Chitosan and Chitin Deacetylase Activity Are Necessary for Development and Virulence of Ustilago maydis.
TL;DR: In this article, the chitin deacetylase (CDA) family of Ustilago maydis has been studied and it was shown that certain combinations of mutations significantly affected virulence with reduced adherence, appressorium formation, penetration and activation of plant defenses.
Abstract: The biotrophic fungus Ustilago maydis harbors a chitin deacetylase (CDA) family of six active genes as well as one pseudogene which are differentially expressed during colonization. This includes one secreted soluble CDA (Cda4) and five putatively glycosylphosphatidylinositol (GPI)-anchored CDAs, of which Cda7 belongs to a new class of fungal CDAs. Here, we provide a comprehensive functional study of the entire family. While budding cells of U. maydis showed a discrete pattern of chitosan staining, biotrophic hyphae appeared surrounded by a chitosan layer. We purified all six active CDAs and show their activity on different chitin substrates. Single as well as multiple cda mutants were generated and revealed a virulence defect for mutants lacking cda7 We implicated cda4 in production of the chitosan layer surrounding biotrophic hyphae and demonstrated that the loss of this layer does not reduce virulence. By combining different cda mutations, we detected redundancy as well as specific functions for certain CDAs. Specifically, certain combinations of mutations significantly affected virulence concomitantly with reduced adherence, appressorium formation, penetration, and activation of plant defenses. Attempts to inactivate all seven cda genes simultaneously were unsuccessful, and induced depletion of cda2 in a background lacking the other six cda genes illustrated an essential role of chitosan for cell wall integrity.IMPORTANCE The basidiomycete Ustilago maydis causes smut disease in maize, causing substantial losses in world corn production. This nonobligate pathogen penetrates the plant cell wall with the help of appressoria and then establishes an extensive biotrophic interaction, where the hyphae are tightly encased by the plant plasma membrane. For successful invasion and development in plant tissue, recognition of conserved fungal cell wall components such as chitin by the plant immune system needs to be avoided or suppressed. One strategy to achieve this lies in the modification of chitin to chitosan by chitin deacetylases (CDAs). U. maydis has seven cda genes. This study reveals discrete as well as redundant contributions of these genes to virulence as well as to cell wall integrity. Unexpectedly, the inactivation of all seven genes is not tolerated, revealing an essential role of chitosan for viability.
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TL;DR: A comprehensive review of fungal effector biology can be found in this article, with a focus on the versatile virulence functions of effectors in promoting pathogen infection and colonization.
Abstract: Phytopathogenic fungi secrete a large arsenal of effector molecules, including proteinaceous effectors, small RNAs, phytohormones and derivatives thereof. The pathogenicity of fungal pathogens is primarily determined by these effectors that are secreted into host cells to undermine innate immunity, as well as to facilitate the acquisition of nutrients for their in planta growth and proliferation. After conventional and non-conventional secretion, fungal effectors are translocated into different subcellular compartments of the host cells to interfere with various biological processes. In extracellular spaces, apoplastic effectors cope with physical and chemical barriers to break the first line of plant defenses. Intracellular effectors target essential immune components on the plasma membrane, in the cytosol, including cytosolic organelles, and in the nucleus to suppress host immunity and reprogram host physiology, favoring pathogen colonization. In this review, we comprehensively summarize the recent advances in fungal effector biology, with a focus on the versatile virulence functions of fungal effectors in promoting pathogen infection and colonization. A perspective of future research on fungal effector biology is also discussed.
29 citations
TL;DR: The critical roles that secreted GHs from plant-associated fungi and oomycetes play in plant–microbe interactions are highlighted, an overview of existing knowledge gaps are provided and future directions are summarized.
