Engineering Broad-Spectrum Bacterial Blight Resistance by Simultaneously Disrupting Variable TALE-Binding Elements of Multiple Susceptibility Genes in Rice

摘要

Xanthomonas oryzae pv.oryzae(Xoo),the causal agent of bacterial blight of rice,employs the transcription activator-like effectors(TALEs)to induce the expression of the OsSWEET family of putative sugar trans-porter genes,which function in conferring disease susceptibility(S)in rice plants.To engineer broad-spectrum bacterial blight resistance,we used CRISPR/Cas9-mediated gene editing to disrupt the TALE-binding elements(EBEs)of two S genes,OsSWEET11 and OsSWEET14,in rice cv.Kitaake,which harbors the recessive resistance allele of Xa25/OsSWEET13.The engineered rice line MS14K exhibited broad-spectrum resistance to most Xoo strains with a few exceptions,suggesting that the compatible strains may contain new TALEs.We identified two PthXo2-like TALEs,Tal5LN18 and Tal7PXO61,as major virulence factors in the compatible Xoo strains LN18 and PXO61,respectively,and found that Xoo encodes at least five types of PthXo2-like effectors.Given that PthXo2/PthXo2.1 target OsSWEET13 for transcriptional acti-vation,the genomes of 3000 rice varieties were analyzed for EBE variationsin the OsSWEET13 promoter,and 10 Xa25-like haplotypes were identified.We found that Tal5LN18 and Tal7PXO61 bind slightly different EBE sequences in the OsSWEET13 promoter to activate its expression.CRISPR/Cas9 technology was then used to generate InDels in the EBE of the OsSWEET13 promoter in MS14K to creat a new germplasm with three edited OsSWEET EBEs and broad-spectrum resistance against all Xoo strains tested.Collec-tively,our findings illustrate how to disarm TALE-S co-evolved loci to generate broad-spectrum resistance through the loss of effector-triggered susceptibility in plants.