The involvement of the Type Six Secretion System (T6SS) in the virulence of Ralstonia solanacearum on brinjal

Abstract

Ralstonia solanacearum is an important soil-borne plant pathogen which causes bacterial wilt in a large number of crops. Bacterial Type Six Secretion System (T6SS) is known to participate in pathogenesis, bacterial interaction and inter-bacterial competition. Contribution of T6SS in the virulence of R. solanacearum on eggplant (Solanum melongena L) is studied. In this study, five T6SS gene (ompA, vgrG3, hcp, tssH and tssM) mutants have been developed by insertional mutagenesis and the virulence of the mutants was evaluated on eggplant. In general, the T6SS mutants showed significant reduction of wilt on eggplant. R. solanacearum mutant of ompA gene significantly reduced the wilt from day five through day eight in petiole inoculation. In soil drench inoculation, R. solanacearum mutant of vgrG3 gene reduced the wilt on eggplant and was significantly different throughout the experimental period. Other mutants, viz., tssH, tssM and hcp, also reduced the wilt during the initial stages of disease development. This is the first report on the role of T6SS genes, ompA, vgrG3, hcp and tssH on virulence of R. solanacearum.

This is a preview of subscription content, log in to check access.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

T6SS:

Type Six Secretion System

RSSC:

Ralstonia solanacearum Species complex

EPS:

Extracellular Polysaccharide

T2SS:

Type Two Secretion System

T3SS:

Type three Secretion system

References

  1. Asolkar T, Ramesh R (2018a) Identification of virulence factors and type III effectors of phylotype I, Indian Ralstonia solanacearum strains Rs-09-161 and Rs-10-244. J Genet 97:55–66. https://doi.org/10.1007/s12041-018-0894-z

    CAS  Article  PubMed  Google Scholar 

  2. Asolkar T, Ramesh R (2018b) Development of T3SS mutants (hrpB− and hrcV−) of Ralstonia solanacearum, evaluation of virulence attenuation in brinjal and tomato-A pre-requisite to validate T3Es of R. solanacearum. Indian J Microbiol 58:372–380. https://doi.org/10.1007/s12088-018-0736-y

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Assefa M, Dawit W, Lencho A, Hunduma T (2015) Assessment of wilt intensity and identification of causal fungal and bacterial pathogens on hot pepper (Capsicum annuum L.) in BakoTibbe and Nonno districts of west Shewa zone. Ethiopia Int J Phytopathol 4:21–28

    Article  Google Scholar 

  4. Badr S, Li Y, Duan K (2016) Comparison of the structure, regulation and functions between type three and type six secretion system in gram-negative bacteria. J Med Microb Diagn 5:2161–2703. https://doi.org/10.4172/2161-0703.1000243

    Article  Google Scholar 

  5. Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I (2009) Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC genomics 10:104. https://doi.org/10.1186/1471-2164-10-104

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Brito B, Aldon D, Barberis P, Boucher C, Genin S (2002) A signal transfer system through three compartments transduces the plant cell contact-dependent signal controlling Ralstonia solanacearum HRP genes. Mol Plant Microbe Interact 15:109–119. https://doi.org/10.1094/MPMI.2002.15.2.109

    CAS  Article  PubMed  Google Scholar 

  7. Cascales E, Cambillau C (2012) Structural biology of type VI secretion systems. Philos Trans Royal Soc B 367:1102–1111. https://doi.org/10.1098/rstb.2011.0209

    CAS  Article  Google Scholar 

  8. Choi CH, Lee JS, Lee YC, Park TI, Lee JC (2008) Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiol 8:216. https://doi.org/10.1186/1471-2180-8-216

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Cornelis GR (2006) The type III secretion injectisome. Nat Rev Microbiol 4:811-825. https://doi.org/10.1038/nrmicro1526

    CAS  Article  PubMed  Google Scholar 

  10. Elphinstone JG (2005) The current bacterial wilt situation: a global overview. In: Prior P, Hayward AC, Allen C (eds) Bacterial wilt disease and the Ralstonia solanacearum species complex. APS Press, St. Paul MN, pp 9–28

    Google Scholar 

  11. Fegan M, Prior P (2005) How complex is the Ralstonia solanacearum species complex. In: Prior P, Hayward AC, Allen C (eds) Bacterial wilt disease and the Ralstonia solanacearum species complex. APS Press, Madison, Wisconsin, USA, pp 449–461

