Bacterial blight (BB) and bacterial leaf streak (BLS), caused by  Xanthomonas oryzae pv.  oryzae ( Xoo ) and  Xanthomonas oryzae pv.  oryzicola ( Xoc ), respectively, are the two most devastating bacterial diseases of rice worldwide. Both bacterial pathogens infect rice plants relying on type III secreted transcriptional activation-like effectors (TALEs) that bind to specific effector binding elements (EBEs) in the promoter of susceptibility ( S ) genes, and activate its expressions for disease development (Chen  et al ., 2010). To counteract BB, rice has evolved a unique type of executor resistance ( R ) genes ( Xa7 ,  Xa10 ,  Xa23 ,  etc .), which can specifically trap certain  Xoo TALEs via EBEs within their promoters and trigger strong hypersensitive response (HR). Previously, we verified that gene correction of  xa23 with EBE_AvrXa23 restored its function in triggering defence responses against  Xoo invasion (Wei  et al ., 2021). Considering  Xa23 -mediated resistance to BB has been overcome by new  Xoo isolates in the field in recent years, and no natural  R genes against  Xoc have been identified in rice; here, we investigated whether an EBE-stacking-in-the-promoter strategy could be employed in the molecular rice breeding for durable and broad-spectrum resistance to both BB and BLS by genome-editing technology.

Sequencing analysis of the  xa23  locus in the commercial rice cultivar Nangeng 46 (N46) revealed that it shared an identical coding region with  Xa23  in CBB23, whereas it lacked the complete EBE_AvrXa23 sequence in the promoter (Figure 1a). We presumed that introducing multiple EBEs, which were responsive to TALEs from various  Xoo  and  Xoc  strains, into the  xa23  ^N46  locus by genome editing would render host broad-spectrum and durable resistance to both pathogens. Thus, 10 EBEs (Table S1) responding to PthXo1, PthXo3, AvrXa23, Tal9a_BLS256,  etc . were selected and constructed into a 220-bp EBE _{Xoc / Xoo}  array (Figure 1a; Figure S1). It was synthesized, PCR amplified using a pair of chemically modified primers and used as the DNA donor (Figure S1) for targeted DNA insertion in the  xa23  ^N46  promoter through CRISPR/Cas9-induced non-homologous end joining (NHEJ) repair pathway as described previously (Lu  et al ., 2020).

Plant Biotechnology Journal，IF=13

https://doi.org/10.1111/pbi.14233