Do Lytic Polysaccharide Monooxygenases Aid in Plant Pathogenesis and Herbivory?

Lytic polysaccharide monooxygenases (LPMOs), copper-dependent enzymes mainly found in fungi, bacteria, and viruses, are responsible for enabling plant infection and degradation processes. Since their discovery 10 years ago, significant progress has been made in understanding the major role these enzymes play in biomass conversion. The recent discovery of additional LPMO families in fungi and oomycetes (AA16) as well as insects (AA15) strongly suggests that LPMOs might also be involved in biological processes such as overcoming plant defenses. In this review, we aim to give a comprehensive overview of the potential role of different LPMO families from the perspective of plant defense and their multiple implications in devising new strategies for achieving crop protection from plant pathogens and insect pests.

An AA9-LPMO containing a CBM1 domain in Aspergillus nidulans is active on cellulose and cleaves cello-oligosaccharides.

Lytic polysaccharide monooxygenases (LPMOs) are copper dependent enzymes that carry out oxidative cleavage of cellulose and other polysaccharides. Aspergillus nidulans, an ascomycete fungus that contains multiple AA9 LPMOs in the genome, offers an excellent model system to study their activity during the oxidative degradation of biomass. AN1602, a dual domain AA9-LPMO in A. nidulans appended with a carbohydrate-binding module, CBM1, was expressed in Pichia pastoris for analyzing oxidative cleavage on cellulosic substrates. The mass spectral and HPAEC analyses showed that the enzyme cleaves phosphoric acid swollen cellulose (PASC) in the presence of a reducing agent, yielding a range of cello-oligosaccharides. In addition to the polymeric substrate cellulose, AN1602 is also active on soluble cellohexaose, a property that is restricted to only a few characterized LPMOs. Product analysis of AN1602 cleaved cellohexaose revealed that C4 was the sole site of oxidation. The sequence and predicted structure of the catalytic domain of AN1602 matched very closely to known C4 cellohexaose active enzymes.

A family of AA9 lytic polysaccharide monooxygenases in Aspergillus nidulans is differentially regulated by multiple substrates and at least one is active on cellulose and xyloglucan.

Fungal genomes contain multiple genes encoding AA9 lytic polysaccharide monooxygenases (LPMOs), a recently discovered class of enzymes known to be active on cellulose and expressed when grown on biomass. Because of extensive genetic and biochemical data already available, Aspergillus nidulans offers an excellent model system to study the need for multiple AA9 LPMOs and their activity during oxidative degradation of biomass. We provide the first report on regulation of the entire family of AA9 LPMOs in A. nidulans over a range of polysaccharides including xylan, xyloglucan, pectin, glucan, and cellulose. We have successfully cloned and expressed AN3046, an AA9 LPMO in A. nidulans that is active on cellulose. Additionally, we performed mass spectral analyses that show the enzyme is active on the hemicellulose xyloglucan. The AN3046 LPMO showed synergy with other hydrolases in degrading sorghum stover. Our data showing activity of the overexpressed LPMO on cellulose and xyloglucan provides further evidence for the breadth of substrates acted on by AA9 LPMOs.

Identification of the Abundant Hydroxyproline-Rich Glycoproteins in the Root Walls of Wild-Type Arabidopsis, an ext3 Mutant Line, and Its Phenotypic Revertant.

Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding EXTENSIN 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes.

Enzymatic activity and substrate specificity of the recombinant tomato ß-galactosidase 1.

The open reading frame of tomato ß-galactosidase 1 was expressed in yeast, and the enzymatic properties and substrate specificity were investigated. The enzyme had peak activity at pH 5.0 and 40-50°C. TBG1 was active on ß-(1,3)- and ß-(1,6)-galactobiose and lactose. TBG1 released galactose from lupin galactan, tomato fruit alkali soluble pectin, arabinogalactan, gum arabic and methyl ß-(1,6)-galactohexaoside, but not from labeled ß-(1,4)-galactoheptaose. TBG1 was assessed for its ability to degrade three galactosyl-containing cell wall fractions purified from different development and ripening stages of tomato fruit. TBG1 released galactose from all of the fractions from all of the stages tested. TBG1 activity was highest on the hemicellulose fraction at the 10 and 20d after pollination stage. This result is not correlated the with TBG1 expression pattern. TBG1 might act on a small but specific set of polysaccharide containing galactose.

A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover.

BACKGROUND: Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study. RESULTS: A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance. CONCLUSIONS: Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.

Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber.

BACKGROUND: Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra- and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. RESULTS: Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U.mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2:1 or 4:1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber. CONCLUSION: Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.

