Multifaceted roles of plant glycosyl hydrolases during pathogen infections: more to discover.

MAIN CONCLUSION: Carbohydrates are hydrolyzed by a family of carbohydrate-active enzymes (CAZymes) called glycosidases or glycosyl hydrolases. Here, we have summarized the roles of various plant defense glycosidases that possess different substrate specificities. We have also highlighted the open questions in this research field. Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates. Compared to those of all other sequenced organisms, plant genomes contain a remarkable diversity of glycosidases. Plant glycosidases exhibit activities on various substrates and have been shown to play important roles during pathogen infections. Plant glycosidases from different GH families have been shown to act upon pathogen components, host cell walls, host apoplastic sugars, host secondary metabolites, and host N-glycans to mediate immunity against invading pathogens. We could classify the activities of these plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. Here, we have provided comprehensive information on the catalytic activities, GH family classification, subcellular localization, domain structure, functional roles, and microbial strategies to regulate the activities of defense-related plant GHs. We have also emphasized the research gaps and potential investigations needed to advance this topic of research.

A GH81-type ß-glucan-binding protein enhances colonization by mutualistic fungi in barley.

Cell walls are important interfaces of plant-fungal interactions, acting as robust physical and chemical barriers against invaders. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other's cell walls. These enzymes release various sugar oligomers into the apoplast, some of which activate host immunity via surface receptors. Recent characterization of cell walls from plant-colonizing fungi has emphasized the abundance of ß-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated ß-glucan laminarin. Thereby, we identified a plant glycoside hydrolase family 81-type glucan-binding protein (GBP) as a ß-glucan interactor. Mutation of GBP1 and its only paralog, GBP2, in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of colonization was accompanied by enhanced responses at the host cell wall, including an extension of callose-containing cell wall appositions. Moreover, GBP mutation in barley also reduced fungal biomass in roots by the hemibiotrophic pathogen Bipolaris sorokiniana and inhibited the penetration success of the obligate biotrophic leaf pathogen Blumeria hordei. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi-a role that has potentially been appropriated by barley-adapted pathogens.

Chemiluminescence-Based Assay to Monitor Early Oxidative Bursts in Soybean (Glycine max) Lateral Roots.

The reactive oxygen species (ROS) burst assay is a valuable tool for studying pattern-triggered immunity (PTI) in plants. During PTI, the interaction between pathogen recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) leads to the rapid production of ROS in the apoplastic space. The resultant ROS can be measured using a chemiluminescent approach that involves the usage of horseradish peroxidase and luminol. Although several methods and protocols are available to detect early ROS bursts in leaf tissues, no dedicated method is available for root tissues. Here, we have established a reliable method to measure the PAMP-triggered ROS burst response in soybean lateral roots. In plants, lateral roots are the potential entry and colonization sites for pathogens in the rhizosphere. We have used important PAMPs such as chitohexaose, flagellin 22 peptide fragment, and laminarin to validate our method. In addition, we provide a detailed methodology for the isolation and application of fungal cell wall components to monitor the oxidative burst in soybean lateral roots. Furthermore, we provide methodology for performing ROS burst assays in soybean leaf discs with laminarin and fungal cell walls. This approach could also be applied to leaf and root tissues of other plant species to study the PTI response upon elicitor treatment. © 2023 Wiley Periodicals LLC. Basic Protocol: Reactive oxygen species (ROS) burst assay in soybean lateral root tissues Alternate Protocol: ROS burst assay in soybean leaf discs Support Protocol: Isolating fungal cell wall fractions.

Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging ß-glucan decasaccharide to subvert immune responses.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) ß-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved ß-1,3;1,6-glucan decasaccharide (ß-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight ß-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by ß-1,3-endoglucanases due to the presence of three ß-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of ß-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.

The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function.

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.

Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides.

Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted ß-galactosidase 1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O-glycan. In counter defense, P. syringae pathovars evade host immunity by using BGAL1-resistant O-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.

A Genotypic Comparison Reveals That the Improvement in Nitrogen Remobilization Efficiency in Oilseed Rape Leaves Is Related to Specific Patterns of Senescence-Associated Protease Activities and Phytohormones.

