Skip to main content
SpringerLink
Log in

GunA of Sinorhizobium (Ensifer) fredii HH103 is a T3SS-secreted cellulase that differentially affects symbiosis with cowpea and soybean

  • Regular Article
  • Published:
Plant and Soil Aims and scope Submit manuscript

Abstract

Aims

The symbiosis between Sinorhizobium fredii HH103 and its host legumes is influenced by the type 3 secretion system (T3SS), which delivers proteins (effectors) directly into the host cells to promote infection. GunA, one of the predicted HH103 effectors, potentially codes for a cellulase. In this work we tried to characterise GunA and elucidate its role in symbosis with soybean and cowpea.

Methods

A GunA::HA fusion protein was constructed to study T3SS-dependent secretion. Cellulase activity of GunA was measured and gunA::uidA-GFP and gunA::cyA fusions were constructed to monitor gunA expression in nodules and to study translocation to the host cells, respectively. Finally, the symbiotic performance of a gunA mutant was studied in soybean and cowpea.

Results

GunA from S. fredii HH103 shows cellulase activity and is secreted through the T3SS in response to the inducer flavonoid genistein. Interestingly, at the beggining of the symbiotic process, GunA was partially responsible for the induction of the expression of the soybean GmPR1 gene, a gene used as a marker for plant defense responses. However, GunA was also detected in soybean and cowpea developed nodules. Finally, nodulation assays indicate that GunA is beneficial for symbiosis with soybean but detrimental with cowpea.

Conclusion

Secretion of GunA through the S. fredii HH103 T3SS clearly and differentially impacts the symbiotic performance of this strain with soybean and cowpea. GunA, or its cellulase activity, is recognised by soybean root cells very early in the symbiotic process but, curiously, its secretion can also be detected in mature nodules. This suggests different symbiotic roles at different symbiotic stages that need to be further elucidated.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1: Analysis of secretion of GunA to the extracellular medium in response to the inducer flavonoid genistein.
Fig. 2: Cellulase activity in a CMC Congo red agar assay.
Fig. 3: Expression of the Sinorhizobium fredii HH103 Rif R gunA gene in cowpea and soybean nodules.
Fig. 4: GmPR1 gene expression in soybean roots.

Similar content being viewed by others

References

  • Bellincampi D, Cervone F, Lionetti V (2014) Plant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions. Front Plant Sci 5:228

    Article  PubMed  PubMed Central  Google Scholar 

  • Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198

    CAS  PubMed  Google Scholar 

  • Caldelari Baumberger I, Fraefel N, Göttfert M, Hennecke H (2003) New NodW- or NifA-regulated Bradyrhizobium japonicum genes. Mol Plant-Microbe Interact 16:342–351

    Article  PubMed  Google Scholar 

  • Deakin WJ, Broughton WJ (2009) Symbiotic use of pathogenic strategies: rhizobial protein secretion systems. Nat Rev Microbiol 7:312–320

    Article  CAS  PubMed  Google Scholar 

  • de Lyra MCCP, López-Baena FJ, Madinabeitia N, Vinardell JM, Espuny MR, Cubo MT, Bellogín RA, Ruiz-Sainz JE, Ollero FJ (2006) Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean. Int Microbiol 9:125–133

    Google Scholar 

  • Denoux C, Galletti R, Mammarella N, Gopalan S, Werck D, de Lorenzo G, Ferrari S, Ausubel FM, Dewdney J (2008) Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings. Mol Plant 1:423–445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Downie JA (2010) The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev 34:150–170

    Article  CAS  PubMed  Google Scholar 

  • Fåhraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381

    PubMed  Google Scholar 

  • Gage DJ (2004) Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiol Mol Biol Rev 68:280–300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Galan JE, Waksman G (2018) Protein-injection machines in bacteria. Cell 172:1306–1318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227

    Article  CAS  Google Scholar 

  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

    Article  CAS  Google Scholar 

  • Jiménez-Guerrero I, Pérez-Montaño F, Medina C, Ollero FJ, López-Baena FJ (2015a) NopC is a Rhizobium-specific type 3 secretion system effector secreted by Sinorhizobium (Ensifer) fredii HH103. PLoS One 10:e0142866

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiménez-Guerrero I, Pérez-Montaño F, Monreal JA, Preston GM, Fones H, Vioque B, Ollero FJ, López-Baena FJ (2015b) Rhizobial type 3 secretion system effectors suppress the early soybean defense responses induced by its natural symbiont Sinorhizobium (Ensifer) fredii HH103. Mol Plant Microbe Interact 28:790–799

    Article  CAS  PubMed  Google Scholar 

  • Jiménez-Guerrero I, Pérez-Montaño F, Medina C, Ollero FJ, López-Baena FJ (2017) The Sinorhizobium (Ensifer) fredii HH103 nodulation outer protein NopI is a determinant for efficient nodulation of soybean and cowpea. Appl Environ Microbiol 83:e02770–e02716

