The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene

Abstract

The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under environmental stresses. This is the first report on the successful improvement of a rhizobium with a chaperone gene.

Can stress response genes be used to improve the symbiotic performance of rhizobia?

“…More interestingly the symbiotic effectiveness of the clpB -overproducing strain increased ∼60% at pH 5 and ∼83% at pH 7, compared to the wild-type strain. This improved symbiotic phenotype may be related to an increased expression of symbiosis genes, as detected for the nodulation genes nodA and nodC [116].…”

“…The first successful improvement of a rhizobium with a chaperone gene was achieved using the chickpea nodulating strain M. mediterraneum UPM-Ca36 T modified with extra-copies of the clpB gene [116]. The nodulation kinetics analysis showed a higher rate of nodule development as well as a higher number of nodules in plants inoculated with the clpB -transformed strain.…”

Mediterranean Native Leguminous Plants: A Reservoir of Endophytic Bacteria with Potential to Enhance Chickpea Growth under Stress Conditions

“…Chickpea ( Cicer arietinum L.) was chosen for these experiments due to its importance in terms of a food legume crop and source of protein worldwide, whose production is limited by several biotic and abiotic stresses [44]. Moreover, two compatible mesorhizobia strains, namely Mesorhizobium ciceri LMS-1 [47] and M. mediterraneum UPM-Ca36 T [48] strains, were selected in order to evaluate whether the co-inoculation effect with the endophytic bacteria is dependent on the Mesorhizobium strain used. Based on plant growth-promoting characteristics, three pseudomonads strains, Pseudomonas sp.…”

“…Chickpea seeds were surface sterilized and germinated as described [48]. One seed was planted per plastic pot (±300 mL) previously filled with sterilized vermiculite.…”

traG Gene Is Conserved across Mesorhizobium spp. Able to Nodulate the Same Host Plant and Expressed in Response to Root Exudates

“…Nevertheless, the involvement of other bacterial genes in legume-rhizobia symbioses has been described. Some studies have shown that genes commonly found among prokaryotes and involved in a diversity of cellular mechanisms have also a role in legume- Rhizobium symbiosis, as, for example, stress response genes [6–8], quorum sensing, or secretion system genes [9–12].…”