The Monothiol Glutaredoxin Grx4 Influences Iron Homeostasis and Virulence in Ustilago maydis.

The corn smut fungus, Ustilago maydis, is an excellent model for studying biotrophic plant-pathogen interactions, including nutritional adaptation to the host environment. Iron acquisition during host colonization is a key aspect of microbial pathogenesis yet less is known about this process for fungal pathogens of plants. Monothiol glutaredoxins are central regulators of key cellular functions in fungi, including iron homeostasis, cell wall integrity, and redox status via interactions with transcription factors, iron-sulfur clusters, and glutathione. In this study, the roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis were investigated by constructing strains expressing a conditional allele of grx4 under the control of the arabinose-inducible, glucose-repressible promoter Pcrg1. The use of conditional expression was necessary because Grx4 appeared to be essential for U. maydis. Transcriptome and genetic analyses with strains depleted in Grx4 revealed that the protein participates in the regulation of iron acquisition functions and is necessary for the ability of U. maydis to cause disease on maize seedlings. Taken together, this study supports the growing appreciation of monothiol glutaredoxins as key regulators of virulence-related phenotypes in pathogenic fungi.

Induction of Aspergillus fumigatus zinc cluster transcription factor OdrA/Mdu2 provides combined cellular responses for oxidative stress protection and multiple antifungal drug resistance.

IMPORTANCE: An overexpression screen of 228 zinc cluster transcription factor encoding genes of A. fumigatus revealed 11 genes conferring increased tolerance to antifungal drugs. Out of these, four oxidative stress and drug tolerance transcription factor encoding odr genes increased tolerance to oxidative stress and antifungal drugs when overexpressed. This supports a correlation between oxidative stress response and antifungal drug tolerance in A. fumigatus. OdrA/Mdu2 is required for the cross-tolerance between azoles, polyenes, and oxidative stress and activates genes for detoxification. Under oxidative stress conditions or when overexpressed, OdrA/Mdu2 accumulates in the nucleus and activates detoxifying genes by direct binding at their promoters, as we describe with the mdr1 gene encoding an itraconazole specific efflux pump. Finally, this work gives new insights about drug and stress resistance in the opportunistic pathogenic fungus A. fumigatus.

Fungal COP9 signalosome assembly requires connection of two trimeric intermediates for integration of intrinsic deneddylase.

The conserved eight-subunit COP9 signalosome (CSN) is required for multicellular fungal development. The CSN deneddylase cooperates with the Cand1 exchange factor to control replacements of E3 ubiquitin cullin RING ligase receptors, providing specificity to eukaryotic protein degradation. Aspergillus nidulans CSN assembles through a heptameric pre-CSN, which is activated by integration of the catalytic CsnE deneddylase. Combined genetic and biochemical approaches provided the assembly choreography within a eukaryotic cell for native fungal CSN. Interactomes of functional GFP-Csn subunit fusions in pre-CSN deficient fungal strains were compared by affinity purifications and mass spectrometry. Two distinct heterotrimeric CSN subcomplexes were identified as pre-CSN assembly intermediates. CsnA-C-H and CsnD-F-G form independently of CsnB, which connects the heterotrimers to a heptamer and enables subsequent integration of CsnE to form the enzymatically active CSN complex. Surveillance mechanisms control accurate Csn subunit amounts and correct cellular localization for sequential assembly since deprivation of Csn subunits changes the abundance and location of remaining Csn subunits.

Verticillium dahliae Vta3 promotes ELV1 virulence factor gene expression in xylem sap, but tames Mtf1-mediated late stages of fungus-plant interactions and microsclerotia formation.

Verticillium transcription activator of adhesion 3 (Vta3) is required for plant root colonization and pathogenicity of the soil-borne vascular fungus Verticillium dahliae. RNA sequencing identified Vta3-dependent genetic networks required for growth in tomato xylem sap. Vta3 affects the expression of more than 1,000 transcripts, including candidates with predicted functions in virulence and morphogenesis such as Egh16-like virulence factor 1 (Elv1) and Master transcription factor 1 (Mtf1). The genes encoding Elv1 and Mtf1 were deleted and their functions in V. dahliae growth and virulence on tomato (Solanum lycopersicum) plants were investigated using genetics, plant infection experiments, gene expression studies and phytohormone analyses. Vta3 contributes to virulence by promoting ELV1 expression, which is dispensable for vegetative growth and conidiation. Vta3 decreases disease symptoms mediated by Mtf1 in advanced stages of tomato plant colonization, while Mtf1 induces the expression of fungal effector genes and tomato pathogenesis-related protein genes. The levels of pipecolic and salicylic acids functioning in tomato defense signaling against (hemi-) biotrophic pathogens depend on the presence of MTF1, which promotes the formation of resting structures at the end of the infection cycle. In summary, the presence of VTA3 alters gene expression of virulence factors and tames the Mtf1 genetic subnetwork for late stages of plant disease progression and subsequent survival of the fungus in the soil.

