Engineering xa5 Alleles Defines Core Functional Domain and Dual Resistance Mechanism Against Rice Bacterial Blight

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae ( Xoo ), is a major threat to global rice production. The recessive resistance gene xa5 (encoding TFIIAγ5 V39E ) confers broad-spectrum resistance but has been underutilized in breeding due to its recessive inheritance and incomplete mechanistic understanding. To overcome this limitation, we employed CRISPR-Cas9 and prime editing to engineer novel alleles at the xa5 locus. We created seven variant lines and identified four that conferred strong and stable resistance under the tested field conditions against multiple Xoo strains across multiple growing seasons, including those carrying premature termination at residue 42 (CR-+T, CR-+G) and those with substitutions at position 39 (V39E in PE-TC-AG, V39D in PE-T-A). Critically, these resistant lines showed seed germination, seedling vigor, yield, and grain quality comparable to those of the wild type under field conditions. Using these genetic resources, we dissected the molecular basis of xa5 -mediated resistance. We identified a nine-amino-acid region (residues 33‒41) as the core determinant for susceptibility and uncovered a dual resistance mechanism: loss of interaction with most transcription activator-like effectors (TALEs), thereby preventing the induction of host susceptibility genes, coupled with the retention of specific recognition of TALE Avrxa5. This specific TFIIAγ5 V39E -Avrxa5 interaction upregulates defense-related genes, including OsPRa and OsPRb , and triggers a defense response characterized by H 2 O 2 accumulation and catalase suppression. This retained recognition provides an additional defense layer beyond the canonical loss-of-susceptibility mechanism. We further propose a model in which recessive inheritance results from protein-level competition, wherein wild-type TFIIAγ5 outcompetes the V39E variant for Avrxa5 binding, thereby suppressing defense activation. Collectively, our study establishes a molecular framework for xa5 -mediated recessive resistance and, more importantly, delivers novel, high-performance xa5 alleles as promising genetic resources for breeding durable blight-resistant rice.