scholarly journals A Novel Core Effector Vp1 Promotes Fungal Colonization and Virulence of Ustilago maydis

2021 ◽  
Vol 7 (8) ◽  
pp. 589
Author(s):  
Cuong V. Hoang ◽  
Chibbhi K. Bhaskar ◽  
Lay-Sun Ma
Keyword(s):  
Reverse Genetics ◽  
Signal Sequence ◽  
Protein Localization ◽  
Sequence Divergence ◽  
In Silico Analysis ◽  
Ustilago Maydis ◽  
Effector Proteins ◽  
Fungal Colonization ◽  
Smut Fungi ◽  
Fungal Virulence

The biotrophic fungus Ustilago maydis secretes a plethora of uncharacterized effector proteins and causes smut disease in maize. Among the effector genes that are up-regulated during the biotrophic growth in maize, we identified vp1 (virulence promoting 1), which has an expression that was up-regulated and maintained at a high level throughout the life cycle of the fungus. We characterized Vp1 by applying in silico analysis, reverse genetics, phenotypic assessment, microscopy, and protein localization and provided a fundamental understanding of the Vp1 protein in U. maydis. The reduction in fungal virulence and colonization in the vp1 mutant suggests the virulence-promoting function of Vp1. The deletion studies on the NLS (nuclear localization signal) sequence and the protein localization study revealed that the C-terminus of Vp1 is processed after secretion in plant apoplast and could localize to the plant nucleus. The Ustilago hordei ortholog UhVp1 lacks NLS localized in the plant cytoplasm, suggesting that the orthologs might have a distinct subcellular localization. Further complementation studies of the Vp1 orthologs in related smut fungi revealed that none of them could complement the virulence function of U. maydis Vp1, suggesting that UmVp1 could acquire a specialized function via sequence divergence.

scholarly journals A cell surface-exposed protein complex with an essential virulence function in Ustilago maydis

2021 ◽  
Author(s):  
Nicole Ludwig ◽  
Stefanie Reissmann ◽  
Kerstin Schipper ◽  
Carla Gonzalez ◽  
Daniela Assmann ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Complex ◽  
Pathogenic Bacteria ◽  
Constitutive Expression ◽  
Pathogenic Fungi ◽  
Ustilago Maydis ◽  
Effector Proteins ◽  
Host Cells ◽  
Smut Fungi ◽  
Living Plant

AbstractPlant pathogenic fungi colonizing living plant tissue secrete a cocktail of effector proteins to suppress plant immunity and reprogramme host cells. Although many of these effectors function inside host cells, delivery systems used by pathogenic bacteria to translocate effectors into host cells have not been detected in fungi. Here, we show that five unrelated effectors and two membrane proteins from Ustilago maydis, a biotrophic fungus causing smut disease in corn, form a stable protein complex. All seven genes appear co-regulated and are only expressed during colonization. Single mutants arrest in the epidermal layer, fail to suppress host defence responses and fail to induce non-host resistance, two reactions that likely depend on translocated effectors. The complex is anchored in the fungal membrane, protrudes into host cells and likely contacts channel-forming plant plasma membrane proteins. Constitutive expression of all seven complex members resulted in a surface-exposed form in cultured U. maydis cells. As orthologues of the complex-forming proteins are conserved in smut fungi, the complex may become an interesting fungicide target.

scholarly journals Cross-species analysis between the maize smut fungi Ustilago maydis and Sporisorium reilianum highlights the role of transcriptional plasticity of effector orthologs for virulence and disease

2020 ◽  
Author(s):  
Weiliang Zuo ◽  
Deepak K Gupta ◽  
Jasper RL Depotter ◽  
Marco Thines ◽  
Gunther Doehlemann
Keyword(s):  
Ustilago Maydis ◽  
Effector Proteins ◽  
Differentially Expressed ◽  
Transcriptional Level ◽  
Smut Fungi ◽  
List Type ◽  
Sequencing Analysis ◽  
Distinct Disease ◽  
Sporisorium Reilianum ◽  
Transcriptional Plasticity

