Berberine bridge enzyme-like oxidases of cellodextrins and mixed-linked ß-glucans control seed coat formation.

Plants have evolved various resistance mechanisms to cope with biotic stresses that threaten their survival. The BBE23 member (At5g44360/BBE23) of the Arabidopsis berberine bridge enzyme-like (BBE-l) protein family (Arabidopsis thaliana) has been characterized in this paper in parallel with the closely related and previously described CELLOX (At4g20860/BBE22). In addition to cellodextrins, both enzymes, renamed here as CELLODEXTRIN OXIDASE 2 and 1 (CELLOX2 and CELLOX1), respectively, oxidize the mixed-linked ß-1→3/ß-1→4-glucans (MLGs), recently described as capable of activating plant immunity, reinforcing the view that the BBE-l family includes members that are devoted to the control of the homeostasis of potential cell wall-derived damage-associated molecular patterns (DAMPs). The 2 putatively paralogous genes display different expression profiles. Unlike CELLOX1, CELLOX2 is not expressed in seedlings or adult plants and is not involved in immunity against Botrytis cinerea. Both are instead expressed in a concerted manner in the seed coat during development. Whereas CELLOX2 is expressed mainly during the heart stage, CELLOX1 is expressed at the immediately later stage, when the expression of CELLOX2 decreases. Analysis of seeds of cellox1 and cellox2 knockout mutants shows alterations in the coat structure: the columella area is smaller in cellox1, radial cell walls are thicker in both cellox1 and cellox2, and the mucilage halo is reduced in cellox2. However, the coat monosaccharide composition is not significantly altered, suggesting an alteration of the organization of the cell wall, thus reinforcing the notion that the architecture of the cell wall in specific organs is determined not only by the dynamics of the synthesis/degradation of the main polysaccharides but also by its enzymatic oxidation.

Radical cation scavenging activity of berberine bridge enzyme-like oligosaccharide oxidases acting on short cell wall fragments.

Oligogalacturonide-oxidases (OGOXs) and cellodextrin-oxidase (CELLOX) are plant berberine bridge enzyme-like oligosaccharide-oxidases (OSOXs) that oxidize, respectively, oligogalacturonides (OGs) and cellodextrins (CDs), thereby inactivating their elicitor nature and concomitantly releasing H2O2. Little is known about the physiological role of OSOX activity. By using an ABTS·+-reduction assay, we identified a novel reaction mechanism through which the activity of OSOXs on cell wall oligosaccharides scavenged the radical cation ABTS·+ with an efficiency dependent on the type and length of the oxidized oligosaccharide. In contrast to the oxidation of longer oligomers such as OGs (degree of polymerization from 10 to 15), the activity of OSOXs on short galacturonan- and cellulose-oligomers (degree of polymerization ≤ 4) successfully counteracted the radical cation-generating activity of a fungal laccase, suggesting that OSOXs can generate radical cation scavenging activity in the apoplast with a power proportional to the extent of degradation of the plant cell wall, with possible implications for redox homeostasis and defense against oxidative stress.

Molecular dynamics simulations and kinetic measurements provide insights into the structural requirements of substrate size-dependent specificity of oligogalacturonide oxidase 1 (OGOX1).

Oligogalacturonides (OGs) are pectin fragments released from the breakdown of the homogalacturonan during pathogenesis that act as Damage-Associated Molecular Patterns. OG-oxidase 1 (OGOX1) is an Arabidopsis berberine bridge enzyme-like (BBE-l) oligosaccharide oxidase that oxidizes OGs, impairing their elicitor activity and concomitantly releasing H2O2. The OG-oxidizing activity of OGOX1 is markedly pH-dependent, with optimum pH around 10, and is higher towards OGs with a degree of polymerization higher than two. Here, the molecular determinants of OGOX1 responsible for the binding of OGs with different lengths have been investigated through molecular dynamics simulations and enzyme kinetics studies. OGOX1 was simulated in complex with OGs with different degree of polymerization such as di-, tri-, tetra- and penta-galacturonide, in water solution at alkaline pH. Our simulations revealed that, among the four OGOX1/OG combinations, the penta-galacturonide (OG5) showed the best conformation in the active site to be efficiently oxidized by OGOX1. The optimal conformation can be stabilized by salt-bridges formed between the carboxyl groups of OG5 and five positively charged amino acids of OGOX1, highly conserved in all OGOX paralogs. Our results suggest that these interactions limit the mobility of OG5 as well as longer OGs, contributing to maintain the terminal monomer of OGs in the optimal orientation in order to be oxidized by the enzyme. In accordance with these results, the enzyme efficiency (Kcat/KM) of OGOX1 on OG5 (40.04) was found to be significantly higher than that on OG4 (13.05) and OG3 (0.6).

