Role of plant compounds in the modulation of the conjugative transfer of pRet42a

Abstract

One of the most studied mechanisms involved in bacterial evolution and diversification is conjugative transfer (CT) of plasmids. Plasmids able to transfer by CT often encode beneficial traits for bacterial survival under specific environmental conditions. Rhizobium etli CFN42 is a Gram-negative bacterium of agricultural relevance due to its symbiotic association with Phaseolus vulgaris through the formation of Nitrogen-fixing nodules. The genome of R. etli CFN42 consists of one chromosome and six large plasmids. Among these, pRet42a has been identified as a conjugative plasmid. The expression of the transfer genes is regulated by a quorum sensing (QS) system that includes a traI gene, which encodes an acyl-homoserine lactone (AHL) synthase and two transcriptional regulators (TraR and CinR). Recently, we have shown that pRet42a can perform CT on the root surface and inside nodules. The aim of this work was to determine the role of plant-related compounds in the CT of pRet42a. We found that bean root exudates or root and nodule extracts induce the CT of pRet42a in the plant rhizosphere. One possibility is that these compounds are used as nutrients, allowing the bacteria to increase their growth rate and reach the population density leading to the activation of the QS system in a shorter time. We tested if P. vulgaris compounds could substitute the bacterial AHL synthesized by TraI, to activate the conjugation machinery. The results showed that the transfer of pRet42a in the presence of the plant is dependent on the bacterial QS system, which cannot be substituted by plant compounds. Additionally, individual compounds of the plant exudates were evaluated; among these, some increased and others decreased the CT. With these results, we suggest that the plant could participate at different levels to modulate the CT, and that some compounds could be activating genes in the conjugation machinery.

Fig 1. CT frequency in response to presence of different plants and conditions.

A. Evaluation of CT frequencies of pRet42a in presence or absence of P. vulgaris and on the root surface, in media without C and N sources. B. Evaluation of CT frequencies of pRet42a in presence or absence of P. vulgaris, and on the root surface, in media supplemented with C and N. C. Evaluation of CT frequencies of pRet42a in media, in presence of P. vulgaris, Z. mays and M. sativa. D. Evaluation of CT frequencies of pRet42a on the roots of P. vulgaris, Z. mays and M. sativa. All the experiments were performed at 1, 10- and 20-days post-inoculation (dpi). ND, not detected.

Fig 2. CT frequency in response to plant exudates and extracts.

A. Evaluation of CT frequencies of pRet42a in response to root exudates from P. vulgaris and M. sativa. B. Evaluation of CT frequencies of pRet42a in response to extracts of root and nodules of P. vulgaris plants. Statistical analysis was performed by one-way ANOVA (p value < 0.05) with Dunnett´s multiple comparisons test. Key: ** (p value = 0.0088), *** (p value = 0.0001). **** (p value < 0.0001). For statistical analyses, each treatment was compared to the control condition.

Fig 3. CT frequency in response to different flavonoids.

Evaluation of CT frequencies of pRet42a in response to naringenin (N), genistein (G), quercetin (Q), acetosyringone (A), luteolin (L), apigenin (Ap), gallic acid (AG) and all the flavonoids together (All), at 2 μM (A), 20 μM (B) and 50 μM (C). D. Evaluation of conjugation frequencies of pRet42a in response to different concentrations of apigenin. Statistical analysis was performed by one-way ANOVA (p value < 0.05) with Dunnett´s multiple comparisons test. Key: In A, ** (p value = 0.0047), **** (p value < 0.0001). In C, ** (p value < 0.003). In D, * (p value = 0.0135), ** (p value = 0.0002), **** (p value < 0.0001). For statistical analyses, each treatment was compared to the control condition (C).

Fig 4. CT frequency of a QS regulatory mutant, in the media in presence of P. vulgaris plants and on the root surface.

Evaluation of CT frequencies of pRet42a (CFNX187) and a traI mutant of this plasmid (CFNX669) in the media in presence or absence of P. vulgaris, and on the root surface at 1, 10 and 20 dpi. ND, not detected.

Fig 5. pRet42a CT frequency in response to different environmental conditions.

A. Different oxygen concentrations. B. Nutrient composition of the media. C. Solid or liquid media. Statistical comparison was performed through a t test (p value < 0.05) in comparison to the control condition. Key: ** (p value = 0.0015), **** (p value < 0.0001).

Fig 6. Scheme of possible pathways involved in pRet42a CT modulation.

The essential regulatory system (Top) figure shows the effect of the AHLs (orange circles) produced by the different replicons of R. etli CFN42 on the activation of pRet42a transfer. Despite that transfer responds to different AHLs, the interaction between AHLs produced by TraI and TraR/CinR regulators is essential for pRet42a transfer. In the Modulatory System (Bottom), we propose a mechanism for the effect of flavonoids, other plant compounds and environmental signals as low oxygen conditions. For flavonoids and other plant compounds (triangles), we propose an interaction with pRet42d encoded NodD regulators, or with other LysR-like regulators present in other replicons. Since NodD-flavonoids triggers Nod Factors expression, a similar mechanism could be involved in the modulation of CT of pRet42a. For the other signals, as O₂ concentration, an unknown protein (star) could sense the signal and then modulate transfer genes expression. These “star” proteins might be the hypothetical proteins located between Dtr and Mpf genes. The dotted line indicates that the contained replicons could produce AHLs (Top) or LysR-like proteins (Bottom). Green arrows indicate activation or production; red arrows indicate repression.