Fructans as microbe- or damage-associated molecular patterns: Evolutionary perspectives and apoplastic dynamics in Cichorium intybus and Eruca sativa.

In order to sustain the necessary crop production rates, which are necessary to meet the demands of our growing global population, agriculture still relies on the extensive use of pesticides. Nevertheless, the harmful effects pesticides on health and environment are well known. A lot of research effort goes into the discovery and development of more sustainable alternatives to reduce the negative effects of biotic and abiotic stresses. One of these alternatives, priming of the plant immune system using natural compounds, has received a lot of attention in the last two decades. A priming stimulus allows plants to respond better and faster against future stresses. In line with integrated pest management strategies, the use of priming agents in combination with other biocontrol methods and pesticides can help reduce the extensive use of the latter. To find potential natural priming compounds, it is important to look to the physiology of the plant and study compounds that have a natural function against stress exposure. Besides amino acids, organic acids and other compounds, several carbohydrates have been tested as priming agents, often with good results. A role of sugars in plant immune responses was more clearly defined in the sweet immunity concept. By extension, some oligo- and polysaccharides should also be considered, including fructans.

Fructans are oligo-and polysaccharides built from Fru residues. They are found in ca. 15% of flowering plant species, where Fru moieties are added to Suc by different fructosyltransferases. On the other hand, fructan hydrolysis relies on plant fructan exohydrolases (FEHs). Fructans are synthesized and stored in the vacuole of the plant. Based on linkage type between the Fru residues different fructan types are found in plants. Inulin (β-2,1) is mostly found in dicots, while levan (β-2,6) and graminan (mixed linkage type) are more common in monocots. Several studies have indicated positive effects of fructans against abiotic stress, exemplified by many transgenic studies, as well as comparison studies between fructan and non-fructan accumulators. Recent studies also highlighted their potential use as priming agent, showing promising effects of plant inulin and levan in lettuce and apple, respectively. In Arabidopsis thaliana levan oligosaccharides (LOS) of microbial origin, low degree of polymerization (DP) levan produced by partial hydrolysis of higher DP levan, proved effective in reducing susceptibility of the plant to Botrytis cinerea infection. Since microbial fructan production, mainly of the levan-type, is common in bacteria and produced as an exopolysaccharide, this fructan may be sensed by the host plant as a microbe-associated molecular pattern (MAMP), triggering defence responses. Different fructans can activate toll-like receptors in animals to stimulate immune responses. Because of the high DP nature of microbial fructans, it is expected that low DP, more mobile LOS are produced as a primary signal. This production may rely on plant and microbial fructanase enzymes, playing a central role in apoplastic fructan dynamics.

In a first part of the thesis we identified the most promising fructan priming agents and studied their influence on carbohydrate metabolism in the plant. Two plant species were chosen, rocket (Eruca sativa) and chicory (Cichorium intybus), both with agricultural value. Rocket belongs to the Brassicaceae, allowing us to compare our results with the previous results obtained in A. thaliana. Chicory was chosen as a fructan-accumulating plant, as it produces high amount of inulin in the taproot and has served as a model plant organism in past fructan research. This allowed us to study not only the effect of non-self fructan priming, but also the effect of exogenous priming with endogenous fructans in the case of chicory. In this species, inulin-type FOS (fructooligosaccharides) may be sensed as damage-associated molecular patterns (DAMPs). B. cinerea was chosen as a fungal pathogen to infect both host species. This necrotrophic pathogen has a very broad host range and has economically devastating effects.

Since previous research on priming in these species is limited or absent, priming (leaf spraying) and disease assays were optimised using the necessary control treatments. Then, a broad array of different fructan types was used, some commercially available, while others were prepared and/or further purified in the lab. Besides testing higher DP fructans, enzymatic hydrolysis was used to produce FOS and LOS. A sulfated levan, as well as sulfated LOS produced from partial hydrolysis, were also considered due to the positive effects observed in animal studies. As hypothesized, mostly low DP fructans could induce an increased resistance to B. cinerea after leaf priming. Most importantly, microbial LOS priming greatly improved disease resistance in both rocket and chicory. FOS priming was only effective in chicory, which may indicate DAMP-specific type responses that are absent in rocket. Inulin and branched fructans had no clear positive effects, while microbial levan marginally improved resistance. Sulfated LOS also improved resistance in both species. Sulfated LOS induced a direct ROS burst in the plant, typical for elicitors, while non-sulfated LOS and FOS behaved as a genuine priming compound. In addition, sulfated LOS significantly inhibited growth of B. cinerea on plate, while no negative effects were observed for LOS. In rocket, total leaf sugars increased significantly after LOS priming as well as subsequent infection. In both species, LOS priming induced a temporal peak in apoplastic small soluble sugars. This indicates that during LOS priming, LOS and/or glucose dependent signalling may be activated for downstream immune responses. Although similar dynamics were observed for FOS, high levels of Glc and FOS in the apoplast may benefit B. cinerea, since this fungus can hydrolyse and utilise inulin-type fructan.

In the second part of the thesis, we focussed on two fructanase enzymes. First, we characterized a novel chicory FEH. So far, no 6-FEH was identified from chicory, although such enzymes had been discovered in non-fructan plants such as A. thaliana and sugar beet. After a detailed study on protein sequence alignments, we identified a potential 6-FEH that was wrongly annotated as a cell wall invertase. The enzyme was expressed in Pichia pastoris and characterized on a functional level. The enzyme had high affinity towards levan-type fructan and a confirmed apoplastic localization. We showed that it can hydrolyse a typical fructan profile produced from microbial levan synthesis. Based on these data it is proposed that this enzyme has a key role in the apoplastic production of LOS from microbial levan, which may be central in interactions of the plant with beneficial microbes in the rhizosphere context. In depth modelling studies and in vitro assays provided important insights into the substrate specificity and catalytic behaviour of this enzyme. Second, we characterized a B. cinerea inulinase that we identified from the genome. This endo-inulinase is different from previously characterized fungal inulinases, since it harbours an additional N-terminal domain, resembling an extra β-sandwich. We expressed this form, as well as a shortened form missing the extra domain. The results indicated a strong difference in kinetic behaviour, with the long form following Hill kinetics, while Michaelis-Menten kinetics were observed for the short form. Using this B. cinerea inulinase, in depth modelling and docking studies were performed to gain important insights into structural differences between endo- and exo-type fructanase reactions. After a similar analysis of microbial levanases, an updated terminology is proposed for fructanase enzymes.

In conclusion, the results of the thesis highlight low DP fructans, LOS and FOS, as promising priming agents to improve plant resistance to biotic stress. Probably all higher plants are able to sense LOS as a MAMP through yet unidentified receptors, while the inulin accumulator chicory likely evolved an extra receptor to detect endogenous inulin-type FOS as a DAMP, leaking out of damaged cells. The observed carbohydrate dynamics after LOS priming will allow future studies to go deeper into understanding the specific signalling pathways involved. Furthermore, detailed investigation of key fructan-degrading enzymes highlights the importance of apoplastic 6-FEHs in plant-microbe interactions, and new mechanistic insights into fructanase reaction mechanisms obtained from studying the B. cinerea inulinase provide a central starting point for further research going deeper into the unravelling of the interaction and signalling cascades involved in sweet priming of plants and beneficial plant bacteria (e.g. Bacilli; emerging levan detour concepts emerging from sucrose in root exudates). All these fundamental insights will boost the development of priming and/or biological control micro-organism formulations, reducing the widespread use of agrochemicals.