Robust Cre recombinase activity in the biotrophic smut fungus Ustilago maydis enables efficient conditional null mutants in planta.

Site-specific recombinases have been used in higher eukaryotes, especially in animals, for a broad range of applications, including chromosomal translocations, large deletions, site-specific integration, and tissue-specific as well as conditional knock-outs. The application of site-specific recombination has also been demonstrated in simple eukaryotes like fungi and protozoa. However, its use in fungal research, especially in phytopathogenic fungi, has often been limited to "recycle" the marker genes used in transformation experiments. We show that Cre recombinase can be used for conditional gene deletions in the phytopathogenic fungus Ustilago maydis. Conditional gene knock-outs can be generated via the transcriptional control of the recombinase by U. maydis promoters specifically activated during the biotrophic phase of fungal growth, enabling gene deletions at defined developmental stages inside the plant tissue. Also, we show that a tamoxifen-activated Cre-recombinase allows the tight control necessary for the induced deletion of essential genes by the addition of tamoxifen. These tools will be helpful to address the function of genes under both axenic and in planta conditions for the U. maydis-maize pathosystem and should pave the way for similar approaches in other plant pathosystems.

Unfolded Protein Response and Scaffold Independent Pheromone MAP Kinase Signaling Control Verticillium dahliae Growth, Development, and Plant Pathogenesis.

Differentiation, growth, and virulence of the vascular plant pathogen Verticillium dahliae depend on a network of interconnected cellular signaling cascades. The transcription factor Hac1 of the endoplasmic reticulum-associated unfolded protein response (UPR) is required for initial root colonization, fungal growth, and vascular propagation by conidiation. Hac1 is essential for the formation of microsclerotia as long-time survival resting structures in the field. Single endoplasmic reticulum-associated enzymes for linoleic acid production as precursors for oxylipin signal molecules support fungal growth but not pathogenicity. Microsclerotia development, growth, and virulence further require the pheromone response mitogen-activated protein kinase (MAPK) pathway, but without the Ham5 scaffold function. The MAPK phosphatase Rok1 limits resting structure development of V.dahliae, but promotes growth, conidiation, and virulence. The interplay between UPR and MAPK signaling cascades includes several potential targets for fungal growth control for supporting disease management of the vascular pathogen V.dahliae.

Conditional gene expression reveals stage-specific functions of the unfolded protein response in the Ustilago maydis-maize pathosystem.

Ustilago maydis is a model organism for the study of biotrophic plant-pathogen interactions. The sexual and pathogenic development of the fungus are tightly connected since fusion of compatible haploid sporidia is prerequisite for infection of the host plant, maize (Zea mays). After plant penetration, the unfolded protein response (UPR) is activated and required for biotrophic growth. The UPR is continuously active throughout all stages of pathogenic development in planta. However, since development of UPR deletion mutants stops directly after plant penetration, the role of an active UPR at later stages of development remained to be determined. Here, we established a gene expression system for U. maydis that uses endogenous, conditionally active promoters to either induce or repress expression of a gene of interest during different stages of plant infection. Integration of the expression constructs into the native genomic locus and removal of resistance cassettes were required to obtain a wild-type-like expression pattern. This indicates that genomic localization and chromatin structure are important for correct promoter activity and gene expression. By conditional expression of the central UPR regulator, Cib1, in U. maydis, we show that a functional UPR is required for continuous plant defence suppression after host infection and that U. maydis relies on a robust control system to prevent deleterious UPR hyperactivation.

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

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.

Signal peptide peptidase activity connects the unfolded protein response to plant defense suppression by Ustilago maydis.

