The effect of quorum sensing signals on nodulation of Medicago truncatula

Abstract

N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviours in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognised by plants, and this may influence their interactions with bacteria. This thesis tested whether the exposure to AHLs affects the nodule-forming, nitrogen-fixing symbiosis between legume hosts and rhizobia. It used the legume, Medicago truncatula, and its symbiont, Sinorhizobium meliloti, as this model symbiosis has been well characterised on a molecular and cellular basis. In addition, previous studies have characterised the identities and roles of AHLs from S. meliloti during nodulation. First, protocols were established to grow M. truncatula plants under conditions conducive to symbiosis and, at the same time, to minimise growth of bacteria growing in and on roots. This was important as bacteria can destroy AHLs and could thus interfere with externally applied AHLs. M. truncatula was found to harbour culturable bacteria that were derived from the inside of the seed coat and were recalcitrant to surface sterilisation. A protocol using an antibiotic treatment of the seeds to minimise bacterial growth, and an axenic system growing M. truncatula seedlings on large agar plates was chosen for subsequent experiment. M. truncatula seedlings were exposed to a range of synthetic AHLs derived either from its specific symbiont, S. meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. Increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone (HSL), while the other AHLs did not result in significant changes to nodule numbers. The increase in nodule numbers was dependent on AHL concentrations and was repeatedly observed at concentrations of 1 M and above. No evidence for altered nodule invasion by the rhizobia was found. 3-oxo-C14-HSL ‘primed’ Medicago plants before inoculation with rhizobia, indicating that the increase in nodule numbers occurs at early stages and as a direct effect on the plant and not on the rhizobia. Increased nodule numbers following 3-oxo-C14-HSL lactone treatment were not under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-HSL were not found in the ethylene-insensitive sickle mutant. Gene expression analysis further suggested that viii this AHL affects the expression of ethylene-related genes during nodulation. It was concluded that plant perception of the S. meliloti-specific 3-oxo-C14-HSL influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism. A comparison of M. truncatula with M. sativa (alfalfa), Trifolium repens (white clover) and Lotus japonicus (Lotus) showed that the observed effects of AHLs on nodule numbers, at least at the concentration chosen, were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. In M. truncatula, the effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. This result suggests a very specific effect of AHLs on nodulation, possibly via modulation of ethylene-controlled infection, but not on general root developmental processes. During the investigation of protocols to eliminate bacterial contamination, it was discovered that nodulation phenotypes in response to AHL exposure strongly depended on the presence of plant-associated bacteria. Therefore, the composition and possible role of the M. truncatula-associated microbiome was further investigated. High throughput sequencing showed that the antibiotic treatment significantly reduced the presence and composition of the microbiome. Interestingly, application of 3-oxo-C14-HSL also significantly altered the microbiome, but this effect was very specific for bacteria belonging to the genus Pantoea. Only in the absence, but not in the presence of the majority of the plant microbiome, did M. truncatula show increased nodulation in response to 3-oxo-C14-HSL, and this was associated with increased expression of early nodulation genes. These results suggest that the bacterial community of Medicago affects nodulation responses towards AHLs, particularly towards to 3-oxo-C14-HSL, likely by interfering with AHL stability, perception or plant responses. In summary, this thesis showed that plant perception of AHLs alters symbiosis-specific phenotypes, suggesting that AHLs are not only important to regulate bacterial behaviours during nodulation, but also that the plant has evolved mechanisms to respond to specific AHLs from its symbiont, likely by reducing ethylene signalling or synthesis to enhance the number of nodules.