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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. [ABSTRACT FROM AUTHOR]

The vascular plant pathogenic fungus Verticillium dahliae has to adapt to environmental changes outside and inside its host. V. dahliae harbors homologs of Neurospora crassa clock genes. The molecular functions and interactions of Frequency (Frq) and Frq-interacting RNA helicase (Frh) in controlling conidia or microsclerotia development were investigated in V. dahliae JR2. Fungal mutant strains carrying clock gene deletions, an FRH point mutation, or GFP gene fusions were analyzed on transcript, protein, and phenotypic levels as well as in pathogenicity assays on tomato plants. Our results support that the Frq–Frh complex is formed and that it promotes conidiation, but also that it suppresses and therefore delays V. dahliae microsclerotia formation in response to light. We investigated a possible link between the negative element Frq and positive regulator Suppressor of flocculation 1 (Sfl1) in microsclerotia formation to elucidate the regulatory molecular mechanism. Both Frq and Sfl1 are mainly present during the onset of microsclerotia formation with decreasing protein levels during further development. Induction of microsclerotia formation requires Sfl1 and can be delayed at early time points in the light through the Frq–Frh complex. Gaining further molecular knowledge on V. dahliae development will improve control of fungal growth and Verticillium wilt disease. [ABSTRACT FROM AUTHOR]

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. Author summary: Verticillium dahliae causes significant economic losses to a wide range of valuable crops worldwide. The fungus can overwinter in the soil for years until it infects the roots of suitable host plants and colonizes the xylem sap, resulting in plant disease. V. dahliae needs the transcriptional regulator Vta3 for colonization of the tomato xylem sap, where it promotes gene expression of the virulence factor Elv1 but reduces expression of the transcription factor Mtf1 for fungal virulence and development. We discovered that Vta3 induced ELV1 expression is exclusively important for virulence but not necessary for V. dahliae growth or spore production. Vta3 delays Mtf1 action, which triggers plant immune responses and is required for late stages of infection. In summary, the presence of VTA3 alters gene expression of virulence factors and suppresses the Mtf1 subnetwork for late stages of plant disease progression. At the end of the infection cycle, Mtf1 initiates the formation of resting structures that allow the fungus to survive in the soil and later to re-infect host plants. A better understanding of the genetic networks underlying V. dahliae development and virulence is essential for developing new strategies to control growth and distribution of the fungus in fields. [ABSTRACT FROM AUTHOR]

Verticillium dahliae causes economic losses to a wide range of crops as a vascular fungal pathogen. This filamentous ascomycete spends long periods of its life cycle in the plant xylem, a unique environment that requires adaptive processes. Specifically, fungal proteins produced in the xylem sap of the plant host may play important roles in colonizing the plant vasculature and in inducing disease symptoms. RNA sequencing revealed over 1500 fungal transcripts that are significantly more abundant in cells grown in tomato xylem sap compared with pectin-rich medium. Of the 85 genes that are strongly induced in the xylem sap, four genes encode the hydrophobins Vdh1, Vdh2, Vdh4 and Vdh5. Vdh4 and Vhd5 are structurally distinct from each other and from the three other hydrophobins (Vdh1-3) annotated in V. dahliae JR2. Their functions in the life cycle and virulence of V. dahliae were explored using genetics, cell biology and plant infection experiments. Our data revealed that Vdh4 and Vdh5 are dispensable for V. dahliae development and stress response, while both contribute to full disease development in tomato plants by acting at later colonization stages. We conclude that Vdh4 and Vdh5 are functionally specialized fungal hydrophobins that support pathogenicity against plants. [ABSTRACT FROM AUTHOR]

Fungal spores, germlings, and mycelia adhere to substrates, including host tissues. The adhesive forces depend on the substrate and on the adhesins, the fungal cell surface proteins. Attachment is often a prerequisite for the invasion of the host, hence its importance. Adhesion visibly precedes colonization of root surfaces and outer cortex layers, but little is known about the molecular details. We propose that by starting from what is already known from other fungi, including yeast and other filamentous pathogens and symbionts, the mechanism and function of Trichoderma adhesion will become accessible. There is a sequence, and perhaps functional, homology to other rhizosphere-competent Sordariomycetes. Specifically, Verticillium dahliae is a soil-borne pathogen that establishes itself in the xylem and causes destructive wilt disease. Metarhizium species are best-known as insect pathogens with biocontrol potential, but they also colonize roots. Verticillium orthologs of the yeast Flo8 transcription factor, Som1, and several other relevant genes are already under study for their roles in adhesion. Metarhizium encodes relevant adhesins. Trichoderma virens encodes homologs of Som1, as well as adhesin candidates. These genes should provide exciting leads toward the first step in the establishment of beneficial interactions with roots in the rhizosphere. [ABSTRACT FROM AUTHOR]

