BIOCHEMICAL FUNCTIONAL CHARACTERIZATION AND MOLECULAR BIOLOGY OF PLANT INHIBITOR PROTEINS ACTING AGAINST GLYCOSIDE HYDROLASE

Plant cell walls are composed mostly of polysaccharides and it consist of three layers (the primary cell wall, the secondary cell wall and the middle lamella) that are made up of different percentage of cellulose, pectins and hemicelluloses. These latter are composed of a linear backbone made up of (1,4)-β-D-glycans with an equatorial configuration. Based on type of glycans forming the backbone it is possible to distinguish: mannans contain β-(1,4)-linked mannose; in xyloglucan β-1,4 glucans can be substituted with a diverse array of glycosyl and nonglycosyl residues and xylans are composed by β-(1,4)-linked xylose residues. The seeds of many legumes are known to accumulate galactomannan in their endospermic cell walls. In many dicots xyloglucans constitute the major hemicellulose of growing cell walls, comprising ~20% of the dry mass of primary cell walls. Grasses - but not monocots in general - have a reduced xyloglucan content. Cell wall polysaccharide biogenesis includes polymer synthesis, secretion, assembly, and rearrangement during development. All of these modification demands the reversible ‘loosening’ of the cellulose– hemicellulose–pectin network. Glycoside hydrolase (GH) enzymes located in the wall or in the plasma membrane play a crucial role in the degradation of different cell wall polysaccharides. On the other hand, pathogenic microorganisms secrete glycoside hydrolase to penetrate plant cell walls. As a response, plants produce glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan- specific endo-β-1,4-glucanase inhibitor proteins-like (XEGIPs-like) are typical of dicots, they inhibit the hydrolytic activity of a xyloglucan-specific β-1,4-endoglucanase isolated from GH12 family. XEGIPs-like have been found widespread in dicots: they were detected in the medium of cultured tomato cells, purified from carrot callus, isolated from the nectar of ornamental tobacco, when overexpressed they were capable of protecting potato from disease caused by endo-β-1,4- glucanase GH12 from Phytophthora infestans, enhanced in apple in response to infection of Botryosphaeria dothidea, they have distinct roles in defence mechanisms in Humulus lupus. In cereals three types of GHIPs occur in a fairly coordinated fashion throughout grain development and germination: Triticum aestivum L. endoxylanase inhibitors (TAXIs-like), xylanase inhibitor proteins (XIPs-like), thaumatin-like xylanase inhibitors (TLXIs-like). The accumulation of GHIPs during the early stages of germination is consistent with the phenomenon of germination-based resistance and their highest concentrations occur in the aleuronic layer. The apoplastic localization of GHIPs in cereals may be favourable for their action as inhibitors of microbial xylanases GH10 and/or GH11 from Aspergillus niger, Bacillus subtilis and Hypocrea jecorina intruding the host plant. GHIPs homologous are also present in legume (LACGs-like): γ-conglutin is largely expressed and accumulated in Lupinus spp. and Bg7S in Glycine spp. GHIPs have common structural features. In particular, the alignment of the primary structurer showed that the position of the 12 cysteines is fully conserved, so various GHIPs have similar three- dimensional structures. Cys10-Cys11 is located at the C-terminal region of the proteins, the sequence amongst them is called “inhibition loop 1” and a conserved arginine, in XEGIPs-like, or leucine, in TAXI-I, is involved in the bond GH12 or GH11, respectively. In the LAGCs-like it’s also present the loop, but a deletion of about five amino acids involve this region, otherwise highly conserved. The superimposition of TAXI-I 3D structure (PDB accession number: 1T6E) and EDGP 3D structure (PDB accession number: 3VLA) with γ-conglutin 3D structure (PDB accession number: 4PPH) confirm the structural difference of the loop responsible for the interaction with the enzyme as a consequence of the five amino acid long deletion. This may be the cause of an unfavourable local spatial conformation of the protein for the correct interaction with the enzyme. The disulphide bridge Cys9-Cys12 define another region called “inhibition loop 2” where a conserved arginine, in XEGIPs-like, or histidine, in TAXIs-like and LACGs-like, is involved in the bond with a specific GH. The sequence of the IL2 loop of γ-conglutin