Rice apoplastic CBM1-interacting protein counters blast pathogen invasion by binding conserved carbohydrate binding module 1 motif of fungal proteins.

When infecting plants, fungal pathogens secrete cell wall-degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsRMC binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against M. oryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution.

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

Cooperative regulation of PBI1 and MAPKs controls WRKY45 transcription factor in rice immunity.

The U-box type ubiquitin ligase PUB44 positively regulates pattern-triggered immunity in rice. Here, we identify PBI1, a protein that interacts with PUB44. Crystal structure analysis indicates that PBI1 forms a four-helix bundle structure. PBI1 also interacts with WRKY45, a master transcriptional activator of rice immunity, and negatively regulates its activity. PBI1 is degraded upon perception of chitin, and this is suppressed by silencing of PUB44 or expression of XopP, indicating that PBI1 degradation depends on PUB44. These data suggest that PBI1 suppresses WRKY45 activity when cells are in an unelicited state, and during chitin signaling, PUB44-mediated degradation of PBI1 leads to activation of WRKY45. In addition, chitin-induced MAP kinase activation is required for WRKY45 activation and PBI1 degradation. These results demonstrate that chitin-induced activation of WRKY45 is regulated by the cooperation between MAP kinase-mediated phosphorylation and PUB44-mediated PBI1 degradation.

A jacalin-like lectin domain-containing protein of Sclerospora graminicola acts as an apoplastic virulence effector in plant-oomycete interactions.

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant-pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from Sg (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs by protein mass spectrometry analysis of extracellular fluid from Sg-inoculated foxtail millet leaves. SgJRLs consist of a jacalin-like lectin domain and an N-terminal putative secretion signal; SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with green fluorescent protein (GFP) localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defence-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defence responses.

Sugar Composition in Asparagus Spears and Its Relationship to Soil Chemical Properties.

Glycoside hydrolases require carboxyl groups as catalysts for their activity. A retaining xylanase from Streptomyces olivaceoviridis E-86 belonging to glycoside hydrolase family 10 possesses Glu128 and Glu236 that respectively function as acid/base and nucleophile. We previously developed a unique mutant of the retaining xylanase, N127S/E128H, whose deglycosylation is triggered by azide. A crystallographic study reported that the transient formation of a Ser-His catalytic dyad in the reaction cycle possibly reduced the azidolysis reaction. In the present study, we engineered a catalytic dyad with enhanced stability by site-directed mutagenesis and crystallographic study of N127S/E128H. Comparison of the Michaelis complexes of N127S/E128H with pNP-X2 and with xylopentaose showed that Ser127 could form an alternative hydrogen bond with Thr82, which disrupts the formation of the Ser-His catalytic dyad. The introduction of T82A mutation in N127S/E128H produces an enhanced first-order rate constant (6 times that of N127S/E128H). We confirmed the presence of a stable Ser-His hydrogen bond in the Michaelis complex of the triple mutant, which forms the productive tautomer of His128 that acts as an acid catalyst. Because the glycosyl azide is applicable in the bioconjugation of glycans by using click chemistry, the enzyme-assisted production of the glycosyl azide may contribute to the field of glycobiology.

Gtgen3A, a novel plant GH3 ß-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana.

Gentiobiose, a ß-1,6-linked glycosyl-disaccharide, accumulates abundantly in Gentianaceae and is involved in aspects of plant development, such as fruits ripening and release of bud dormancy. However, the mechanisms regulating the amount of gentio-oligosaccharide accumulation in plants remain obscure. The present study aimed to identify an enzyme that modulates gentio-oligosaccharide amount in gentian (Gentiana triflora). A protein responsible for gentiobiose hydrolysis, GtGen3A, was identified by partial purification and its peptide sequence analysis. The enzyme had a molecular mass of ∼67 kDa without a secretory signal peptide sequence. Sequence analysis revealed that GtGen3A could be a ß-glucosidase member belonging to glycoside hydrolase family 3 (GH3). GtGen3A showed a homology to GH3 ß-glucan exohydrolases, ExoI of Hordeum vulgare, and ExgI from Zea mays, which preferentially hydrolyzed ß-1,3- and ß-1,4-linked oligosaccharides. The purified recombinant GtGen3A (rGtGen3A) produced in Escherichia coli showed optimal reaction at pH 6.5 and 20°C. The rGtGen3A liberated glucose from ß-1,2-, ß-1,3-, ß-1,4-, and ß-1,6-linked oligosaccharides, and showed the highest activity toward gentiotriose among the substrates tested. Kinetic analysis also revealed that rGtGen3A preferentially hydrolyzed gentiotriose. Virus-induced gene silencing of Gtgen3A in gentian plantlets resulted in predominant accumulation of gentiotriose rather than gentiobiose. Furthermore, the expression level of Gtgen3A was almost similar to the amount of gentiobiose in field-grown gentians. These findings suggest that the main function of GtGen3A is the hydrolysis of gentiotriose to gentiobiose, and that GtGen3A plays a role in modulating gentiobiose amounts in gentian.

