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Heterologous expression of nifA or nodD genes improves chickpea-Mesorhizobium symbiotic performance

  • José Rodrigo da-Silva
  • Esther Menéndez
  • Fernando Eliziário
  • Pedro F. Mateos
  • Ana AlexandreEmail author
  • Solange Oliveira
Regular Article

Abstract

Aims

The aim of this study was to investigate whether the overexpression of NifA and NodD regulators contribute to the symbiotic improvement of chickpea mesorhizobia.

Methods

The native strains V-15b, ST-2, and PMI-6 were transformed with extra copies of nifA or nodD genes and several plants trial were performed.

Results

Plant growth assays showed that nifA overexpression was able to improve the symbiotic effectiveness of V-15b, while nodD overexpression lead to the improvement of ST-2 and PMI-6. Hydroponic assays showed that plants inoculated with V15bnifA+ and PMI6nodD+ started developing nodules earlier than those inoculated with the corresponding control strains. In addition, the number of nodules was always higher in plants inoculated with the strains overexpressing the symbiotic genes. Analysis of histological sections of nodules formed by V15bnifA+ showed a more developed fixation zone when compared with control. On the other hand, nodules induced by PMI6nodD+ did not show a senescent zone, which was observed in nodules from plants inoculated with the control strain. Plants inoculated with PMI6nodD+ and ST2nodD+ showed a higher number of infection threads than the corresponding control inoculations.

Conclusion

These results indicate that overexpressing nifA and nodD may be an important tool to achieve the improvement of the symbiotic performance of mesorhizobia.

Keywords

Overexpression Nodulation Nitrogen fixation Legume Rhizobia Symbiotic effectiveness 

Notes

Acknowledgements

The authors thank Dr. Alvaro Peix (IRNASA-CSIC) for providing pMP4661 plasmid and Dr. Doroteia Campos for her help with the real-time PCR experiments performed in the Molecular Biology Laboratory-ICAAM. The authors also thank G. Mariano for technical assistance.

Funding information

This work was financed by FEDER Funds through the Operational Program for Competitiveness Factors—COMPETE and National Funds through FCT (Fundação para a Ciência e a Tecnologia), under the Strategic Project UID/AGR/00115/2013, Project n° FCOMP-01-0124-FEDER-028316 (PTDC/BIA-EVF/4158/2012), Project POCI-01-0145-FEDER-016810 (PTDC/AGR-PRO/2978/2014) and InAlentejo ALENT-07-0262-FEDER-001871. J. Rodrigo da-Silva acknowledges a PhD fellowship (1254-13-8) from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

Supplementary material

11104_2019_3950_MOESM1_ESM.docx (1.6 mb)
ESM 1 (DOCX 1642 kb)

