Plant Chitinases and Their Roles in Resistance to Fungal Diseases

Chitinases are enzymes that hydrolyze the N-acetylglucosamine polymer chitin, and they occur in diverse plant tissues over a broad range of crop and noncrop species. The enzymes may be expressed constitutively at low levels but are dramatically enhanced by numerous abiotic agents (ethylene, salicylic acid, salt solutions, ozone, UV light) and by biotic factors (fungi, bacteria, viruses, viroids, fungal cell wall components, and oligosaccharides). Different classes of plant chitinases are distinguishable by molecular, biochemical, and physicochemical criteria. Thus, plant chitinases may differ in substrate-binding characteristics, localization within the cell, and specific activities. Because chitin is a structural component of the cell wall of many phytopathogenic fungi, extensive research has been conducted to determine whether plant chitinases have a role in defense against fungal diseases. Plant chitinases have different degrees of antifungal activity to several fungi in vitro. In vivo, although rapid accumulation and high levels of chitinases (together with numerous other pathogenesis-related proteins) occur in resistant tissues expressing a hypersensitive reaction, high levels also can occur in susceptible tissues. Expression of cloned chitinase genes in transgenic plants has provided further evidence for their role in plant defense. The level of protection observed in these plants is variable and may be influenced by the specific activity of the enzyme, its localization and concentration within the cell, the characteristics of the fungal pathogen, and the nature of the host-pathogen interaction. The expression of chitinase in combination with one or several different antifungal proteins should have a greater effect on reducing disease development, given the complexities of fungal-plant cell interactions and resistance responses in plants. The effects of plant chitinases on nematode development in vitro and in vivo are worthy of investigation.     

Conserved processes and lineage-specific proteins in fungal cell wall evolution

The cell wall is a defining organelle that differentiates fungi from its sister clades in the opisthokont superkingdom. With a sensitive technique to align low-complexity protein sequences, we have identified 187 cell wall-related proteins in Saccharomyces cerevisiae and determined the presence or absence of homologs in 17 other fungal genomes. There were both conserved and lineage-specific cell wall proteins, and the degree of conservation was strongly correlated with protein function. Some functional classes were poorly conserved and lineage specific: adhesins, structural wall glycoprotein components, and unannotated open reading frames. These proteins are primarily those that are constituents of the walls themselves. On the other hand, glycosyl hydrolases and transferases, proteases, lipases, proteins in the glycosyl phosphatidyl-inositol-protein synthesis pathway, and chaperones were strongly conserved. Many of these proteins are also conserved in other eukaryotes and are associated with wall synthesis in plants. This gene conservation, along with known similarities in wall architecture, implies that the basic architecture of fungal walls is ancestral to the divergence of the ascomycetes and basidiomycetes. The contrasting lineage specificity of wall resident proteins implies diversification. Therefore, fungal cell walls consist of rapidly diversifying proteins that are assembled by the products of an ancestral and conserved set of genes.     

Fungal effector proteins: past, present and future

The pioneering research of Harold Flor on flax and the flax rust fungus culminated in his gene-for-gene hypothesis. It took nearly 50 years before the first fungal avirulence ( Avr ) gene in support of his hypothesis was cloned. Initially, fungal Avr genes were identified by reverse genetics and map-based cloning from model organisms, but, currently, the availability of many sequenced fungal genomes allows their cloning from additional fungi by a combination of comparative and functional genomics. It is believed that most Avr genes encode effectors that facilitate virulence by suppressing pathogen-associated molecular pattern-triggered immunity and induce effector-triggered immunity in plants containing cognate resistance proteins. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either on the plasma membrane or inside the plant cell. Indirect recognition of an effector (also known as the guard model) implies that the virulence target of an effector in the host (the guardee) is guarded by the resistance protein (the guard) that senses manipulation of the guardee, leading to activation of effector-triggered immunity. In this article, we review the literature on fungal effectors and some pathogen-associated molecular patterns, including those of some fungi for which no gene-for-gene relationship has been established.     

Evolutionary dynamics in the fungal polarization network,a mechanistic perspective

Polarity establishment underlies proper cell cycle completion across virtually all organisms. Much progress has been made in generating an understanding of the structural and functional components of this process, especially in model species. Here we focus on the evolutionary dynamics of the fungal polarization protein network in order to determine general components and mechanistic principles, species- or lineage-specific adaptations and the evolvability of the network. The currently available genomic and proteomic screens in a variety of fungal species have shown three main characteristics: (1) certain proteins, processes and functions are conserved throughout the fungal clade; (2) orthologous functions can never be assumed, as various cases have been observed of homologous loci with dissimilar functions; (3) species have, typically, various species- or lineage-specific proteins incorporated in their polarization network. Further large-scale comparative and experimental studies, including those on non-model species representing the great fungal diversity, are needed to gain a better understanding of the evolutionary dynamics and generalities of the polarization network in fungi.     