SummaryThe constitution and regulation of effector repertoires determines and shapes the outcome of the interaction with the host. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize, but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights to pathogen evolution and host adaption.We tracked the infection progress of U. maydis and S. reilianum in maize leaves, characterized two distinct infection stages for cross species RNA-sequencing analysis and identified 207 out of 335 one-to-one effector orthologs being differentially regulated during host colonization, while transcriptional plasticity of the effector orthologs correlated with the distinct disease development strategies.By using CRISPR-Cas9 mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes contributes to species specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/sr1007) showed diverged protein function during speciation, providing a possible case for neofunctionalization.Together, we showed the diversification of effector genes in related pathogens can be caused both by plasticity on the transcriptional level, as well as through neofunctionalization of the encoded effector proteins.

scholarly journals Identification and characterization of two transmembrane proteins required for virulence of Ustilago maydis

2021 ◽  
Author(s):  
Paul Weiland ◽  
Florian Altegoer
Keyword(s):  
Virulence Factors ◽  
Biochemical Analysis ◽  
Transmembrane Proteins ◽  
Ustilago Maydis ◽  
Effector Proteins ◽  
Smut Fungi ◽  
Initial Characterization ◽  
Associated Proteins ◽  
Identification And Characterization

AbstractSmut fungi comprise a large group of biotrophic phytopathogens infecting important crops such as wheat and corn. Through the secretion of effector proteins, the fungus actively suppresses plant immune reactions and modulates its host’s metabolism. Consequently, how soluble effector proteins contribute to virulence is already characterized in a range of phytopathogens. However, membrane-associated virulence factors have been much less studied to date. Here, we investigated six transmembrane (TM) proteins that show elevated gene expression during biotrophic development of the maize pathogen Ustilago maydis. We show that two of the six proteins, named Vmp1 and Vmp2 (virulence-associated membrane protein), are essential for the full virulence of U. maydis. The deletion of the corresponding genes lead to a substantial attenuation in the virulence of U. maydis. Furthermore, both are conserved in various related smuts and contain no domains of known function. Our biochemical analysis clearly shows that Vmp1 and Vmp2 are membrane-associated proteins, potentially localizing to the U. maydis plasma membrane. Mass photometry and light scattering suggest that Vmp1 mainly occurs as a monomer, while Vmp2 is dimeric. Notably, the large and partially unstructured C-terminal domain of Vmp2 is crucial for virulence while not contributing to dimerization. Taken together, we here provide an initial characterization of two membrane proteins as virulence factors of U. maydis.

scholarly journals Identification and Characterization of Two Transmembrane Proteins Required for Virulence of Ustilago maydis

2021 ◽  
Vol 12 ◽  
Author(s):  
Paul Weiland ◽  
Florian Altegoer
Keyword(s):  
Virulence Factors ◽  
Biochemical Analysis ◽  
Transmembrane Proteins ◽  
Ustilago Maydis ◽  
Effector Proteins ◽  
Smut Fungi ◽  
Initial Characterization ◽  
Associated Proteins ◽  
Identification And Characterization

Smut fungi comprise a large group of biotrophic phytopathogens infecting important crops such as wheat and corn. Through the secretion of effector proteins, the fungus actively suppresses plant immune reactions and modulates its host’s metabolism. Consequently, how soluble effector proteins contribute to virulence is already characterized in a range of phytopathogens. However, membrane-associated virulence factors have been much less studied to date. Here, we investigated six transmembrane (TM) proteins that show elevated gene expression during biotrophic development of the maize pathogen Ustilago maydis. We show that two of the six proteins, named Vmp1 and Vmp2 (virulence-associated membrane protein), are essential for the full virulence of U. maydis. The deletion of the corresponding genes leads to a substantial attenuation in the virulence of U. maydis. Furthermore, both are conserved in various related smuts and contain no domains of known function. Our biochemical analysis clearly shows that Vmp1 and Vmp2 are membrane-associated proteins, potentially localizing to the U. maydis plasma membrane. Mass photometry and light scattering suggest that Vmp1 mainly occurs as a monomer, while Vmp2 is dimeric. Notably, the large and partially unstructured C-terminal domain of Vmp2 is crucial for virulence while not contributing to dimerization. Taken together, we here provide an initial characterization of two membrane proteins as virulence factors of U. maydis.

