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

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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.

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Abbreviations

BB:

bacterial blight

NIL:

near-isogenic line

Xoo :

Xanthomonas oryzae pv. oryzae

References

  • Bai, J., Choi, S. H., & Ponciano, G. (2000). Xanthomonas oryzae pv. oryzae avirulence genes contribute differently and specifically to pathogen aggressiveness. Molecular Plant–Microbe Interactions, 13(12), 1322–1329. doi:10.1094/MPMI.2000.13.12.1322.

    Article  PubMed  CAS  Google Scholar 

  • Cottage, A., Yang, A., Maunders, H., de Lacy, R. C., & Ramsay, N. A. (2001). Identification of DNA sequences flanking T-DNA Insertions by PCR-walking. Plant Molecular Biology Reporter, 19, 321–327. doi:10.1007/BF02772830.

    Article  CAS  Google Scholar 

  • Davies, D. R., Goryshin, I. Y., Reznikoff, W. S., & Rayment, I. (2000). Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. Science, 289, 77–85. doi:10.1126/science.289.5476.77.

    Article  PubMed  CAS  Google Scholar 

  • Ezuka, A., & Kaku, H. (2000). A historical review of bacterial blight of rice. Bulletin of the National Institute of Agrobiological Resources in Japan, 15, 53–54.

    Google Scholar 

  • Flor, H. H. (1971). Current status of the gene-for-gene concept. Annual Reviews of Phytopathology, 9, 275–276. doi:10.1146/annurev.py.09.090171.001423.

    Article  Google Scholar 

  • Gu, K. Y., Yang, B., & Tian, D. S. (2005). R gene expression induced by a type-III effector triggers disease resistance in rice. Nature, 435, 1122–1125. doi:10.1038/nature03630.

    Article  PubMed  CAS  Google Scholar 

  • Guilhabert, M. R., Hoffman, L. M., Mills, D. A., & Kirkpatrick, B. C. (2001). Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes. Molecular Plant–Microbe Interactions, 14, 701–706. doi:10.1094/MPMI.2001.14.6.701.

    Article  PubMed  CAS  Google Scholar 

  • Hoffman, L. M., Jendrisak, J. J., Meis, R. J., Goryshin, I. Y., & Reznikoff, S. W. (2000). Transposome insertional mutagenesis and direct sequencing of microbial genomes. Genetica, 108, 19–24. doi:10.1023/A:1004083307819.

    Article  PubMed  CAS  Google Scholar 

  • Hopkins, C. M., White, F. F., Choi, S. H., Guo, A., & Leach, J. E. (1992). Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae. Molecular Plant–Microbe Interactions, 5, 451–459.

    PubMed  CAS  Google Scholar 

  • Kauffman, H., Reddy, A. P. K., Hsieh, S. P. Y., & Merca, S. D. (1973). An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Disease Reporter, 57, 537–541.

    Google Scholar 

  • Leach, J. E., White, F. W., Rhoads, M. L., & Leung, H. (1990). A repetitive DNA sequence differentiates Xanthomonas campestris pv. oryzae from other pathovars of Xanthomonas campestris. Molecular Plant–Microbe Interactions, 3, 238–246.

    CAS  Google Scholar 

  • Lee, D. H., Kim, M. D., & Lee, W. H. (2004). Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant Escherichia coli. Applied Microbiology and Biotechnology, 63, 549–552. doi:10.1007/s00253-003-1370-z.

    Article  PubMed  CAS  Google Scholar 

  • Li, P., Long, J. Y., Huang, Y. C., Zhang, Y., & Wang, J. S. (2004). AvrXa3: a novel member of avrBs3 gene family from Xanthomonas oryzae pv. oryzae has a dual function. Progress in Natural Science, 14, 767–773.

    Google Scholar 

  • Mew, T. W. (1987). Current status and future prospects of research on bacterial blight of rice. Annual Review of Phytopathology, 25, 359–382. doi:10.1146/annurev.py.25.090187.002043.

    Article  Google Scholar 

  • Niño-Liu, D. O., Ronald, P. C., & Bogdanove, A. J. (2006). Xanthomonas oryzae pathovars: model pathogens of a model crop. Molecular Plant Pathology, 7, 303–324. doi:10.1111/j.1364-3703.2006.00344.x.

    Article  Google Scholar 

  • Park, Y. J., Song, E. S., Kim, Y. T., Noh, T. H., Kang, H. W., & Lee, B. M. (2007). Analysis of virulence and growth of a purine auxotrophic mutant of Xanthomonas oryzae pathovar oryzae. FEMS Microbiology Letters, 276(1), 55–59. doi:10.1111/j.1574-6968.2007.00909.x.