Molecular dialogues between Trichoderma and roots: Role of the fungal secretome

Trichoderma species are opportunistic fungi residing primarily in soil, tree bark and on wild mushrooms. Trichoderma is capable of killing other fungi and penetrating plant roots, and is commonly used as both a biofungicide and inducer of plant defence against pathogens. These fungi also exert other beneficial effects on plants including growth promotion and tolerance to abiotic stresses, primarily mediated by their intimate interactions with roots. In root–microbe interactions (both beneficial and harmful), fungal secreted proteins play a crucial role in establishing contact with the roots, fungal attachment, root penetration and triggering of plant responses. In Trichoderma–root interactions, the sucrose present in root exudates has been demonstrated to be important in fungal attraction. Attachment to roots is mediated by hydrophobin-like proteins, and secreted swollenins and plant cell wall degrading enzymes facilitate internalization of the fungal hyphae. During the early stage of penetration, suppression of plant defence is vital to successful initial root colonisation; this is mediated by small soluble cysteine-rich secreted proteins (effector-like proteins). Up to this stage, Trichoderma's behaviour is similar to that of a plant pathogen invading root structures. However, subsequent events like oxidative bursts, the synthesis of salicylic acid by the plants, and secretion of elicitor-like proteins by Trichoderma spp. differentiate this fungus from pathogens. These processes induce immunity in plants that help counter subsequent invasion by plant pathogens and insects. In this review, we present an inventory of soluble secreted proteins from Trichoderma that might play an active role in beneficial Trichoderma–plant interactions, and review the function of such proteins where known.     

Endoplasmic reticulum localized PerA is required for cell wall integrity,azole drug resistance,and virulence in Aspergillus fumigatus

GPI‐anchoring is a universal and critical post‐translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI‐anchored, and disruption of GPI‐anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI‐anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β‐glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow‐derived macrophages relative to wild type. Given the structural specificity of fungal GPI‐anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.     

The role of the cell wall in fungal pathogenesis

Fungal infections are a serious health problem. In recent years, basic research is focusing on the identification of fungal virulence factors as promising targets for the development of novel antifungals. The wall, as the most external cellular component, plays a crucial role in the interaction with host cells mediating processes such as adhesion or phagocytosis that are essential during infection. Specific components of the cell wall (called PAMPs) interact with specific receptors in the immune cell (called PRRs), triggering responses whose molecular mechanisms are being elucidated. We review here the main structural carbohydrate components of the fungal wall (glucan, mannan and chitin), how their biogenesis takes place in fungi and the specific receptors that they interact with. Different model fungal pathogens are chosen to illustrate the functional consequences of this interaction. Finally, the identification of the key components will have important consequences in the future and will allow better approaches to treat fungal infections.     

农业生态系统中的AM真菌多样性

农业生态系统复杂庞大,是由如麦田生态系统、水稻田生态系统、果园生态系统、草地生态系统、保护地生态系统等组成的一个复合生态系统。重点介绍农业生态系统中丛枝菌根(AM)和AM真菌多样性,探讨农业生态系统中调控AM真菌多样性的途径以及今后研究的动向。     

Pn-AMPs,the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato,Lycopersicum esculentum

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.     

Botrytis cinerea Protein O-Mannosyltransferases Play Critical Roles in Morphogenesis,Growth, and Virulence

Protein O-glycosylation is crucial in determining the structure and function of numerous secreted and membrane-bound proteins. In fungi, this process begins with the addition of a mannose residue by protein O-mannosyltransferases (PMTs) in the lumen side of the ER membrane. We have generated mutants of the three Botrytis cinerea pmt genes to study their role in the virulence of this wide-range plant pathogen. B. cinerea PMTs, especially PMT2, are critical for the stability of the cell wall and are necessary for sporulation and for the generation of the extracellular matrix. PMTs are also individually required for full virulence in a variety of hosts, with a special role in the penetration of intact plant leaves. The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs. Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves. Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs. Since plants do no possess these enzymes, PMTs constitute a promising target in the development of novel control strategies against B. cinerea.     

真菌草酸的代谢和作用研究进展

草酸(oxalic acid)是一种重要的生物代谢产物,广泛分布于植物、动物和微生物中,在不同的生命体中发挥重要功能.本文回顾了国内外关于真菌草酸的相关研究进展.许多真菌能够分泌草酸,包括植物病原真菌、食药用真菌及工业真菌等.草酸作为一种简单的二元羧酸,在真菌中主要通过三羧酸循环途径、乙醛酸循环途径和草酰乙酸途径合成....     

LysM结构域及其与植物-真菌相互作用的关系

在长期的进化过程中,植物与真菌之间形成了复杂而又紧密的联系,其中最主要的就是侵染与防御的关系。植物的抗病性由于涉及农作物、林木的生长与产量,逐渐成为研究热点。在植物免疫系统中,对病原真菌的识别是一个重要环节。目前认为在这一过程中,LysM结构域起到了极为关键的作用。植物细胞膜上有含LysM结构域的识别受体,该受体可以结合真菌细胞壁上的几丁质,并将信号传递到胞内,从而启动免疫反应。在真菌中,同样具有含LysM结构域的基因,主要是一类效应因子。它们可能参与真菌在侵染过程中的"伪装",以逃避植物的识别。该文以LysM结构域在植物-真菌相互作用中扮演的角色为着眼点,讨论有关研究的意义与趋势,并对如何利用LysM结构域的相关研究进行有效的抗病育种提出了新的设想。     

Bcl-2 proteins link programmed cell death with growth and morphogenetic adaptations in the fungal plant pathogen Colletotrichum gloeosporioides

Proteins belonging to the Bcl-2 family regulate apoptosis in mammals by controlling mitochondria efflux of cytochrome c and other apoptosis-related proteins. Homologues of human Bcl-2 proteins are found in different metazoan organisms where they play a similar role, while they seem to be absent in plants and fungi. Nonetheless, Bcl-2 protein members can induce or prevent yeast cell death, suggesting that enough functional conservation exists between apoptotic machineries of mammals and fungi. Here we show that induction or prevention of apoptosis by Bcl-2 proteins in the fungal plant pathogen Colletotrichum gloeosporioides is tightly linked with growth and morphogenetic adaptation that occur throughout the entire fungal life cycle. Isolates expressing the pro-apoptotic Bax protein underwent cell death with apoptotic characteristics, and showed alterations in conidial germination that are associated with pathogenic and non-pathogenic life styles. Isolates expressing the anti-apoptotic Bcl-2 protein had prolonged longevity, were protected from Bax-induced cell death, and exhibited enhanced stress resistance. These isolates also had enhanced mycelium and conidia production, and were hyper virulent to host plants. Our findings show that apoptotic-associated machinery regulates morphogenetic switches, which are critical for proper responses and adaptation fungi to different environments.     

Fungal apoptosis: function, genes and gene function

Cells of all living organisms are programmed to self-destruct under certain conditions. The most well known form of programmed cell death is apoptosis, which is essential for proper development in higher eukaryotes. In fungi, apoptotic-like cell death occurs naturally during aging and reproduction, and can be induced by environmental stresses and exposure to toxic metabolites. The core apoptotic machinery in fungi is similar to that in mammals, but the apoptotic network is less complex and of more ancient origin. Only some of the mammalian apoptosis-regulating proteins have fungal homologs, and the number of protein families is drastically reduced. Expression in fungi of animal proteins that do not have fungal homologs often affects apoptosis, suggesting functional conservation of these components despite the absence of protein-sequence similarity. Functional analysis of Saccharomyces cerevisiae apoptotic genes, and more recently of those in some filamentous species, has revealed partial conservation, along with substantial differences in function and mode of action between fungal and human proteins. It has been suggested that apoptotic proteins might be suitable targets for novel antifungal treatments. However, implementation of this approach requires a better understanding of fungal apoptotic networks and identification of the key proteins regulating apoptotic-like cell death in fungi.     

Proteomic analysis of Fusarium solani isolated from the Asian longhorned beetle, Anoplophora glabripennis

Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were not detected While little is known about the role of filamentous fungi and their associations with insects, these findings suggest that this isolate has the endogenous potential to degrade lignocellulose and extract nutrients from woody tissue.     

Perspectives in the biological function and the technological application of polygalacturonases

Polygalacturonases (PG) have evolved in the past years from a pectinase “simply” being used for food processing to an important parameter in plant–fungal interaction. PG-inhibiting proteins (PGIP) that are synthesised in plants as a specific response to PGs of pathogenic fungi, have become a focus as a possible target in resistance breeding, and PGIPs are also a concern as an inhibiting factor in food processing. Plant PGs have been identified as a major factor in fruit ripening, and PG-deficient transgenic plants have been bred. Mainly fungal PGs are used in industrial processes for juice clarification and the range of enzymes is being extended through new recombinant and non-recombinant fungal strains. Finally, novel fields of application can be envisaged for PGs in the production of oligogalacturonides as functional food components. Here we aim to highlight the various fields where PGs are encountered and where they are of biological or technological importance. Received: 22 June 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999     

The structure and synthesis of the fungal cell wall

The fungal cell wall is a dynamic structure that protects the cell from changes in osmotic pressure and other environmental stresses, while allowing the fungal cell to interact with its environment. The structure and biosynthesis of a fungal cell wall is unique to the fungi, and is therefore an excellent target for the development of anti-fungal drugs. The structure of the fungal cell wall and the drugs that target its biosynthesis are reviewed. Based on studies in a number of fungi, the cell wall has been shown to be primarily composed of chitin, glucans, mannans and glycoproteins. The biosynthesis of the various components of the fungal cell wall and the importance of the components in the formation of a functional cell wall, as revealed through mutational analyses, are discussed. There is strong evidence that the chitin, glucans and glycoproteins are covalently cross-linked together and that the cross-linking is a dynamic process that occurs extracellularly.