Role of the Fungal Cell Wall in Pathogenesis and Antifungal Resistance

Study of the fungal cell wall is currently an area of very active research. The relevance of the fungal cell wall for cell survival, and pathogenicity has been well established. The view of the cell wall as a tough and impenetrable structure has been left behind, and it is now conceived as a plastic shield that undergoes structural changes depending on the surrounding environmental conditions and morphological states. The fungal cell wall is also the source of most of the pathogen-associated molecular patterns that immune cells recognize, and thus facilitates establishment of a protective antifungal immunity. Paradoxically, fungi, through their cell wall, possess disguising mechanisms to avoid immune recognition. This review gathers the current knowledge about the cell wall of Candida albicans, Aspergillus fumigatus and Paracoccidioides brasiliensis, stressing the importance of the fungal cell wall for pathogenesis, immune recognition, and as a source of targets for antifungal drugs.     

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.     

Comparison of Rhizosphere Bacterial Communities in Arabidopsis thaliana Mutants for Systemic Acquired Resistance

Systemic acquired resistance (SAR) is an inducible systemic plant defense against a broad spectrum of plant pathogens, with the potential to secrete antimicrobial compounds into the soil. However, its impact on rhizosphere bacteria is not known. In this study, we examined fingerprints of bacterial communities in the rhizosphere of the model plant Arabidopsis thaliana to determine the effect of SAR on bacterial community structure and diversity. We compared Arabidopsis mutants that are constitutive and non-inducible for SAR and verified SAR activation by measuring pathogenesis-related protein activity via a β-glucoronidase (GUS) reporter construct driven by the β-1-3 glucanase promoter. We used terminal restriction fragment length polymorphism (T-RFLP) analysis of MspI- and HaeIII-digested 16S rDNA to estimate bacterial rhizosphere community diversity, with Lactobacillus sp. added as internal controls. T-RFLP analysis showed a clear rhizosphere effect on community structure, and diversity analysis of both rhizosphere and bulk soil operational taxonomic units (as defined by terminal restriction fragments) using richness, Shannon–Weiner, and Simpson’s diversity indices and evenness confirmed that the presence of Arabidopsis roots significantly altered bacterial communities. This effect of altered soil microbial community structure by plants was also seen upon multivariate cluster analysis of the terminal restriction fragments. We also found visible differences in the rhizosphere community fingerprints of different Arabidopsis SAR mutants; however, there was no clear decrease of rhizosphere diversity because of constitutive SAR expression. Our study suggests that SAR can alter rhizosphere bacterial communities, opening the door to further understanding and application of inducible plant defense as a driving force in structuring soil bacterial assemblages.     

Terpene Down-Regulation Triggers Defense Responses in Transgenic Orange Leading to Resistance against Fungal Pathogens

