Fungal Pathogenesis Research Articles

Measuring Stress Phenotypes in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human fungal pathogen capable of surviving in a wide range of environments and hosts. It has been developed as a model organism to study fungal pathogenesis due to its fully sequenced haploid genome and optimized gene deletion and mutagenesis protocols. These methods have greatly aided indetermining the relationship between Cryptococcus genotype and phenotype. Furthermore, the presence of congenic mata and matα strains associated with a defined sexual cycle has helped further understand cryptococcal biology. Several in vitro stress conditions have been optimized to closely mimic the stress that yeast encounter in the environment or within the infected host. These conditions have proven to be extremely useful in elucidating the role of several genes in allowing yeast to adapt and survive in hostile external environments. This chapter describes various in vitro stress conditions that could be used to test the sensitivity of different mutant strains, as well as the protocol for preparing them. We have also included a list of mutants that could be used as a positive control strain when testing the sensitivity of the desired strain to a specific stress.

Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and susceptibility to powdery mildew.

Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.

Accumulation of endogenous free radicals is required to induce titan-like cell formation in Cryptococcus neoformans.

Cryptococcus neoformans is an excellent model to investigate fungal pathogenesis. This yeast can produce "titan cells," which are cells of an abnormally larger size that contribute to the persistence of the yeast in the host. In this work, we have used a new approach to characterize them by identifying drugs that inhibit this process. We have used a repurposing off-patent drug library, combined with an automatic method to image and analyze fungal cell size. In this way, we have identified many compounds that inhibit this transition. Interestingly, several compounds were antioxidants, allowing us to confirm that endogenous ROS and mitochondrial changes are important for titan cell formation. This work provides new evidence of the mechanisms required for titanization. Furthermore, the future characterization of the inhibitory mechanisms of the identified compounds by the scientific community will contribute to better understand the role of titan cells in virulence.

Genome-wide expression QTL mapping reveals the highly dynamic regulatory landscape of a major wheat pathogen

BackgroundIn agricultural ecosystems, outbreaks of diseases are frequent and pose a significant threat to food security. A successful pathogen undergoes a complex and well-timed sequence of regulatory changes to avoid detection by the host immune system; hence, well-tuned gene regulation is essential for survival. However, the extent to which the regulatory polymorphisms in a pathogen population provide an adaptive advantage is poorly understood.ResultsWe used Zymoseptoria tritici, one of the most important pathogens of wheat, to generate a genome-wide map of regulatory polymorphism governing gene expression. We investigated genome-wide transcription levels of 146 strains grown under nutrient starvation and performed expression quantitative trait loci (eQTL) mapping. We identified cis-eQTLs for 65.3% of all genes and the majority of all eQTL loci are within 2kb upstream and downstream of the transcription start site (TSS). We also show that polymorphism in different gene elements contributes disproportionally to gene expression variation. Investigating regulatory polymorphism in gene categories, we found an enrichment of regulatory variants for genes predicted to be important for fungal pathogenesis but with comparatively low effect size, suggesting a separate layer of gene regulation involving epigenetics. We also show that previously reported trait-associated SNPs in pathogen populations are frequently cis-regulatory variants of neighboring genes with implications for the trait architecture.ConclusionsOverall, our study provides extensive evidence that single populations segregate large-scale regulatory variation and are likely to fuel rapid adaptation to resistant hosts and environmental change.

Long noncoding RNAs emerge from transposon-derived antisense sequences and may contribute to infection stage-specific transposon regulation in a fungal phytopathogen

