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.

Oligogalacturonide application increases resistance to Fusarium head blight in durum wheat.

Fusariosis causes substantial yield losses in the wheat crop worldwide and compromises food safety because of the presence of toxins associated with the fungal disease. Among the current approaches to crop protection, the use of elicitors able to activate natural defense mechanisms in plants is a strategy gaining increasing attention. Several studies indicate that applications of plant cell-wall-derived elicitors, such as oligogalacturonides (OGs) derived from partial degradation of pectin, induce local and systemic resistance against plant pathogens. The aim of this study was to establish the efficacy of OGs in protecting durum wheat (Triticum turgidum subsp. durum), which is characterized by an extreme susceptibility to Fusarium graminearum. To evaluate the functionality of OGs, spikes and seedlings of cv. Svevo were inoculated with OGs, F. graminearum spores, and a co-treatment of both. Results demonstrated that OGs are active elicitors of wheat defenses, triggering typical immune marker genes and determining regulation of fungal genes. Moreover, bioassays on spikes and transcriptomic analyses on seedlings showed that OGs can regulate relevant physiological processes in Svevo with dose-dependent specificity. Thus, the OG sensing system plays an important role in fine tuning immune signaling pathways in durum wheat.

The Cotton Wall-Associated Kinase GhWAK7A Mediates Responses to Fungal Wilt Pathogens by Complexing with the Chitin Sensory Receptors.

Plant receptor-like kinases (RLKs) are important players in response to pathogen infections. Verticillium and Fusarium wilts, caused by Verticillium dahliae (Vd) and Fusarium oxysporum f. sp vasinfectum (Fov), respectively, are among the most devastating diseases in cotton (Gossypium spp). To understand the cotton response to these soil-borne fungal pathogens, we performed a genome-wide in silico characterization and functional screen of diverse RLKs for their involvement in cotton wilt diseases. We identified Gossypium hirsutum GhWAK7A, a wall-associated kinase, that positively regulates cotton response to both Vd and Fov infections. Chitin, the major constituent of the fungal cell wall, is perceived by lysin-motif-containing RLKs (LYKs/CERK1), leading to the activation of plant defense against fungal pathogens. A conserved chitin sensing and signaling system is present in cotton, including chitin-induced GhLYK5-GhCERK1 dimerization and phosphorylation, and contributes to cotton defense against Vd and Fov Importantly, GhWAK7A directly interacts with both GhLYK5 and GhCERK1 and promotes chitin-induced GhLYK5-GhCERK1 dimerization. GhWAK7A phosphorylates GhLYK5, which itself does not have kinase activity, but requires phosphorylation for its function. Consequently, GhWAK7A plays a crucial role in chitin-induced responses. Thus, our data reveal GhWAK7A as an important component in cotton response to fungal wilt pathogens by complexing with the chitin receptors.

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.

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.

An oligogalacturonide-derived molecular probe demonstrates the dynamics of calcium-mediated pectin complexation in cell walls of tip-growing structures.

Pectic homogalacturonan (HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell-to-cell adhesion. Current immunological methods enable only steady-state detection of egg box formation in situ. Here we present a tool for efficient real-time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium-mediated binding of fluorescently tagged long oligogalacturonides (OGs) with endogenous de-esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real-time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip-growing structures.

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.

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.

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.

AtPME17 is a functional Arabidopsis thaliana pectin methylesterase regulated by its PRO region that triggers PME activity in the resistance to Botrytis cinerea.

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid-ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.

Changes in the microsomal proteome of tomato fruit during ripening.

The variations in the membrane proteome of tomato fruit pericarp during ripening have been investigated by mass spectrometry-based label-free proteomics. Mature green (MG30) and red ripe (R45) stages were chosen because they are pivotal in the ripening process: MG30 corresponds to the end of cellular expansion, when fruit growth has stopped and fruit starts ripening, whereas R45 corresponds to the mature fruit. Protein patterns were markedly different: among the 1315 proteins identified with at least two unique peptides, 145 significantly varied in abundance in the process of fruit ripening. The subcellular and biochemical fractionation resulted in GO term enrichment for organelle proteins in our dataset, and allowed the detection of low-abundance proteins that were not detected in previous proteomic studies on tomato fruits. Functional annotation showed that the largest proportion of identified proteins were involved in cell wall metabolism, vesicle-mediated transport, hormone biosynthesis, secondary metabolism, lipid metabolism, protein synthesis and degradation, carbohydrate metabolic processes, signalling and response to stress.

