scholarly journals The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

2021 ◽  
Vol 7 (2) ◽  
pp. 86
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
Mating Partner ◽  
Fungal Virulence ◽  
Functional Studies ◽  
Infection Structures ◽  
Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

scholarly journals The Ustilago hordei-barley interaction is a versatile system to characterize fungal effectors

2020 ◽  
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
Mating Partner ◽  
Fungal Virulence ◽  
Functional Studies ◽  
Infection Structures ◽  
Expression Platform

AbstractObligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence/pathogenicity. In this study, we present the U. hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We have generated U. hordei solopathogenic strains, which form infectious filaments without presence of compatible mating partner. Solopathogenic strains are suitable as heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization were analyzed by transmission electron microscopy, which shows that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel Yu ◽  
Megan A Outram ◽  
Emma Creen ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel S Yu ◽  
Megan A Outram ◽  
Emma Crean ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

scholarly journals The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and insights into Kex2 protease processing of fungal effectors

2020 ◽  
Author(s):  
Megan A. Outram ◽  
Yi-Chang Sung ◽  
Daniel Yu ◽  
Bayantes Dagvadorj ◽  
Sharmin A. Rima ◽  
...  
Keyword(s):  
Crystal Structure ◽  
In Silico ◽  
Plant Pathogens ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
List Type ◽  
Fungal Virulence ◽  
Parastagonospora Nodorum ◽  
Wheat Lines ◽  
Kex2 Protease

SummaryPlant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3.We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi.The β-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici.We propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence.

scholarly journals Transcription factor lineages in plant-pathogenic fungi, connecting diversity with fungal virulence

2021 ◽  
Author(s):  
Evan John ◽  
Kar-Chun Tan ◽  
Richard Peter Oliver ◽  
Karam Singh
Keyword(s):  
Plant Pathogens ◽  
Pathogenic Fungi ◽  
Plant Pathogenic Fungi ◽  
Fungal Virulence ◽  
Diverse Range ◽  
Dna Binding Domains ◽  
Functional Studies ◽  
Binding Domains ◽  
Plant Pathogenicity ◽  
Host Infection

Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, controlling development and virulence in plant pathogenic fungi. Recent research has established regulatory roles for several taxonomically conserved fungal TFs, but the evolution of specific virulence regulators is not well understood. This study sought to explore the representation of TFs across a taxonomically-diverse range of fungi, with a focus on plant pathogens. A significant trend was observed among the obligate, host-associated pathogens, which possess a reduced overall TF repertoire, alluding to a lack of pressure for maintaining diversity. A novel orthology-based analysis is then presented that refined TF classifications, traditionally based on the nature of the DNA-binding domains. Using this analysis, cases of TF over/underrepresentation across fungal pathogen lineages are systematically highlighted. Specific examples are then explored and discussed that included the TF orthologues of Ste12, Pf2 and EBR1, plus phytotoxic secondary-metabolite cluster regulators, which all presented novel and distinct evolutionary insights. Ultimately, as the examples presented demonstrate, this resource can be interrogated to guide functional studies that seek to characterise virulence-specific regulators and shed light on the factors underpinning plant pathogenicity.

scholarly journals Comparative Genomics and Functional Studies of Wheat BED-NLR Loci

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1406
Author(s):  
Clemence Marchal ◽  
Georg Haberer ◽  
Manuel Spannagl ◽  
Cristobal Uauy ◽  
Keyword(s):  
Cell Death ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Functional Studies ◽  
Network Analyses ◽  
Binding Factor ◽  
Localization Signal ◽  
Integrated Domains ◽  
Genomic Regions