Characterization of two 1,3-ß-glucan-modifying enzymes from Penicillium sumatraense reveals new insights into 1,3-ß-glucan metabolism of fungal saprotrophs.

BACKGROUND: 1,3-ß-glucan is a polysaccharide widely distributed in the cell wall of several phylogenetically distant organisms, such as bacteria, fungi, plants and microalgae. The presence of highly active 1,3-ß-glucanases in fungi evokes the biological question on how these organisms can efficiently metabolize exogenous sources of 1,3-ß-glucan without incurring in autolysis. RESULTS: To elucidate the molecular mechanisms at the basis of 1,3-ß-glucan metabolism in fungal saprotrophs, the putative exo-1,3-ß-glucanase G9376 and a truncated form of the putative glucan endo-1,3-ß-glucosidase (ΔG7048) from Penicillium sumatraense AQ67100 were heterologously expressed in Pichia pastoris and characterized both in terms of activity and structure. G9376 efficiently converted laminarin and 1,3-ß-glucan oligomers into glucose by acting as an exo-glycosidase, whereas G7048 displayed a 1,3-ß-transglucanase/branching activity toward 1,3-ß-glucan oligomers with a degree of polymerization higher than 5, making these oligomers more recalcitrant to the hydrolysis acted by exo-1,3-ß-glucanase G9376. The X-ray crystallographic structure of the catalytic domain of G7048, solved at 1.9 Å of resolution, consists of a (ß/α)8 TIM-barrel fold characteristic of all the GH17 family members. The catalytic site is in a V-shaped cleft containing the two conserved catalytic glutamic residues. Molecular features compatible with the activity of G7048 as 1,3-ß-transglucanase are discussed. CONCLUSIONS: The antagonizing activity between ΔG7048 and G9376 indicates how opportunistic fungi belonging to Penicillium genus can feed on substrates similar for composition and structure to their own cell wall without incurring in a self-deleterious autohydrolysis.

Berberine Bridge Enzyme-like Oligosaccharide Oxidases Act as Enzymatic Transducers Between Microbial Glycoside Hydrolases and Plant Peroxidases.

Oligogalacturonide (OG)-oxidase 1 (OGOX1) and cellodextrin (CD)-oxidase (CELLOX) are plant berberine bridge enzyme-like oligosaccharide oxidases that oxidize OGs and CDs, cell-wall fragments with the nature of damage-associated molecular patterns. The oxidation of OGs and CDs attenuates their elicitor activity and concomitantly releases H2O2. By using a multiple enzyme-based assay, we demonstrate that the H2O2 generated downstream of the combined action between a fungal polygalacturonase and OGOX1 or an endoglucanase and CELLOX can be directed by plant peroxidases (PODs) either towards a reaction possibly involved in plant defense, such as the oxidation of monolignol or a reaction possibly involved in a developmental event, such as the oxidation of auxin (indole-3-acetic acid), pointing to OGOX1 and CELLOX as enzymatic transducers between microbial glycoside hydrolases and plant PODs. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A molecular dynamics-guided mutagenesis identifies two aspartic acid residues involved in the pH-dependent activity of OG-OXIDASE 1.