The corn smut fungus Ustilago maydis requires the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum (ER) during the biotrophic interaction with its host plant Zea mays (maize). Crosstalk between the UPR and pathways controlling pathogenic development is mediated by protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Cib1/Clp1 complex formation results in mutual modification of the connected regulatory networks thereby aligning fungal proliferation in planta, efficient effector secretion with increased ER stress tolerance and long-term UPR activation in planta. Here we address UPR-dependent gene expression and its modulation by Clp1 using combinatorial RNAseq/ChIPseq analyses. We show that increased ER stress resistance is connected to Clp1-dependent alterations of Cib1 phosphorylation, protein stability and UPR gene expression. Importantly, we identify by deletion screening of UPR core genes the signal peptide peptidase Spp1 as a novel key factor that is required for establishing a compatible biotrophic interaction between U. maydis and its host plant maize. Spp1 is dispensable for ER stress resistance and vegetative growth but requires catalytic activity to interfere with the plant defense, revealing a novel virulence specific function for signal peptide peptidases in a biotrophic fungal/plant interaction.

Transcripts and tumors: regulatory and metabolic programming during biotrophic phytopathogenesis.

Biotrophic fungal pathogens of plants must sense and adapt to the host environment to complete their life cycles. Recent transcriptome studies of the infection of maize by the biotrophic pathogen Ustilago maydis are providing molecular insights into an ordered program of changes in gene expression and the deployment of effectors as well as key features of nutrient acquisition. In particular, the transcriptome data provide a deeper appreciation of the complexity of the transcription factor network that controls the biotrophic program of invasion, proliferation, and sporulation. Additionally, transcriptome analysis during tumor formation, a key late stage in the life cycle, revealed features of the remodeling of host and pathogen metabolism that may support the formation of tremendous numbers of spores. Transcriptome studies are also appearing for other smut species during interactions with their hosts, thereby providing opportunities for comparative approaches to understand biotrophic adaptation.

Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

A conserved co-chaperone is required for virulence in fungal plant pathogens.

The maize pathogenic fungus Ustilago maydis experiences endoplasmic reticulum (ER) stress during plant colonization and relies on the unfolded protein response (UPR) to cope with this stress. We identified the U. maydis co-chaperone, designated Dnj1, as part of this conserved cellular response to ER stress. ∆dnj1 cells are sensitive to the ER stressor tunicamycin and display a severe virulence defect in maize infection assays. A dnj1 mutant allele unable to stimulate the ATPase activity of chaperones phenocopies the null allele. A Dnj1-mCherry fusion protein localizes in the ER and interacts with the luminal chaperone Bip1. The Fusarium oxysporum Dnj1 ortholog contributes to the virulence of this fungal pathogen in tomato plants. Unlike the human ortholog, F. oxysporum Dnj1 partially rescues the virulence defect of the Ustilago dnj1 mutant. By enabling the fungus to restore ER homeostasis and maintain a high secretory activity, Dnj1 contributes to the establishment of a compatible interaction with the host. Dnj1 orthologs are present in many filamentous fungi, but are absent in budding and fission yeasts. We postulate a conserved and essential role during virulence for this class of co-chaperones.

Unfolded protein response in filamentous fungi-implications in biotechnology.

The unfolded protein response (UPR) represents a mechanism to preserve endoplasmic reticulum (ER) homeostasis that is conserved in eukaryotes. ER stress caused by the accumulation of potentially toxic un- or misfolded proteins in the ER triggers UPR activation and the induction of genes important for protein folding in the ER, ER expansion, and transport from and to the ER. Along with this adaptation, the overall capacity for protein secretion is markedly increased by the UPR. In filamentous fungi, various approaches to employ the UPR for improved production of homologous and heterologous proteins have been investigated. As the effects on protein production were strongly dependent on the expressed protein, generally applicable strategies have to be developed. A combination of transcriptomic approaches monitoring secretion stress and basic research on the UPR mechanism provided novel and important insight into the complex regulatory cross-connections between UPR signalling, cellular physiology, and developmental processes. It will be discussed how this increasing knowledge on the UPR might stimulate the development of novel strategies for using the UPR as a tool in biotechnology.

The SPF27 homologue Num1 connects splicing and kinesin 1-dependent cytoplasmic trafficking in Ustilago maydis.

The conserved NineTeen protein complex (NTC) is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.

Crosstalk between the unfolded protein response and pathways that regulate pathogenic development in Ustilago maydis.