References

  1. Agarwal G et al (2012) Comparative analysis of Kabuli Chickpea transcriptome with Desi and Wild Chickpea provides a rich resource for development of functional markers. PLoS One 7:e52443.  https://doi.org/10.1371/journal.pone.0052443 CrossRefGoogle Scholar
  2. Alexandre A, Brígido C, Laranjo M, Rodrigues S, Oliveira S (2009) A survey of chickpea rhizobia diversity in Portugal reveals the predominance of species distinct from Mesorhizobium ciceri and Mesorhizobium mediterraneum. Microb Ecol 58:930–941.  https://doi.org/10.1007/s00248-009-9536-6 CrossRefGoogle Scholar
  3. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198.  https://doi.org/10.1099/00221287-84-1-188 Google Scholar
  4. Bloemberg GV, Wijfjes AH, Lamers GE, Stuurman N, Lugtenberg BJ (2000) Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities. Mol Plant-Microbe Interact: MPMI 13:1170–1176.  https://doi.org/10.1094/mpmi.2000.13.11.1170 CrossRefGoogle Scholar
  5. Bosworth AH et al (1994) Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression. Appl Environ Microbiol 60:3815–3832Google Scholar
  6. Brígido C, Alexandre A, Oliveira S (2012a) Transcriptional analysis of major chaperone genes in salt-tolerant and salt-sensitive mesorhizobia. Microbiol Res 167:623–629.  https://doi.org/10.1016/j.micres.2012.01.006 CrossRefGoogle Scholar
  7. Brígido C, Robledo M, Menendez E, Mateos PF, Oliveira S (2012b) A ClpB chaperone knockout mutant of Mesorhizobium ciceri shows a delay in the root nodulation of chickpea plants. Mol Plant-Microbe Interact 25:1594–1604.  https://doi.org/10.1094/mpmi-05-12-0140-r CrossRefGoogle Scholar
  8. Broughton WJ, Jabbouri S, Perret X (2000) Keys to symbiotic harmony. J Bacteriol 182:5641–5652.  https://doi.org/10.1128/JB.182.20.5641-5652.2000 CrossRefGoogle Scholar
  9. Caetano-Anollés G, Wall LG, De Micheli AT, Macchi EM, Bauer WD, Favelukes G (1988) Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti. Plant Physiol 86:1228–1235.  https://doi.org/10.1104/pp.86.4.1228
  10. Chengtao Y, Guanqiao Y, Shanjiong SS, Jiabi Z (2004) Functional difference between Sinorhizobium meliloti NifA and Enterobacter cloacae NifA. Sci China C Life Sci 47:44–51.  https://doi.org/10.1360/02yc0268 CrossRefGoogle Scholar
  11. Cooper JE (2004) Multiple responses of rhizobia to flavonoids during legume root infection. Adv Bot Res 41:1–62.  https://doi.org/10.1016/S0065-2296(04)41001-5 CrossRefGoogle Scholar
  12. Covell S, Ellis RH, Roberts EH, Summerfield RJ (1986) The influence of temperature on seed germination rate in grain legumes: I. A comparison of chickpea, lentil, soyabean and cowpea at constant temperatures. J Exp Bot 37:705–715.  https://doi.org/10.1093/jxb/37.5.705 CrossRefGoogle Scholar
  13. D’Haeze W, Holsters M (2002) Nod factor structures, responses, and perception during initiation of nodule development. Glycobiology 12:79R–105R.  https://doi.org/10.1094/MPMI-20-2-0129 CrossRefGoogle Scholar
  14. del Cerro P et al (2015) Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation. BMC Genomics 16:251.  https://doi.org/10.1186/s12864-015-1458-8 CrossRefGoogle Scholar
  15. Dupont L, Alloing G, Pierre O, El Msehli S, Hopkins J, Hérouart D, Frendo P (2012) The legume root nodule: from symbiotic nitrogen fixation to senescence. In: Nagata T (ed) Senescence. InTech, pp 137–168.  https://doi.org/10.5772/34438
  16. Finan TM, Kunkel B, De Vos GF, Signer ER (1986) Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167:66–72.  https://doi.org/10.1128/jb.167.1.66-72.1986 CrossRefGoogle Scholar
  17. Fischer HM (1994) Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev 58:352–386Google Scholar
  18. Fischer H-M, Alvarez-Morales A, Hennecke H (1986) The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobium japonicum nifA gene is involved in control of nif gene expression and formation of determinate symbiosis. EMBO J 5:1165–1173CrossRefGoogle Scholar
