hypothesis shows that a single plant R gene product recognizes the responding avirulence protein.However,resistance varieties are usually overcome by the emergence of virulent isolates shortly after their release.It has been hypothesized that the gain-of-virulence in M.oryzae is mainly due to the mutations in Avr genes.Therefore,the genetic diversification of avirulence genes plays an important role in utilization of R genes.In the previous study,we identified two functional haplotypes of the avirulence gene Avr Pii,defined as Avr Pii-J and Avr Pii-C,respectively.To further elucidate the genetic diversification of Avr Pii,we analysed genetic structure and recombinants of Avr Pii gene in six M.oryzae populations collected from Guangdong,Hunan,Guizhou,Liaoning,Jilin,and Heilongjiang procvince,respectively,as well as 23 chosen isolates,which are pathogenic on grass species other than rice.The main results are as follow:1.Genetic diversity of the Avr Pii geneAll 360 isolates were inoculated to monogenic line IRBLi-F5 which containing Pii.The frequencies of compatible interaction(susceptible)among the six M.oryzae populations were 91.7%(GD),46.7%(HN),65.0%(GZ),60.0%(LN),86.7%(JL)and 90.0%(HLJ),respectively.Since the interaction between rice and M.oryzae follows the “gene-for-gene” theory,it is suggested that Avr Pii genes are mainly distributed in Hunan,Guizhou,and Liaoning population,while they are less distributed in Guangdong,Jilin and Heilongjiang population.To further investigation of genetic diversity in Avr Pii locus,four pairs of Avr Pii-specific primers were applied to amplify Avr Pii from tested isolates.The results show that Avr Pii-J only present in southern China(Guangdong,Hunan,and Guizhou),but not in northeast China(Liaoning,Jilin and Heilongjiang).Avr Pii-C presents in all six M.oryzae populations and shows no obvious geographical differentiation.In addition,23 isolates that pathogenic on grass species carry Avr Pii-J only.These results suggest that Avr Pii-J may be a more primitive haplotype produced in the southern China rice growing area,while Avr Pi-C is a widely distributed haplotype later differentiated.To further elucidate the allelic differentiation of Avr Pii,all isolates containing Avr Pii were selected and re-sequenced.Avr Pii can be divided into 4 genotypes based on SNPs in coding region and the transposon composition of 3' untranslated region,of which there is one genotype for Avr Pii-J and three for Avr Pii-C.Integrated with phenotype and genotype,360 isolates were divided into 4 types: A/1-C,A/1-J,A/1-C/J and V/0.The southern China rice growing area contains all 4 types,while the northern China rice growing area contains only 2 types(A/1-C and V/0).The analysis showed that the M.oryzae isolates carrying one or both Avr Pii functional haplotypes,should be avirulent to the monogenic line IRBLi-F5 containing Pii.In addition,compared to the northern rice growing area,differentiation of Avr Pii in the southern rice growing area should be earlier and the genetic diversity is significant higher.2.The 3' untranslated region of Avr Pii is more important for its avirulent functionTo verify the function of the upstream and downstream sequences of the coding region,four recombinants were constructed using the wild-type Avr Pii via domain-swapping of two haplotypes.All constructs were introduced into Pii-virulent isolate CHL357 via Agrobacterium-mediated transformation.The results showed that the phenotypes of the two 5' UTR exchange recombinants were moderately virulent(MV),whereas the phenotypes of the two 3' UTR exchange recombinants were virulent(V),indicating that the 3' UTR of Avr Pii may be more important for its avirulent function.