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.Plants are sessile organisms that produce and emit a vast array of volatile organic compounds (VOCs) to communicate between parts of the same plant and with other plants. It is generally accepted that the original role of these compounds in nature is related to defense functions (Degenhardt et al., 2003). Most VOCs are terpenoids, fatty acid degradation compounds, phenylpropanoids, and amino acid-derived products. Among these, terpenoids are likely to be the most abundant and expensive to produce (Gershenzon, 1994). Terpenoids are isoprenoid-derived compounds synthesized through the condensation of C5 isoprene units, a process that is catalyzed by a wide diversity of terpene synthases using geranyl diphosphate (GDP), farnesyl diphosphate (FDP), and geranylgeranyl diphosphate (GGDP) as substrates. These reactions give rise to the C5 hemiterpenes, the C10 monoterpenes, the C15 sesquiterpenes, and the C20 diterpenes (Dudareva et al., 2006).In green tissues, volatile terpenoid synthesis is either induced upon wounding or occurs constitutively; terpenes can be then stored in specific organs or tissues where they would be most effective in defense responses, such as leaf trichomes, resin ducts and lacticifers, pockets near the epidermis, or secretory cavities in Citrus spp. (Langenheim, 1994; Turner et al., 2000; Trapp and Croteau, 2001; Voo et al., 2012). Genetic engineering experiments have demonstrated that specific terpenoid compounds emitted by leaves can intoxicate, repel, or deter herbivores (Aharoni et al., 2003; Wu et al., 2006), or they may attract the natural predators and parasitoids of damaging herbivores to protect plants from further damage (Kappers et al., 2005; Schnee et al., 2006). These terpenoids are naturally found in complex mixtures, and it has been proposed that they can act synergistically, as in conifer resin, for simultaneous protection against pests and pathogens (Phillips and Croteau, 1999). Although fatty acid degradation products (such as jasmonates) and phenylpropanoids (such as salicylates) as well as their volatile and nonvolatile precursors are clearly involved in many induced defense responses against pests and pathogens (Glazebrook, 2005), much less is known regarding the participation of terpenoid volatiles in the defense against microorganisms in plants and about the possible interactions of these terpenoids with phytohormones.In contrast to their function in leaves, when released from flowers and mature fruits, the main function of terpenoid volatiles is in the attraction of pollinators (Pichersky and Gershenzon, 2002; Kessler et al., 2008; Junker and Blüthgen, 2010; Schiestl, 2010) and seed-dispersing animals (Lomáscolo et al., 2010; Rodríguez et al., 2011b), respectively. Fruit maturation and ripening are usually associated with large increases in the synthesis and accumulation of specific flavored volatiles, which are proposed to function as signals for seed dispersal (Auldridge et al., 2006; Goff and Klee, 2006; Rodríguez et al., 2013).Upon wounding, plant responses to biotic stresses are orchestrated locally and systemically by signaling molecules. Among these molecules, the jasmonates regulate defenses against arthropod herbivores and necrotroph fungal pathogens as well as biotrophic pathogens, such as some mildews (Ellis and Turner, 2001; Stintzi et al., 2001; Kessler et al., 2004; Li et al., 2005; Wasternack, 2007; Browse and Howe, 2008). In addition to jasmonates, molecules such as salicylic acid (SA) and ethylene appear to regulate distinct defense pathways and are major synergistic (Mur et al., 2006) or antagonistic (De Vos et al., 2005) regulators of plant innate immunity. Plants produce a specific blend of these alarm signals after pathogen or pest attacks, and the production of these molecules varies greatly in quantity, composition, and timing. These signals activate differential sets of defense-related genes that eventually determine the nature of the defense response against the attacker (Reymond and Farmer, 1998; Rojo et al., 2003; De Vos et al., 2005). All genes that encode enzymes involved in the biosynthesis of jasmonates are jasmonic acid (JA) inducible (Wasternack, 2006), indicating that JA biosynthesis is regulated by positive feedback. The precursor for the biosynthesis of JA is α-linolenic acid. The activity of the 13-lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) enzymes converts α-linolenic acid to cis-(+)-12-oxophytodienoic acid (OPDA). OPDA REDUCTASE3 catalyzes the reduction of OPDA (and dinor-OPDA) to oxo-pentenyl-cycloheptane-octanoic acid, which, in turn, undergoes three rounds of β-oxidation leading to jasmonyl-CoA formation. Jasmonyl-CoA is then cleaved by a putative thioesterase yielding (+)-7-iso-JA, which equilibrates to the more stable (−)-JA (Wasternack and Kombrink, 2010).The exogenous application of jasmonates on plants and the