Identification and enzymatic characterization of an endo-1,3-ß-glucanase from Euglena gracilis.

Euglena produces paramylon as a storage polysaccharide, and is thought to require ß-1,3-glucan degrading enzymes to release and utilize the accumulated carbohydrate. To investigate ß-1,3-glucan degradation in Euglena, endo-1,3-ß-glucanases were partially purified from Euglena gracilis by hydrophobic, gel filtration and anion-exchange chromatography. Tryptic digests and mass-spectrometric analysis identified three proteins in the purified fraction as a member of glycoside hydrolase family (GH) 17 and two members of GH81. These genes were cloned from an Euglena cDNA pool by PCR. EgCel17A fused with a histidine-tag at the carboxy terminus was heterologously produced by Aspergillus oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40kDa by SDS-PAGE, which was identical to that deduced from its amino acid sequence. The enzyme showed hydrolytic activity towards ß-1,3-glucans such as laminarin and paramylon. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0-5.5 and 60°C after 1h incubation or 50°C after 20h incubation. The enzyme had a Km of 0.21mg/ml and a Vmax of 40.5units/mg protein for laminarin degradation at pH 5.0 and 50°C. Furthermore, EgCel17A catalyzed a transglycosylation reaction by which reaction products with a higher molecular weight than the supplied substrates were initially generated; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble ß-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-ß-glucanase to be identified from E. gracilis.

Fungal hemicellulose-degrading enzymes cause physical property changes concomitant with solubilization of cell wall polysaccharides.

MAIN CONCLUSION: Physical properties of wheat coleoptile segments decreased after treatment with hemicellulose-degrading enzymes, indicating that hemicellulosic polysaccharides function to control the strength of primary cell walls. Changes in the physical properties of plant cell walls, a viscoelastic structure, are thought to be one of the growth-limiting factors for plants and one of the infection-affecting factors for fungi. To study the significance of hemicellulosic polysaccharides that form cross-bridges between cellulose microfibrils in controlling cell wall strength in monocot plants, the effects of hemicellulose degradation by recombinant Magnaporthe oryzae xylanase and 1,3-1,4-ß-glucanase, and recombinant Aspergillus oryzae xyloglucanase on the physical properties and polysaccharide solubilization were investigated using wheat (Triticum aestivum L.) coleoptiles. Treatments with xylanase or 1,3-1,4-ß-glucanase significantly decreased the viscosity and elasticity of wheat coleoptile segments. In addition, xyloglucanase treatment slightly decreased the viscoelasticity. Furthermore, 1,3-1,4-ß-glucan polymer was solubilized during hydrolysis with xylanase and xyloglucanase, even though neither enzyme had hydrolytic activity towards 1,3-1,4-ß-glucan. These results suggest that xylan and xyloglucan interact with 1,3-1,4-ß-glucan and that the composites and hemicellulosic polysaccharides form inter-molecular bridges. Degradation of these bridges causes decreases in the physical properties, resulting in increased extensibility of the cell walls. These findings provide a testable model in which wheat coleoptile cell walls are loosened by the degradation of hemicellulosic polysaccharides and hemicellulose-degrading enzymes play a significant role in loosening the walls during fungal infection.

The gentio-oligosaccharide gentiobiose functions in the modulation of bud dormancy in the herbaceous perennial Gentiana.

Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [ß-D-Glcp-(1→6)-D-Glc] and gentianose [ß-D-Glcp-(1→6)-D-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of ß-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.

Purification and characterization of UDP-arabinopyranose mutase from Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[(14)C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.

Polysaccharide-inducible endoglucanases from Lentinula edodes exhibit a preferential hydrolysis of 1,3-1,4-ß-glucan and xyloglucan.