Phanerochaete chrysosporium produces a diverse array of extracellular enzymes when grown on sorghum.

In an effort to understand how fungi degrade biomass, we grew Phanerochaete chrysosporium on sorghum stover and chronicled the growth of the fungus over the course of 14 days. The fungal mass grew steadily until the fifth day, reaching 0.06 mg of cells per milligram of dry mass, which fell by the seventh day and stayed at nearly the same level until day 14. After 1 day, hemicellulases, cellulases, and polygalacturonases were detected in the extracellular fluid at 1.06, 0.34, and 0.20 U/ml, respectively. Proteomic studies performed with the extracellular fluid using liquid chromatography­tandem mass spectrometry identified 57, 116, and 102 degradative enzymes targeting cellulose, hemicellulose, pectin, lignin, proteins, and lipids on days 1, 7, and 14, respectively. Significant concentrations of breakdown products of the sorghum polysaccharides were detected in the extracellular fluid indicating that the enzymes were breaking the polysaccharides, and after 14 days, almost 39% of the sorghum sugars had been used by the fungus. Our results suggest that P. chrysosporium produces a set of enzymes to degrade the components of lignocellulose from the beginning of its growth, but modifies the complement of enzymes it secretes over time to adapt to the particular substrate available.

Xylan decomposition by Aspergillus clavatus endo-xylanase.

Agricultural and forest waste products are abundant and low-cost biomass sources useful in renewable fuel energy and feedstock preparation. Hydrolysis of a major biomass component, hemicellulose, is accomplished by the action of endo-xylanases. Reaction products vary in composition and degree of polymerization as a function of both feedstock and the enzyme activities utilized, ranging from monomeric sugars to complex branched polysaccharides. The study herein describes heterologous expression in Aspergillus awamori of a betabeta-(1-4) endo-xylanase isolated from the whole-genome DNA sequence of A. clavatus along with a comprehensive biochemical and functional analysis of the enzyme, including substrate preference and hydrolysis patterns. The A. clavatus xylanase promotes incomplete hydrolysis of xylan substrates resulting in xylobiose, xylotriose and xylotetraose. Incomplete degradation resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitating digestion and absorption of nutrients.

Enzymatic activity and substrate specificity of recombinant tomato beta-galactosidases 4 and 5.

The open reading frames of tomato beta-galactosidase (TBG) 4 and 5 cDNAs were expressed in yeast, and the enzymes properties and substrate specificities were investigated. The two enzymes had peak activities between pH 4-4.5 and 37-45 degrees C. TBG4 specifically hydrolyzed beta-(1-->4) and 4-linked galactooligosaccharides. TBG5 had a strong preference to hydrolyze beta-(1-->3) and beta-(1-->6)-linked galactooligosaccharides. Exo-beta-galactanase activity of the TBG enzymes was measured by determining the release of galactosyl residues from native tomato cell wall fractions throughout fruit development and ripening. Both TBGs released galactose from all of the fractions and stages tested. TBG4 activity was highest using chelator soluble pectin and alkali soluble pectin at the turning stage of ripening. Using aminopyrene trisulfonate labeled substrates, TBG4 was the only enzyme with strong exo-beta-(1-->4)-galactanase activity on 5 mer or greater galactans. TBG4 and TBG5 were both able to degrade galactosylated rhamnogalacturonan. Neither enzyme was able to degrade galactosylated xyloglucan.

Isolation and identification of oligomers from partial degradation of lime fruit cutin.

Complementary degradative treatments with low-temperature hydrofluoric acid and methanolic potassium hydroxide have been used to investigate the protective biopolymer cutin from Citrus aurantifolia (lime) fruits, augmenting prior enzymatic and chemical strategies to yield a more comprehensive view of its molecular architecture. Analysis of the resulting soluble oligomeric fragments with one- and two-dimensional NMR and MS methods identified a new dimer and three trimeric esters of primary alcohols based on 10,16-dihydroxyhexadecanoic acid and 10-oxo-16-hydroxyhexadecanoic acid units. Whereas only 10-oxo-16-hydroxyhexadecanoic acid units were found in the oligomers from hydrofluoric acid treatments, the dimer and trimer products isolated to date using diverse degradative methods included six of the seven possible stoichiometric ratios of monomer units. A novel glucoside-linked hydroxyfatty acid tetramer was also identified provisionally, suggesting that the cutin biopolymer can be bound covalently to the plant cell wall. Although the current findings suggest that the predominant molecular architecture of this protective polymer in lime fruits involves esters of primary and secondary alcohols based on long-chain hydroxyfatty acids, the possibility of additional cross-linking to enhance structural integrity is underscored by these and related findings of nonstandard cutin molecular architectures.