    Article  PubMed  PubMed Central  Google Scholar 

  • Jimenéz-Zurdo JI, Mateos PF, Dazzo FB, Martínez-Molina E (1996) Cell-bound cellulase and polygalacturonase production by Rhizobium and Bradyrhizobium species. Soil Biol Biochem 28:917–921

    Article  Google Scholar 

  • Krause A, Doerfel A, Göttfert M (2002) Mutational and transcriptional analysis of the type III secretion system of Bradyrhizobium japonicum. Mol Plant-Microbe Interact 5:1228–1235

    Article  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • López-Baena FJ, Monreal JA, Pérez-Montaño F, Guasch-Vidal B, Bellogín RA, Vinardell JM, Ollero FJ (2009) The absence of Nops secretion in Sinorhizobium fredii HH103 increases GmPR1 expression in Williams soybean. Mol Plant-Microbe Interact 22:1445–1454

    Article  CAS  PubMed  Google Scholar 

  • López-Baena FJ, Ruiz-Sainz JE, Rodríguez-Carvajal MA, Vinardell JM (2016) Bacterial molecular signals in the Sinorhizobium fredii-soybean symbiosis. Int J Mol Sci 17:E755

    Article  CAS  PubMed  Google Scholar 

  • López-Baena FJ, Vinardell JM, Pérez-Montaño F, Crespo-Rivas JC, Bellogín RA, Espuny MR, Ollero FJ (2008) Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103. Microbiology 154:1825–1836

    Article  CAS  PubMed  Google Scholar 

  • Margaret I, Becker A, Blom J, Bonilla I, Goesmann A, Göttfert M, Lloret J, Mittard-Runte V, Rückert C, Ruiz-Sainz JE, Vinardell JM, Weidner S (2011) Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean. J Biotechnol 155:11–19

    Article  CAS  PubMed  Google Scholar 

  • Mazarei M, Elling AA, Maier TR, Puthoff DP, Baum TJ (2007) GmEREBP1 is a transcription factor activating defense genes in soybean and Arabidopsis. Mol Plant-Microbe Interact 20:107–119

    Article  CAS  PubMed  Google Scholar 

  • Murray JD (2011) Invasion by invitation: rhizobial infection in legumes. Mol Plant-Microbe Interact 24:631–639

    Article  CAS  PubMed  Google Scholar 

  • Okazaki S, Kaneko T, Sato S, Saeki K (2013) Hijacking of leguminous nodulation signalling by the rhizobial type III secretion system. Proc Natl Acad Sci U S A 110:17131–17136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pallen MJ, Chaudhuri RR, Henderson IR (2003) Genomic analysis of secretion systems. Curr Opin Microbiol 6:519–527

    Article  CAS  PubMed  Google Scholar 

  • Pérez-Montaño F, Jiménez-Guerrero I, Acosta-Jurado S, Navarro-Gómez P, Ollero FJ, Ruiz-Sainz JE, López-Baena FJ, Vinardell JM (2016) A transcriptomic analysis of the effect of genistein on Sinorhizobium fredii HH103 reveals novel rhizobial genes putatively involved in symbiosis. Sci Rep 6:31592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pessi G, Ahrens CH, Rehrauer H, Lindemann A, Hauser F, Fischer HM, Hennecke H (2007) Genome-wide transcript analysis of Bradyrhizobium japonicum bacteroids in soybean root nodules. Mol Plant-Microbe Interact 20:1353–1363

    Article  CAS  PubMed  Google Scholar 

  • Poole P, Ramachandran V, Terpolilli J (2018) Rhizobia: from saprophytes to endosymbionts. Nat Rev Microbiol 16:291–303. https://doi.org/10.1038/nrmicro.2017.171

    Article  CAS  Google Scholar 

  • Robledo M, Rivera L, Menéndez E, Martínez-Hidalgo P, Rivas R, Velázquez E, Dazzo FB, Martínez-Molina E, Mateos PF (2015) Role of Rhizobium cellulase CelC2 in host root colonization and infection. In: de Bruijn FJ (ed) Biological Nitrogen Fixation. Wiley, USA, pp 525–532

    Chapter  Google Scholar 

  • Rodrigues JA, López-Baena FJ, Ollero FJ, Vinardell JM, Espuny MR, Bellogín RA, Ruiz-Sainz JE, Thomas JR, Sumpton D, Ault J, Thomas-Oates J (2007) NopM and NopD are rhizobial nodulation outer proteins: identification using LC-MALDI and LC-ESI with a monolithic capillary column. J Proteome Res 6:1029–1037

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor (USA)

    Google Scholar 

  • Schechter LM, Guenther J, Olcay EA, Jang S, Krishnan HB (2010) Translocation of NopP by Sinorhizobium fredii USDA257 into Vigna unguiculata root nodules. Appl Environ Microbiol 76:3758–3761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schirrmeister J, Friedrich L, Wenzel M, Hoppe M, Wolf C, Göttfert M, Zehner S (2011) Characterization of the self-cleaving effector protein NopE1 of Bradyrhizobium japonicum. J Bacteriol 193:3733–3739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Simon R (1984) High frequency mobilization of gram-negative bacterial replicons by the in vivo constructed Tn5-mob transposon. Mol Gen Genet 196:413–420