  19. García-Fraile P et al (2012) Rhizobium promotes non-legumes growth and quality in several production steps: towards a biofertilization of edible raw vegetables healthy for humans. PLoS One 7:e38122.  https://doi.org/10.1371/journal.pone.0038122 CrossRefGoogle Scholar
  20. Garg N, Geetanjali (2007) Symbiotic nitrogen fixation in legume nodules: process and signaling. A review. Agron Sustain Dev:27.  https://doi.org/10.1051/agro:2006030
  21. Gay-Fraret J, Ardissone S, Kambara K, Broughton WJ, Deakin WJ, Le Quéré A (2012) Cyclic-β-glucans of Rhizobium (Sinorhizobium) sp. strain NGR234 are required for hypo-osmotic adaptation, motility, and efficient symbiosis with host plants. FEMS Microbiol Lett 333:28–36.  https://doi.org/10.1111/j.1574-6968.2012.02595.x
  22. Gibson AH (1987) Evaluation of nitrogen fixation by legumes in the greenhouse and growth chamber. In: Elkan GH (ed) Symbiotic nitrogen fixation technology. Marcel Dekker, Inc, New York, pp 321–363Google Scholar
  23. Glick BR (2015) Beneficial plant-bacterial interactions. Springer, WaterlooCrossRefGoogle Scholar
  24. Gong ZY, He ZS, Zhu JB, Yu GQ, Zou HS (2006) Sinorhizobium meliloti nifA mutant induces different gene expression profile from wild type in Alfalfa nodules. Cell Res 16:818–829.  https://doi.org/10.1038/sj.cr.7310096 CrossRefGoogle Scholar
  25. Gong Z, Zhu J, Yu G, Zou H (2007) Disruption of nifA gene influences multiple cellular processes in Sinorhizobium meliloti. J Genet Genomics 34:783–789.  https://doi.org/10.1016/s1673-8527(07)60089-7 CrossRefGoogle Scholar
  26. Harper JE, Gipson AH (1984) Differential nodulation tolerance to nitrate among legume species. Crop Sci 24:797–801.  https://doi.org/10.2135/cropsci1984.0011183X002400040040x
  27. Heidstra R, Nilsen G, Martinez-Abarca F, van Kammen A, Bisseling T (1997) Nod factor-induced expression of leghemoglobin to study the mechanism of NH4NO3 inhibition on root hair deformation. Mol Plant-Microbe Interact 10:215–220.  https://doi.org/10.1094/MPMI.1997.10.2.215 CrossRefGoogle Scholar
  28. Hosie AH, Allaway D, Poole PS (2002) A monocarboxylate permease of Rhizobium leguminosarum is the first member of a new subfamily of transporters. J Bacteriol 184:5436–5448CrossRefGoogle Scholar
  29. Hubber AM, Sullivan JT, Ronson CW (2007) Symbiosis-induced cascade regulation of the Mesorhizobium loti R7A VirB/D4 type IV secretion system. Mol Plant-Microbe Interact: MPMI 20:255–261.  https://doi.org/10.1094/mpmi-20-3-0255 CrossRefGoogle Scholar
  30. Janczarek M, Rachwał K, Marzec A, Grządziel J, Palusińska-Szysz M (2015) Signal molecules and cell-surface components involved in early stages of the legume-rhizobium interactions. Appl Soil Ecol 85:94–113.  https://doi.org/10.1016/j.apsoil.2014.08.010 CrossRefGoogle Scholar
  31. Jieping Z, Xiaomi D, Ling X, Jiabi Z, Shanjiong S, Guanqiao Y (2002) Extra-copy nifA enhances the nodulation efficiency of Sinorhizobium fredii. Chin Sci Bull 47:565–567.  https://doi.org/10.1360/02tb9130 CrossRefGoogle Scholar
  32. Kamboj DV, Bhatia R, Pathak DV, Sharma PK (2010) Role of nodD gene product and flavonoid interactions in induction of nodulation genes in Mesorhizobium ciceri. Physiol Mol Biol Plants 16:69–77.  https://doi.org/10.1007/s12298-010-0009-7 CrossRefGoogle Scholar
  33. Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191–197.  https://doi.org/10.1016/0378-1119(88)90117-5 CrossRefGoogle Scholar
  34. Krause A, Doerfel A, Gottfert M (2002) Mutational and transcriptional analysis of the type III secretion system of Bradyrhizobium japonicum. Mol Plant-Microbe Interact: MPMI 15:1228–1235.  https://doi.org/10.1094/mpmi.2002.15.12.1228 CrossRefGoogle Scholar
