European Journal of Plant Pathology

, Volume 134, Issue 3, pp 551–560 | Cite as

Characterization of pilP, a gene required for twitching motility, pathogenicity, and biofilm formation of Acidovorax avenae subsp. avenae RS-1

  • He Liu
  • Wen-Xiao Tian
  • Muhammad Ibrahim
  • Bin Li
  • Guo-Qing Zhang
  • Bo ZhuEmail author
  • Guan-Lin XieEmail author


The Gram-negative bacterium Acidovorax avenae subsp. avenae is the causal agent of bacterial brown stripe (BBS), which can cause severe diseases in many plants, including rice, with huge economic importance. Type IV pili (TFP) are hair-like appendages involved in several bacterial activities such as bacterial surface motility, surface adherence, colonization, biofilm formation, and virulence. The aim of our study is to characterize the association of A. avenae subsp. avenae TFP with BBS in rice. We generated a transposon (Tn5) mutant library. Then, an insertional mutagenesis on the background of this bacterium was identified as reduced pathogenicity. The confirmed inserted genetic region was into gene pilP, which encodes a TFP assembly protein. The pilP-deficient mutant strain seriously affected the motility twitching ability, biofilm formation and virulence. Collectively, our results clearly indicated that the pilP gene and TFP in A. avenae subsp. avenae play a key role in plant pathogenicity, twitching motility, and biofilm formation.


Acidovorax avanae subspecies avanae Type IV pili Twitching motility Virulence Biofilm formation 



We extend our thanks to Saul Burdman (Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel) for kindly providing us the suicide vector pJP5603. This work was supported by the Postdoctoral Research Foundation of China (317000-X91102), Special Fund for Agro-scientific Research in the Public Interest (201003029 and 201003066), the National Natural Science Foundation of China (30871655), and Zhejiang Agri. Dep. (Ji-fa 2008-97).