Abstract: During host colonization, plant-associated microbes, including fungi and oomycetes, deliver a collection of glycoside hydrolases (GHs) to their cell surfaces and surrounding extracellular environments. The number and type of GHs secreted by each organism is typically associated with their lifestyle or mode of nutrient acquisition. Secreted GHs of plant-associated fungi and oomycetes serve a number of different functions, with many of them acting as virulence factors (effectors) to promote microbial host colonization. Specific functions involve, for example, nutrient acquisition, the detoxification of antimicrobial compounds, the manipulation of plant microbiota, and the suppression or prevention of plant immune responses. In contrast, secreted GHs of plant-associated fungi and oomycetes can also activate the plant immune system, either by acting as microbe-associated molecular patterns (MAMPs), or through the release of damage-associated molecular patterns (DAMPs) as a consequence of their enzymatic activity. In this review, we highlight the critical roles that secreted GHs from plant-associated fungi and oomycetes play in plant–microbe interactions, provide an overview of existing knowledge gaps and summarize future directions.
23 citations
TL;DR: In this article, the inhibitory effects of volatile organic compounds (VOCs) produced by B. subtilis CF-3 on Colletotrichum gloeosporioides, a major destructive phytopathogen of litchi anthracnose, were analyzed using proteomics and transcriptomics.
Abstract: Bacillus subtilis is commonly used as a biocontrol bacterium owing to its strong antifungal activity, broad-spectrum inhibition, and general safety. In this study, the inhibitory effects of volatile organic compounds (VOCs) produced by B. subtilis CF-3 on Colletotrichum gloeosporioides, a major destructive phytopathogen of litchi anthracnose, were analyzed using proteomics and transcriptomics. Differentially expressed genes (DEGs) and proteins (DEPs) indicated that the inhibition of C. gloeosporioides by B. subtilis CF-3 VOCs downregulated the expression of genes related to cell membrane fluidity, cell wall integrity, energy metabolism, and production of cell wall-degrading enzymes. Particularly, those important DEGs and DEPs related to the ergosterol biosynthetic and biosynthesis of unsaturated fatty acids are most significantly influenced. 2,4-di-tert-butylphenol, a characteristic component of B. subtilis CF-3 VOCs, also showed a similar effect on C. gloeosporioides. Our results provide a theoretical basis for the potential application of B. subtilis CF-3 in the postharvest protection of fruits and vegetables.
14 citations
TL;DR: Attention is placed on aspects of fungal cell wall biogenesis during plant infection, with emphasis on the maize leaf anthracnose and stalk rot fungus, Colletotrichum graminicola.
Abstract: The genus Colletotrichum harbors many plant pathogenic species, several of which cause significant yield losses in the field and post harvest. Typically, in order to infect their host plants, spores germinate, differentiate a pressurized infection cell, and display a hemibiotrophic lifestyle after plant invasion. Several factors required for virulence or pathogenicity have been identified in different Colletotrichum species, and adaptation of cell wall biogenesis to distinct stages of pathogenesis has been identified as a major pre-requisite for the establishment of a compatible parasitic fungus–plant interaction. Here, we highlight aspects of fungal cell wall biogenesis during plant infection, with emphasis on the maize leaf anthracnose and stalk rot fungus, Colletotrichum graminicola.
12 citations
TL;DR: In this paper, RNAi silencing of PxCDA resulted in a dramatic reduction in fungal growth that was linked to a rapid elicitation of chitin-triggered immunity.
Abstract: Fungicide resistance is a serious problem for agriculture. This is particularly apparent in the case of powdery mildew fungi. Therefore, there is an urgent need to develop new agrochemicals. Chitin is a well-known elicitor of plant immunity, and fungal pathogens have evolved strategies to overcome its detection. Among these strategies, chitin deacetylase (CDA) is responsible for modifying immunogenic chitooligomers and hydrolysing the acetamido group in the N-acetylglucosamine units to avoid recognition. In this work, we tested the hypothesis that CDA can be an appropriate target for antifungals using the cucurbit powdery mildew pathogen Podosphaera xanthii. According to our hypothesis, RNAi silencing of PxCDA resulted in a dramatic reduction in fungal growth that was linked to a rapid elicitation of chitin-triggered immunity. Similar results were obtained with treatments with carboxylic acids such as EDTA, a well-known CDA inhibitor. The disease-suppression activity of EDTA was not associated with its chelating activity since other chelating agents did not suppress disease. The binding of EDTA to CDA was confirmed by molecular docking studies. Furthermore, EDTA also suppressed green and grey mould-causing pathogens applied to oranges and strawberries, respectively. Our results conclusively show that CDA is a promising target for control of phytopathogenic fungi and that EDTA could be a starting point for fungicide design.
