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.

Fig 1. Analysis of clpB gene transcription by semiquantitative RT-PCR in M. mediterraneum Ca36pPHU (lane 1, light gray bar) and Ca36pPHUclpB (lane 2, dark gray bar) strains.

The relative clpB transcript abundance was normalized against the amplification of a fragment of 16S rRNA gene. For the quantification of RT-PCR analysis, data are presented as the mean and standard error values of three independent biological replicates. Different letters (a, b) correspond to statistical significant differences (P < 0.05).

Fig 2. Growth curves of M. mediterraneum UPMCa36^T wild-type strain (Ca36WT) and its derivatives (Ca36pPHU and Ca36pPHUclpB) under control and heat shock conditions.

Bacterial growth at 28°C (A). Bacterial growth after a heat shock of 48°C during 30 min, followed by growth at 28°C (B).

Fig 3. Nodulation kinetics of chickpea plants inoculated with Ca36WT, Ca36pPHU or Ca36pPHUclpB strains during 24 days after inoculation.

Each point represents the mean and standard error values of eight plants per treatment.

Fig 4. Results obtained from a plant growth assay performed under control (pH7) and stress conditions (pH5).

The chickpea plants were inoculated with the M. mediterraneum UPM-Ca36^T wild-type strain (Ca36WT) and Ca36pPHUclpB. SDW—Shoot dry weight (A). RDW—Root dry weight (B). NN—Number of Nodules (C). AWN—Average Weight per Nodule (D). SE-Symbiotic Effectiveness (E). Data correspond to the mean and standard error of five plant replicates (n = 5) per treatment. Different letters (a-g) correspond to statistical significant differences (P<0.05). Dark grey bars correspond to results obtained under control conditions (pH7). Light grey bars correspond to results obtained in plants subjected to pH stress conditions (pH5).

Fig 5. Microscopic analysis of root hair curling of chickpea plants inoculated with Ca36WT, Ca36pPHU or Ca36pPHUclpB.

This analysis was performed in the third and fourth days after inoculation. Plants inoculated with the Ca36WT strain (A). Plants inoculated with the Ca36pPHU strain (B). Plants inoculated with the Ca36pPHUclpB strain (C). Scale bar: 0.053 μm.

Fig 6. Nodule development in chickpea.

Portions of nodulated roots inoculated with the Ca36pPHU (A), wild-type Ca36WT (C, E), or Ca36pPHUclpB strains (B, D, F) are shown. A and B, Stereophotomicrographs of sections of embedded nodules stained with Toluidine Blue. C to F, Photomicrographs of embedded nodules stained with Toluidine Blue. A to D, bright field light microscopy. E and F, Phase contrast microscopy. A and B, whole nodules (asterisks indicate meristematic zones). C and D, meristematic zones at higher magnification. E and F, infection zones (Black arrows indicate particles inside uninfected cells; White arrows indicate particles inside infected cells). Scale Bars: 400 μm (A and B); 100 μm (C and D); 50 μm (E and F).

Fig 7. Analysis of nodA and nodC gene transcription by semiquantitative RT-PCR in the Ca36pPHU (lane 1, light gray bars) and clpB-transformed (lane 2, dark gray bars) strains.

To normalize the relative nodA and nodC transcripts abundance the amplification of a fragment of 16S rRNA gene was also performed. Data in the graph correspond to the mean and standard error values of three independent biological replicates. Different letters (a, b) correspond to statistical significant differences (P < 0.05).