BackgroundThe genome of the obligate biotrophic phytopathogenic barley powdery mildew fungus Blumeria hordei is inflated due to highly abundant and possibly active transposable elements (TEs). In the absence of the otherwise common repeat-induced point mutation transposon defense mechanism, noncoding RNAs could be key for regulating the activity of TEs and coding genes during the pathogenic life cycle.ResultsWe performed time-course whole-transcriptome shotgun sequencing (RNA-seq) of total RNA derived from infected barley leaf epidermis at various stages of fungal pathogenesis and observed significant transcript accumulation and time point-dependent regulation of TEs in B. hordei. Using a manually curated consensus database of 344 TEs, we discovered phased small RNAs mapping to 104 consensus transposons, suggesting that RNA interference contributes significantly to their regulation. Further, we identified 5,127 long noncoding RNAs (lncRNAs) genome-wide in B. hordei, of which 823 originated from the antisense strand of a TE. Co-expression network analysis of lncRNAs, TEs, and coding genes throughout the asexual life cycle of B. hordei points at extensive positive and negative co-regulation of lncRNAs, subsets of TEs and coding genes.ConclusionsOur work suggests that similar to mammals and plants, fungal lncRNAs support the dynamic modulation of transcript levels, including TEs, during pivotal stages of host infection. The lncRNAs may support transcriptional diversity and plasticity amid loss of coding genes in powdery mildew fungi and may give rise to novel regulatory elements and virulence peptides, thus representing key drivers of rapid evolutionary adaptation to promote pathogenicity and overcome host defense.

An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway.

The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.

Mycoviruses: Antagonistic Potential, Fungal Pathogenesis, and Their Interaction with Rhizoctonia solani.

Mycoviruses, or fungal viruses, are prevalent in all significant fungal kingdoms and genera. These low-virulence viruses can be used as biocontrol agents to manage fungal diseases. These viruses are divided into 19 officially recognized families and 1 unclassified genus. Mycoviruses alter sexual reproduction, pigmentation, and development. Spores and fungal hypha spread mycoviruses. Isometric particles mostly encapsulate dsRNA mycoviruses. The widespread plant-pathogenic fungus Rhizoctonia solani, which has caused a rice sheath blight, has hosted many viruses with different morphologies. It causes significant crop diseases that adversely affect agriculture and the economy. Rice sheath blight threatens the 40% of the global population that relies on rice for food and nutrition. This article reviews mycovirology research on Rhizoctonia solani to demonstrate scientific advances. Mycoviruses control rice sheath blight. Hypovirulence-associated mycoviruses are needed to control R. solani since no cultivars are resistant. Mycoviruses are usually cryptic, but they can benefit the host fungus. Phytopathologists may use hypovirulent viruses as biological control agents. New tools are being developed based on host genome studies to overcome the intellectual challenge of comprehending the interactions between viruses and fungi and the practical challenge of influencing these interactions to develop biocontrol agents against significant plant pathogens.

Pathogenic fungi shape the fungal community, network complexity, and pathogenesis in kiwifruit.

Kiwifruit decay caused by endophytic fungi is affected by exogenous pathogens that trigger changes in fungal community composition and interact with the endophytic fungal community. Four fungal pathogens of kiwifruit were identified. These were Aspergillus japonicus, Aspergillus flavus, Botryosphaeria dothidea, and Penicillium oxalicum. Except for P. oxalicum, the remaining three species represent newly described pathogens of kiwifruit. All four fungal species caused disease and decay in mature kiwifruit. Results of the fungal community analysis indicated that three pathogens that A. japonicus, A. flavus and P. oxalicum were the most dominant, however, other fungal species that did not cause disease symptoms were also present. Positive interactions between fungal species were found in asymptomatic, symptomatic, and infected kiwifruit. The ability of all four pathogens to infect kiwifruit was confirmed in an inoculation experiment. The presence of any one of the four identified pathogens accelerated decay development and limited the postharvest longevity of harvested kiwifruit. Results of the study identified and confirmed the ability of four fungal species to infect and cause decay in harvested kiwifruit. Changes in the structure and composition of the kiwifruit microbiome during the decay process were also characterized. This provides a foundation for the further study of the microbiome of kiwifruit and their involvement in postharvest diseases.

Alternative Non-Mammalian Animal and Cellular Methods for the Study of Host-Fungal Interactions.