7 citations
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TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
225,085 citations
TL;DR: Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose.
5,395 citations
TL;DR: It is argued that nascent fungal infections will cause increasing attrition of biodiversity, with wider implications for human and ecosystem health, unless steps are taken to tighten biosecurity worldwide.
Abstract: The past two decades have seen an increasing number of virulent infectious diseases in natural populations and managed landscapes. In both animals and plants, an unprecedented number of fungal and fungal-like diseases have recently caused some of the most severe die-offs and extinctions ever witnessed in wild species, and are jeopardizing food security. Human activity is intensifying fungal disease dispersal by modifying natural environments and thus creating new opportunities for evolution. We argue that nascent fungal infections will cause increasing attrition of biodiversity, with wider implications for human and ecosystem health, unless steps are taken to tighten biosecurity worldwide.
2,408 citations
Max Planck Society1, University of Marburg2, Broad Institute3, Trent University4, University of Toronto5, California State University, Long Beach6, University of British Columbia7, University of Georgia8, University of Louisville9, Utrecht University10, Saint Joseph's University11, Spanish National Research Council12, Cornell University13, CINVESTAV14, University of Kentucky15, Duke University16, University of Valencia17, Saint Louis University18, Bayer19, Exelixis20, Technische Universität München21
TL;DR: The discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi.
Abstract: Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.
1,120 citations
TL;DR: E-CRISP provides flexible output and experimentoriented design parameters, enabling design of multiple libraries and thereby systematic analysis of the influence of different parameters, and can be used to reevaluate CRISPR constructs for onor off-target sites and targeted genomic loci.
Abstract: encodes an RNA (crRNA), consisting of a guide RNA (gRNA) and transactivating CRISPR RNA parts. A processed crRNA fragment is incorporated into the Cas9 protein, guiding it to the target DNA, where the Cas9 nuclease introduces a double-strand break9,10. The CRISPR-Cas system has been successfully used in human induced pluripotent stem cells, mice, zebrafish and flies, among other organisms, to disrupt gene function. Here we describe E-CRISP, a web application to design gRNA sequences (Fig. 1a). It provides flexible output and experimentoriented design parameters, enabling design of multiple libraries and thereby systematic analysis of the influence of different parameters. E-CRISP identifies target sequences complementary to the gRNA ending in a 3ʹ protospacer-adjacent motif (PAM), N(G or A)G, which is required for the recruited Cas9 nuclease to cut the DNA double strand. E-CRISP uses a fast indexing approach to find binding sites and a binary interval tree for rapid annotation of putative gRNA target sites (Supplementary Note 1). Using these algorithms, it is feasible to create genome-scale libraries for several organisms in a few hours. For instance, to design a library covering the Drosophila melanogaster genome requires less than 1 h (Supplementary Fig. 1 and Supplementary Table 1). Off-target effects and target-site homology are evaluated by E-CRISP using the alignment program Bowtie2 (Supplementary Note 2). Designs are shown in the output if the number of offtargets does not exceed a user-specified threshold. If more than one design is found targeting a desired locus, designs are ranked according to on-target specificity and number of off-targets. E-CRISP can also be used to reevaluate CRISPR constructs for onor off-target sites and targeted genomic loci. As an example, we searched for designs to target let-7 for gene disruption in zebrafish, fly, worm and human (Fig. 1b). We found at least one gRNA design per locus. In worm, fly and human, the cuts are located at the site that is transformed to mature microRNA and thus should lead to mutations blocking its proper function. In zebrafish the cut is located in the predicted hairpin structure. E-CRISP is available for twelve organisms and can be easily extended. E-CRISP will help to further develop and deploy the acKnoWLedGments This work was supported by the Wellcome Trust through a Senior Research Fellowship to J.R. (084229), a core grant to the Wellcome Trust Centre for Cell Biology (092076), a European Research Council grant (233457) to M.T., a Genome Québec International Recruitment Award to M.T. and a Canada Research Chair in Systems and Synthetic Biology to M.T.
726 citations