Dissertation

Co-Option of Pre-Existing Pathways During Rhizobium-Legume Symbiosis Evolution

Lillo, Alessandra

2012

Overview

Fixed nitrogen is one of the most limiting factors for plant growth. The major biological source of fixed nitrogen in ecosystems is provided by nitrogen-fixing bacteria that are able to break the triple bond of N2 molecules and convert it into ammonium via an enzymatic (nitrogenase) activity (Kouchi et al., 2010; Zehr et al., 2003). One of the most important nitrogen-fixing systems is the rhizobium root nodule symbiosis. In this interaction phylogenetically diverse nitrogen fixing rhizobium bacteria colonize nodules on the root of their host plants and find the appropriate conditions to provide fixed nitrogen in exchange for carbohydrates (Kouchi et al., 2010). This efficient endosymbiosis is restricted to two taxonomic lineages: the legume family (Fabaceae) and the genus Parasponia in the Cannabis family (Cannabaceae) (Soltis et al., 1999; Soltis et al., 1995). The ability of these species to achieve a rhizobium endosymbiosis guarantees them a reliable source of nitrogen. In this Thesis I have studied the legume-rhizobium symbiosis, starting from the idea that part of pre-existing signalling pathways have been coopted during evolution of this mutualistic interaction. It is well known that parts of the signalling pathway that is essential for the more ancestral mycorrhizal symbiosis are recruited to support the rhizobium symbiosis (Kouchi et al., 2010; Ivanov et al., 2012). In line with this, it is hypothesized that also other ancestral pathways are recruited during evolution of nodulation. For example, the debate on whether nodule and lateral root formation share parts of their developmental programs has been going on for long (Nutman, 1948; Hirsch et al., 1997; Mathesius et al., 2000; Wopereis et al., 2000; Gonzalez-Rizzo et al., 2006). In Chapter 2 we studied lateral root primordium formation in the model legumes Medicago truncatula (Medicago) and Lotus japonicus (Lotus). Although it is commonly believed that exclusively pericycle cells give rise to the lateral root primordium, similar as seen in Arabidopsis thaliana (Arabidopsis) (Oldroyd et al., 2011; Hirsch et al., 1997), we provide morphological evidence that in the studied legume species this is not the case. In both, Lotus and Medicago, also root cortical cell divisions occur during lateral root formation. Furthermore, we found a striking correlation in the cell layers that are recruited during lateral root and nodule primordium formation. This supports the hypothesis that at least parts of the lateral root developmental program have been recruited during evolution of symbiotic root nodules. Gene duplications -of which a whole genome duplication (WGD) is the most dramatic variant- are known as important driving forces in evolution of new traits. 56 to 65 million years ago an ancestral legume species within the Papilionoidae subfamily (Papilionoids) experienced a WGD event and subsequently gave rise to several major phylogenetic crowns (Lavin et al., 2005). Three of these major lineages are represented by Medicago, Lotus and soybean (Glycine max) (Fawcett et al., 2009; Cannon et al., 2010; Young et al., 2011). I hypothesize that among the orthologous gene pairs maintained in these 3 species are genes that are essential for nodulation. Such genes are yet unidentified by forward genetic screens, because of their (partial) redundancy. I adopted a phylogenetic strategy to identify new candidate genes involved in the legume-Rhizobium symbiosis (Chapter 3, 4 & 5). In a targeted approach, we focussed on the cytokinin phosphorelay pathway, since cytokinin is well known to be involved during nodule organogenesis. This resulted in the identification of one gene pair encoding type-A Response Regulators (RRs). Both these genes, named MtRR9 and MtRR11 in Medicago, are rapidly activated upon rhizobial signalling, whereas all other type-A RR genes are not. Constitutive expression of these type-A RRs is sufficient to trigger cortical cell divisions, suggesting a positive regulatory role for these proteins in root nodule formation. Yet the illustrated role for MtRR9 and MtRR11 in rhizobial symbiosis provides a proof of principle of this method to identify gene pairs involved in legume specific characters (Chapter 3). An unbiased search for paralogous gene pairs revealed two conserved gene duplications in the NADPH oxidases gene family (Young et al., 2011; De Mita & Geurts, unpublished). NADPH oxidases are reactive oxygen species (ROS) producing enzymes. Based on expression pattern it has been speculated that these enzymes are involved in legume-Rhizobium symbiosis (Marino et al., 2010; Chapter 4). So far, such hypothesis was not supported by experimental data. We identified two sets of duplicated genes that have been maintained after the Papilionoid specific WGD (I. MtRBOHA and MtRBOHG and II. MtRBOHE, MtRBOHB, MtRBOHC and MtRBOHD) and aimed to provide support for a symbiotic function for (some of) these genes. In Chapter 4, we show that MtRBOHA and MtRBOHG are redundant, yet essential during symbiosis. In nodules both proteins are associated to rhizobial infection threads at putative sides of bacterial release. MtRBOHA and MtRBOHG are essential during, or just after rhizobium release from infection threads, and their activity sustains the life span of infected cells. Although both genes seem to have redundant functions in symbiosis, they are maintained after the WGD in 3 different legume species. This suggests that there is positive selection to maintain both copies. Though it remains unclear whether this is due to sub- and/or neo-functionalization or due to gene dosage effects (Chapter 4 & 6). The second paralogous gene pair encoding NADPH oxidases underwent additional duplication resulting in 4 genes; MtRBOHE, MtRBOHB, MtRBOHC and MtRBOHD. These genes are phylogenetically positioned in the same orthology group as Arabidopsis AtRHD2. AtRHD2 is a key regulator of root hair tip growth. In Chapter 5 we show that MtRBOHC expression is quickly upregulated upon Rhizobium induced signalling. We postulate that the root hair tip-growth machinery is conserved among species, and that this mechanism is co-opted to support rhizobium root hair based infection. To test this hypothesis MtrbohC knockout mutant was analysed. This mutant did not display an obvious phenotype; symbiotic nor non-symbiotic. This indicates that MtRBOHC is most probably redundant in function.

• Publisher: ProQuest Dissertations & Theses
• Subject: Botany / Flowers & plants / Genetics / Genomics / Phylogenetics
• ISBN: 9798516039515