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Bacterial leaf blight (BLB), among the most serious diseases in rice production, is caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23, the broadest resistance gene against BLB in rice, is widely used in rice breeding. In this study, the rice variety CBB23 carrying the Xa23 resistance gene was inoculated with AH28 and PXO99A to identify differentially expressed genes (DEGs) associated with the resistance. Transcriptome sequencing of the infected leaves showed 7997 DEGs between the two strains at different time points, most of which were up-regulated, including cloned rice anti-blight, peroxidase, pathology-related, protein kinase, glucosidase, and other coding genes, as well as genes related to lignin synthesis, salicylic acid, jasmonic acid, and secondary metabolites. Additionally, the DEGs included 40 cloned, five NBS-LRR, nine SWEET family, and seven phenylalanine aminolyase genes, and 431 transcription factors were differentially expressed, the majority of which belonged to the WRKY, NAC, AP2/ERF, bHLH, and MYB families. Metabolomics analysis showed that a large amount of alkaloid and terpenoid metabolite content decreased significantly after inoculation with AH28 compared with inoculation with PXO99A, while the content of amino acids and their derivatives significantly increased. This study is helpful in further discovering the pathogenic mechanism of AH28 and PXO99A in CBB23 rice and provides a theoretical basis for cloning and molecular mechanism research related to BLB resistance in rice.

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Using genetic resistance against bacterial blight (BB) caused by Xanthomonas oryzae pathovar oryzae (Xoo) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm against Xoo. Here, we use an improved prime-editing system to implement two new strategies for BB resistance. Knock-in of TAL effector binding elements (EBE) derived from the BB susceptible gene SWEET14 into the promoter of a dysfunctional executor R gene xa23 reaches 47.2% with desired edits including biallelic editing at 18% in T0 generation that enables an inducible TALE-dependent BB resistance. Editing the transcription factor TFIIA gene TFIIAγ5 required for TAL effector-dependent BB susceptibility recapitulates the resistance of xa5 at an editing efficiency of 88.5% with biallelic editing rate of 30% in T0 generation. The engineered loci provided resistance against multiple Xoo strains in T1 generation. Whole-genome sequencing detected no OsMLH1dn-associated random mutations and no off-target editing demonstrating high specificity of this PE system. This is the first-ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock-in of 30-nucleotides cis-regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolving Xoo strains and protect it from epidemics.

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INTRODUCTION: Xa23 as an executor mediates broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), which contains a matching avirulence gene avrXa23, in rice for bacterial leaf blight (BLB). avrXa23 encodes a transcription activator-like effector (TALE) protein which binds to the EBE (effector-binding element) of the Xa23 promoter. It is unclear whether the considerable pressure of Xa23 leads to an emerging Xoo strain that overcomes Xa23 resistance. OBJECTIVES: This study aimed to uncover new Xoo isolate(s) that overcome Xa23-mediated resistance and to investigate how the pathogen evades the resistance. METHODS: Totally 185 Xoo isolates were used to screen possibly compatible strain(s) with Xa23-containing rice CBB23 by pathogenicity test. Genome Sequencing, Southern blot, tal gene cloning, Western blot, qRT-PCR and electrophoretic mobility shift assays (EMSA) were conducted to determine the mechanism of one Xoo isolate being compatible with Xa23-containing rice. RESULTS: One isolate AH28 from Anhui province is compatible with CBB23. AH28 strain contains an ortholog of avrXa23, tal7b and has 17 tal genes. The 4th RVD (repeat-variable diresidue) in Tal7b are missed and the 5th and 8th RVDs changed from NG and NS to NS and S*, respectively. These alternations made Tal7b unable to bind to the EBE of Xa23 promoter to activate the expression of Xa23 in rice. The ectopic expression of tal7b in a tal-free mutant PH of PXO99A did not alter the virulence of the strain PH, whereas avrXa23 made AH28 from compatibility to incompatibility with Xa23 rice. CONCLUSION: Best to our knowledge, this is the first insight of a naturally-emerging Xoo isolate that overcomes the broad-spectrum resistance of Xa23 by the variable AvrXa23-like TALE Tal7b. The RVD alteration in AvrXa23 may be a common strategy for the pathogen evolution to avoid being "trapped" by the executor R gene.

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Rice production is often affected by biotic and abiotic stressors. The breeding of resistant cultivars is a cost-cutting and environmentally friendly strategy to maintain a sustainable high production level. An elite water-saving and drought-resistant rice (WDR), Hanhui3, is susceptible to blast and bacterial blight (BB). This study was conducted to introgress three resistance genes (Pi2, xa5, and Xa23) for blast and BB into Hanhui3, using marker-assisted selection (MAS) for the foreground selection and a whole-genome single-nucleotide polymorphism (SNP) array for the background selection. As revealed by the whole-genome SNP array, the recurrent parent genome (RPG) recovery of the improved NIL was 94.2%. The resistance levels to blast and BB of the improved NIL and its derived hybrids were higher than that of the controls. In addition, the improved NIL and its derived hybrids retained the desired agronomic traits from Hanhui3, such as yield. The improved NIL could be useful to enhance resistance against biotic stressors and produce stable grain yields in Oryza sativa subspecies indica rice breeding programs.