    Google Scholar 

  12. Gallique M, Decoin V, Barbey C, Rosay T, Feuilloley MG, Orange N, Merieau A (2017) Contribution of the Pseudomonas fluorescens MFE01 type VI secretion system to biofilm formation. PLoS ONE 12:e0170770. https://doi.org/10.1371/journal.pone.0170770

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Genin S, Denny TP (2012) Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol 50:67–89. https://doi.org/10.1146/annurev-phyto-081211-173000

    CAS  Article  PubMed  Google Scholar 

  14. Green ER, Mecsas J (2016) Bacterial secretion systems–an overview. Microbiol Spectr 4:1–32. https://doi.org/10.1128/microbiolspec.VMBF-0012-2015

    CAS  Article  Google Scholar 

  15. Huang Q, Allen C (2000) Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants. Physiol Mol Plant Path 57:77–83. https://doi.org/10.1006/pmpp.2000.0283

    CAS  Article  Google Scholar 

  16. Kang Y, Liu H, Genin S, Schell MA, Denny TP (2002) Ralstonia solanacearumrequires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence. Mol Microbiol 46:427–437. https://doi.org/10.1046/j.1365-2958.2002.03187.x

    CAS  Article  PubMed  Google Scholar 

  17. Lee JS, Choi CH, Kim JW, Lee JC (2010) Acinetobacter baumannii outer membrane protein A induces dendritic cell death through mitochondrial targeting. J Microbiol 48:387–392. https://doi.org/10.1007/s12275-010-0155-1

    CAS  Article  PubMed  Google Scholar 

  18. Lin YM, Chou IC, Wang JF, Ho FI, Chu YJ, Huang PC et al (2008) Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis. Mol Plant Microbe interact 21:1261–1270. https://doi.org/10.1094/MPMI-21-9-1261

    CAS  Article  PubMed  Google Scholar 

  19. Liu H, Kang Y, Genin S, Schell MA, Denny TP (2001) Twitching motility of Ralstonia solanacearum requires a type IV pilus system. Microbiology 147:3215–3229. https://doi.org/10.1099/00221287-147-12-3215

    CAS  Article  PubMed  Google Scholar 

  20. Liu H, Zhang S, Schell MA, Denny TP (2005) Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence. Mol Plant Microbe Interact 18:1296–1305. https://doi.org/10.1094/MPMI-18-1296

    CAS  Article  PubMed  Google Scholar 

  21. Lossi NS, Manoli E, Forster A, Dajani R, Pape T, Freemont P, Filloux A (2013) The HsiB1C1 (TssB/TssC) complex of the Pseudomonas aeruginosa type VI secretion system forms a bacteriophage tail sheath-like structure. J Biol Chem 288:7536–7548. https://doi.org/10.1074/jbc.M112.439273

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Mansfield J, Genin S, Magori S et al (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629. https://doi.org/10.1111/J.1364-3703.2012.00804.X

    Article  PubMed  PubMed Central  Google Scholar 

  23. Meng F, Yao J, Allen C (2011) A MotN mutant of Ralstonia solanacearum is hypermotile and has reduced virulence. J Bacteriol 193:2477–2486. https://doi.org/10.1128/JB.01360-10

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Pautsch A, Schulz GE (1998) Structure of the outer membrane protein A transmembrane domain. Nat Struct Mol Biol 5:1013–1017. https://doi.org/10.1038/2983

    CAS  Article  Google Scholar 

  25. Pukatzki S, Ma AT, Sturtevant D et al (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Nat Acad Sci 103:1528–1533. https://doi.org/10.1073/pnas.0510322103

    CAS  Article  PubMed  Google Scholar 

  26. Pukatzki S, McAuley SB, Miyata ST (2009) The type VI secretion system: translocation of effectors and effector-domains. Curr Opin Microbiol 12:11–17. https://doi.org/10.1016/j.mib.2008.11.010

    CAS  Article  PubMed  Google Scholar 

  27. Ramesh R, Gaitonde S, Achari G, Asolkar T et al (2014) Genome Sequencing of Ralstonia solanacearumBiovar 3, Phylotype I, Strains Rs-09-161 and Rs-10-244, Isolated from Eggplant and Chili in India. Genome Announcement 2:1–2. https://doi.org/10.1128/genomeA.00323-14