The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway regulating endoplasmic reticulum (ER) homeostasis during ER stress, which results, for example, from an increased demand for protein secretion. Here, we characterize the homologs of the central UPR regulatory proteins Hac1 (for Homologous to ATF/CREB1) and Inositol Requiring Enzyme1 in the plant pathogenic fungus Ustilago maydis and demonstrate that the UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timing of UPR is required for virulence, since premature activation interferes with the b-dependent switch from budding to filamentous growth. In addition, we found crosstalk between UPR and the b target Clampless1 (Clp1), which is essential for cell cycle release and proliferation in planta. The unusual C-terminal extension of the U. maydis Hac1 homolog, Cib1 (for Clp1 interacting bZIP1), mediates direct interaction with Clp1. The interaction between Clp1 and Cib1 promotes stabilization of Clp1, resulting in enhanced ER stress tolerance that prevents deleterious UPR hyperactivation. Thus, the interaction between Cib1 and Clp1 constitutes a checkpoint to time developmental progression and increased secretion of effector proteins at the onset of biotrophic development. Crosstalk between UPR and the b mating-type regulated developmental program adapts ER homeostasis to the changing demands during biotrophy.

The Ustilago maydis forkhead transcription factor Fox1 is involved in the regulation of genes required for the attenuation of plant defenses during pathogenic development.

Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host plant, Zea mays. The pathogenic stage of U. maydis is initiated by the fusion of two haploid cells, resulting in the formation of a dikaryotic hypha that invades the plant cell. The switch from saprophytic, yeast-like cells to the biotrophic hyphae requires the complex regulation of a multitude of biological processes to constitute the compatible host-fungus interaction. Transcriptional regulators involved in the establishment of the infectious dikaryon and penetration of the host tissue have been identified; however, regulators required during the post-penetration stages remained to be elucidated. In this study, we report the identification of a U. maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumor development. The Deltafox1 hyphae induce the accumulation of H(2)O(2) in and around infected cells and a maize defense response phenotypically represented by the encasement of proliferating hyphae in a cellulose-containing matrix. The phenotype can be attributed to the fox1-dependent deregulation of several effector genes that are linked to pathogenic development and host defense suppression.

The Ustilago maydis Clp1 protein orchestrates pheromone and b-dependent signaling pathways to coordinate the cell cycle and pathogenic development.

Regulation of the cell cycle and morphogenetic switching during pathogenic and sexual development in Ustilago maydis is orchestrated by a concerted action of the a and b mating-type loci. Activation of either mating-type locus triggers the G2 cell cycle arrest that is a prerequisite for the formation of the infectious dikaryon; this cell cycle arrest is released only after penetration of the host plant. Here, we show that bW, one of the two homeodomain transcription factors encoded by the b mating-type locus, and the zinc-finger transcription factor Rbf1, a master regulator for pathogenic development, interact with Clp1 (clampless 1), a protein required for the distribution of nuclei during cell division of the dikaryon. In addition, we identify Cib1, a previously undiscovered bZIP transcription factor required for pathogenic development, as a Clp1-interacting protein. Clp1 interaction with bW blocks b-dependent functions, such as the b-dependent G2 cell cycle arrest and dimorphic switching. The interaction of Clp1 with Rbf1 results in the repression of the a-dependent pheromone pathway, conjugation tube formation, and the a-induced G2 cell cycle arrest. The concerted interaction of Clp1 with Rbf1 and bW coordinates a- and b-dependent cell cycle control and ensures cell cycle release and progression at the onset of biotrophic development.

The transcription factor Rbf1 is the master regulator for b-mating type controlled pathogenic development in Ustilago maydis.

In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.

The Clp1 protein is required for clamp formation and pathogenic development of Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bE and bW, encoded by the b-mating-type locus. We have identified a b-dependently induced gene, clampless1 (clp1), that is required for the proliferation of dikaryotic filaments in planta. We show that U. maydis hyphae develop structures functionally equivalent to clamp cells that participate in the distribution of nuclei during cell division. In clp1 mutant strains, dikaryotic filaments penetrate the plant cuticle, but development is stalled before the first mitotic division, and the clamp-like structures are not formed. Although clp1 is immediately activated upon b-induction on the transcriptional level, nuclear-localized Clp1 protein is first observed at the stage of plant penetration prior to the first cell division. Induced expression of clp1 strongly interferes with b-dependent gene regulation and blocks b-dependent filament formation and b-dependent cell cycle arrest. We speculate that the Clp1 protein inhibits the activity of the bE/bW heterodimer to facilitate the cell cycle progression during hyphal growth.