existence of mutant and/or transgenic plants altered in JA biosynthesis or signaling have led to altered susceptibility or resistance to pathogens. Impaired JA biosynthesis or signaling is generally associated with decreased levels of defensive compounds, including VOCs, and reduced plant biomass and/or fitness under insect attack (Howe et al., 1996; Halitschke and Baldwin, 2004). For example, Arabidopsis (Arabidopsis thaliana) mutants defective in JA perception (e.g. coronatine-insensitive1 [coi1]) or biosynthesis (e.g. aos and defective in anther dehiscence1) are susceptible to pathogen infections (Feys et al., 1994; Xie et al., 1998; Park et al., 2002; Turner et al., 2002). In contrast, mutants (e.g. constitutive expression of vegetative storage protein1 and Arabidopsis Ser/Thr phosphatase of type 2C1) with constitutive or wound-induced activation of the JA pathway exhibit enhanced resistance to fungal pathogens and pests and phenotypes characteristic of JA-treated plants (Ellis and Turner, 2001; Ellis et al., 2002; Schweighofer et al., 2007).Sweet orange (Citrus sinensis) is a perennial tree species that is exposed to recurrent biotic and abiotic challenges during its decades of growth in orchards. Orange fruits undergo a nonclimacteric maturation process in which the biochemistry, physiology, and structure of the organ are altered to complete the release of mature seeds. These changes typically include fruit growth and texture modification; color change through the degradation of chlorophylls and a parallel induction of carotenogenesis in the peel (flavedo) and pulp; flavonoid accumulation in the pulp; increases and decreases in the sugar and acid contents, respectively; and global accumulation and selective emission of volatile terpenoids (Spiegel-Roy and Goldschmidt, 1996). In nature, d-limonene accumulates gradually in the oil glands of the peel during fruit development and reaches its maximum level shortly before the breaker stage, followed by a steady decline during maturation (Attaway et al., 1967; Kekelidze et al., 1989; Rodríguez et al., 2011b). The high amount of d-limonene that accumulates in orange peels has a tremendous metabolic cost, suggesting an important biological role for this terpene and other related compounds in the interactions between fruits and the biotic environment.Previously, we examined the biological role of d-limonene by manipulating oil gland chemistry via the antisense (AS) overexpression of a d-limonene synthase gene from Satsuma mandarin (Citrus unshiu) in orange fruits. Compared with empty vector (EV) controls, fruit peels from AS transformants showed a dramatic reduction in d-limonene accumulation; decreased levels of other monoterpenes, sesquiterpenes, and monoterpene aldehydes; and increased levels of monoterpene alcohols. When challenged with the necrotroph fungus Penicillium digitatum, the causal agent of green mold rot, AS-transformed fruits were highly resistant to fungal infection. Full susceptibility to P. digitatum infection was restored when AS fruits were supplemented with d-limonene but not other monoterpene alcohols, indicating that d-limonene accumulation in the orange peel was required for the successful progress of this plant-pathogen interaction (Rodríguez et al., 2011a, 2011b). Green mold rot is the most important postharvest disease of citrus fruit worldwide, accounting for up to 60% to 80% of total losses during postharvest life of the fruit. P. digitatum is considered to be a specialist pathogen of citrus fruits that efficiently infects the peel through injuries in which ubiquitous fungal spores germinate and rapidly colonize the surrounding areas (Droby et al., 2008). The control of this pathogen relies heavily on the use of synthetic chemicals, but concerns regarding their potential negative effects on human health and also the generation of fungicide-resistant strains have encouraged finding alternatives, such as the generation of citrus trees with fruits that are genetically resistant to the pathogen.In this work, to better understand the mechanism underlying the constitutive resistance to P. digitatum conferred by the reduction of limonene in AS orange fruits, we analyzed the pattern of fruit growth and the morphological and biochemical developmental characteristics and performed a global analysis of gene expression using a 20K citrus microarray. The study is supplemented by examining the possible involvement of key hormone signals and isoprenoid precursors in the fruit peel. We report here that the reduced level of d-limonene in AS fruits is tightly associated with the constitutive activation of defense response signaling cascades. Our results establish, to our knowledge for the first time, a correlation between increased volatile terpene content and the decline of defense responses in a fleshy fruit during maturation, which would facilitate necrotroph fungal infections in citrus fruits.     