is more similar to the sequence of the IL2 loop of TAXI-I, rather than to the one of XEGIPs. In particular, a His residue, considered a key amino acid for the inhibitory activity of TAXI-I is also present in γ-conglutin sequence, but not in XEGIPs. γ-conglutin was expressed in Pichia pastoris. Unexpectedly, this recombinant γ-conglutin (rγc) was able to inhibit a GH11 enzyme, but not GH12. In lupin, γ-conglutin is naturally cleaved in two subunits, whereas in P. pastoris it is not. Most likely, the proteolytic processing influences the structural conformation of γ-conglutin and small local rearrangements may be the cause of the observed activity. Also a set of γ-conglutin mutants was designed upon TAXIs-like and XEGIPs-like sequences and expressed in Pichia pastoris. The mutants were able to modulate the inhibition capacity. The enzymatic assays and the bioinformatics analysis confirmed that the presence of IL1 is not strictly required to manifest inhibition, even if the specifically inserted amino acid stretches enhanced the activity. On the other hand, histidine in IL2 is confirmed to be necessary and sufficient to manifest the inhibitory competence of rγc. The LACGs-like among GHIPs remains the less characterized. For this reason, we undertook experiments aimed to study the inhibitory specificity of wild type γ-conglutin. Inhibitory capacity was tested against under different condition using selected GH2 and GH5 members. GH11 and GH12 were not tested since previous results evidenced no capacity. In Arabidopsis thaliana, which is a small flowering plant widely used as a model organism in plant biology, we have found five genes coding for GHIPs belonging to each of three aforementioned groups. At1g03220 and At1g03230 are closer related to XEGIPs-like proteins: the cysteines pathway is totally conserved, they show arginine in the inhibition loop 1 and inhibition loop 2, which interact with glycoside hydrolases family 12, and along the sequences there are some potential glycosylation sites. At5g19110 is the unique putative proteins that shows leucine and histidine in the inhibition loop 1 and inhibition loop 2, respectively, the same region used by TAXIs-like to interact with glycoside hydrolases family GH11. At5g19100 and At5g19120 show the characteristic LACGs-like deletion at the IL1 and only in At5g19100 sequence at the IL2 we recover the histidine like both LACGs-like and TAXIs-like. We have studied the expression of these genes to deep into the biology of the plant response to pathogen attack. The results of this set of experiment contributed to unveil the role of each. The seeds were germinated in Petri dishes with MS medium and held in growth chambers with constant temperature of 21°C, under long-day photoperiod, for different times (0-23-48-96-144 hours) and in different conditions simulating biotic stresses. Western-blot with antibody anti-Cγ show the presence of a similar γ-conglutin protein in A. thaliana seeds at 96h of germination in MS medium and then incubate in a chitosan (150 mg/L) solution for 3h. For the first time we show that proteolytic processing of LACGs-like occurs in organism other than legume. In parallel, total RNAs were extracted and RT-qPCR has been set up to quantify the relative expression levels of gene expression. The GHIPs homologues genes after 48, 96 and 144 hours of germination aren’t expressed under basal condition. Seeds germinated for 96 hours and then exposed to incubation with aqueous solution or 150mg/l chitosan solution (mimic a biotic stress) shown an expression of gene, greater if the biotic stress was applied at seeds contaminated. In the seeds germinated in dishes that after 96 hours shown a spontaneous growth of Paenibacillus polymyxa, an endophytic bacterium exploited as biocontrolling agent. In this case the expression of all analysed genes increased. In another experimental set we tested the direct effect of GH2, GH5, GH11 treatment on A. thaliana seedling 7 days old. In this contest, it has been evaluated both the expression of the five selected genes and the effects on the enzymes due to plants exudates. By and large, this work describes new experimental findings that open new interesting scenarios to better understand some physiological aspects of the plant defence mechanisms, and provides insight of the structural basis of GHIPs inhibitory activity, specificity and repercussions on cellular responses to pathogens attacks.