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

  1. Sanya National Center of Technology Innovation for Saline-Alkali Tolerant Rice, School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China; Sanya Institute, Hainan Academy of Agricultural Sciences, Sanya 572025, China; Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences / State Key Laboratory of Tropical Crop Breeding / Key Laboratory of Biology & Genetic Resources of Rubber Tree, Ministry of Agriculture and Rural Affairs, Haikou 571101, China; State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Hangzhou 311400, China; State Key Laboratory of Crop Gene Resources and Breeding, Beijing 100081, China
  • Contact: XIA Zhihui
  • Supported by:
    The study was supported by the Project of Sanya Yazhou Bay Science and Technology City, China (Grant No. SCKJ-JYRC-2023-16), the National Natural Science Foundation of China (Grant No. 31071173), the opening Foundation of State Key Laboratory of Crop Gene Resources and Breeding, China (Grant No. CGRB-2024-08), and the Project of Hainan Province Postgraduate Innovative Research, China (Grant No. Qhyb2023-88).

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Abstract: Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a major threat to global rice production. The recessive resistance gene xa5 (encoding TFIIAγ5V39E) 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 TFIIAγ5V39E-Avrxa5 interaction upregulates defense-related genes, including OsPRa and OsPRb, and triggers a defense response characterized by H2O2 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.

Key words:  , rice bacterial blight, xa5 gene, transcription activator-like effector, genome editing, recessive resistance