Three genes encoding glycoside hydrolase family 12 (GH12) enzymes from Lentinula edodes, namely Lecel12A, Lecel12B, and Lecel12C, were newly cloned by PCR using highly conserved sequence primers. To investigate enzymatic properties, recombinant enzymes encoded by L. edodes DNAs and GH12 genes from Postia placenta (PpCel12A and PpCel12B) and Schizophyllum commune (ScCel12A) were prepared in Brevibacillus choshinensis. Recombinant LeCel12A, PpCel12A, and PpCel12B, which were grouped in GH12 subfamily 1, preferentially hydrolyzed 1,3-1,4-ß-glucan. By contrast, LeCel12B, LeCel12C, and ScCel12A, members of the subfamily 2, exhibited specific hydrolysis of xyloglucan. These results suggest that two subfamilies of GH12 are separated based on the substrate specificity. Transcript levels of L. edodes genes increased 72 h after growth of L. edodes mycelia cells in the presence of plant cell wall polymers such as xyloglucan, 1,3-1,4-ß-glucan, and cellulose. These results suggest that L. edodes GH12 enzymes have evolved to hydrolyze 1,3-1,4-ß-glucan and xyloglucan, which might enhance hyphal extension and nutrient acquisition.

Degradation and synthesis of ß-glucans by a Magnaporthe oryzae endotransglucosylase, a member of the glycoside hydrolase 7 family.

BACKGROUND: Plant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition. RESULTS: A novel endotransglucosylase catalyzes cleavage and transfer of ß-glucans and decreases the physical strength of plant cell walls. CONCLUSION: Endotransglucosylation causes depolymerization and polymerization of ß-glucans, depending on substrate molecular size. SIGNIFICANCE: Enzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-ß-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing ß-1,4-linkages, in which 1,3-1,4-ß-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into ß-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of ß-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-ß-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize ß-glucans, depending on the size of the acceptor substrate.

A novel glycosylphosphatidylinositol-anchored glycoside hydrolase from Ustilago esculenta functions in ß-1,3-glucan degradation.

A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative ß-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 ß-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward ß-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify ß-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of ß-1,3-glucan after its release in the extracellular medium.

Characterization of ß-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom).

We purified and cloned a ß-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of ß-N-acetylgalactosaminides and ß-N-acetylglucosaminides, indicating that LeHex20A is a ß-N-acetylhexosaminidase classified into EC 3.2.1.52. The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 ß-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.

Identification, cloning, and characterization of ß-glucosidase from Ustilago esculenta.

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 ß-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 ß-glucosidase, a gene encoding a putative GH3 ß-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 ß-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from ß-1,3-, ß-1,4-, and ß-1,6-linked oligosaccharides, and from 1,3-1,4-ß-glucan and laminarin polysaccharides, indicating that UeBgl3A is a ß-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.

Biochemical characterization of Magnaporthe oryzae ß-glucosidases for efficient ß-glucan hydrolysis.

ß-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0-5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on ß-1,3-linked oligosaccharides than on ß-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-ß-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-ß-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-ß-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.

Characterization of endo-1,3-1,4-ß-glucanases in GH family 12 from Magnaporthe oryzae.

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze ß-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-ß-glucans, such as barley ß-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-ß-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-ß-glucan during infection and spore formation.

Characterization of a cellobiohydrolase (MoCel6A) produced by Magnaporthe oryzae.

Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), ß-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.

Anionic derivatives of xyloglucan function as acceptor but not donor substrates for xyloglucan endotransglucosylase activity.

Tamarind xyloglucan was oxidised by reaction with sodium hypochlorite in the presence of 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO). Galactose residues and non-xylosylated glucose residues were thus converted into galacturonic and glucuronic acid residues, respectively, producing an anionic polysaccharide. Acid hydrolysis of oxidised xyloglucan yielded two aldobiouronic acids, deduced to be beta-D: -GalpA-(1-->2)-D-Xyl and beta-D: -GlcpA-(1-->4)-D-Glc. Anionic xyloglucan had a decreased ability to hydrogen-bond to cellulose and to complex with iodine. It was almost totally resistant to digestion by cellulase [endo-(1-->4)-beta-glucanase] and did not serve as a donor substrate for xyloglucan endotransglucosylase (XET) activity. Like several other anionic polysaccharides, it promoted XET activity when unmodified (non-ionic) xyloglucan was used as donor substrate. Anionic xyloglucan may mimic polyanions whose presence in the plant cell wall promotes the action of endogenous XTH proteins. NaOCl with TEMPO oxidised the heptasaccharide, XXXG, to form XXX-glucarate, which did serve as an acceptor substrate although at a rate approximately fourfold less than XXXG itself. Anionic derivatives of xyloglucan, acting as acceptor but not donor substrates, may be valuable tools for exploring the biological roles of XTHs in the integration versus the re-structuring of xyloglucan in the plant cell wall.

A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose.

Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants.