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

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 transporter 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, Tal5 LN18 and Tal7 PXO61 , 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 activation, the genomes of 3000 rice varieties were analyzed for EBE variationsin the OsSWEET13 promoter, and 10 Xa25 -like haplotypes were identified. We found that Tal5 LN18 and Tal7 PXO61 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. Collectively, 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.