During the infection, plant cells secrete different OG-oxidase (OGOX) paralogs, defense flavoproteins that oxidize the oligogalacturonides (OGs), homogalacturonan fragments released from the plant cell wall that act as Damage Associated Molecular Patterns. OGOX-mediated oxidation inactivates their elicitor nature, but on the other hand makes OGs less hydrolysable by microbial endo-polygalacturonases (PGs). Among the different plant defense responses, apoplastic alkalinization can further reduce the degrading potential of PGs by boosting the oxidizing activity of OGOXs. Accordingly, the different OGOXs so far characterized showed an optimal activity at pH values greater than 8. Here, an approach of molecular dynamics (MD)-guided mutagenesis succeeded in identifying the amino acids responsible for the pH dependent activity of OGOX1 from Arabidopsis thaliana. MD simulations indicated that in alkaline conditions (pH 8.5), the residues Asp325 and Asp344 are engaged in the formation of two salt bridges with Arg327 and Lys415, respectively, at the rim of enzyme active site. According to MD analysis, the presence of such ionic bonds modulates the size and flexibility of the cavity used to accommodate the OGs, in turn affecting the activity of OGOX1. Based on functional properties of the site-directed mutants OGOX1.D325A and OGOX.D344A, we demonstrated that Asp325 and Asp344 are major determinants of the alkaline-dependent activity of OGOX1.

A microalgal-based preparation with synergistic cellulolytic and detoxifying action towards chemical-treated lignocellulose.

High-temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the ß-glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non-axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline-treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by-products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose-derived sugars by C. vulgaris under mixotrophic growth.

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting.

Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus, was constitutively expressed in N. tabacum by targeting the enzyme both to the apoplast and to the protein storage vacuole. The apoplast targeting failed to isolate plants expressing the recombinant enzyme despite a large number of transformants being screened. On the opposite side, the targeting of the cellobiohydrolase to the protein storage vacuole led to several transgenic lines expressing CBM3GH5, with an enzyme yield of up to 0.08 mg g DW-1 (1.67 Units g DW-1) in the mature leaf tissue. The analysis of CBM3GH5 activity revealed that the enzyme accumulated in different plant organs in a developmental-dependent manner, with the highest abundance in mature leaves and roots, followed by seeds, stems and leaf ribs. Notably, both leaves and stems from transgenic plants were characterized by an improved temperature-dependent saccharification profile.

Industrial Use of Cell Wall Degrading Enzymes: The Fine Line Between Production Strategy and Economic Feasibility.

Cell Wall Degrading Enzymes (CWDEs) are a heterogeneous group of enzymes including glycosyl-hydrolases, oxidoreductases, lyases, and esterases. Microbes with degrading activities toward plant cell wall polysaccharides are the most relevant source of CWDEs for industrial applications. These organisms secrete a wide array of CWDEs in amounts strictly necessary for their own sustenance, nonetheless the production of CWDEs from wild type microbes can be increased at large-scale by using optimized fermentation strategies. In the last decades, advances in genetic engineering allowed the expression of recombinant CWDEs also in lab-domesticated organisms such as E. coli, yeasts and plants, dramatically increasing the available options for the large-scale production of CWDEs. The optimization of a CWDE-producing biofactory is a hard challenge that biotechnologists tackle by testing different expression strategies and expression-hosts. Although both the yield and production costs are critical factors to produce biomolecules at industrial scale, these parameters are often disregarded in basic research. This review presents the main characteristics and industrial applications of CWDEs directed toward the cell wall of plants, bacteria, fungi and microalgae. Different biofactories for CWDE expression are compared in order to highlight strengths and weaknesses of each production system and how these aspects impact the final enzyme cost and, consequently, the economic feasibility of using CWDEs for industrial applications.

Dampening the DAMPs: How Plants Maintain the Homeostasis of Cell Wall Molecular Patterns and Avoid Hyper-Immunity.

Several oligosaccharide fragments derived from plant cell walls activate plant immunity and behave as typical damage-associated molecular patterns (DAMPs). Some of them also behave as negative regulators of growth and development, and due to their antithetic effect on immunity and growth, their concentrations, activity, time of formation, and localization is critical for the so-called "growth-defense trade-off." Moreover, like in animals, over accumulation of DAMPs in plants provokes deleterious physiological effects and may cause hyper-immunity if the cellular mechanisms controlling their homeostasis fail. Recently, a mechanism has been discovered that controls the activity of two well-known plant DAMPs, oligogalacturonides (OGs), released upon hydrolysis of homogalacturonan (HG), and cellodextrins (CDs), products of cellulose breakdown. The potential homeostatic mechanism involves specific oxidases belonging to the family of berberine bridge enzyme-like (BBE-like) proteins. Oxidation of OGs and CDs not only inactivates their DAMP activity, but also makes them a significantly less desirable food source for microbial pathogens. The evidence that oxidation and inactivation of OGs and CDs may be a general strategy of plants for controlling the homeostasis of DAMPs is discussed. The possibility exists of discovering additional oxidative and/or inactivating enzymes targeting other DAMP molecules both in the plant and in animal kingdoms.

