Pathogen-induced plant proteins

Pathogen-induced plant proteins are classified by their functional characteristics: (a) involvement in plant cell signaling; (b) inhibition of enzymes excreted by the pathogens; (c) stabilization of plant cell walls or ability to trigger apoptosis; (d) enzymatic activity producing lysis of cell walls of pathogenic fungi and bacteria; (e) enzymatic activity in metabolic pathways of phenylpropane and terpene phytoalexins; and (f) ability to affect the pathogens directly, by disturbing the function of their cell membranes or by inactivating their ribosomes. Examples of transgenic plants with increased immunity against pathogens are also provided.     

Plant pathogens as a source of diverse enzymes for lignocellulose digestion

The plant cell wall is a major barrier that many plant pathogens must surmount for successful invasion of their plant hosts. Full genome sequencing of a number of plant pathogens has revealed often large, complex, and redundant enzyme systems for degradation of plant cell walls. Recent surveys have noted that plant pathogenic fungi are highly competent producers of lignocellulolytic enzymes, and their enzyme activity patterns reflect host specificity. We propose that plant pathogens may contribute to biofuel production as diverse sources of accessory enzymes for more efficient conversion of lignocellulose into fermentable sugars.     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as ''acid growth'', by which we mean they are more extensible at low pH (< 5) ¹. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism ^{2, 3}. Auxin activates a H⁺ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins ⁴, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This ''acid growth'' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins ⁵. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 °C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.Open in a separate windowClick here to view.^{(58M, flv)}     

Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane

Many plant‐ and animal‐pathogenic Gram‐negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel‐like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two‐step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two‐domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin‐rich middle lamella by the bacterial pilus. One‐domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin‐type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly.     

Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae)

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts (Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.     

The expansin superfamily

The expansin superfamily of plant proteins is made up of four families, designated α-expansin, β-expansin, expansin-like A and expansin-like B. α-Expansin and β-expansin proteins are known to have cell-wall loosening activity and to be involved in cell expansion and other developmental events during which cell-wall modification occurs. Proteins in these two families bind tightly to the cell wall and their activity is typically assayed by their stimulation of cell-wall extension and stress relaxation; no bona fide enzymatic activity has been detected for these proteins. α-Expansin proteins and some, but not all, β-expansin proteins are implicated as catalysts of 'acid growth', the enlargement of plant cells stimulated by low extracellular pH. A divergent group of β-expansin genes are expressed at high levels in the pollen of grasses but not of other plant groups. They probably function to loosen maternal cell walls during growth of the pollen tube towards the ovary. All expansins consist of two domains; domain 1 is homologous to the catalytic domain of proteins in the glycoside hydrolase family 45 (GH45); expansin domain 2 is homologous to group-2 grass pollen allergens, which are of unknown biological function. Experimental evidence suggests that expansins loosen cell walls via a nonenzymatic mechanism that induces slippage of cellulose microfibrils in the plant cell wall.     

Isolation, heterologous expression and characterization of an endo-polygalacturonase produced by the phytopathogen Burkholderia cepacia

Endo-polygalacturonases (endoPGs) belong to the glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond present in the smooth regions of pectins. Pectic substances are among the principal macromolecular components of the primary plant cell walls and are subjected to enzymatic degradation not only in the course of important physiological processes such as plant senescence and ripening, but also during infection events by plant pathogens. Here we report, for the first time, the isolation and the purification of an endoPG (PehA) from the supernatant of the plant pathogen Burkholderia cepacia strain ATCC 25416. In order to obtain adequate amounts of protein required for structural and functional studies, the gene coding for pehA was PCR-amplified and cloned in Escherichia coli cells. The recombinant protein was purified to homogeneity and characterized. PehA exhibited a pI value of 8.0 and an optimal activity at pH 3.5. Far-UV circular dichroism (CD) measurements show that PehA assumes a beta-helix fold super-secondary structural motif.     

Determination of Subunit Composition of Clostridium cellulovorans Cellulosomes That Degrade Plant Cell Walls

Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.     

Nep1-like proteins from plant pathogens: recruitment and diversification of the NPP1 domain across taxa

An emerging group of proteins found in many plant pathogens are related to their ability to cause plant cell death. These proteins may be identified by the presence of a common NPP1 (necrosis-inducing Phytophthora protein) domain, and have collectively been named NLPs (Nep1-like proteins). The NLPs are distinguished by their wide distribution across taxa and their broad spectrum of activity against dicotyledonous plants. The function of NLPs is not known but there is strong evidence that they may act as positive virulence factors, accelerating disease and pathogen growth in plant hosts. Interest in NLPs is gaining momentum as more members of this protein family are discovered in more species of plant pathogens.     

