The effect of the M truncatula root microbiome on root phenotype and

ethyle-regulated gene expression

We aimed to (i) investigate the effect of the root microbiome on phenotypic responses of M. truncatula exposed to 3-oxo-C14-HSL and (ii) determine the expression of nodulation and ethylene-regulated genes in M. truncatula exposed to 3-oxo-C14-HSL in the presence/absence of the root microbiome. The M. truncatula microbiome did affect plant responses toward several AHLs. The long acyl AHL increased nodule number while short acyl-side AHL did not. Conversely, C10-HSL, 3-oxo-C12-HSL and 3-oxo- C14-HSL showed a significant increase on nodule numbers in the absence of the microbiome, but this effect was lost in the presence of the micobiome. Thus, 3-oxo-C14- HSL was not the only compound that significantly increased nodule numbers as seen in previous Chapters (Chapters 3 and 4). Considering that very long acyl-side chain AHLs are specifically produced by rhizobia is not surprising that Medicago is able to distinguish these AHLs and respond specifically as Medicago has been shown to perceive and distinguish different AHLs from its symbiont and from an opportunistic pathogen (Mathesius et al., 2003). On the other hand, short acyl-side chain AHLs, C4- HSL and C6-HSL, showed a significant increase in nodule numbers with the presence of the microbiome but no effect in the absence of the microbiome. Short acyl-side chain AHLs are less specific to bacteria synthetising them, and this might explain the lesser response of M. truncatula to these AHLs. For example, C4-HSL is sythetised by P. aeruginosa (Pearson et al., 1995), C6-HSL by S. meliloti AK631 (Teplitski et al., 2003), Pantoea sp. (Morohoshi et al., 2007) and Yersinia pseudotuberculosis (Atkinson et al., 1999) among others. 3-oxo-C6-HSL is produced by many bacteria, including Erwinia carotovora, Pantoea sp., Vibrio fischeri and Yersinia enterocolitica (Atkinson et al., 2006; Fray et al., 1999; Dong et al., 2000). Thus, it might be possible that the Medicago root microbiome modulates nodule numbers by altering these compounds (i.e. enzymatic degradation of AHLs) which might, in turn, influence the root microbiome. The M. truncatula root architecture and also the plant biomass were affected by the microbiome and/or the AHL treatment. However, nodule numbers was the most affected phenotype of all.

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Root phenotypic responses in non-nodulated Medicago seedlings were specifically altered in response to the type of AHL and the phenotype measured. Root length at 1

M and 10 M showed significant changes, which depend on the AHL concentration. For example, C10-HSL significantly decreased root length in the presence of the microbiome at 10 M but not at 1 M. However, none of these effects were quantitatively large.

The increment of nodule numbers observed in M. truncatula roots exposed to 3-oxo- C14-HSL only occurred in the absence of the microbiome and after the AHL treatment. Taking into consideration that ethylene might be involved in Medicago responses towards AHLs, particularly 3-oxo-C14-HSL, it is also possible that reductions in bacterial numbers (due to the antibiotic treatment) decreases ethylene due to a downregulation of plant defence signalling including ethylene induced stress genes. This hypothesis is supported by the findings of Hontzeas et al., (2004), who showed that the plant growth promoting rhizobacteria Enterobacter cloacae UW4 (ACC deaminase- containing bacteria) decreased the expression of ethylene-induced stress genes. On the other hand, Iniguez et al., (2005) showed that ethylene was involved in the decrease of bacterial endophytic colonisation in monocotyledons and dicotyledons. This premise was demonstrated by experiments done with the ethylene-insensitive mutant of M. truncatula, sickle (skl), which harboured more endophytes than the wild type (Iniguez et al., 2005). In addition applications of ethylene early during seedling development resulting in the lowest endophytic colonisation of roots and hypocotyls of wt M. truncatula, suggesting that this response occurs at early stages of the plant-bacterial colonisation. Furthermore, ethylene has been found to participate in plant innate immunity processes such as induced systemic resistance (ISR) in the plant defence pathway at very early stages (Mersmann et al., 2010). In the present study, the nodule numbers of the skl mutant with and without antibiotic treatment changed significantly. The highest nodule number per plant was achieved by the skl seedlings exposed to 3- oxo-C14-HSL in the absence of the microbiome but not in seedlings exposed to 3-oxo- C14-HSL with the microbiome. It is also possible that the skl mutant may not have the same bacterial population composition of the wt M. truncatula (A17) as it has been reported that microbial communities are shaped by different genotypes even within the same specie (Berendsen et al., 2012).