    Article  PubMed  CAS  Google Scholar 

  • Rajeshwari, R., & Sonti, R. V. (2000). Stationary-Phase Variation Due to Transposition of Novel Insertion Elements in Xanthomonas oryzae pv. oryzae. Journal of Bacteriology, 182(17), 4797–4802. doi:10.1128/JB.182.17.4797-4802.2000.

    Article  PubMed  CAS  Google Scholar 

  • Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  • Sun, Q., Wu, W., Qian, W., Hu, J., Fang, R., & He, C. (2003). High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system. FEMS Microbiology Letters, 226, 145–150. doi:10.1016/S0378-1097(03)00583-4.

    Article  PubMed  CAS  Google Scholar 

  • Swings, J., Van Den Mooter, M., Vayterin, L., Hoste, B., Gillis, M., Mew, T. W., et al. (1990). Reclassification of the causal agents of bacterial blight (Xanthomonas campestris pv. oryzae) and bacterial leaf streak (Xanthomonas campestris pv. oryzicola) of rice as pathovars of Xanthomonas oryzae (ex Ishiyama 1922) sp. nov., nom. rev. International Journal of Systematic Bacteriology, 40, 309–311.

    Article  Google Scholar 

  • Vera Cruz, C. M., Bai, J., Ona, I., Leung, H., Nelson, R. J., Mew, T. W., et al. (2000). Predicting durability of a disease resistance gene based on an assessment of the fitness loss and epidemiological consequences of avirulence gene mutation. Proceedings of the National Academy of Sciences of the United States of America, 97, 13500–13505. doi:10.1073/pnas.250271997.

    Article  PubMed  CAS  Google Scholar 

  • Voelker, L. L., & Dybvig, K. (1998). Transposon mutagenesis. Methods in Molecular Biology (Clifton, N.J.), 104, 235–238.

    CAS  Google Scholar 

  • Wang, C. L., Cheng, L. T., Zeng, C. Z., Zhang, Q. Y., Liu, P. Q., Liu, Y. G., et al. (2006). Chromosome walking for fine mapping of Xa23 gene locus by using genomic libraries. Chinese Journal of Rice Science, 20(4), 355–360.

    CAS  Google Scholar 

  • Yang, B., Sugio, A., & White, F. F. (2006). Os8N3 is a host disease-susceptibility gene for bacterial blight of rice. Proceedings of the National Academy of Sciences of the United States of America, 103(27), 10503–10508. doi:10.1073/pnas.0604088103.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Q., Lin, S. C., Zhao, B. Y., Wang, C. L., Yang, W. C., Zhou, Y. L., et al. (1998). Identifying of a new gene for resistance to bacterial blight from O. rufipogon. Rice Genetics Newsletter, 15, 138–142.

    Google Scholar 

  • Zhang, Q., Wang, C. L., Zhao, K. J., Zhao, Y. L., Caslana, V. C., Zhu, X. D., et al. (2001). The effectiveness of advanced rice lines with new resistance gene Xa23 to rice bacterial blight. Rice Genetics Newsletter, 18, 71–73.

    CAS  Google Scholar 

  • Zhu, W. G., Yang, B., Chittoor, J. M., Johnson, L. B., & White, F. F. (1998). AvrXa10 contains an acidic transcriptional activation domain in the functionally conserved C-terminus. Molecular Plant–Microbe Interactions, 11, 824–832. doi:10.1094/MPMI.1998.11.8.824.

    Article  PubMed  CAS  Google Scholar 

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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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Author notes

  1. Ying-Lun Fan

    Present address: Liaocheng University, Liaocheng, 252059, Shandong Province, People’s Republic of China

Authors and Affiliations

  1. National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing, 100081, People’s Republic of China

    Chun-Lian Wang, An-Bi Xu, Ying Gao, Ying-Lun Fan, Yun-Tao Liang, Chong-Ke Zheng, Liang-Qing Sun & Kai-Jun Zhao

  2. Hainan University, Haikou, 571101, Hainan, Province, People’s Republic of China

    An-Bi Xu

  3. Institute of Tropical Bioscience and Biotechnology/National Key Biotechnology Laboratory for Tropical Crops, Chinese Academy of Tropical Agriculture Sciences (CATAS), Haikou, 571101, Hainan Province, People’s Republic of China

    Wen-Quan Wang

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  1. Chun-Lian Wang
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Correspondence to Kai-Jun Zhao.

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The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.

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Wang, CL., Xu, AB., Gao, Y. et al. Generation and characterisation of Tn5-tagged Xanthomonas oryzae pv. oryzae mutants that overcome Xa23-mediated resistance to bacterial blight of rice. Eur J Plant Pathol 123, 343–351 (2009). https://doi.org/10.1007/s10658-008-9371-5

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