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Acta Agronomica Sinica ›› 2025, Vol. 51 ›› Issue (2): 334-346.doi: 10.3724/SP.J.1006.2025.32006

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and functional study of OgXa13 in Oryza meyeriana

ZHANG Zheng-Kang1(), SU Yan-Hong1(), RUAN Sun-Mei1, ZHANG Min1, ZHANG Pan1, ZHANG Hui2, ZENG Qian-Chun2, LUO Qiong1,*()   

  1. 1Yunnan State Key Laboratory of Biological Resources Conservation and Utilization, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    2College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
  • Received:2023-02-12 Accepted:2024-10-25 Online:2025-02-12 Published:2024-11-13
  • Contact: E-mail: qiongbf@aliyun.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    NSFC Yunnan United Fund(U2102219);NSFC Yunnan United Fund(U1302261)

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Abstract:

Bacterial blight is one of the most devastating bacterial diseases in rice production. The identification and utilization of resistance genes in rice breeding is the most economical and effective method for controlling this disease. Oryza meyeriana represents a valuable genetic resource due to its high resistance, and even immunity, to bacterial blight. In this study, we cloned the full-length cDNA and an 8908 bp genomic sequence of OgXa13, a homolog of OsXa13, from Oryza meyeriana using transcriptome and genome sequencing. Sequence analysis revealed that the gene structure of OgXa13 consists of five exons and four introns, mirroring the structure of rice OsXa13, and its core promoter sequence is identical to that of the rice susceptibility gene OsXa13. A total of 21 amino acid differences were observed between OgXa13 and OsXa13, with four key substitutions located in the MtN3.1 domain. Overexpression of OgXa13 and its introduction into TP309 plants via genetic transformation significantly enhanced resistance to bacterial blight. It is hypothesized that the amino acid sequence differences contribute to the distinct functions of the OgXa13 and OsXa13 proteins, suggesting that OgXa13 could be utilized as a dominant resistance gene in rice breeding. Furthermore, knockout of the OsXa13 gene using CRISPR/Cas9 in Nipponbare T1 homozygous lines also significantly enhanced resistance to bacterial blight, demonstrating that editing the susceptibility gene OsXa13 through CRISPR/Cas9 is an effective strategy for improving resistance. This study provides a valuable genetic resource and new insights for breeding rice with enhanced resistance to bacterial blight.

Key words: rice, Oryza meyeriana, bacterial blight resistance, gene cloning, OgXa13

Fig. 1

Locations of the four gRNA target sites on the OsXa13 gene PAM: CRISPR/Cas identification of specific short DNA sequences, marked in red with PAM sequences."

Table 1

Oligo sequence"

Oligo名称
Oligo name		 正向序列
Forward sequence (5′-3′)		 反向互补序列
Reverse complementary sequence (5′-3′)
Oligo-Target1 TGTGTGTCACCCTCTCCGGTGTTGC AAACGCAACACCGGAGAGGGTGACA
Oligo-Target2 TGTGTGGGGTACAGCTCGGTGCCGT AAACACGGCACCGAGCTGTACCCCA
Oligo-Target3 TGTGTGCTACCTCGTCTACGCGCCG AAACCGGCGCGTAGACGAGGTAGCA
Oligo-Target4 TGTGTGAGCCGAGGAACACGGCCGT AAACACGGCCGTGTTCCTCGGCTCA

Table 2

Primers used for the detection of transgenic plants"