Nucleotide-binding leucine-rich-repeat (LRR) receptors (NLRs) with non-canonical integrated domains (NLR-IDs) are widespread in plant genomes. Zinc-finger BED (named after the Drosophila proteins Boundary Element-Associated Factor and DNA Replication-related Element binding Factor, named BED hereafter) are among the most frequently found IDs. Five BED-NLRs conferring resistance against bacterial and fungal pathogens have been characterized. However, it is unknown whether BED-NLRs function in a manner similar to other NLR-IDs. Here, we used chromosome-level assemblies of wheat to explore the Yr7 and Yr5a genomic regions and show that, unlike known NLR-ID loci, there is no evidence for a NLR-partner in their vicinity. Using neighbor-network analyses, we observed that BED domains from BED-NLRs share more similarities with BED domains from single-BED proteins and from BED-containing proteins harboring domains that are conserved in transposases. We identified a nuclear localization signal (NLS) in Yr7, Yr5, and the other characterized BED-NLRs. We thus propose that this is a feature of BED-NLRs that confer resistance to plant pathogens. We show that the NLS was functional in truncated versions of the Yr7 protein when expressed in N. benthamiana. We did not observe cell-death upon the overexpression of Yr7 full-length, truncated, and ‘MHD’ variants in N. benthamiana. This suggests that either this system is not suitable to study BED-NLR signaling or that BED-NLRs require additional components to trigger cell death. These results define novel future directions to further understand the role of BED domains in BED-NLR mediated resistance.

scholarly journals Fungal Pathogens Affecting the Production and Quality of Medical Cannabis in Israel

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 882
Author(s):  
Shachar Jerushalmi ◽  
Marcel Maymon ◽  
Aviv Dombrovsky ◽  
Stanley Freeman
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Pathogenic Fungi ◽  
Plant Diseases ◽  
Soilborne Pathogens ◽  
Medical Cannabis ◽  
Plant Parts ◽  
Commercial Cultivation

The use of and research on medical cannabis (MC) is becoming more common, yet there are still many challenges regarding plant diseases of this crop. For example, there is a lack of formal and professional knowledge regarding fungi that infect MC plants, and practical and effective methods for managing the casual agents of disease are limited. The purpose of this study was to identify foliar, stem, and soilborne pathogens affecting MC under commercial cultivation in Israel. The predominant major foliage pathogens were identified as Alternaria alternata and Botrytis cinerea, while the common stem and soilborne pathogens were identified as Fusarium oxysporum and F. solani. Other important fungi that were isolated from foliage were those producing various mycotoxins that can directly harm patients, such as Aspergillus spp. and Penicillium spp. The sampling and characterization of potential pathogenic fungi were conducted from infected MC plant parts that exhibited various disease symptoms. Koch postulates were conducted by inoculating healthy MC tissues and intact plants with fungi isolated from infected commercially cultivated symptomatic plants. In this study, we report on the major and most common plant pathogens of MC found in Israel, and determine the seasonal outbreak of each fungus.

scholarly journals Conservation of a gene cluster reveals novel cercosporin biosynthetic mechanisms and extends production to the genus Colletotrichum

2017 ◽  
Author(s):  
Ronnie de Jonge ◽  
Malaika K. Ebert ◽  
Callie R. Huitt-Roehl ◽  
Paramita Pal ◽  
Jeffrey C. Suttle ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Gene Cluster ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Pathogenic Fungi ◽  
Gene Clusters ◽  
Phylogenomic Analysis ◽  
First Case ◽  
Horizontal Transfers ◽  
Fungal Genus

AbstractSpecies in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean and other major food crops. Here we sequenced the genome of the sugar beet pathogen C. beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae. Although cercosporin biosynthesis has been thought to-date to rely on an eight gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein (CFP) previously shown to be involved with cercosporin auto-resistance, and four additional genes required for cercosporin biosynthesis including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Finally, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide new insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.Significance StatementSpecies in the fungal genus Cercospora cause diseases in many important crops worldwide. Their success as pathogens is largely due to the secretion of cercosporin during infection. We report that the cercosporin toxin biosynthesis (CTB) cluster is ancient and was horizontally transferred to diverse fungal pathogens on an unprecedented scale. Since these analyses revealed genes adjacent to the established CTB cluster, we evaluated their role in C. beticola to show that four are necessary for cercosporin biosynthesis. Finally, we confirmed that the apple pathogen Colletotrichum fioriniae produces cercosporin, the first case outside the family Mycosphaerellaceae. Other Colletotrichum plant pathogens also harbor the CTB cluster, which points to a wider concern that this toxin may play in virulence and human health.