Additionally to the ethylene mediated phenotypic responses, we investigated whether they were also detectable at a molecular level. We assessed the expression levels of

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different nodulation and ethylene-regulated genes. All nodulation and ethylene- repressed genes were significantly modulated by the AHL treatment. Interestingly, RIP1 and ENOD11 gene expression were significantly regulated by the presence of the microbiome, AHL exposure and their interaction. Furthermore, the expression of the early nodulins and ethylene-repressed genes RIP1 and ENOD11 was significantly upregulated in Medicago plants only exposed to 3-oxo-C14-HSL in the absence of the microbiome indicating that the plants perceive less ethylene. The significant upregulation of RIP1 and ENOD11 genes correlates with the significant increase in nodule numbers observed in plants exposed to 3-oxo-C14-HSL in the absence of the microbiome. As these genes are ethylene-repressed and nodulation inducible, these results indicate that, Medicago nodulation responses to 3-oxo-C14-HSL and the microbiome are ethylene-dependent. RIP1 and ENODs are involved in the modulation of the root hair curling and infection thread formation (Ramu et al. 2002; Peleg- Grossman et al. 2007). Thus, 3-oxo-C14-HSL application in the absence of the microbiome might regulate nodule numbers through increased infection thread formation. The upregulation of NIN, ERN1, ENOD11 and RIP1 indicates that initially there is a positive regulation of the Nod factor signalling pathway and reduced ethylene- perception, which in turn, could correlate with more nodule numbers. NIN gene expression was regulated in an AHL-dependent and antibiotic-independent manner. Thus, the modulation of NIN gene expression is controlled specifically by the AHL exposure. NIN has been proposed to be involved in the activation of cortical cell division, which leads to nodule formation (Vernié et al. 2015). Therefore, the upregulation of NIN gene expression in response to 3-oxo-C14-HSL might enhance cortical cell division, resulting in more nodule numbers. ERN1 gene expression was significantly modulated by 3-oxo-C14-HSL and the antibiotic treatment. However, the interaction between AHL and antibiotic treatment was not significant. skl plants significantly increased nodule numbers by 3-oxo-C14-HSL with antibiotics but only compared to the non-antibiotic treatments. The skl response and the upregulation of ERN1 and NIN in the presence of the 3-oxo-C14-HSL independently of the presence of the microbiome suggest that the microbiome might not interfere with the ethylene pathway. This suggests that there is an ethylene-independent effect of the microbiome on nodule numbers. On the other hand, ACO gene expression showed a significant antibiotic treatment effect, but not an AHL effect or a significant interaction between treatments suggesting that the microbiome represses ethylene synthesis. Therefore, we cannot strongly conclude that the microbiome mediates ethylene signalling in M.

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truncatula. Further studies incorporating more genes will be needed to stablish definitive conclusions about how the root microbiome affects nodule number responses to 3-oxo-C14-HSL in M. truncatula. It would also be interesting to evaluate Medicago nodulation responses to AHLs, in particular whether 3-oxo-C14-HSL application increases the number of infection threads and cortical cell divisions will be needed to investigate the mechanism behind this nodulation response. In addition, it would be interesting to measure ethylene concentration inside the infected roots to test whether AHL application directly affects ethylene concentration during the early stages of nodulation.