Combined resistance to oxidative stress and reduced antenna size enhance light-to-biomass conversion efficiency in Chlorella vulgaris cultures.

BACKGROUND: Microalgae are efficient producers of lipid-rich biomass, making them a key component in developing a sustainable energy source, and an alternative to fossil fuels. Chlorella species are of special interest because of their fast growth rate in photobioreactors. However, biological constraints still cast a significant gap between the high cost of biofuel and cheap oil, thus hampering perspective of producing CO2-neutral biofuels. A key issue is the inefficient use of light caused by its uneven distribution in the culture that generates photoinhibition of the surface-exposed cells and darkening of the inner layers. Efficient biofuel production, thus, requires domestication, including traits which reduce optical density of cultures and enhance photoprotection. RESULTS: We applied two steps of mutagenesis and phenotypic selection to the microalga Chlorella vulgaris. First, a pale-green mutant (PG-14) was selected, with a 50% reduction of both chlorophyll content per cell and LHCII complement per PSII, with respect to WT. PG-14 showed a 30% increased photon conversion into biomass efficiency vs. WT. A second step of mutagenesis of PG-14, followed by selection for higher tolerance to Rose Bengal, led to the isolation of pale-green genotypes, exhibiting higher resistance to singlet oxygen (strains SOR). Growth in photobioreactors under high light conditions showed an enhanced biomass production of SOR strains with respect to PG-14. When compared to WT strain, biomass yield of the pale green + sor genotype was enhanced by 68%. CONCLUSIONS: Domestication of microalgae like Chlorella vulgaris, by optimizing both light distribution and ROS resistance, yielded an enhanced carbon assimilation rate in photobioreactor.

Design of a highly thermostable hemicellulose-degrading blend from Thermotoga neapolitana for the treatment of lignocellulosic biomass.

The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-ß-galactanase and an α-l-arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (kcat values of ˜177 and ˜133 s-1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases - endo-1,4-ß-xylanase (XynA), endo-1,4-ß-mannanase (ManB/Man5A) and ß-glucosidase (GghA) - to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases.

An Arabidopsis berberine bridge enzyme-like protein specifically oxidizes cellulose oligomers and plays a role in immunity.

The plant cell wall is the barrier that pathogens must overcome to cause a disease, and to this end they secrete enzymes that degrade the various cell wall components. Due to the complexity of these components, several types of oligosaccharide fragments may be released during pathogenesis and some of these can act as damage-associated molecular patterns (DAMPs). Well-known DAMPs are the oligogalacturonides (OGs) released upon degradation of homogalacturonan and the products of cellulose breakdown, i.e. the cellodextrins (CDs). We have previously reported that four Arabidopsis berberine bridge enzyme-like (BBE-like) proteins (OGOX1-4) oxidize OGs and impair their elicitor activity. We show here that another Arabidopsis BBE-like protein, which is expressed coordinately with OGOX1 during immunity, specifically oxidizes CDs with a preference for cellotriose (CD3) and longer fragments (CD4-CD6). Oxidized CDs show a negligible elicitor activity and are less easily utilized as a carbon source by the fungus Botrytis cinerea. The enzyme, named CELLOX (cellodextrin oxidase), is encoded by the gene At4 g20860. Plants overexpressing CELLOX display an enhanced resistance to B. cinerea, probably because oxidized CDs are a less valuable carbon source. Thus, the capacity to oxidize and impair the biological activity of cell wall-derived oligosaccharides seems to be a general trait of the family of BBE-like proteins, which may serve to homeostatically control the level of DAMPs to prevent their hyperaccumulation.