Detection and identification of rhamnogalacturonan lyase activity in intercellular spaces of expanding cotton cotyledons.

Rhamnogalacturonan lyase (RG lyase) activity has been detected and its relative activity measured in vivo during the expansion of cotton (Gossypium hirsutum L.) cotyledons. Rhamnogalacturonan (RG) oligomers labeled with a fluorescent tag were injected into the intercellular spaces of cotton cotyledons and, after incubation, the digested substrate was rinsed out. Enzyme digestion products were detected and identified by capillary zone electrophoresis. Rhamnogalacturonan lyase products were identified as such by co-migration with the digestion products of linear RG oligomers when the oligomers were treated with fungal RG lyase but not when treated with fungal RG hydrolase. In addition, reaction of plant RG lyase digestion products of RG oligomers with I(2)/KI, which selectively removes unsaturated galactopyranosyluronic acid (GaLap) residues formed at the non-reducing end of the oligomer, converted the plant digestion products into RG oligomers that co-migrated with fungal RG hydrolase products. The activity of the enzyme in the intercellular spaces of cotton cotyledons is very low and could be detected most easily when not >0.03 nmol of substrate was injected in a approximately 0.7-cm(2) area and incubated in vivo for 2-6 h. Rhamnogalacturonan lyase activity was the highest in rapidly expanding 3- to 4-day-old cotyledons and gradually decreased during the slow-down in expansion over the next 2-3 days. The RG lyase activity was also detected when the APTS (8-aminopyrene-1,3,6-trisulfonic acid, trisodium salt)-labeled substrates were introduced into intercellular spaces by infiltration instead of injection, indicating that the activity was not induced by wounding or released into the apoplast by cell damage. An exo-RG galacturonohydrolase activity was also found, but RG hydrolase and exo-RG rhamnohydrolase were not detected.

Changes in homogalacturonans and enzymes degrading them during cotton cotyledon expansion.

Changes in homogalacturonans (HGs) and enzymes degrading them have been investigated during cotton (Gossypium hirsutum L.) cotyledon expansion. Using an in vivo assay for pectin-degrading enzymes that involves fluorescent labeled oligomers of GalA as substrate and capillary electrophoresis for product analysis, we found that endo- and exo-polygalacturonases are present in the cotyledon extracellular spaces, and there are dramatic changes in the levels of both activities as the cotyledons change their rate of expansion. Capacity for endo-polygalacturonase activity was highest during the initial stages of cotyledon expansion. However, for exo-polygalacturonase activity it was highest in the later stages of expansion. Cell walls were prepared from 3-, 5-, and 7-day-old cotton cotyledons and treated with liquid HF at -23 degrees C. This treatment cleaves the glycosidic linkages of most neutral sugars in the walls without degrading HGs. HGs with a relatively high degree of esterification can then be solubilized with water, and those with low esterification can be solubilized with concentrated imidazole buffer. The majority of HGs were obtained in the water extracts. The degrees of esterification were 57%, 47%, and 47% in water extracts and 34%, 25%, and 27% in imidazole extracts, in 3-, 5-, and 7-day-old cotton cotyledons, respectively. Using a PA100 ion-exchange column, the members of a GalA homologous series up to approximately 70 residues can be separated. The results from HG molecular length distribution analysis indicated that the HG at 3 days was on average shorter than that in the older cotyledons, perhaps reflecting the higher level of endo-polygalacturonase activity at this stage of more rapid growth.

Variation of taxane content in needles of Taxus x media cultivars with different growth characteristics.

Needles from 17 different Taxus x media cultivars, belonging to 4 groups showing different growth characteristics, were analyzed using high performance liquid chromatography for their content of 10-deacetylbaccatin III, baccatin III, cephalomannine and paclitaxel (Taxol). The 4 Taxus x media cultivar groups were: 1.) medium to fast growing and upright form; 2.) slow growing and upright form; 3.) fast growing and spreading form; and 4.) slow growing and spreading form. The purpose of this study was to identify yew cultivars of fast growth rate, upright growth and high taxane content in their needles. The highest content of paclitaxel was found in 'Coleana' of group 1 (378 microg/g of the extracted dry weight). Three cultivars in group 1, 'Coleana', 'Stovekenii' and 'Hicksii', make good candidates for taxane extraction because of their high paclitaxel and 10-deacetylbaccatin III content, fast biomass accumulation and upright growing form. They are also good starting materials to develop alternative methods for the production of paclitaxel and its analogous compounds through modern biotechnology approaches.

Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls.

To facilitate analysis of plant cell wall polysaccharide structure and composition, we cloned 74 genes encoding polysaccharide-degrading enzymes from Aspergillus nidulans, Aspergillus fumigatus, and Neurospora crassa and expressed the genes as secreted proteins with C-terminal Myc and 6x His tags. Most of the recombinant enzymes were active in enzyme assays, and optima for pH and temperature were established. A subset of the enzymes was used to fragment polysaccharides from the irregular xylem 9 (irx9) mutant of Arabidopsis. The analysis revealed a decrease in the abundance of xylan in the mutant, indicating that the IRX9 gene, which encodes a putative family 43 glycosyltransferase, is required for xylan synthesis.

Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans.

An oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from the filamentous fungus Aspergillus nidulans was cloned and expressed in Pichia pastoris as a secreted histidine-tagged protein and purified by affinity chromatography. The enzyme acts on xyloglucan oligomers and releases the first two glycosyl residue segments from the reducing end, provided that neither the first glucose nor the xylose attached to the third glucose residue from the reducing end is not further substituted. The enzyme has a specific activity of 7 U/mg at the pH optimum of 3 and at the temperature optimum of 42 degrees C.

Negative subtraction hybridization: an efficient method to isolate large numbers of condition-specific cDNAs.

BACKGROUND: The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by switching Aspergillus nidulans from growth on glucose to growth on selected polysaccharides. Approximately 4,700 contigs from 12,320 ESTs were already available from a cDNA library representing transcripts isolated from glucose-grown A. nidulans during asexual development. Our goals were to expand the cDNA collection without repeated sequencing of previously identified ESTs and to find as many transcripts as possible that are specifically induced in complex polysaccharide metabolism. RESULTS: We have devised a Negative Subtraction Hybridization (NSH) method and tested it in A. nidulans. NSH entails screening a plasmid library made from cDNAs prepared from cells grown under a selected physiological condition with labeled cDNA probes prepared from another physiological condition. Plasmids with inserts that failed to hybridize to cDNA probes through two rounds of screening (i.e. negatives) indicate that they are transcripts present at low concentration in the labeled probe pool. Thus, these transcripts will be predominantly condition-specific, along with some rare transcripts. In a screen for transcripts induced by switching the carbon source from glucose to 12 selected polysaccharides, 3,532 negatives were isolated from approximately 100,000 surveyed colonies using this method. Negative clones were end-sequenced and assembled into 2,039 contigs, of which 1,722 were not present in the previously characterized glucose-grown cDNA library. Single-channel microarray hybridization experiments confirmed that the majority of the negatives represented genes that were differentially induced by a switch from growth in glucose to one or more of the polysaccharides. CONCLUSIONS: The Negative Subtraction Hybridization method described here has several practical benefits. This method can be used to screen any existing cDNA library, including full-length and pooled libraries, and does not rely on PCR or sequence information. In addition, NSH is a cost-effective method for the isolation of novel, full-length cDNAs for differentially expressed transcripts or enrichment of rare transcripts.

Differences in the methyl ester distribution of homogalacturonans from near-isogenic wheat lines resistant and susceptible to the wheat stem rust fungus.

Plants possess an efficient nonself surveillance system triggering induced disease resistance mechanisms upon molecular recognition of microbial invaders. Successful pathogens have evolved strategies to evade or counteract these mechanisms, e.g., by the generation of suppressors. Pectic fragments produced during host cell wall degradation can act as endogenous suppressors of the hypersensitive response in wheat leaves. We have isolated and characterized homogalacturonans from cell walls of two wheat cultivars susceptible to the stem rust fungus, Puccinia graminis f. sp. tritici, namely cvs. Prelude and Marquis, and from near-isogenic lines of both cultivars containing the Sr5-gene for hypersensitive rust resistance. Two independent approaches were used to compare their methyl esterification: i) immunochemistry using the monoclonal antibodies JIM5, JIM7, PAM1, and LM7 and ii) chromatography of oligogalacturonides representing stretches of contiguous nonmethyl-esterified GalA residues. The results clearly indicate a significant difference in the homogalacturonans from susceptible and resistant wheat lines. The difference can best be explained by assuming a nonrandom and more blockwise distribution of the methyl esters in the homogalacturonans of susceptible wheat cultivars as compared with a presumably more random distribution in the near-isogenic resistant lines. Possible consequences of this difference for the enzymatic generation of endogenous suppressors are discussed.