Heterologous expression of nifA or nodD genes improves chickpea-Mesorhizobium symbiotic performance
The aim of this study was to investigate whether the overexpression of NifA and NodD regulators contribute to the symbiotic improvement of chickpea mesorhizobia.
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.
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.
These results indicate that overexpressing nifA and nodD may be an important tool to achieve the improvement of the symbiotic performance of mesorhizobia.
KeywordsOverexpression Nodulation Nitrogen fixation Legume Rhizobia Symbiotic effectiveness
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.
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).
- 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
- 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
- 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
- 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
- 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
- Fischer HM (1994) Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev 58:352–386Google Scholar
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- 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
- 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