scholarly journals Pseudozyma saprotrophic yeasts have retained a large effector arsenal, including functional Pep1 orthologs

2018 ◽  
Author(s):  
Rahul Sharma ◽  
Bilal Ökmen ◽  
Gunther Doehlemann ◽  
Marco Thines
Keyword(s):  
Ustilago Maydis ◽  
Effector Proteins ◽  
Genetic Complementation ◽  
Smut Fungi ◽  
High Confidence ◽  
Positive Effects ◽  
Effector Gene ◽  
Sexual Recombination ◽  
Plant Surfaces ◽  
Plant Parasitic

SummaryThe basidiomycete smut fungi are predominantly plant parasitic, causing severe losses in some crops. Most species feature a saprotrophic haploid yeast stage, and several smut fungi are only known from this stage, with some isolated from habitats without suitable hosts, e.g. from Antarctica. Thus, these species are generally believed to be apathogenic, but recent findings that some of these might have a plant pathogenic sexual counterpart, casts doubts on the validity of this hypothesis. Here, four Pseudozyma genomes were re-annotated and compared to published smut pathogens and the well-characterised effector gene Pep1 from these species was checked for its ability to complement a Pep1 deletion strain of Ustilago maydis. It was found that 113 high-confidence putative effector proteins were conserved among smut and Pseudozyma genomes. Among these were several validated effector proteins, including Pep1. By genetic complementation we show that Pep1 homologs from the supposedly apathogenic yeasts restore virulence in Pep1-deficient mutants Ustilago maydis. Thus, it is concluded that Pseudozyma species have retained a suite of effectors. This hints at the possibility that Pseudozyma species have kept an unknown plant pathogenic stage for sexual recombination or that these effectors have positive effects when colonising plant surfaces.

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.

Ultrastructure of meiosis and the spindle pole body cycle in freeze-substituted basidia of the smut fungi Ustilago maydis and Ustilago avenae

1992 ◽  
Vol 70 (3) ◽  
pp. 629-638 ◽  
Author(s):  
Kerry O'Donnell
Keyword(s):  
Electron Microscopy ◽  
Spindle Pole Body ◽  
Ustilago Maydis ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Smut Fungi ◽  
Late Prophase ◽  
Prophase I ◽  
Pole Body ◽  
Middle Piece

Meiosis in the smut fungi Ustilago maydis and Ustilago avenae (Basidiomycota, Ustilaginales) was studied by electron microscopy of serial-sectioned freeze substituted basidia. At prophase I, a spindle pole body composed of two globular elements connected by a middle piece was attached to the extranuclear surface of each nucleus. Astral and spindle microtubules were initiated at each globular element at late prophase I to prometaphase I. During spindle initiation, the middle piece disappeared and interdigitating half-spindles entered the nucleoplasm, which was surrounded by discontinuous nuclear envelope together with perinuclear endoplasmic reticulum. Kinetochore pairs at metaphase I were analyzed to obtain a karyotype for each species. The meiotic spindle pole body replicational cycle is described. Key words: electron microscopy, freeze-substitution, meiosis, Ustilago, spindle pole body.