In the study of fungal pathogenesis, alternative methods have gained prominence due to recent global legislation restricting the use of mammalian animals in research. The principle of the 3 Rs (replacement, reduction, and refinement) is integrated into regulations and guidelines governing animal experimentation in nearly all countries. This principle advocates substituting vertebrate animals with other invertebrate organisms, embryos, microorganisms, or cell cultures. This review addresses host-fungus interactions by employing three-dimensional (3D) cultures, which offer more faithful replication of the in vivo environment, and by utilizing alternative animal models to replace traditional mammals. Among these alternative models, species like Caenorhabditis elegans and Danio rerio share approximately 75% of their genes with humans. Furthermore, models such as Galleria mellonella and Tenebrio molitor demonstrate similarities in their innate immune systems as well as anatomical and physiological barriers, resembling those found in mammalian organisms.

Fungi's Swiss Army Knife: Pleiotropic Effect of Melanin in Fungal Pathogenesis during Cattle Mycosis.

Fungal threats to public health, food security, and biodiversity have escalated, with a significant rise in mycosis cases globally. Around 300 million people suffer from severe fungal diseases annually, while one-third of food crops are decimated by fungi. Vertebrate, including livestock, are also affected. Our limited understanding of fungal virulence mechanisms hampers our ability to prevent and treat cattle mycoses. Here we aim to bridge knowledge gaps in fungal virulence factors and the role of melanin in evading bovine immune responses. We investigate mycosis in bovines employing a PRISMA-based methodology, bioinformatics, and data mining techniques. Our analysis identified 107 fungal species causing mycoses, primarily within the Ascomycota division. Candida, Aspergillus, Malassezia, and Trichophyton were the most prevalent genera. Of these pathogens, 25% produce melanin. Further research is required to explore the involvement of melanin and develop intervention strategies. While the literature on melanin-mediated fungal evasion mechanisms in cattle is lacking, we successfully evaluated the transferability of immunological mechanisms from other model mammals through homology. Bioinformatics enables knowledge transfer and enhances our understanding of mycosis in cattle. This synthesis fills critical information gaps and paves the way for proposing biotechnological strategies to mitigate the impact of mycoses in cattle.

The role of the Mediator complex in fungal pathogenesis and response to antifungal agents.

Mediator is a complex of polypeptides that plays a central role in the recruitment of RNA polymerase II to promoters and subsequent transcriptional activation in eukaryotic organisms. Studies have now shown that Mediator has a role in regulating expression of genes implicated in virulence and antifungal drug resistance in pathogenic fungi. The roles of specific Mediator subunits have been investigated in several species of pathogenic fungi, particularly in the most pathogenic yeast Candida albicans. Uniquely, pathogenic yeast also present several interesting examples of divergence in Mediator structure and function, most notably in C. glabrata, which possesses two orthologues of Med15, and in C. albicans, which has a massively expanded family of Med2 orthologues known as the TLO gene family. This review highlights specific examples of recent progress in characterizing the role of Mediator in pathogenic fungi.

Fungal co-expression network analyses identify pathogen gene modules associated with host insect invasion.

The broad host range fungal insect pathogen, Beauveria bassiana, has been commercialized as an alternative to chemical insecticides for pest control worldwide. B. bassiana represents a unique model system with which to examine host-pathogen interactions, and a wide range of genes and processes have been studied. However, significant aspects of virulence, particularly on the genomic scale, remain poorly studied. Here, we have combined available transcriptomes with three newly generated data sets for a combined total analysis of 76 deep-sequenced samples covering growth, development, stress responses, and infection during the life cycle of B. bassiana. Co-expression network analyses resulted in the identification of gene modules enriched during two critical stages of the infection process, namely (i) cuticle penetration and (ii) in vivo hyphal body (dimorphic transition) growth capable of avoiding innate and humoral immune defenses. These analyses identify unique signatures of metabolism, signaling, secondary metabolite production, host defense suppression, membrane reorganization, effector production, and secretion for each stage, including genetic regulators and epigenetic patterns. These data provide a comprehensive framework for understanding and probing fungal adaptations to its pathogenic life cycle and expand the candidate repertoire for continued dissection of the host-pathogen interaction. IMPORTANCE Insect fungal pathogens have evolved unique strategies for overcoming host structural and immunological defenses that span from the sclerotized cuticle to innate and humoral cellular responses. Two critical stages of the infection process involve (i) cuticle penetration and (ii) immune evasion within the insect hemocoel. A set of 76 global transcriptomic data for B. bassiana that include the cuticle penetration and hemocoel growth stages were analyzed for patterns (gene modules) of expression, yielding unique insights into these different life stages. These analyses integrate gene networks involved in fungal development, stress response and pathogenesis to further the systematic understanding of the global processes integral to the unique adaptation employed by fungal pathogens of insects.

