Screening Of Rice PTI-related Genes Against Rice Blast And Generation Of Blast-resistant Pi21 Gene Edited Lines

Rice blast,caused by the fungal pathogen Magnaporthe oryzae,is one of the most destructive diseases worldwide.The blast resistance controlled by the major R genes is easy to break down,due to the changes of the blast fungus strains.Therefore,developing new strategies and new technologies for rice blast resistance breeding has become an urgent requirement in rice breeding.Based on the pathways of pathogen associated molecular patterns(PAMP)-triggered immunity(PTI),we tested the RNAi-transgenic rice lines of a number of rice candidate genes by M.grisea inoculation to screen rice blast resistance genes.At the same time,the Pi21 gene in the rice variety Nipponbare was targeted for CRISPR/Cas9-mediated editing and blast resistant rice lines were obtained.The main results are as following:(1)RNAi transgenic rice lines of a set of M.grisea-responsive rice genes were generated by transformation using the rice variety TJ394.In this study,M.grisea strains 98019A,TMC-1,RO1-1,ROR-1 and 318-2,which showed compatible interaction with the rice variety TJ394,were identified by M.grisea inoculation.The M.grisea strain TMC-1,showing high level of sporulation and susceptible inoculation reaction on TJ394,were further used to distinguish between rice RNAi materials and TJ394 for rice blast resistance.(2)In this study,we focused on the screening of rice blast resistance related genes by M.grisea inoculation of 34 RNAi transgenic rice materials using the micro-chamber method.Through the rice blast fungus inoculation tests,the results showed that the disease indexes of 7 independent transgenic lines of XY23 were 0.6-1.3 lower than TJ394,P<0.05.The disease indexes of 3 independent transgenic lines of XY14 were 0.7-1.1 lower than TJ394,P<0.05.These independent RNAi lines of XY23 and XY14 all exhibited enhanced resistance to M.grisea compared with TJ394,suggesting the two genes may be important rice blast resistance related genes.(3)The Pi21 gene in the blast susceptible rice variety Nipponbare was targeted for gene editing using the CRISPR/Cas9 technology.A set of pi21 recessive mutants exhibited enhanced rice blast resistance.86.7%of T0 transgenic plants contained nucleotide alteration within the Pi21 target sequence.T-DNA-containing T1 plants assayed by PCR of T-DNA sequences were eliminated,and 107 T-DNA-free plants were identified from 23 T1 lines.Restriction digestion and sequencing analysis revealed that 21 out of the 107 rice plants were pi21 homozygous mutants.One of the pi21 mutant lines was futher tested in M.oryzae inoculation,and showed significantly enhanced rice blast resistance compared with the untransformed Nipponbare.qRT-PCR analysis showed that the expression of PRlb?PR2?PR3?PBZl?E2F and PDR was more rapidly and strongly induced by M.oryzae in the pi21 mutant plants than in the susceptible Nipponbare.