  35. Laranjo M, Machado J, Young JPW, Oliveira S (2004) High diversity of chickpea Mesorhizobium species isolated in a Portuguese agricultural region. FEMS Microbiol Ecol 48:101–107CrossRefGoogle Scholar
  36. Laranjo M, Alexandre A, Rivas R, Velázquez E, Young JPW, Oliveira S (2008) Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species. FEMS Microbiol Ecol 66:391–400.  https://doi.org/10.1111/j.1574-6941.2008.00584.x CrossRefGoogle Scholar
  37. Laranjo M, Alexandre A, Oliveira S (2014) Legume growth-promoting rhizobia: an overview on the Mesorhizobium genus. Microbiol Res 169:2–17.  https://doi.org/10.1016/j.micres.2013.09.012 CrossRefGoogle Scholar
  38. Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Prome JC, Denarie J (1990) Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature 344:781–784.  https://doi.org/10.1038/344781a0 CrossRefGoogle Scholar
  39. Limpens E, Bisseling T (2009) Nod factor signal transduction in the Rhizobium–legume symbiosis. In: AMC E, Ketelaar T (eds) Root Hairs. Springer Berlin Heidelberg, Berlin, pp 249–276.  https://doi.org/10.1007/978-3-540-79405-9_10 CrossRefGoogle Scholar
  40. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408.  https://doi.org/10.1006/meth.2001.1262 CrossRefGoogle Scholar
  41. Machado D, Krishnan HB (2003) nodD alleles of Sinorhizobium fredii USDA191 differentially influence soybean nodulation, nodC expression, and production of exopolysaccharides. Curr Microbiol 47:134–137.  https://doi.org/10.1007/s00284-002-3972-6 CrossRefGoogle Scholar
  42. Mercante V, Duarte CM, Sanchez CM, Zalguizuri A, Caetano-Anolles G, Lepek VC (2015) The absence of protein Y4yS affects negatively the abundance of T3SS Mesorhizobium loti secretin, RhcC2, in bacterial membranes. Front Plant Sci 6:12.  https://doi.org/10.3389/fpls.2015.00012 CrossRefGoogle Scholar
  43. Nascimento F, Brígido C, Alho L, Glick BR, Oliveira S (2012) Enhanced chickpea growth-promotion ability of a Mesorhizobium strain expressing an exogenous ACC deaminase gene. Plant Soil 353:221–230.  https://doi.org/10.1007/s11104-011-1025-2 CrossRefGoogle Scholar
  44. Nour SM, Cleyet-Marel J-C, Normand P, Fernandez MP (1995) Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov. Int J Syst Bacteriol 45:640–648.  https://doi.org/10.1099/00207713-45-4-640 CrossRefGoogle Scholar
  45. Novichkov PS et al (2013) RegPrecise 3.0--a resource for genome-scale exploration of transcriptional regulation in bacteria. BMC Genomics 14:745.  https://doi.org/10.1186/1471-2164-14-745 CrossRefGoogle Scholar
  46. Oldroyd GED (2013) Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 11:252–263.  https://doi.org/10.1038/nrmicro2990 CrossRefGoogle Scholar
  47. Paço A, Brígido C, Alexandre A, Mateos PF, Oliveira S (2016) The symbiotic performance of chickpea rhizobia can be improved by additional copies of the clpB chaperone gene. PLoS One 11:e0148221.  https://doi.org/10.1371/journal.pone.0148221 CrossRefGoogle Scholar
  48. Pérez-Montaño F et al (2014) The symbiotic biofilm of Sinorhizobium fredii SMH12, necessary for successful colonization and symbiosis of Glycine max cv Osumi, is regulated by quorum sensing systems and inducing flavonoids via NodD1. PLoS One 9:e105901.  https://doi.org/10.1371/journal.pone.0105901 CrossRefGoogle Scholar
  49. Robledo M, Jimenez-Zurdo JI, Soto MJ, Velazquez E, Dazzo F, Martinez-Molina E, Mateos PF (2011) Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2. Mol Plant-Microbe Interact: MPMI 24:798–807.  https://doi.org/10.1094/mpmi-10-10-0249 CrossRefGoogle Scholar
  50. Roponen I (1970) The effect of darkness on the leghemoglobin content and amino acid levels in the root nodules of pea plants. Physiol Plant 23:452–460.  https://doi.org/10.1111/j.1399-3054.1970.tb06435.x CrossRefGoogle Scholar