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassicanapus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same‐sex mating‐type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage‐specific (LS) region apparently originating from the Verticilliumdahliae‐related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence‐reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent. [ABSTRACT FROM AUTHOR]

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta. Author summary: Verticillium wilt is a destructive disease of many economic important crop plants induced by fungal plant pathogenic Verticillium spp. such as V. dahliae. Developmental processes and the production of secondary metabolites are coordinated by fungal velvet domain proteins. The four V. dahliae velvet domain proteins form the heterodimers Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 during vegetative growth. Vel1, Vel2 and Vel3 coordinate the formation of the microsclerotia resting structures and the production of the metabolite melanin, which is deposited in microsclerotia. The survival of these resting structures in the soil is essential for Verticillium spp. during the monocyclic disease. Microsclerotia germinate in the presence of a suitable host, the resulting hyphae grow to and colonize the root. We discovered that this important process of initial colonization requires the presence of Vel1. Once inside the plant, the fungus distributes in the vascular system by conidia formation, which depends on Vel1 and Vel3. Vel1 is a promising target to control the fungal disease, because it is required for the induction of disease symptoms. [ABSTRACT FROM AUTHOR]

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the MEP1 , NLP2 , and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization. [ABSTRACT FROM AUTHOR]

Many fungi are able to produce resting structures, which ensure survival and protect them against various stresses in their habitat such as exposure to UV light, temperature variations, drought as well as changing pH and nutrient conditions. Verticillium dahliae is a plant pathogenic fungus that forms melanized resting structures, called microsclerotia, for survival of time periods without a host. These highly stress resistant microsclerotia persist in the soil for many years and are therefore problematic for an effective treatment of the fungus. The Verticillium transcription activator of adhesion 1 (Vta1) was initially identified as one of several transcriptional regulators that rescue adhesion in non-adhesive Saccharomyces cerevisiae cells. Vta2 and Vta3 are required for early steps in plant infection and colonization and additionally control microsclerotia formation. Here, we show that Vta1 function is different, because it is dispensable for root colonization and infection. Vta1 is produced in the fungal cell during microsclerotia development. Analysis of the deletion mutant revealed that the absence of Vta1 allows microsclerotia production, but they are colorless and no more melanized. Vta1 is required for melanin production and activates transcription of melanin biosynthesis genes including the polyketide synthase encoding PKS1 and the laccase LAC1. The primary function of Vta1 in melanin production is important for survival of microsclerotia as resting structures of V. dahliae. • Verticillium dahliae Vta1 is a novel transcriptional regulator of melanization. • Vta1 is produced during microsclerotia development. • Deletion of VTA1 results in colorless resting structures. • Overlapping Vta1 and Cmr1 genetic networks represent a dual control of melanization. [ABSTRACT FROM AUTHOR]

Verticillium species represent economically important phytopathogenic fungi with bacteria as natural rhizosphere antagonists. Growth inhibition patterns of Verticillium in different media were compared to saprophytic Aspergillus strains and were significantly more pronounced in various co-cultivations with different Pseudomonas strains. The Brassica napus rhizosphere bacterium Pseudomonas fluorescens DSM8569 is able to inhibit growth of rapeseed ( Verticillium longisporum) or tomato ( Verticillium dahliae) pathogens without the potential for phenazine or 2,4-diacetylphloroglucinol (DAPG) mycotoxin biosynthesis. Bacterial inhibition of Verticillium growth remained even after the removal of pseudomonads from co-cultures. Fungal growth response in the presence of the bacterium is independent of the fungal control genes of secondary metabolism LAE1 and CSN5. The phenazine producer P. fluorescens 2-79 (P_phen) inhibits Verticillium growth especially on high glucose solid agar surfaces. Additional phenazine-independent mechanisms in the same strain are able to reduce fungal surface growth in the presence of pectin and amino acids. The DAPG-producing Pseudomonas protegens CHA0 (P_DAPG), which can also produce hydrogen cyanide or pyoluteorin, has an additional inhibitory potential on fungal growth, which is independent of these antifungal compounds, but which requires the bacterial GacA/GacS control system. This translational two-component system is present in many Gram-negative bacteria and coordinates the production of multiple secondary metabolites. