scholarly journals The Phytophthora infestans Haustorium Is a Site for Secretion of Diverse Classes of Infection-Associated Proteins

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Shumei Wang ◽  
Lydia Welsh ◽  
Peter Thorpe ◽  
Stephen C. Whisson ◽  
Petra C. Boevink ◽  
...  
Keyword(s):  
Cell Wall ◽  
Phytophthora Infestans ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Extracellular Proteins ◽  
Host Cells ◽  
Cysteine Protease Inhibitor ◽  
Plant Interactions ◽  
Content Type

ABSTRACT The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection. IMPORTANCE There are many different classes of proteins secreted from Phytophthora infestans that may influence or facilitate infection. Elucidating where and how they are secreted during infection is an important step toward developing methods to control their delivery processes. We used an inhibitor of conventional secretion to identify the following different classes of infection-associated extracellular proteins: cell wall-degrading and cell wall-modifying enzymes, microbe-associated molecular pattern-like proteins that may elicit immune responses, and apoplastic effectors that are predicted to suppress immunity. In contrast, secretion of a cytoplasmic effector that is translocated into host cells is nonconventional, as it is insensitive to inhibitor treatment. This evidence further supports the finding that proteins that are active in the apoplast and effector proteins that are active in the host cytoplasm are differentially secreted by P. infestans. Critically, it demonstrates that a disease-specific developmental structure, the haustorium, is a major secretion site for diverse protein classes during infection.

scholarly journals The Role of Arabinogalactan Type II Degradation in Plant-Microbe Interactions

2021 ◽  
Vol 12 ◽  
Author(s):  
Maria Guadalupe Villa-Rivera ◽  
Horacio Cano-Camacho ◽  
Everardo López-Romero ◽  
María Guadalupe Zavala-Páramo
Keyword(s):  
Cell Wall ◽  
Plant Defense ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Degradation Products ◽  
Hydrolytic Enzymes ◽  
Pathogenic Fungi ◽  
Plant Interactions ◽  
Root Tips ◽  
Microbe Interactions

Arabinogalactans (AGs) are structural polysaccharides of the plant cell wall. A small proportion of the AGs are associated with hemicellulose and pectin. Furthermore, AGs are associated with proteins forming the so-called arabinogalactan proteins (AGPs), which can be found in the plant cell wall or attached through a glycosylphosphatidylinositol (GPI) anchor to the plasma membrane. AGPs are a family of highly glycosylated proteins grouped with cell wall proteins rich in hydroxyproline. These glycoproteins have important and diverse functions in plants, such as growth, cellular differentiation, signaling, and microbe-plant interactions, and several reports suggest that carbohydrate components are crucial for AGP functions. In beneficial plant-microbe interactions, AGPs attract symbiotic species of fungi or bacteria, promote the development of infectious structures and the colonization of root tips, and furthermore, these interactions can activate plant defense mechanisms. On the other hand, plants secrete and accumulate AGPs at infection sites, creating cross-links with pectin. As part of the plant cell wall degradation machinery, beneficial and pathogenic fungi and bacteria can produce the enzymes necessary for the complete depolymerization of AGs including endo-β-(1,3), β-(1,4) and β-(1,6)-galactanases, β-(1,3/1,6) galactanases, α-L-arabinofuranosidases, β-L-arabinopyranosidases, and β-D-glucuronidases. These hydrolytic enzymes are secreted during plant-pathogen interactions and could have implications for the function of AGPs. It has been proposed that AGPs could prevent infection by pathogenic microorganisms because their degradation products generated by hydrolytic enzymes of pathogens function as damage-associated molecular patterns (DAMPs) eliciting the plant defense response. In this review, we describe the structure and function of AGs and AGPs as components of the plant cell wall. Additionally, we describe the set of enzymes secreted by microorganisms to degrade AGs from AGPs and its possible implication for plant-microbe interactions.

scholarly journals The Role of Brachypodium distachyon Wall-Associated Kinases (WAKs) in Cell Expansion and Stress Responses

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2478
Author(s):  
Xingwen Wu ◽  
Antony Bacic ◽  
Kim L. Johnson ◽  
John Humphries
Keyword(s):  
Cell Wall ◽  
Stress Response ◽  
Plant Cell ◽  
Stress Responses ◽  
Cell Expansion ◽  
Plant Cell Wall ◽  
Brachypodium Distachyon ◽  
Critical Role ◽  
Cell Integrity

The plant cell wall plays a critical role in signaling responses to environmental and developmental cues, acting as both the sensing interface and regulator of plant cell integrity. Wall-associated kinases (WAKs) are plant receptor-like kinases located at the wall—plasma membrane—cytoplasmic interface and implicated in cell wall integrity sensing. WAKs in Arabidopsis thaliana have been shown to bind pectins in different forms under various conditions, such as oligogalacturonides (OG)s in stress response, and native pectin during cell expansion. The mechanism(s) WAKs use for sensing in grasses, which contain relatively low amounts of pectin, remains unclear. WAK genes from the model monocot plant, Brachypodium distachyon were identified. Expression profiling during early seedling development and in response to sodium salicylate and salt treatment was undertaken to identify WAKs involved in cell expansion and response to external stimuli. The BdWAK2 gene displayed increased expression during cell expansion and stress response, in addition to playing a potential role in the hypersensitive response. In vitro binding assays with various forms of commercial polysaccharides (pectins, xylans, and mixed-linkage glucans) and wall-extracted fractions (pectic/hemicellulosic/cellulosic) from both Arabidopsis and Brachypodium leaf tissues provided new insights into the binding properties of BdWAK2 and other candidate BdWAKs in grasses. The BdWAKs displayed a specificity for the acidic pectins with similar binding characteristics to the AtWAKs.

scholarly journals Fermentation and subsequent disposition of 14C-labelled plant cell wall material in the rat

1993 ◽  
Vol 69 (1) ◽  
pp. 189-197 ◽  
Author(s):  
D. F. Gray ◽  
M. A. Eastwood ◽  
W. G. Brydon ◽  
S. C. Fry
Keyword(s):  
Cell Wall ◽  
Gastrointestinal Tract ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Spinacia Oleracea ◽  
Wall Material ◽  
Fermentation Products ◽  
Bacterial Fermentation ◽  
Cell Wall Preparation

A 14C-Iabelled plant cell wall preparation (I4C-PCW) produced from spinach (Spinacia oleracea L.) cell culture exhibits uniform labelling of the major polysaccharide groups (%): pectins 53, hemicellulose 13, cellulose 21, starch 3. This 14C-PCW preparation has been used in rat studies as a marker for plant cell wall metabolism. Metabolism of the 14C-PCW occurred largely over the first 24 h. This was due to fermentation in the caecum. The pectic fraction of the plant cell walls was degraded completely in the rat gastrointestinal tract, but some [14C-]cellulose was still detected after 24 h in the colon. Of the 14C,22% was recovered in the host liver, adipose tissue and skin, 26% excreted as 14CO2 and up to 18%was excreted in the faeces. There was no urinary excretion of 14C. In vitro fermentation using a caecal inocuium showed reduced 14CO2 production, 12% compared with 26% in the intact rat. 14C-PCW is auseful marker to investigate the fate of plant cell wall materials in the gastrointestinal tract. These studies show both bacterial fermentation of the 14C-PCW and host metabolism of the 14C-labelled fermentation products.

scholarly journals Direct Target Network of the Neurospora crassa Plant Cell Wall Deconstruction Regulators CLR-1, CLR-2, and XLR-1

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
James P. Craig ◽  
Samuel T. Coradetti ◽  
Trevor L. Starr ◽  
N. Louise Glass
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Transcription Factors ◽  
Neurospora Crassa ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Nutrient Sensing ◽  
Wall Material ◽  
Content Type ◽  
Genes Encoding

ABSTRACTFungal deconstruction of the plant cell requires a complex orchestration of a wide array of intracellular and extracellular enzymes. InNeurospora crassa, CLR-1, CLR-2, and XLR-1 have been identified as key transcription factors regulating plant cell wall degradation in response to soluble sugars. The XLR-1 regulon was defined using a constitutively active mutant allele, resulting in hemicellulase gene expression and secretion under noninducing conditions. To define genes directly regulated by CLR-1, CLR-2, and XLR-1, we performed chromatin immunoprecipitation and next-generation sequencing (ChIPseq) on epitope-tagged constructs of these three transcription factors. WhenN. crassais exposed to plant cell wall material, CLR-1, CLR-2, and XLR-1 individually bind to the promoters of the most strongly induced genes in their respective regulons. These include promoters of genes encoding cellulases for CLR-1 and CLR-2 (CLR-1/CLR-2) and promoters of genes encoding hemicellulases for XLR-1. CLR-1 bound to its regulon under noninducing conditions; however, this binding alone did not translate into gene expression and enzyme secretion. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest paralog inSaccharomyces cerevisiae, Gal4p. Coimmunoprecipitation studies showed that CLR-1 and CLR-2 act in a homocomplex but not as a CLR-1/CLR-2 heterocomplex.IMPORTANCEUnderstanding fungal regulation of complex plant cell wall deconstruction pathways in response to multiple environmental signals via interconnected transcriptional circuits provides insight into fungus/plant interactions and eukaryotic nutrient sensing. Coordinated optimization of these regulatory networks is likely required for optimal microbial enzyme production.

scholarly journals Immunocytochemical Localization of HrpA and HrpZ Supports a Role for the Hrp Pilus in the Transfer of Effector Proteins from Pseudomonas syringae pv. tomato Across the Host Plant Cell Wall

2001 ◽  
Vol 14 (3) ◽  
pp. 394-404 ◽  
Author(s):  
Ian R. Brown ◽  
John W. Mansfield ◽  
Suvi Taira ◽  
Elina Roine ◽  
Martin Romantschuk
Keyword(s):  
Cell Wall ◽  
Pseudomonas Syringae ◽  
Plant Cell ◽  
Type Iii Secretion ◽  
Plant Cell Wall ◽  
Immunogold Labeling ◽  
Effector Proteins ◽  
Basal Bodies ◽  
Type Iii

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 μm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

scholarly journals Glycoside Hydrolase Genes Are Required for Virulence ofAgrobacterium tumefaciensonBryophyllum daigremontianaand Tomato

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Stephanie L. Mathews ◽  
Haylea Hannah ◽  
Hillary Samagaio ◽  
Camille Martin ◽  
Eleanor Rodriguez-Rassi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Host Cell ◽  
Plant Cell ◽  
Crown Gall ◽  
Plant Cell Wall ◽  
Tumor Formation ◽  
Glycoside Hydrolases ◽  
Plant Host ◽  
Content Type ◽  
Limited Digestion

ABSTRACTAgrobacterium tumefaciensis a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type IV secretion system where the DNA becomes integrated into the host cell chromosomes. The expression of T-DNA in the plant results in tumor formation. Although the binding of the bacteria to plant surfaces has been studied previously, there is little work on possible interactions of the bacteria with the plant cell wall. Seven of the 48 genes encoding putative glycoside hydrolases (Atu2295,Atu2371,Atu3104,Atu3129,Atu4560,Atu4561, andAtu4665) in the genome ofA. tumefaciensC58 were found to play a role in virulence on tomato andBryophyllum daigremontiana. Two of these genes (pglAandpglB;Atu3129andAtu4560) encode enzymes capable of digesting polygalacturonic acid and, thus, may play a role in the digestion of pectin. One gene (arfA;Atu3104) encodes an arabinosylfuranosidase, which could remove arabinose from the ends of polysaccharide chains. Two genes (bglAandbglB;Atu2295andAtu4561) encode proteins with β-glycosidase activity and could digest a variety of plant cell wall oligosaccharides and polysaccharides. One gene (xynA;Atu2371) encodes a putative xylanase, which may play a role in the digestion of xylan. Another gene (melA;Atu4665) encodes a protein with α-galactosidase activity and may be involved in the breakdown of arabinogalactans. Limited digestion of the plant cell wall byA. tumefaciensmay be involved in tumor formation on tomato andB. daigremontiana.IMPORTANCEA. tumefaciensis used in the construction of genetically engineered plants, as it is able to transfer DNA to plant hosts. Knowledge of the mechanisms of DNA transfer and the genes required will aid in the understanding of this process. Manipulation of glycoside hydrolases may increase transformation and widen the host range of the bacterium.A. tumefaciensalso causes disease (crown gall tumors) on a variety of plants, including stone fruit trees, grapes, and grafted ornamentals such as roses. It is possible that compounds that inhibit glycoside hydrolases could be used to control crown gall disease caused byA. tumefaciens.

scholarly journals Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Silvia Hüttner ◽  
Anikó Várnai ◽  
Dejan M. Petrović ◽  
Cao Xuan Bach ◽  
Dang Thi Kim Anh ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Functional Characterization ◽  
Cellulose Microfibrils ◽  
Cell Wall Polysaccharide ◽  
Poor Growth ◽  
Content Type ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

ABSTRACT The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall. IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

scholarly journals Induction of Genes Encoding Plant Cell Wall-Degrading Carbohydrate-Active Enzymes by Lignocellulose-Derived Monosaccharides and Cellobiose in the White-Rot FungusDichomitus squalens

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Sara Casado López ◽  
Mao Peng ◽  
Tedros Yonatan Issak ◽  
Paul Daly ◽  
Ronald P. de Vries ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Expression Patterns ◽  
Fine Tuning ◽  
White Rot ◽  
White Rot Fungus ◽  
Content Type ◽  
Dichomitus Squalens

ABSTRACTFungi can decompose plant biomass into small oligo- and monosaccharides to be used as carbon sources. Some of these small molecules may induce metabolic pathways and the production of extracellular enzymes targeted for degradation of plant cell wall polymers. Despite extensive studies in ascomycete fungi, little is known about the nature of inducers for the lignocellulolytic systems of basidiomycetes. In this study, we analyzed six sugars known to induce the expression of lignocellulolytic genes in ascomycetes for their role as inducers in the basidiomycete white-rot fungusDichomitus squalensusing a transcriptomic approach. This identified cellobiose andl-rhamnose as the main inducers of cellulolytic and pectinolytic genes, respectively, ofD. squalens. Our results also identified differences in gene expression patterns between dikaryotic and monokaryotic strains ofD. squalenscultivated on plant biomass-derived monosaccharides and the disaccharide cellobiose. This suggests that despite conservation of the induction between these two genetic forms ofD. squalens, the fine-tuning in the gene regulation of lignocellulose conversion is differently organized in these strains.IMPORTANCEWood-decomposing basidiomycete fungi have a major role in the global carbon cycle and are promising candidates for lignocellulosic biorefinery applications. However, information on which components trigger enzyme production is currently lacking, which is crucial for the efficient use of these fungi in biotechnology. In this study, transcriptomes of the white-rot fungusDichomitus squalensfrom plant biomass-derived monosaccharide and cellobiose cultures were studied to identify compounds that induce the expression of genes involved in plant biomass degradation.

scholarly journals Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Dai Komiya ◽  
Akane Hori ◽  
Takuya Ishida ◽  
Kiyohiko Igarashi ◽  
Masahiro Samejima ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Substrate Specificity ◽  
Ferulic Acid ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Triad ◽  
Acetyl Xylan Esterase ◽  
Content Type ◽  
Aspergillus Luchuensis

ABSTRACT Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of AlAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

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