引物Primers 引物序列Primer sequences (5′-3′) 用途Purpose
HYG F: GCTTCTGCGGGCGATTTGTGT
R: GGTCGCGGAGGCTATGGATGC		 潮霉素检测引物
Hygromycin detection primer
OgXa13-ox F: TAGTGGAAAAGGAAGGTGGCTCC
R: TGGGGACGAAGTAGAGCGTGAC		 过表达阳性株系鉴定
Identification of positive overexpression lines
OgXa13 F: GGCAAGCTGCTCTAGCCAATAC
R: CAAGGGGAACAGCTAACAAGTGC		 OgXa13基因阳性株系鉴定
Identification of transgenic OgXa13 gene-positive lines
Target1 F: GCTAGAGAGGAAGGCTTAAG
R: CAATGCAATGGTGCAGACAAC		 靶点1测序引物
Target 1 sequencing primer
Target2/3 F: CATCATCTCCTTCCTGGTGTTCC
R: GACGGAGAGCCCGATCGGCATG		 靶点2和靶点3测序引物
Target 2 and target 3 sequencing primers
Target4 F: CATGCCGATCGGGCTCTCCGTC
R: CACCACCTCGACCTTGTGCAC		 靶点4测序引物
Target 4 sequencing primer

Fig. 2

Schematic diagram of gene structure of OgXa13 and OsXa13 A: schematic diagram of the gene structure of OgXa13; B: schematic diagram of the gene structure of OsXa13. Exons are represented by black boxes and introns by white boxes."

Fig. 3

Protein structure analysis of OgXa13 and OsXa13 A: a schematic representation of OgXa13 and OsXa13; the MtN3_slv domain are indicated by gray boxes; the thin black lines indicate the different amino acids and sites of OgXa13 and OsXa13 in the MtN3_slv domain; B: transmembrane prediction map of OgXa13 protein; C: transmembrane prediction map of OsXa13 protein."

Fig. 4

Bacterial blight resistance of TP309 plants overexpressing OgXa13 A: the leaf disease symptoms 14 days after inoculation of OgXa13 positive transgenic plants with leaf blight bacterium PXO99; B: identification of positive transgenic plants with gene-specific primers; C: spot length 14 days after inoculation of OgXa13 positive transgenic plants with bacterial pathogen PXO99; D: percentage of spot length to total leaf length at 14 days after inoculation of OgXa13 positive transgenic plants lines with PXO99; E: OgXa13 expression levels in leaves of positive transgenic T1 generation homozygous plants. *, P > 0.05; **, P < 0.01."

Fig. 5

Bacterial blight resistance of OgXa13 genes transgenic rice plants A: the leaf disease symptoms 14 days after inoculation of positive transgenic plants with leaf blight bacterium PXO99; B: identification of positive transgenic plants with gene-specific primers; C: expression levels of OgXa13 in leaves of positive transgenic T1 generation homozygous plants inoculated with PXO99 for 0 h and 48 h; D: percentage of spot length to total leaf length at 14 days after inoculation of positive transgenic plants lines with PXO99; E: spot length 14 days after inoculation of positive transgenic plants with bacterial pathogen PXO99. *, P > 0.05; **, P < 0.01."

Fig. 6

Bacterial blight resistance of OgXa13 genomic genes transgenic T2 generation rice plants A: the leaf disease symptoms 21 days after inoculation of T2 positive transgenic plants with leaf blight bacterium PXO99; B: identification of positive transgenic plants with gene-specific primers; C: spot length 21 days after inoculation of T2 positive transgenic plants with bacterial pathogen PXO99; D: percentage of spot length to total leaf length at 21 days after inoculation of T2 positive transgenic plants lines with PXO99; E: T2 generation transgenic homozygous plants were inoculated with the Amount of bacterial on 21 days. *, P > 0.05; **, P < 0.01."

Fig. 7

Bacterial blight resistance in CRISPR/Cas9 knockout lines of OsXa13 A: alignment of nucleic acid and amino acid sequences of mutant sites between Nipponbare and T1 knock out lines; B: spot length 14 days after inoculation of Nipponbare and three homozygous knockout lines with PXO99; C: spot length 14 days after inoculation of Nipponbare and three homozygous knockout lines with PXO99; D: percentage of spot length to total leaf length at 14 days after inoculation of Nipponbare and three homozygous knock out lines with PXO99. Nip: Nipponbare; Xa13-KO: knock out lines. *, P > 0.05; * *, P < 0.01."

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