    Article  CAS  PubMed  Google Scholar 

  • Sinha D, Gupta MK, Patel HK, Ranjan A, Sonti RV (2013) Cell wall degrading enzyme induced rice innate immune responses are suppressed by the type 3 secretion system effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae. PLoS One 8:e75867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sory MP, Cornelis GR (1994) Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol 14:583–594

    Article  CAS  PubMed  Google Scholar 

  • Staehelin C, Krishnan HB (2015) Nodulation outer proteins: double-edged swords of symbiotic rhizobia. Biochem J 470:263–274

    Article  PubMed  Google Scholar 

  • Süß C, Hempel J, Zehner S, Krause A, Patschkowski T, Göttfert M (2006) Identification of genistein-inducible and type III-secreted proteins of Bradyrhizobium japonicum. J Biotechnol 126:69–77

    Article  CAS  PubMed  Google Scholar 

  • Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43:777–780

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tu JC (1974) Rhizobial root nodules of soybean as revealed by scanning and transmission electron microscopy. Phytopatol 65:447–454

    Article  Google Scholar 

  • Verma DPS, Zogbi V, Bal AK (1978) A cooperative action of plant and Rhizobium to dissolve the host cell wall during development of root nodule symbiosis. Plant Sci Lett 13:137–142

    Article  Google Scholar 

  • Vinardell JM, Acosta-Jurado S, Zehner S, Göttfert M, Becker A, Baena I, Blom J, Crespo-Rivas JC, Goesmann A, Jaenicke S, Krol E, McIntosh M, Margaret I, Pérez-Montaño F, Schneiker-Bekel S, Serranía J, Szczepanowski R, Buendía AM, Lloret J, Bonilla I, Pühler A, Ruiz-Sainz JE, Weidner S (2015) The Sinorhizobium fredii HH103 genome: a comparative analysis with S. fredii strains differing in their symbiotic behavior with soybean. Mol Plant-Microbe Interact 28:811–824

    Article  CAS  PubMed  Google Scholar 

  • Vincent JM (1970) The modified Fåhræus slide technique. In: Vincent JM (ed) A manual for the practical study of root nodule bacteria. Blackwell scientific publications. United Kingdom, Oxford, pp 144–145

    Google Scholar 

  • Wassem R, Kobayashi H, Kambara K, Le Quéré A, Walker GC, Broughton WJ, Deakin WJ (2008) TtsI regulates symbiotic genes in Rhizobium sp. NGR234 by binding to tts boxes. Mol Microbiol 78:736–748

    Article  CAS  Google Scholar 

  • Zehner S, SchoberG WM, Lang K, Göttfert M (2008) Expression of the Bradyrhizobium japonicum type III secretion system in legume nodules and analysis of the associated tts box promoter. Mol Plant-Microbe Interact 21:1087–1093

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank the Junta de Andalucía (project P11-CVI-7050) and the Spanish Ministerio de Economía y Competitividad (project BIO2016-78409-R). We also would like to thank the Servicio General de Biología (CITIUS) of the Universidad de Sevilla for their technical assistance.

Author information

Authors and Affiliations

  1. Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Avenida de Reina Mercedes, 6, 41012, Seville, Spain

    Irene Jiménez-Guerrero, Francisco Pérez-Montaño, Francisco Javier Ollero, José María Vinardell & Francisco Javier López-Baena

  2. Technische Universität Dresden, Institute of Genetics, Helmholtzstrasse 10, 01062, Dresden, Germany

    Anna Zdyb, Mandy Beutler, Gesa Werner & Michael Göttfert

Authors
  1. Irene Jiménez-Guerrero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francisco Pérez-Montaño
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anna Zdyb
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mandy Beutler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gesa Werner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Göttfert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Francisco Javier Ollero
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. José María Vinardell
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Francisco Javier López-Baena
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francisco Javier López-Baena.

Additional information

Responsible Editor: Ulrike Mathesius

Electronic supplementary material

Supplementary Figure 1

GunA translocation assays. cAMP levels measured in soybean nodules harvested 18 dpi from plants inoculated with the HH103 RifR and the HH103 RifRrhcJ::Ω strains carrying the gunA::cya fusion. The HH103 RifR strain was used as a control. Data shown are the mean (± standard deviation of the mean) for two biological replicates. Each cAMP value was individually compared to that obtained in plants inoculated with the HH103 RifR strain using the Mann-Whitney non-parametrical test. None of the treatments were significantly different at the level α = 5% (p < 0.05). (PNG 64 kb)

High resolution image (TIF 1303 kb)

Supplementary Table 1

(DOCX 21 kb)

Supplementary Table 2

(DOCX 13 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jiménez-Guerrero, I., Pérez-Montaño, F., Zdyb, A. et al. GunA of Sinorhizobium (Ensifer) fredii HH103 is a T3SS-secreted cellulase that differentially affects symbiosis with cowpea and soybean. Plant Soil 435, 15–26 (2019). https://doi.org/10.1007/s11104-018-3875-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11104-018-3875-3

Keywords

Search

Navigation