Identification Of Rice Blast Resistant Pid3Orthologs By Allele Mining And Studies On Artificial Evolution Of Pid3(Crop Gentics And Breeding)

Rice blast, caused by the filamentous ascomycete Magnaporthe oryzae (M. oryzae), is one of the major diseases that drastically damage rice production. The use of plant varieties with resistance (R) genes is, and will continue to be, the most cost-effective and environmentally-friendly way to control this disease. Thus far, more than70blast R genes have been described at the genetic level,24of which have been cloned and characterized. Most of these cloned blast R genes be long to the nucleotide binding-site leucine-rich repeat (NBS-LRR) family, and some have been found to be alleles. We previously cloned Pid3from the indica variety Digu by performing a genome-wide comparison of paired NBS-LRR genes and their pseudogene alleles between two rice genome-sequenced varieties,9311(indica) and Nipponbare (japonica), Pid3alleles in most japonica varieties were identified as pseudogenes due to the presence of a nonsense mutation, but this pseudogene mutation did not occur in the indica varieties that were tested, or in common wild rice species.In this study,41orthologs of Pid3in common wild rice and in cultivated rice were identified using an allele mining approach, and11of which were transformed into the rice blast susceptible variety TP309. After resistance evaluation for transgenic plants,5Pid3orthologs were found to be functional rice blast resistance genes. The ortholog in indica variety9311had the same blast resistance spectrum as Pid3, while the remaining4orthologs had different resistance spectrum between each other, and the ortholog in common wild rice A4had the broadest resistance spectrum as to these blast strains we used. Additional, we found the Pid3ortholog in9311had been broadly used as resistance resources in our Country’s indica rice breeding program, and these Pid3orthologs could be important blast resistance resources for japonica rice breeding in the future.The predicted40-1-PID3and9311-PID3proteins differed solely in the LRR region, thus, specificity differences between orthologs could be determined by the LRR region. Functional analysis in transgenic plants of recombinant orthologs constructed in vitro provided further information:DG38and A438recombinants, encoding the LRR of38-1-Pid3, conferred Pid3and A4-Pid3resistance specificity, respectively;38DG recombinant, encoding the LRR of Pid3, conferred38-1-Pid3resistance specificity;38A4recombinant, encoding the LRR of A4-Pid3, conferred different resistance specificity from38-1-Pid3and A4-Pid3, thus, specificity differences between orthologs could be determined by the CC-NBS domain too. DG15recombinant, encoding the LRR of15-3-Pid3, exhibited dwarf, abnormal morphology phenotype and conferred blast resistance, whereas the point mutate Pid3(D508V), which had a point mutation in the MHD motif, exhibited moderately dwarf, abnormal morphology phenotype and didn’t confer blast resistance, thus we believed that the molecular mechanism between the two mutates was different, although both were considered the same HR phenotypes in other transient expression researches in tobacco leaves.Considering that most of the cloned blast R genes belong to NBS-LRR type, we constructed some recombinants in vitro between Pid3and other NBS-LRR type rice blast R genes to explore artificial evolution of rice blast R genes. Pid3Pi9and Pid3aPi9recombinants encoding the LRR region and ARC-LRR region of broad blast R gene Pi9, respectively, also conferred blast resistance; The analogue Pid3aPbl and Pid3aaPbl recombinants encoding the LRR region and ARC-LRR region of blast resistance QTL Pb1, respectively, had the blast resistance ability too. These results demonstrated the possibility of artificial evolution of rice blast R genes in the future. Meanwhile, in order to explore large-scale artificial evolution of rice blast R genes, we constructed three-fragment recombinants using the multi-site gateway technology. Although the recombinant did not confer blast resistance, we believed this approach would do favor for artificial evolution of rice blast R genes in the future.Finally, we pyramided Pid3orthologs by crossing transgenic plants with each ortholog, and constrained Pid3expression only in rice green tissues not in embryo and endosperm by using tissue specific promoter PD540(-544) for transgenic bio-safety research.