European Journal of Plant Pathology

, Volume 123, Issue 3, pp 343–351 | Cite as

Generation and characterisation of Tn5-tagged Xanthomonas oryzae pv. oryzae mutants that overcome Xa23-mediated resistance to bacterial blight of rice

  • Chun-Lian Wang
  • An-Bi Xu
  • Ying Gao
  • Ying-Lun Fan
  • Yun-Tao Liang
  • Chong-Ke Zheng
  • Liang-Qing Sun
  • Wen-Quan Wang
  • Kai-Jun Zhao
Article
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Abstract

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23.

Keywords

avrXa23 Flanking sequence PXO99 Transposon Virulent mutant Xoo 

Abbreviations

BB

bacterial blight

NIL

near-isogenic line

Xoo

Xanthomonas oryzae pv. oryzae

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Notes

Acknowledgements

Special thanks are given to Q.D. Xin, X.H. Zhang, X. Sun, T.F. Qing and X.J. Yang for the help in the bacterial inoculation. This work was supported by the National High-Technology Research Programme (The “863” Programme) of the Ministry of Science and Technology of China (Grant 2006AA10Z106) and the Core Research Budget of the Non-profit Governmental Research Institution (2060302-23).

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Copyright information

© KNPV 2008

Authors and Affiliations

  • Chun-Lian Wang
    • 1
  • An-Bi Xu
    • 1
    • 2
  • Ying Gao
    • 1
  • Ying-Lun Fan
    • 1
    • 4
  • Yun-Tao Liang
    • 1
  • Chong-Ke Zheng
    • 1
  • Liang-Qing Sun
    • 1
  • Wen-Quan Wang
    • 3
  • Kai-Jun Zhao
    • 1
  1. 1.National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and BreedingMinistry of Agriculture, Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS)BeijingPeople’s Republic of China
  2. 2.Hainan UniversityHaikouPeople’s Republic of China
  3. 3.Institute of Tropical Bioscience and Biotechnology/National Key Biotechnology Laboratory for Tropical CropsChinese Academy of Tropical Agriculture Sciences (CATAS)HaikouPeople’s Republic of China
  4. 4.Liaocheng UniversityLiaochengPeople’s Republic of China

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