真菌菌丝细胞壁提取物对马铃薯块茎组织抗干腐病的诱导效应

以马铃薯(品种'大西洋')块茎切片为材料,研究了马铃薯非亲和菌株粉红单端孢(Trichothecium roseum)菌丝细胞壁提取物处理对马铃薯块茎组织干腐病抗性的诱导效果及其机理.结果表明,150 μg/mL(以中性糖含量为标准)菌丝细胞壁提取物预处理马铃薯切片72 h后进行损伤接种能显著降低干腐病菌(Fusarium sulphureum)的侵染能力,预处理块茎的病斑直径仅为对照的43.6%;菌丝细胞壁提取物处理能显著增强与马铃薯块茎组织抗病相关的过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性.可见,粉红单端孢菌丝细胞壁提取物预处理能显著增强马铃薯块茎组织对干腐病的抗性,而且主要是通过提高马铃薯块茎组织中与抗病性相关酶的活性来实现的.     

壳寡糖诱导水稻过敏性细胞死亡及抗病性的提高

作为真菌细胞壁的主要成分之一的壳寡糖(Oligo-GlcNAc)能够诱导水稻悬浮细胞和幼叶细胞发生过敏性死亡,并伴有H2O2的积累.以1 μg*mL-1壳寡糖处理水稻悬浮细胞12 h后细胞明显死亡;诱导水稻幼叶细胞出现明显的死亡所需壳寡糖浓度为5 μg*mL-1.以壳寡糖处理的水稻抗稻瘟病性也明显增强.     

A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.     

Preparation of Anti-protein and Anti-mannan Antisera against Fungal Cell Wall by Affinity Chromatography

Iranzo, M., Marcilla, A., Elorza, M. V., Mormeneo, S., and Sentandreu, R. 1994. Preparation of anti-protein and anti-mannan antisera against fungal cell wall by affinity chromatography. Experimental Mycology 18, 159-167. A novel and easy chromatographic method has been developed for the isolation of anti-protein and anti-mannan antisera from a population of polyclonal antibodies obtained against Candida albicans and Yarrowia lipolytica cell wall mannoproteins. The technique is based on the immobilization of mannan (to be used as immunoadsorbent) by Affi-Prep H_z resin after the oxidation of neighboring hydroxyl groups of the polysaccharide with sodium periodate. For Y. lipolytica polyclonal antiserum, a single chromatographic step using the homologous mannan was sufficient to obtain an antiprotein antibody preparation free of antimannan antibodies. For C. albicans, three chromatographic processes using homologous and heterologous mannan were needed to obtain a satisfactory antiprotein antiserum. The potential application of the anti-protein antiserum obtained has been demonstrated by indirect immunofluorescence assays of whole cells and electrophoretic analysis of wall proteins in C. albicans and Saccharomyces cerevisiae .     

Slow Growth Induces Heat-Shock Resistance in Normal and Respiratory-deficient Yeast

Yeast cells respond to a variety of environmental stresses, including heat shock and growth limitation. There is considerable overlap in these responses both from the point of view of gene expression patterns and cross-protection for survival. We performed experiments in which cells growing at different steady-state growth rates in chemostats were subjected to a short heat pulse. Gene expression patterns allowed us to partition genes whose expression responds to heat shock into subsets of genes that also respond to slow growth rate and those that do not. We found also that the degree of induction and repression of genes that respond to stress is generally weaker in respiratory deficient mutants, suggesting a role for increased respiratory activity in the apparent stress response to slow growth. Consistent with our gene expression results in wild-type cells, we found that cells growing more slowly are cross-protected for heat shock, i.e., better able to survive a lethal heat challenge. Surprisingly, however, we found no difference in cross-protection between respiratory-deficient and wild-type cells, suggesting induction of heat resistance at low growth rates is independent of respiratory activity, even though many of the changes in gene expression are not.     

Macromolecules Released by a Plant Growth-promoting Rhizobacterium as Elicitors of Systemic Resistance in Tomato to Bacterial and Fungal Pathogens

One of 500 rhizobacteria isolated from soil, rhizosphere and rhizoplane of healthy tomato plants was previously selected in laboratory, greenhouse and field tests as a good inducer of systemic resistance. This plant growth‐promoting rhizobacterium (PGPR) was identified as Bacillus cereus by fatty‐acid analysis. Bacillus cereus bacterial cells were removed from liquid culture by centrifugation and the supernatant repeatedly dialyzed (cut‐off = 12 000 daltons) against distilled water. Dialysates applied to roots protected tomato plants against leaf fungal and bacterial pathogens, evidence that macromolecules synthesized by the PGPR and released into the environment act as elicitors of systemic resistance.     

Arabidopsis Heterotrimeric G-Proteins Play a Critical Role in Host and Nonhost Resistance against Pseudomonas syringae Pathogens

Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gβ and Gγ subunits in response to host and nonhost Pseudomonas pathogens. Plants lacking functional Gα, Gβ and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gβ led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. tabaci whereas overexpression of Gβ, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. maculicola, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gβ mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to P. syringae.