  51. Rouws LF, Simoes-Araujo JL, Hemerly AS, Baldani JI (2008) Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant. Arch Microbiol 189:397–405.  https://doi.org/10.1007/s00203-007-0330-x CrossRefGoogle Scholar
  52. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  53. Sanjuan J, Olivares J (1989) Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency. J Bacteriol 171:4154–4161.  https://doi.org/10.1128/jb.171.8.4154-4161.1989 CrossRefGoogle Scholar
  54. Sanjuan J, Olivares J (1991) Multicopy plasmids carrying the Klebsiella pneumoniae nifA gene enhance Rhizobium meliloti nodulation competitiveness on alfalfa. Mol Plant-Microbe Interact 4:365–369.  https://doi.org/10.1094/mpmi-4-365 CrossRefGoogle Scholar
  55. Schlaman HR, Horvath B, Vijgenboom E, Okker RJ, Lugtenberg BJ (1991) Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae. J Bacteriol 173:4277–4287CrossRefGoogle Scholar
  56. Schlaman HRM, Phillips DA, Kondorosi E (1998) Genetic organization and transcriptional regulation of rhizobial nodulation genes. In: Spaink HP, Kondorosi A, Hooykaas PJJ (eds) The Rhizobiaceae: molecular biology of model plant-associated bacteria. Springer Netherlands, Dordrecht, pp 361–386.  https://doi.org/10.1007/978-94-011-5060-6_19 CrossRefGoogle Scholar
  57. Spaink HP (2000) Root nodulation and infection factors produced by rhizobial bacteria. Annu Rev Microbiol 54.  https://doi.org/10.1146/annurev.micro.54.1.257
  58. Sullivan JT, Brown SD, Ronson CW (2013) The NifA-RpoN regulon of Mesorhizobium loti strain R7A and its symbiotic activation by a novel LacI/GalR-family regulator. PLoS One 8:e53762.  https://doi.org/10.1371/journal.pone.0053762 CrossRefGoogle Scholar
  59. Theunis M, Kobayashi H, Broughton WJ, Prinsen E (2004) Flavonoids, NodD1, NodD2, and nod-box NB15 modulate expression of the y4wEFG locus that is required for indole-3-acetic acid synthesis in Rhizobium sp. strain NGR234. Mol Plant-Microbe Interact: MPMI 17:1153–1161.  https://doi.org/10.1094/mpmi.2004.17.10.1153 CrossRefGoogle Scholar
  60. Thudi M et al (2016) Whole genome re-sequencing reveals genome-wide variations among parental lines of 16 mapping populations in chickpea (Cicer arietinum L.). BMC Plant Biol 16:10.  https://doi.org/10.1186/s12870-015-0690-3 CrossRefGoogle Scholar
  61. Vaghela MD, Poshiya VK, Savaliya J, Kavani RH, Davada BK (2009) Genetic variability studies in kabuli chickpea (Cicer arietinum L.). Legum Res 32:191–194Google Scholar
  62. van Brussel AA, Bakhuizen R, van Spronsen PC, Spaink HP, Tak T, Lugtenberg BJ, Kijne JW (1992) Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium. Science (New York, NY) 257:70–72.  https://doi.org/10.1126/science.257.5066.70 CrossRefGoogle Scholar
  63. van Brussel AA, Tak T, Boot KJ, Kijne JW (2002) Autoregulation of root nodule formation: signals of both symbiotic partners studied in a split-root system of Vicia sativa subsp. nigra. Mol Plant-Microbe Interact: MPMI 15:341–349.  https://doi.org/10.1094/mpmi.2002.15.4.341
  64. Van de Velde W et al (2006) Aging in legume symbiosis. A molecular view on nodule senescence in Medicago truncatula. Plant Physiol 141:711–720.  https://doi.org/10.1104/pp.106.078691 CrossRefGoogle Scholar
  65. Vijn I, Martinez-Abarca F, Yang WC, das Neves L, van Brussel A, van Kammen A, Bisseling T (1995) Early nodulin gene expression during Nod factor-induced processes in Vicia sativa. Plant J 8:111–119.  https://doi.org/10.1046/j.1365-313X.1995.08010111.x CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Laboratório de Microbiologia do Solo, Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Instituto de Investigação e Formação Avançada (IIFA)Universidade de ÉvoraÉvoraPortugal
  2. 2.Departamento de Microbiología y Genética, Centro Hispano Luso de Investigaciones Agrarias, Unidad Asociada CSIC-USALUniversidad de SalamancaSalamancaSpain

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