Analysis and functional characterization of two tomato genes involved in the mycorrhization process

Abstract

Arbuscular mycorrhizal (AM) fungi are microscopic fungi that live in symbiosis with the roots of most plants. Among the benefits provided to the plants by this interaction we must highlight a better nutrition and a higher resistance to biotic and abiotic stresses. As both symbionts have coevolved for a long time, probably from the beginning of plant adaptation to the land environment, it is expected that the mechanisms for regulation of mycorrhizal development have also coevolved with plants. In this manner, the development and establishment of the AM symbiosis are fine-tuned regulated by environmental factors including nutritional conditions, as well as by molecular dialog and signalling mechanisms, and by a complex network of transcription factors and cofactors. The understanding of the symbiotic process and the key components for its regulation is essential in order to elucidate by what mechanisms the plant is benefited from the interaction with the AM fungi, and to develop strategies to improve the management of the mycorrhizal associations in order to use them as an alternative to the chemical fertilizers and pesticides. Our team work is focused in the analysis of the molecular processes underlying the AM symbiosis using the tomato plant, which constitutes a model plant for physiological and genetic studies and, in addition, it is a worldwide important crop. In this work, two AM-induced genes from tomato, tsb and SlDLK2, are subjected to their functional characterization and analysis, due to their possible role in the mycorrhization process. Particularly, tsb was chosen because of its possible function in cytoskeleton rearrangements during mycorrhization; while SlDLK2, encoding for a protein from the α,β-hydrolase family, because of its possible role as a relevant hormonal receptor involved in signalling during the mycorrhizal process

First of all, in order to quickly and easily screen the functionality of these genes during mycorrhization, a method for obtaining composite tomato plants using Agrobacterium rhizogenes-mediated transformation was implemented. The resulting plants were composed of a transformed root system and a wild type aerial part. An optimized protocol was successfully set up for the transformation and generation of composite seedlings for mycorrhizal studies, with high success rates and that allows to undoubtedly identifying and selecting tomato cotransformed roots from the beginning until the harvesting time through visual selection using the fluorophore marker DsRed, without the requirement of antibiotics. Three different binary vectors were tested for silencing, overexpressing and promoter-GUS expression studies, that have allowed us to successfully perform a functional analysis of the candidate genes. This method, which have been recently published (Ho-Plágaro et al. 2018), is presented in Chapter 1 of this doctoral dissertation. The research carried out regarding the tsb gene is shown in Chapter 2. Tsb encodes a putative MAP (Microtubule Associated Protein) that belongs to a family of MAPs unique from Solanaceae plants. Some of the members of this MAP family have been previously described as pollen specific and with a function in cytoskeleton rearrangements and the formation of the pollen tube (Zhao et al. 2006; Huang et al. 2007; Liu et al. 2013). Here, through the analysis of tsb-silenced and tsb-overexpressing composite plants, a decreased expression was observed for genes related to arbuscule functionality in the AM tsb-silenced roots compared to the wildtype AM roots. In agreement with this result, tsb-overexpressing roots showed an induction all genes used as markers of arbuscule and fungal activities. Moreover, the cortical microtubule array seems to be perturbed in tsb-overexpressing roots. Microtubules and microfilaments are not only known to determine the structure of the cell and its organelles, but also to be involved in the intracellular transportation mechanisms, because they act as “tracks” for the clathrin vesicle trafficking, the

main means of transport of the cell, consequently determining the endocytosis and exocytotic pathways. The results obtained in this work point to a role of tsb in restructuring the microtubule cytoskeleton in the cortical cells of the roots during mycorrhization and indicate that the action of tsb is related to the specific exocytosis processes for the delivery of proteins and compounds to the periarbuscular membrane and the symbiotic interface to allow arbuscule functionality and activity. The second gene studied in this work is a gene that encodes for a protein belonging to the α.β-hydrolase family, particularly to the DLK2 group. For this reason, it was named as SlDLK2. Curiously, the DLK2 protein group is phylogenetically very close to the strigolactone receptors (D14) and the karrikin receptors (KAI2). Strigolactones are plant hormones that play an important role in presymbiotic signalling during mycorrhization. In addition, the karrikin receptor KAI2 has been recently shown to be essential for the AM symbiosis (Gutjahr et al. 2015). However, DLK2 α.β-hydrolases are little characterized, and no relation has yet been found between DLK2 and the mycorrhization process. In Chapter 3 is presented our research work performed to elucidate if SlDLK2 is a signalling receptor important during MA symbiosis. Composite plants with transformed roots were obtained, and we observed that SlDLK2 silencing showed a significant increase of AM fungal development in the host roots. However, SlDLK2 overexpression gives place to an anomalous arbuscule development, with lack of branching, what suggest that SlDLK2 has a clear role in the development and branching of the arbuscules. Additionally, SlDLK2 has a conserved catalytic triad, responsible of the hydrolytic activity described for the strigolactone and karrikin receptors, and here we have actually probed that SlDLK2 retains some ability to interact and hydrolyse synthetic strigolactones. Although our research have not allowed us to elucidate the chemical structure of the specific ligand for SlDLK2, the

results obtained so far point to the C13 α-ionols derivatives as the possible ligands. Finally, in Chapter 4, in order to clarify the possible signalling role of the SlDLK2 receptor during mycorrhization, transcriptomic alterations and the predicted changes in metabolic pathways in SlDLK2-silenced plants are shown, and the causes and consequences of these changes are discussed. Biggest changes observed were associated to a nutrient starvation and induced defence signature in the SlDLK2-RNAi mycorrhizal roots, probably as a consequence of the higher AM development and/or activity in these plants. However, examination of many other differentially expressed genes supports the idea of SlDLK2 as a negative regulator of mycorrhization, and that SlDLK2 might be important to activate GA signalling, restrict hexose production and carbohydrate supply to the AM fungus, and induce a number of resistance mechanisms, in order to control AM fungal development.(A)