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.

Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana.

Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity.

Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea.

Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2'-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography-mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.

Sensitive detection and measurement of oligogalacturonides in Arabidopsis.

Oligogalacturonides (OGs) are pectin fragments derived from the partial hydrolysis of the plant cell wall pectin; they are elicitors of various defense responses. While their activity is well documented, the detection of OGs produced in planta is still a challenging task. A protocol has been developed for the extraction and analysis of OGs from small samples of Arabidopsis tissues by using fluorescent labeled OGs, which allowed to monitor the efficiency of extraction. An efficient recovery was obtained by using a combination of calcium chelating agents at acidic pH. Off-line coupling of high performance anion exchange chromatography with matrix assisted laser desorption ionization- time of flight-mass spectrometryor nanoESI-Orbitrap-MS/MS was used for the identification and characterization of oligosaccharides. The protocol was successfully applied to detect OGs by using low amounts (50 mg) of Arabidopsis leaves and very low amounts (30 mg) of senescent leaves. The protocol was also successfully used to detect OGs in Arabidopsis cell wall material digested with pectinases. The proposed extraction protocol followed by sensitive and high-resolution analysis methods allowed detection of OGs released from the cell wall in Arabidopsis tissues by using minimal sample material. The protocol may be useful to study OG-triggered plant immunity and cell wall remodeling during Arabidopsis growth and development.

Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth.

Lignocellulosic biomass from agriculture wastes is a potential source of biofuel, but its use is currently limited by the recalcitrance of the plant cell wall to enzymatic digestion. Modification of the wall structural components can be a viable strategy to overcome this bottleneck. We have previously shown that the expression of a fungal polygalacturonase (pga2 from Aspergillus niger) in Arabidopsis and tobacco plants reduces the levels of de-esterified homogalacturonan in the cell wall and significantly increases saccharification efficiency. However, plants expressing pga2 show stunted growth and reduced biomass production, likely as a consequence of an extensive loss of pectin integrity during the whole plant life cycle. We report here that the expression in Arabidopsis of another pectic enzyme, the pectate lyase 1 (PL1) of Pectobacterium carotovorum, under the control of a chemically inducible promoter, results, after induction of the transgene, in a saccharification efficiency similar to that of plants expressing pga2. However, lines with high levels of transgene induction show reduced growth even in the absence of the inducer. To overcome the problem of plant fitness, we have generated Arabidopsis plants that express pga2 under the control of the promoter of SAG12, a gene expressed only during senescence. These plants expressed pga2 only at late stages of development, and their growth was comparable to that of WT plants. Notably, leaves and stems of transgenic plants were more easily digested by cellulase, compared to WT plants, only during senescence. Expression of cell wall-degrading enzymes at the end of the plant life cycle may be therefore a useful strategy to engineer crops unimpaired in biomass yield but improved for bioconversion.

Transgenic expression of a fungal endo-polygalacturonase increases plant resistance to pathogens and reduces auxin sensitivity.

Polygalacturonases (PGs), enzymes that hydrolyze the homogalacturonan of the plant cell wall, are virulence factors of several phytopathogenic fungi and bacteria. On the other hand, PGs may activate defense responses by releasing oligogalacturonides (OGs) perceived by the plant cell as host-associated molecular patterns. Tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants expressing a fungal PG (PG plants) have a reduced content of homogalacturonan. Here, we show that PG plants are more resistant to microbial pathogens and have constitutively activated defense responses. Interestingly, either in tobacco PG or wild-type plants treated with OGs, resistance to fungal infection is suppressed by exogenous auxin, whereas sensitivity to auxin of PG plants is reduced in different bioassays. The altered plant defense responses and auxin sensitivity in PG plants may reflect an increased accumulation of OGs and subsequent antagonism of auxin action. Alternatively, it may be a consequence of perturbations of cellular physiology and elevated defense status as a result of altered cell wall architecture.

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.