Leaf-specific thionins of barley-a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants

A novel class of highly abundant polypeptides with antifungal activity has been detected in cell walls of barley leaves. Similar polypeptides known as thionins occur not only in monocotyledonous but also in various dictoyledonous plants. The leaf-specific thionins of barley are encoded by a complex multigene family, which consists of at least 50-100 members per haploid genome. All of these genes are confined to chromosome 6. The toxicity of these thionins for plant pathogenic fungi and the fact that their synthesis can also be triggered by pathogens strongly suggest that thionins are a naturally occurring, inducible plant protein possibly involved in the mechanism of plant defence against microbial infections.     

Cell wall degrading enzymes,inhibitory proteins,and oligosaccharides participate in the molecular dialogue between plants and pathogens

The wall interface between plants and pathogens plays an important role in the outcome of their interactions. Studying the degradation of plant pectic polysaccharides by microbial pectinases, and of microbial β-glucans by plant glucanases has shown that these polymers are a source of oligosaccharides which elicit defence responses in plants. The extent of degradation appears to be controlled by the presence of inhibitory proteins which counteract enzyme hydrolysis. Thus, plant cell walls participate in the molecular dialogue established between plants and pathogens.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Oxalate,germins, and higher-plant pathogens

Earlier surveys (1, B. G. Lane. [1991] FASEB J. 5, 2983-2901; 2, B. G. Lane. [1994] FASEB J. 8, 294-301) helped to uproot entrenched views of plant oxalate as a static substance. It is now recognized that oxalate oxidases (OXOs) found in the "true cereals" (barley, maize, oat, rice, rye, wheat), the so-called germin OXOs (G-OXOs), or simply germins, are involved in cereal defence responses to invasion by fungal pathogens and that they show promise of being valuable agents of plant defence in dicotyledons, where they are not found naturally. G-OXOs have very peculiar properties: (a) their water-soluble oligomeric structures and enzymic activity are stable during SDS-PAGE and nitrocellulose blotting, (b) their undenatured water-soluble forms are refractory to the action of broad-specificity proteases, (c) their water-insoluble forms occur abundantly (approximately 50%) in the extracellular matrix (cell walls) of wheat, and probably in varying amounts in the cell walls of other true cereals. Transfer of the wheat G-OXO coding element to dicotyledons has been found, in all cases so far examined, to result in improved resistance to fungal pathogens. The possible nature of the improved resistance is discussed in relation to (a) generation of microcidal concentrations of hydrogen peroxide when the G-OXOs act on oxalate, (b) elicitation of hypersensitive cell death at lower concentrations of hydrogen peroxide, (c) formation of effective barriers against predator penetration by the hydrogen-peroxide-mediated lignification of cell walls, and (d) destruction of oxalate, which is an inhibitor of the hypersensitive response, a strategy of particular importance in the case of ubiquitous predator organisms such as Sclerotinia sclerotiorum, which secrete high concentrations of oxalate as a toxin.     

Structural-functional features of plant isoperoxidases

Current data on structural-functional features of plant peroxidases and their involvement in functioning of the pro-/antioxidant system responding to stress factors, especially those of biotic origin, are analyzed. The collection of specific features of individual isoforms allows a plant to withstand an aggressive influence of the environment. Expression of some genes encoding different isoperoxidases is regulated by pathogens (and their metabolites), elicitors, and hormone-like compounds; specific features of this regulation are considered in detail. It is suggested that isoperoxidases interacting with polysaccharides are responsible for a directed deposition of lignin on the cell walls, and this lignin in turn is concurrently an efficient strengthening material and protects the plants against pathogens.     

Determination of subunit composition of Clostridium cellulovorans cellulosomes that degrade plant cell walls

Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.     

Novel role for pectin methylesterase in Arabidopsis: a new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production

Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.     

Fg12 ribonuclease secretion contributes to Fusarium graminearum virulence and induces plant cell death

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean; its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light‐dependent manner. Fg12 possessed ribonuclease (RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death‐promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.     

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation¹ and other products such as biocomposite materials⁷. Plant biomass remains one of the greatest untapped reserves on the planet⁴. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin⁶ and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant⁴. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization³, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units⁵. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II².