Detailed characterisation of invasive aspergillosis in a murine model of X-linked chronic granulomatous disease shows new insights in infections caused by Aspergillus fumigatus versus Aspergillus nidulans.

Invasive aspergillosis (IA) is the most prevalent infectious complication in patients with chronic granulomatous disease (CGD). Yet, understanding of fungal pathogenesis in the CGD host remains limited, particularly with regards to A. nidulans infection. We have used a murine model of X-linked CGD to investigate how the pathogenesis of IA varies between A. fumigatus and A. nidulans, comparing infection in both X-linked CGD (gp91-/-) mice and their parent C57BL/6 (WT) mice. A 14-colour flow cytometry panel was used to assess the cell dynamics over the course of infection, with parallel assessment of pulmonary cytokine production and lung histology. We observed a lack of association between pulmonary pathology and infection outcome in gp91-/- mice, with no significant mortality in A. nidulans infected mice. An overwhelming and persistent neutrophil recruitment and IL-1 release in gp91-/- mice following both A. fumigatus and A. nidulans infection was observed, with divergent macrophage, dendritic cell and eosinophil responses and distinct cytokine profiles between the two infections. We have provided an in-depth characterisation of the immune response to pulmonary aspergillosis in an X-linked CGD murine model. This provides the first description of distinct pulmonary inflammatory environments in A. fumigatus and A. nidulans infection in X-linked CGD and identifies several new avenues for further research.

A New Insight into 6-Pentyl-2H-pyran-2-one against Peronophythora litchii via TOR Pathway.

The litchi downy blight disease of litchi caused by Peronophythora litchii accounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetes P. litchii through integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP against P. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation of P. litchii strains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, including PlCytochrome C and the transcription factors PlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, including PlSpm1 and PlrhoH12 in P. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization of PlYY1 targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity of P. litchii. These findings suggest that 6PP-mediated activation of PlYY1 expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence of P. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.

Herbicide 2,4-dichlorophenoxyacetic acid interferes with MAP kinase signaling in Fusarium graminearum and is inhibitory to fungal growth and pathogenesis

Plant hormones are important for regulating growth, development, and plant-pathogen interactions. Some of them are inhibitory to growth of fungal pathogens but the underlying mechanism is not clear. In this study, we found that hyphal growth of Fusarium graminearum was significantly reduced by high concentrations of IAA and its metabolically stable analogue 2,4-dichlorophenoxyacetic acid (2,4-D). Besides inhibitory effects on growth rate, treatments with 2,4-D also caused significant reduction in conidiation, conidium germination, and germ tube growth. Treatments with 2,4-D had no obvious effect on sexual reproduction but significantly reduced TRI gene expression, toxisome formation, and DON production. More importantly, treatments with 2,4-D were inhibitory to infection structure formation and pathogenesis at concentrations higher than 100 µM. The presence of 1000 µM 2,4-D almost completely inhibited plant infection and invasive growth. In F. graminearum, 2,4-D induced ROS accumulation and FgHog1 activation but reduced the phosphorylation level of Gpmk1 MAP kinase. Metabolomics analysis showed that the accumulation of a number of metabolites such as glycerol and arabitol was increased by 2,4-D treatment in the wild type but not in the Fghog1 mutant. Transformants expressing the dominant active FgPBS2S451D T455D allele were less sensitive to 2,4-D and had elevated levels of intracellular glycerol and arabitol induced by 2,4-D in PH-1. Taken together, our results showed that treatments with 2,4-D interfere with two important MAP kinase pathways and are inhibitory to hyphal growth, DON biosynthesis, and plant infection in F. graminearum.