Efficiency and diversity of protein localization by random signal sequences

1990 ◽  
Vol 10 (6) ◽  
pp. 3163-3173
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Protein Localization ◽  
Random Signal ◽  
Perinuclear Region ◽  
Hybrid Protein ◽  
Random Sequences ◽  
Beta Galactosidase

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

scholarly journals The Unfolded Protein Response Regulates Pathogenic Development of Ustilago maydis by Rok1-Dependent Inhibition of Mating-Type Signaling

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Lara Schmitz ◽  
Melina Ayaka Schwier ◽  
Kai Heimel
Keyword(s):  
Unfolded Protein Response ◽  
Mating Type ◽  
Ustilago Maydis ◽  
Mitogen Activated Protein Kinase ◽  
In Planta ◽  
Fungal Virulence ◽  
Content Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Fungal pathogens require the unfolded protein response (UPR) to maintain protein homeostasis of the endoplasmic reticulum (ER) during pathogenic development. In the corn smut fungus Ustilago maydis, pathogenic development is controlled by the a and b mating-type loci. The UPR is specifically activated after plant penetration and required for efficient secretion of effectors and suppression of the plant defense response. The interaction between the UPR regulator Cib1 and the central developmental regulator Clp1 modulates the pathogenic program and triggers fungal colonization of the host plant. By contrast, when activated before plant penetration, the UPR interferes with fungal virulence by reducing expression of bE and bW, the central regulators of pathogenic development encoded by the b mating-type locus. Here, we show that this inhibitory effect results from UPR-mediated suppression of the pheromone response pathway upstream of the b regulatory network. UPR activity prompts dephosphorylation of the pheromone-responsive mitogen-activated protein kinase (MAPK) Kpp2, reducing activity of the pheromone response factor Prf1 that regulates expression of bE and bW. Deletion of the dual specificity phosphatase rok1 fully suppressed UPR-dependent inhibition of Kpp2 phosphorylation, formation of infectious filaments, and fungal virulence. Rok1 determines the activity of mating-type signaling pathways and thus the degree of fungal virulence. We propose that UPR-dependent regulation of Rok1 aligns ER physiology with fungal aggressiveness and effector gene expression during biotrophic growth of U. maydis in the host plant. IMPORTANCE The unfolded protein response (UPR) is crucial for endoplasmic reticulum (ER) homeostasis and disease development in fungal pathogens. In the plant-pathogenic fungus Ustilago maydis, the UPR supports fungal proliferation in planta and effector secretion for plant defense suppression. In this study, we uncovered that UPR activity, which is normally restricted to the biotrophic stage in planta, inhibits mating and the formation of infectious filaments by Rok1-dependent dephosphorylation of the pheromone responsive mitogen-activated protein kinase (MAPK) Kpp2. This observation is relevant for understanding how the fungal virulence program is regulated by cellular physiology. UPR-mediated control of mating-type signaling pathways predicts that effector gene expression and the virulence potential are controlled by ER stress levels.

scholarly journals Comparative secretome of Magnaporthe oryzae identified proteins involved in virulence and cell wall integrity

2020 ◽  
Author(s):  
Ning Liu ◽  
Linlu Qi ◽  
Manna Huang ◽  
Deng Chen ◽  
Changfa Yin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Magnaporthe Oryzae ◽  
Fungal Pathogens ◽  
Protein Localization ◽  
Potential Effect ◽  
Cell Wall Integrity ◽  
Blast Disease ◽  
Secreted Proteins ◽  
Interspecies Interactions ◽  
Fungal Virulence

AbstractPlant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. Only a few secreted proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. ALG3 is an α-1, 3-mannosyltransferase function in N-glycan synthesis for secreted N-glycosylated proteins, and the Δalg3 mutants show strong defects in cell wall integrity and fungal virulence, indicating a potential effect on the secretion of multiple proteins. In this study, we compared the secretome of wild type and Δalg3 mutants, and identified 51 proteins that require ALG3 for proper secretion. These are predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. The tested secreted proteins localized at the apoplast region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of candidate proteins was significantly changed in the Δalg3 mutant, leading to the reduction of protein secretion and abnormal protein localization. Furthermore, we tested the function of two genes, one is a previously reported M. oryzae gene Invertase 1 (INV1) encoding a secreted invertase, and the other one is a gene encoding an Acid mammalian chinitase (AMCase). The fungal virulence was significantly reduced and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Elucidation of the comparative secretome of M. oryzae improves our understanding of the proteins that require ALG3 for secretion, and of their function in fungal virulence and cell wall integrity.

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