scholarly journals Identification and Phenotypic Characterization of Hsp90 Phosphorylation Sites That Modulate Virulence Traits in the Major Human Fungal Pathogen Candida albicans

2021 ◽  
Vol 11 ◽  
Author(s):  
Leenah Alaalm ◽  
Julia L. Crunden ◽  
Mark Butcher ◽  
Ulrike Obst ◽  
Ryann Whealy ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Environmental Stress ◽  
Stress Responses ◽  
Fungal Pathogens ◽  
Phosphorylation Site ◽  
Pathogenic Fungi ◽  
Phenotypic Characterization ◽  
Fungal Virulence ◽  
Post Translational Modifications ◽  
Virulence Traits

The highly conserved, ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. In human pathogenic fungi, which kill more than 1.6 million patients each year worldwide, Hsp90 governs cellular morphogenesis, drug resistance, and virulence. Yet, our understanding of the regulatory mechanisms governing fungal Hsp90 function remains sparse. Post-translational modifications are powerful components of nature’s toolbox to regulate protein abundance and function. Phosphorylation in particular is critical in many cellular signaling pathways and errant phosphorylation can have dire consequences for the cell. In the case of Hsp90, phosphorylation affects its stability and governs its interactions with co-chaperones and clients. Thereby modulating the cell’s ability to cope with environmental stress. Candida albicans, one of the leading human fungal pathogens, causes ~750,000 life-threatening invasive infections worldwide with unacceptably high mortality rates. Yet, it remains unknown if and how Hsp90 phosphorylation affects C. albicans virulence traits. Here, we show that phosphorylation of Hsp90 is critical for expression of virulence traits. We combined proteomics, molecular evolution analyses and structural modeling with molecular biology to characterize the role of Hsp90 phosphorylation in this non-model pathogen. We demonstrated that phosphorylation negatively affects key virulence traits, such as the thermal stress response, morphogenesis, and drug susceptibility. Our results provide the first record of a specific Hsp90 phosphorylation site acting as modulator of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitations as antifungal drug targets.

scholarly journals Remote homology clustering identifies lowly conserved families of effector proteins in plant-pathogenic fungi

2021 ◽  
Vol 7 (9) ◽  
Author(s):  
Darcy A. B. Jones ◽  
Paula M. Moolhuijzen ◽  
James K. Hane
Keyword(s):  
Fungal Pathogens ◽  
Pathogenic Fungi ◽  
Comparison Method ◽  
Fungal Species ◽  
Effector Proteins ◽  
Plant Diseases ◽  
Plant Host ◽  
Homology Inference ◽  
Developing Area ◽  
Inference Methods

Plant diseases caused by fungal pathogens are typically initiated by molecular interactions between ‘effector’ molecules released by a pathogen and receptor molecules on or within the plant host cell. In many cases these effector-receptor interactions directly determine host resistance or susceptibility. The search for fungal effector proteins is a developing area in fungal-plant pathology, with more than 165 distinct confirmed fungal effector proteins in the public domain. For a small number of these, novel effectors can be rapidly discovered across multiple fungal species through the identification of known effector homologues. However, many have no detectable homology by standard sequence-based search methods. This study employs a novel comparison method (RemEff) that is capable of identifying protein families with greater sensitivity than traditional homology-inference methods, leveraging a growing pool of confirmed fungal effector data to enable the prediction of novel fungal effector candidates by protein family association. Resources relating to the RemEff method and data used in this study are available from https://figshare.com/projects/Effector_protein_remote_homology/87965.

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