Ethanol mediates the interaction between Caenorhabditis elegans and the nematophagous fungus Purpureocillium lavendulum.

Accurately recognizing pathogens by the host is vital for initiating appropriate immune response against infecting microorganisms. Caenorhabditis elegans has no known receptor to recognize pathogen-associated molecular pattern. However, recent studies showed that nematodes have a strong specificity for transcriptomes infected by different pathogens, indicating that they can identify different pathogenic microorganisms. However, the mechanism(s) for such specificity remains largely unknown. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum can infect the intestinal tract of the nematode C. elegans and the infection led to the accumulation of reactive oxygen species (ROS) in the infected intestinal tract, which suppressed fungal growth. Co-transcriptional analysis revealed that fungal genes related to anaerobic respiration and ethanol production were up-regulated during infection. Meanwhile, the ethanol dehydrogenase Sodh-1 in C. elegans was also up-regulated. Together, these results suggested that the infecting fungi encounter hypoxia stress in the nematode gut and that ethanol may play a role in the host-pathogen interaction. Ethanol production in vitro during fungal cultivation in hypoxia conditions was confirmed by gas chromatography-mass spectrometry. Direct treatment of C. elegans with ethanol elevated the sodh-1 expression and ROS accumulation while repressing a series of immunity genes that were also repressed during fungal infection. Mutation of sodh-1 in C. elegans blocked ROS accumulation and increased the nematode's susceptibility to fungal infection. Our study revealed a new recognition and antifungal mechanism in C. elegans. The novel mechanism of ethanol-mediated interaction between the fungus and nematode provides new insights into fungal pathogenesis and for developing alternative biocontrol of pathogenic nematodes by nematophagous fungi. IMPORTANCE Nematodes are among the most abundant animals on our planet. Many of them are parasites in animals and plants and cause human and animal health problems as well as agricultural losses. Studying the interaction of nematodes and their microbial pathogens is of great importance for the biocontrol of animal and plant parasitic nematodes. In this study, we found that the model nematode Caenorhabditis elegans can recognize its fungal pathogen, the nematophagous fungus Purpureocillium lavendulum, through fungal-produced ethanol. Then the nematode elevated the reactive oxygen species production in the gut to inhibit fungal growth in an ethanol dehydrogenase-dependent manner. With this mechanism, novel biocontrol strategies may be developed targeting the ethanol receptor or metabolic pathway of nematodes. Meanwhile, as a volatile organic compound, ethanol should be taken seriously as a vector molecule in the microbial-host interaction in nature.

MoJMJD6, a Nuclear Protein, Regulates Conidial Germination and Appressorium Formation at the Early Stage of Pathogenesis in Magnaporthe oryzae.

In plant-pathogen interactions, Magnaporthe oryzae causes blast disease on more than 50 species of 14 monocot plants, including important crops such as rice, millet, and most 15 recently wheat. M. oryzae is a model fungus for studying plant-microbe interaction, and the main source for fungal pathogenesis in the field. Here we report that MoJMJD6 is required for conidium germination and appressorium formation in M. oryzae. We obtained MoJMJD6 mutants (ΔMojmjd6) using a target gene replacement strategy. The MoJMD6 deletion mutants were delayed for conidium germination, glycogen, and lipid droplets utilization and consequently had decreased virulence. In the ΔMojmjd6 null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3) of the genome were unaffected. Taken together, our results indicated that MoJMJD6 function as a nuclear protein which plays an important role in conidium germination and appressorium formation in the M. oryzae. Our work provides insights into MoJMJD6-mediated regulation in the early stage of pathogenesis in plant fungi.

Insights and Perspectives on the Role of Proteostasis and Heat Shock Proteins in Fungal Infections.