    Article  Google Scholar 

  28. Ramesh R, Achari G, Asolkar T, Dsouza M, Singh N (2016) Management of bacterial wilt of brinjal using wild brinjal (Solanum torvum Sw) as root stock. Indian Phytopathol 69:2–6

    Google Scholar 

  29. Safni I, Cleenwerck I, DeVos P, Fegan M, Sly L, Kappler U (2014) Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov'. Int J Syst Evol Microbiol 64:3087–3103. https://doi.org/10.1099/ijs.0.066712-0

    CAS  Article  PubMed  Google Scholar 

  30. Saile E, McGarvey J, Schell M, Denny T (1997) Role of extracellular polysaccharide and endoglucanase in root invasion and colonization of tomato plants by Ralstonia solanacearum. Phytopathology 87:1264–1271. https://doi.org/10.1094/PHYTO.1997.87.12.1264

    CAS  Article  PubMed  Google Scholar 

  31. Shrivastava S, Mande SS (2008) Identification and functional characterization of gene components of Type VI Secretion system in bacterial genomes. PLoS ONE 3:e2955. https://doi.org/10.1371/journal.pone.0002955

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Silverman JM, Brunet YR, Cascales E, Mougous JD (2012) Structure and regulation of the type VI secretion system. Annu Rev Microbiol 66:453–472. https://doi.org/10.1146/annurev-micro-121809-151619

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Tans-Kersten J, Brown D, Allen C (2004) Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment. Mol Plant Microbe Interact 17:686–695. https://doi.org/10.1094/MPMI.2004.17.6.686

    CAS  Article  PubMed  Google Scholar 

  34. Wang J, Zhou Z, He F, Ruan Z, Jiang Y, Hua X, Yu Y (2018) The role of the type VI secretion system vgrG gene in the virulence and antimicrobial resistance of Acinetobacter baumannii ATCC 19606. PLoS ONE 13:e0192288. https://doi.org/10.1371/journal.pone.0192288

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Wicker E, Grassart L, Coranson-Beaudu R, Mian D, Guilbaud C, Prior P (2004) Emerging strains of Ralstonia solanacearum in Martinique (French West Indies): a case study for epidemiology of bacterial wilt. In: Momol MT, Ji P, Jones JB (eds) Proceedings of the first International Symposium on Tomato Diseases, International Society for Horticultural Science, Bruggen, Belgium 695:145–152. https://doi.org/10.17660/ActaHortic.2005.695.16

  36. Zhang L, Xu J, Xu J, Chen K, He L, Feng J (2012) TssM is essential for virulence and required for type VI secretion in Ralstonia solanacearum. J Plant Dis Prot 119:125–134. https://doi.org/10.1007/BF03356431

    CAS  Article  Google Scholar 

  37. Zhang L, Xu J, Xu J, Zhang H, He L, Feng J (2014) TssB is essential for virulence and required for type VI secretion system in Ralstonia solanacearum. Microb Pathog 74:1–7. https://doi.org/10.1016/j.micpath.2014.06.006

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgement

The financial support for this work was provided by Indian Council of Agricultural Research, New Delhi, India through “Outreach project on Phytophthora, Fusarium and Ralstonia diseases of horticultural and field crops”- (PhytoFuRa). The authors are grateful to Director, ICAR- Central Coastal Agricultural Research Institute, Old Goa for providing other necessary facilities.

Author information

Affiliations

Authors

Contributions

TA and RR have made major contributions to the concept and design of the study, analysis and interpretation of the data, and writing of the manuscript. TA executed the experiment.

Corresponding author

Correspondence to Raman Ramesh.

Ethics declarations

Conflict of interest

On behalf of all the authors, the corresponding author states that there is no conflict of interest.

Ethical approval

This article does not contain any studies with human or animal subjects.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1455 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Asolkar, T., Ramesh, R. The involvement of the Type Six Secretion System (T6SS) in the virulence of Ralstonia solanacearum on brinjal. 3 Biotech 10, 324 (2020). https://doi.org/10.1007/s13205-020-02311-4

Download citation

Keywords

  • Ralstonia solanacearum
  • T6SS
  • Mutants
  • Virulence
  • Eggplant
  • Bacterial wilt