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Bacterial blight (BB) is an important constraint on achieving a high and stable rice grain yield. An increasing number of BB resistance (R) genes have been identified and cloned to increase the available options for rice disease resistance breeding. However, it is necessary to understand the distribution of R genes in rice varieties for rational distribution and breeding. Here, we genotyped five R genes, i.e. Xa4, Xa7, Xa21, Xa23, and Xa27, in seventy main cultivars from Guangdong Province, South China using the corresponding specific markers. Our results showed that 61 varieties carried Xa4, only three varieties carried Xa27, and Xa7, Xa21, or Xa23 was not detected in all tested varieties. Notably, only 33 varieties exhibited resistance to pathotype IV Xoo strains. These results indicate that Xa4 is no longer suitable for widespread use in rice breeding, although Xa4 is widely present in tested varieties. Remarkably, the strongly virulent BB strains of pathotype IX evolved quickly in southern China, and Xa23 was found to effectively confer resistance against the pathotype IX strains. Subsequently, we successfully bred two novel inbred rice varieties as also being restorer lines and two photoperiod- and thermo-sensitive genic male sterility (P/TGMS) lines using the broad-spectrum resistance gene Xa23 through marker-assisted selection (MAS) combined with phenotypic selection. All of the developed lines and derived hybrids exhibited enhanced resistance to BB with excellent yield performance. Our research may potentially facilitate both of the inbred and hybrid rice disease resistance breeding.

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Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is an overwhelming disease in rice-growing regions worldwide. Our previous studies revealed that the executor R gene Xa23 confers broad-spectrum disease resistance to all naturally occurring biotypes of Xoo. In this study, comparative transcriptomic profiling of two near-isogenic lines (NILs), CBB23 (harboring Xa23) and JG30 (without Xa23), before and after infection of the Xoo strain, PXO99A, was done by RNA sequencing, to identify genes associated with the resistance. After high throughput sequencing, 1645 differentially expressed genes (DEGs) were identified between CBB23 and JG30 at different time points. Gene Ontlogy (GO) analysis categorized the DEGs into biological process, molecular function, and cellular component. KEGG analysis categorized the DEGs into different pathways, and phenylpropanoid biosynthesis was the most prominent pathway, followed by biosynthesis of plant hormones, flavonoid biosynthesis, and glycolysis/gluconeogenesis. Further analysis led to the identification of differentially expressed transcription factors (TFs) and different kinase responsive genes in CBB23, than that in JG30. Besides TFs and kinase responsive genes, DEGs related to ethylene, jasmonic acid, and secondary metabolites were also identified in both genotypes after PXO99A infection. The data of DEGs are a precious resource for further clarifying the network of Xa23-mediated resistance.

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The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE)-associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113 amino acid protein that shares 50% identity with the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23 but differs in promoter region by lacking the TALE binding element (EBE) for AvrXa23. XA23 can trigger a strong hypersensitive response in rice, tobacco, and tomato. Our results provide the first evidence that plant genomes have an executor R gene family of which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in the pathogen.

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The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE) associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from the wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113-amino acid protein that shares 50% identity to the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23, but differs in promoter region by lacking the TALE binding-element (EBE) for AvrXa23. XA23 can trigger strong hypersensitive response in rice, tobacco and tomato. Our results provide the first evidence that plant genomes have an executor R gene family in which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in pathogen.

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Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is not only a disease devastating rice production worldwide, but also an ideal model system for the study of the interaction between plants and their bacterial pathogens. The rice near-isogenic line (NIL) CBB23, derived from a cross between a wild rice Oryza rufipogon accession (RBB16) and a susceptible indica rice variety (Jingang 30), is highly resistant to all field Xoo strains tested so far. Although the BB resistance of CBB23 has been widely used in rice breeding programmes, the mechanism of its extremely broad-spectrum resistance remains unknown. Here, we report the molecular cloning of an avirulence gene, designated as avrXa23, from Xoo strain PXO99(A) . We validate that AvrXa23, a novel transcription activator-like effector, specifically triggers the broad-spectrum BB resistance in CBB23. The prevalence of avrXa23 in all 38 Xoo strains surveyed may explain the broad-spectrum feature of BB resistance in CBB23. The results will significantly facilitate the molecular cloning of the corresponding resistance (R) gene in the host, and provide new insights into our understanding of the molecular mechanism for broad-spectrum disease resistance in plants.

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