Fungi are a diverse group of eukaryotic organisms that infect humans, animals, and plants. To successfully colonize their hosts, pathogenic fungi must continuously adapt to the host's unique environment, e.g., changes in temperature, pH, and nutrient availability. Appropriate protein folding, assembly, and degradation are essential for maintaining cellular homeostasis and survival under stressful conditions. Therefore, the regulation of proteostasis is crucial for fungal pathogenesis. The heat shock response (HSR) is one of the most important cellular mechanisms for maintaining proteostasis. It is activated by various stresses and regulates the activity of heat shock proteins (HSPs). As molecular chaperones, HSPs participate in the proteostatic network to control cellular protein levels by affecting their conformation, location, and degradation. In recent years, a growing body of evidence has highlighted the crucial yet understudied role of stress response circuits in fungal infections. This review explores the role of protein homeostasis and HSPs in fungal pathogenicity, including their contributions to virulence and host-pathogen interactions, as well as the concerted effects between HSPs and the main proteostasis circuits in the cell. Furthermore, we discuss perspectives in the field and the potential for targeting the components of these circuits to develop novel antifungal therapies.

Suppression of Chitin-Triggered Immunity by Plant Fungal Pathogens: A Case Study of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii.

Fungal pathogens are significant plant-destroying microorganisms that present an increasing threat to the world's crop production. Chitin is a crucial component of fungal cell walls and a conserved MAMP (microbe-associated molecular pattern) that can be recognized by specific plant receptors, activating chitin-triggered immunity. The molecular mechanisms underlying the perception of chitin by specific receptors are well known in plants such as rice and Arabidopsis thaliana and are believed to function similarly in many other plants. To become a plant pathogen, fungi have to suppress the activation of chitin-triggered immunity. Therefore, fungal pathogens have evolved various strategies, such as prevention of chitin digestion or interference with plant chitin receptors or chitin signaling, which involve the secretion of fungal proteins in most cases. Since chitin immunity is a very effective defensive response, these fungal mechanisms are believed to work in close coordination. In this review, we first provide an overview of the current understanding of chitin-triggered immune signaling and the fungal proteins developed for its suppression. Second, as an example, we discuss the mechanisms operating in fungal biotrophs such as powdery mildew fungi, particularly in the model species Podosphaera xanthii, the main causal agent of powdery mildew in cucurbits. The key role of fungal effector proteins involved in the modification, degradation, or sequestration of immunogenic chitin oligomers is discussed in the context of fungal pathogenesis and the promotion of powdery mildew disease. Finally, the use of this fundamental knowledge for the development of intervention strategies against powdery mildew fungi is also discussed.

A glycoside hydrolase 12 protein from Cytospora chrysosperma triggers plant immunity but is not essential to virulence

Phytopathogens secrete numerous effectors that facilitate their infection and colonization processes. However, the pathogenic mechanism of effectors in Cytospora chrysosperma, the causal agent of canker disease in many woody plants, remains poorly understood. In this study, we identified five glycoside hydrolase family 12 (GH12) effector genes in C. chrysosperma genome, all of which were significantly upregulated during the infection stages. Among them, CcEG1, which contains an additional carbohydrate-binding module family 1 domain (CBM1) at the C-terminal, was selected for further analysis. Transient expression studies showed that CcEG1 was localized to the apoplastic region of Nicotiana benthamiana and acted as an elicitor to induce cell death, and activate the expression of genes involved in salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) signaling pathways. Furthermore, the GH12 domain (position 43–249) was sufficient for cell death-inducing activity, rather than the CBM1 domain. Additionally, the leucine-rich repeat (LRR) receptor-like kinases NbBAK1 and NbSOBIR1 were required for defense responses triggered by CcEG1. Intriguingly, deletion of CcEG1 did not affect fungal pathogenicity, growth, response to hydrogen peroxide and cell wall integrity agents, but affected cellulase utilization. In conclusion, our results suggest that CcEG1 induces coreceptors NbBAK1- and NbSOBIR1- dependent plant immunity, increasing our understanding about fungal pathogenesis during the interaction between C. chrysosperma and its host.