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  1. Averhoff, B., & Friedrich, A. (2003). Type IV pili-related natural transformation systems: DNA transport in mesophilic and thermophilic bacteria. Archive Microbiology, 180, 385–393.CrossRefGoogle Scholar
  2. Bahar, O., Goffer, T., & Burdman, S. (2009). Type IV pili are required for virulence, twitching motility, and biofilm formation of Acidovorax avenae subsp citrulli. Molecular Plant-Microbe Interaction, 22, 909–920.CrossRefGoogle Scholar
  3. Balasingham, S. V., Collins, R. F., Assalkhou, R., Homberset, H., Frye, S. A., Derrick, J. P., & Tonjum, T. (2007). Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitides. Journal of Bacteriology, 189, 5716–5727.PubMedCrossRefGoogle Scholar
  4. Belete, B., Lu, H., & Wozniak, D. J. (2008). Pseudomonas aeruginosa AlgR regulates type IV pilus biosynthesis by activating transcription of the fimU-pilVWXY1Y2E operon. Journal of Bacteriology, 190, 2023–2030.PubMedCrossRefGoogle Scholar
  5. Drake, S. L., Sandstedt, S. A., & Koomey, M. (1997). PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high molecular mass multimer. Molecular Microbiology, 23, 657–668.PubMedCrossRefGoogle Scholar
  6. Farinha, M. A., Conway, B. D., Glasier, L. M. G., Ellert, N. W., Irvin, R. T., Sherburne, R., & Paranchych, W. (1994). Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice. Infection and Immunity, 62, 4118–4123.PubMedGoogle Scholar
  7. Gao, G. J., Lu, H. M., Huang, L. F., & Hua, Y. J. (2005). Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans. Chinese Science Bulletin, 50, 311–316.Google Scholar
  8. Helander, I. M., Nurmiaho-Lassila, E. L., Ahvenainen, R., Rhoades, J., & Roller, S. (2001). Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. International Journal of Food Microbiology, 71, 235–244.PubMedCrossRefGoogle Scholar
  9. Horiuchi, T., & Komano, T. (1998). Mutational analysis of plasmid R64 thin pilus prepilin: the entire prepilin sequence is required for processing by type IV prepilin peptidase. Journal of Bacteriology, 180, 4613–4620.PubMedGoogle Scholar
  10. Jin, F., Conrad, J. C., Gibiansky, M. L., & Wong, G. C. L. (2011). Bacteria use type-IV pili to slingshot on surfaces. Proceedings of the National Academy of Sciences USA, 108, 12617–12622.CrossRefGoogle Scholar
  11. Kadota, I., Ohuchi, A., & Nishiyama, K. (1991). Serological properties and specificity of Pseudomonas avenae Manns 1909 the causal agent of bacterial brown stripe of rice. Annals of the Phytopathological Society of Japan, 57, 268–273.CrossRefGoogle Scholar
  12. Kang, Y., Liu, H., Genin, S., Schell, M. A., & Denny, T. P. (2002). Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence. Molecular Microbiology, 46, 427–437.PubMedCrossRefGoogle Scholar
  13. Klausen, M., Heydorn, A., Ragas, P., Lambertsen, L., Aaes-Jørgensen, A., Molin, S., & Tolker-Nielsen, T. (2003). Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Molecular Microbiology, 48, 1511–1524.PubMedCrossRefGoogle Scholar
  14. Li, Y., Hao, G., Galvani, C. D., Meng, Y., Fuente, L. D. L., Hoch, H. C., & Burr, T. J. (2007). Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell–cell aggregation. Microbiology, 153, 719–726.PubMedCrossRefGoogle Scholar
  15. Martin, P. R., Watson, A. A., McCaul, T. F., & Mattick, J. S. (1995). Characterization of a five–cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa. Molecular Microbiology, 16, 497–508.PubMedCrossRefGoogle Scholar
  16. Mattick, J. S. (2002). Type IV pili and twitching motility. Annual Review of Microbiology, 56, 289–314.PubMedCrossRefGoogle Scholar
  17. Meng, Y., Li, Y., Galvani, C. D., Hao, G., Turner, J. N., Burr, T. J., & Hoch, H. C. (2005). Upstream migration of Xylella fastidiosa via pilus-driven twitching motility. Journal of Bacteriology, 187, 5560–5567.PubMedCrossRefGoogle Scholar
  18. Peeters, E., Nelis, H. J., & Coenye, T. (2008). Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. Journal of Microbiological Methods, 72, 157–165.PubMedCrossRefGoogle Scholar
  19. Penfold, R. J., & Pemberton, J. M. (1992). An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene, 118, 145–146.PubMedCrossRefGoogle Scholar
  20. Pujol, C., Eugène, E., Marceau, M., & Nassif, X. (1999). The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion. Proceedings of the National Academy of Sciences USA, 96, 4017–4022.CrossRefGoogle Scholar
  21. Reimers, P. J., & Leach, J. E. (1991). Race-specific resistance to Xanthomonas oryzae pv. oryzae conferred by bacterial blight resistance gene Xa-10 in rice (Oryza sativa) involves accumulation of a lignin-like substance in host tissues. Physiological and Molecular Plant Pathology, 38, 39–55.Google Scholar
  22. Rigel, N. W., & Silhavy, T. J. (2011). Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria. Current Opinion in Microbiology, 15, 1–5.Google Scholar
  23. Ruehl, W. W., Marrs, C., Beard, M. K., Shokooki, V., Hinojoza, J. R., Banks, S., Bieber, D., & Mattick, J. S. (1993). Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro. Molecular Microbiology, 7, 285–288.PubMedCrossRefGoogle Scholar
  24. 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
  25. Saul, B., Ofir, B., Jennifer, K. P., & Fuente, L. D. L. (2011). Involvement of Type IV Pili in pathogenicity of plant pathogenic bacteria. Genes, 2, 706–735.CrossRefGoogle Scholar
  26. Schaada, N. W., Postnikovaa, E., Sechlera, A., Claflinb, L. E., Vidaverc, A. K., Jonesd, J. B., et al. (2008). Reclassification of subspecies of Acidovorax avenae as A. avenae (Manns 1905) emend., A. cattleyae (Pavarino, 1911) comb. nov., A. citrulli (Schaad et al., 1978) comb. nov., and proposal of A. oryzae sp. nov. Systematic and Applied Microbiology, 31, 434–446.Google Scholar
  27. Shakya, D. D., & Chung, H. S. (1983). Detection of Pseudomonas avenae in rice seed. Seed Science and Technology, 11, 583–587.Google Scholar
  28. Simon, R., Priefer, U., & Puhler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nature Biotechnology, 1, 784–791.CrossRefGoogle Scholar
  29. Smith, M. D., & Guild, W. R. (1980). Improved method for conjugative transfer by filter mating of Streptococcus pneumoniae. Journal of Bacteriology, 144, 457–459.PubMedGoogle Scholar
  30. Song, W. Y., Kim, H. M., Hwang, C. Y., & Schaad, N. W. (2004). Detection of Acidovorax avenae ssp. avenae in rice seeds using BIO-PCR. Journal of Phytopathology, 152, 667–676.Google Scholar
  31. Tammam, S., Sampaleanu, L. M., Koo, J., Sundaram, P., Ayers, M., Chong, P. A., Forman-Kay, J. D., Burrows, L. L., & Howell, P. L. (2011). Characterization of the PilN, PilO and PilP type IVa pilus subcomplex. Molecular Microbiology, 82, 1496–1514.PubMedCrossRefGoogle Scholar
  32. Willems, A., Goor, M., Thielemans, S., Gillis, M., Kersters, K., & De Ley, J. (1992). Transfer of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci. International Journal of Systematic Bacteriology, 42, 107–119.Google Scholar
  33. Xie, G. L., Sun, X. L., & Mew, T. W. (1998). Characterization of Acidovorax avenae subsp. avenae from rice seeds. Chinese Journal of Rice Science, 12, 165–171.Google Scholar
  34. Xie, G. L., Zhang, G. Q., Liu, H., Tian, W. X., Li, B., Zhu, B., et al. (2011). Genome sequence of the rice pathogenic bacterium Acidovorax avenae subsp. avenae RS-1. Journal of Bacteriology, 193, 5013–5014.Google Scholar
  35. Young, G. M., Smith, M. J., Minnich, S. A., & Miller, V. L. (1999). The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility. Journal of Bacteriology, 181, 2823–2833.PubMedGoogle Scholar
  36. Yu, Y. H., Streubel, J., Balzergue, S., Champion, A., Boch, J., Koebnik, R., Feng, J. X., Verdier, V., & Szurek, B. (2011). Colonization of rice leaf blades by an African Strain of Xanthomonas oryzae pv. oryzae depends on a new TAL effector that induces the rice Nodulin-3 Os11N3 gene. Molecular Plant-Microbe Interaction, 24, 1102–1113.CrossRefGoogle Scholar

Copyright information

© KNPV 2012

Authors and Affiliations

  1. 1.State Key Laboratory of Rice Biology and Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of BiotechnologyZhejiang UniversityHangzhouChina
  2. 2.State Key Laboratory of Rice Biology and Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of BiotechnologyZhejiang UniversityHangzhouChina

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