Oilseed rape (Brassica napus L.) is an oleoproteaginous crop characterized by low N use efficiency (NUE) that is mainly related to a weak Nitrogen Remobilization Efficiency (NRE) during the sequential leaf senescence of the vegetative stages. Based on the hypothesis that proteolysis efficiency is crucial for the improvement of leafNRE, our objective was to characterize key senescence-associated proteolytic mechanisms of two genotypes (Ténor and Samouraï) previously identified with contrasting NREs. To reach this goal, biochemical changes, protease activities and phytohormone patterns were studied in mature leaves undergoing senescence in two genotypes with contrasting NRE cultivated in a greenhouse under limiting or ample nitrate supply. The genotype with the higher NRE (Ténor) possessed enhanced senescence processes in response to nitrate limitation, and this led to greater degradation of soluble proteins compared to the other genotype (Samouraï). This efficient proteolysis is associated with (i) an increase in serine and cysteine protease (CP) activities and (ii) the appearance of new CP activities (RD21-like, SAG12-like, RD19-like, cathepsin-B, XBCP3-like and aleurain-like proteases) during senescence induced by N limitation. Compared to Samouraï, Ténor has a higher hormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes senescence, particularly under low N conditions, and this is correlated with the stronger protein degradation and serine/CP activities observed during senescence. Short statement: The improvement in N recycling during leaf senescence in a genotype of Brassica napus L. characterized by a high nitrogen remobilization efficiency is related to a high phytohormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes leaf senescence and is correlated with an increase or the induction of specific serine and cysteine protease activities.

Multiplex Fluorescent, Activity-Based Protein Profiling Identifies Active α-Glycosidases and Other Hydrolases in Plants.

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-glycosidase inhibitors in a broad range of plant species. To demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active α-glycosidases and other hydrolases can be analyzed through ABPP in model and nonmodel plants.

Low Protease Content in Medicago truncatula Cell Cultures Facilitates Recombinant Protein Production.

Medicago truncatula is an established model for studying legume biology. More recently, it has also been exploited as a Molecular Farming platform for the production of recombinant proteins, with the successful expression of fungal and human proteins in plants and cell suspension cultures of this species. One of the challenges that now must be overcome is the degradation of final products during production and downstream processing stages. In the M. truncatula genome, there are more than 400 putative protease-encoding genes, but to date, the proteolytic content of Medicago cell cultures has not been studied. In this report, the proteolytic activities that can potentially hamper the successful production of recombinant proteins in this system are evaluated. The potential proteases responsible for the degradation of target proteins are identified. Interestingly, the number of proteases found in Medicago spent medium is considerably lower than that of the well-established tobacco bright yellow 2 (BY-2) system. Papain-like cysteine proteases are found to be the major contributors to recombinant protein degradation in Medicago. This knowledge is used to engineer a cell line with reduced endogenous protease activity by expressing a selective protease inhibitor, further improving this expression platform.

Proteomic Investigations of Proteases Involved in Cotyledon Senescence: A Model to Explore the Genotypic Variability of Proteolysis Machinery Associated with Nitrogen Remobilization Efficiency during the Leaf Senescence of Oilseed Rape.

Oilseed rape is characterized by a low nitrogen remobilization efficiency during leaf senescence, mainly due to a lack of proteolysis. Because cotyledons are subjected to senescence, it was hypothesized that contrasting protease activities between genotypes may be distinguishable early in the senescence of cotyledons. To verify this assumption, our goals were to (i) characterize protease activities in cotyledons between two genotypes with contrasting nitrogen remobilization efficiency (Ténor and Samouraï) under limiting or ample nitrate supply; and (ii) test the role of salicylic acid (SA) and abscisic acid (ABA) in proteolysis regulation. Protease activities were measured and identified by a proteomics approach combining activity-based protein profiling with LC-MS/MS. As in senescing leaves, chlorophyll and protein contents decrease in senescing cotyledons and are correlated with an increase in serine and cysteine protease activities. Two RD21-like and SAG-12 proteases previously associated with an efficient proteolysis in senescing leaves of Ténor are also detected in senescing cotyledons. The infiltration of ABA and SA provokes the induction of senescence and several cysteine and serine protease activities. The study of protease activities during the senescence of cotyledons seems to be a promising experimental model to investigate the regulation and genotypic variability of proteolysis associated with efficient N remobilization.

Proteasome Activity Profiling Uncovers Alteration of Catalytic ß2 and ß5 Subunits of the Stress-Induced Proteasome during Salinity Stress in Tomato Roots.

The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic ß subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic ß subunits. Here, we investigated the existence of stress proteasomes upon abiotic stress (salt stress) in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each ß subunit except for two genes encoding ß2. Using proteasome activity profiling on tomato roots during salt stress, we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of cell viability. This stress-induced active proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the detected stress-induced proteasome is not caused by an altered composition of subunits in active proteasomes, but involves an increased molecular weight of both labeled ß2 and ß5 subunits, and an additional acidic pI shift for labeled ß5, whilst labeled ß1 remains mostly unchanged. Treatment with phosphatase or glycosidases did not affect the migration pattern. This stress-induced proteasome may play an important role in PCD during abiotic stress.

Inhibitor Discovery by Convolution ABPP.