Biomass from microalgae: the potential of domestication towards sustainable biofactories.

Interest in bulk biomass from microalgae, for the extraction of high-value nutraceuticals, bio-products, animal feed and as a source of renewable fuels, is high. Advantages of microalgal vs. plant biomass production include higher yield, use of non-arable land, recovery of nutrients from wastewater, efficient carbon capture and faster development of new domesticated strains. Moreover, adaptation to a wide range of environmental conditions evolved a great genetic diversity within this polyphyletic group, making microalgae a rich source of interesting and useful metabolites. Microalgae have the potential to satisfy many global demands; however, realization of this potential requires a decrease of the current production costs. Average productivity of the most common industrial strains is far lower than maximal theoretical estimations, suggesting that identification of factors limiting biomass yield and removing bottlenecks are pivotal in domestication strategies aimed to make algal-derived bio-products profitable on the industrial scale. In particular, the light-to-biomass conversion efficiency represents a major constraint to finally fill the gap between theoretical and industrial productivity. In this respect, recent results suggest that significant yield enhancement is feasible. Full realization of this potential requires further advances in cultivation techniques, together with genetic manipulation of both algal physiology and metabolic networks, to maximize the efficiency with which solar energy is converted into biomass and bio-products. In this review, we draft the molecular events of photosynthesis which regulate the conversion of light into biomass, and discuss how these can be targeted to enhance productivity through mutagenesis, strain selection or genetic engineering. We outline major successes reached, and promising strategies to achieving significant contributions to future microalgae-based biotechnology.

Four Arabidopsis berberine bridge enzyme-like proteins are specific oxidases that inactivate the elicitor-active oligogalacturonides.

Recognition of endogenous molecules acting as 'damage-associated molecular patterns' (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper-accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth-defence trade-off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper-accumulation. By combining affinity chromatography on acrylamide-trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo-protein, OGOX1 is a sulphite-sensitive H2 O2 -producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD-binding proteins [Berberine Bridge Enzyme-like (BBE-like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE-like enzymes in Arabidopsis are OG oxidases (OGOX1-4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity.

Methods of Isolation and Characterization of Oligogalacturonide Elicitors.

Oligogalacturonides (OGs) are pectic fragments derived from the partial degradation of homogalacturonan in the plant cell wall and able to elicit plant defence responses. Recent methodological advances in the isolation of OGs from plant tissues and their characterization have confirmed their role as bona fide plant Damage-Associated Molecular Patterns. Here, we describe the methods for the isolation of OGs from Arabidopsis leaf tissues and for the characterization of OG structure and biological activity.

Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns.

Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP-PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense.

An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens.

Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens.

A single amino-acid substitution allows endo-polygalacturonase of Fusarium verticillioides to acquire recognition by PGIP2 from Phaseolus vulgaris.

Polygalacturonases (PGs) are secreted by phytopathogenic fungi to degrade the plant cell wall homogalacturonan during plant infection. To counteract Pgs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs) that slow down fungal infection and defend cell wall integrity. PGIPs favour the accumulation of oligogalacturonides, which are homogalacturonan fragments that act as endogenous elicitors of plant defence responses. We have previously shown that PGIP2 from Phaseolus vulgaris (PvPGIP2) forms a complex with PG from Fusarium phyllophilum (FpPG), hindering the enzyme active site cleft from substrate. Here we analyse by small angle X-ray scattering (SAXS) the interaction between PvPGIP2 and a PG from Colletotrichum lupini (CluPG1). We show a different shape of the PG-PGIP complex, which allows substrate entry and provides a structural explanation for the different inhibition kinetics exhibited by PvPGIP2 towards the two isoenzymes. The analysis of SAXS structures allowed us to investigate the basis of the inability of PG from Fusarium verticilloides (FvPG) to be inhibited by PvPGIP2 or by any other known PGIP. FvPG is 92.5% identical to FpPG, and we show here, by both loss- and gain-of-function mutations, that a single amino acid site acts as a switch for FvPG recognition by PvPGIP2.

Structural resolution of the complex between a fungal polygalacturonase and a plant polygalacturonase-inhibiting protein by small-angle X-ray scattering.

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.