Activity-based protein profiling (ABPP) has emerged as a powerful proteomic approach to study the active proteins in their native environment by using chemical probes that label active site residues in proteins. Traditionally, ABPP is classified as either comparative or competitive ABPP. In this protocol, we describe a simple method called convolution ABPP, which takes benefit from both the competitive and comparative ABPP. Convolution ABPP allows one to detect if a reduced signal observed during comparative ABPP could be due to the presence of inhibitors. In convolution ABPP, the proteomes are analyzed by comparing labeling intensities in two mixed proteomes that were labeled either before or after mixing. A reduction of labeling in the mix-and-label sample when compared to the label-and-mix sample indicates the presence of an inhibitor excess in one of the proteomes. This method is broadly applicable to detect inhibitors in proteomes against any proteome containing protein activities of interest. As a proof of concept, we applied convolution ABPP to analyze secreted proteomes from Pseudomonas syringae-infected Nicotiana benthamiana leaves to display the presence of a beta-galactosidase inhibitor.

Activity-based protein profiling of hydrolytic enzymes induced by gibberellic acid in isolated aleurone layers of malting barley.

During barley germination, the aleurone layer secretes most of the enzymes required to degrade the endosperm, many of which are yet to be characterized. We used activity-based protein profiling (ABPP) to detect a range of active enzymes extracted from aleurone layers isolated from grains of a commercial malting barley variety incubated with or without gibberellic acid (GA). Enzymes found to be induced by GA were putative aleurains, cathepsin-B-like proteases and serine hydrolases. By using an inhibitory sugar panel, a specific active retaining ß-glycosidase in the barley aleurone was identified as a putative xylanase. Our results show that ABPP can be used rapidly to identify a variety of active enzyme isoforms in cereal aleurone without the need for enzyme purification.

Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

Characterization of senescence-associated protease activities involved in the efficient protein remobilization during leaf senescence of winter oilseed rape.

Oilseed rape (Brassica napus L.) is a crop plant characterized by a poor nitrogen (N) use efficiency that is mainly due to low N remobilization efficiency during the sequential leaf senescence of the vegetative stage. As a high leaf N remobilization efficiency was strongly linked to a high remobilization of proteins during leaf senescence of rapeseed, our objective was to identify senescence-associated protease activities implicated in the protein degradation. To reach this goal, leaf senescence processes and protease activities were investigated in a mature leaf becoming senescent in plants subjected to ample or low nitrate supply. The characterization of protease activities was performed by using in vitro analysis of RuBisCO degradation with or without inhibitors of specific protease classes followed by a protease activity profiling using activity-dependent probes. As expected, the mature leaf became senescent regardless of the nitrate treatment, and nitrate limitation enhanced the senescence processes associated with an enhanced degradation of soluble proteins. The characterization of protease activities revealed that: (i) aspartic proteases and the proteasome were active during senescence regardless of nitrate supply, and (ii) the activities of serine proteases and particularly cysteine proteases (Papain-like Cys proteases and vacuolar processing enzymes) increased when protein remobilization associated with senescence was accelerated by nitrate limitation. Short statement: Serine and particularly cysteine proteases (both PLCPs and VPEs) seem to play a crucial role in the efficient protein remobilization when leaf senescence of oilseed rape was accelerated by nitrate limitation.

Beta galactosidases in Arabidopsis and tomato - a mini review.

Beta galactosidases (BGALs) are glycosyl hydrolases that remove terminal ß-D-galactosyl residues from ß-D-galactosides. There are 17 predicted BGAL genes in the genomes of both Arabidopsis (BGAL1-17) and tomato (TBG1-17). All tested BGALs have BGAL activity but their distinct expression profiles and ancient phylogenetic separation indicates that these enzymes fulfil diverse, non-redundant roles in plant biology. The majority of these BGALs are predicted to have signal peptide and thought to act during cell wall-related biological processes. Interestingly, deletion of BGAL6 and BGAL10 in Arabidopsis causes reduced mucilage release during seed imbibition and shorter siliques respectively, whereas TBG4 depletion by RNAi decreases in fruit softening in tomato. The majority of plant BGALs remain to be characterized.

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

Broad-range glycosidase activity profiling.

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.

Reevaluation of the reliability and usefulness of the somatic homologous recombination reporter lines.

A widely used approach for assessing genome instability in plants makes use of somatic homologous recombination (SHR) reporter lines. Here, we review the published characteristics and uses of SHR lines. We found a lack of detailed information on these lines and a lack of sufficient evidence that they report only homologous recombination. We postulate that instead of SHR, these lines might be reporting a number of alternative stress-induced stochastic events known to occur at transcriptional, posttranscriptional, and posttranslational levels. We conclude that the reliability and usefulness of the somatic homologous recombination reporter lines requires revision. Thus, more detailed information about these reporter lines is needed before they can be used with confidence to measure genome instability, including the complete sequences of SHR constructs, the genomic location of reporter genes and, importantly, molecular evidence that reconstituted gene expression in these lines is indeed a result of somatic recombination.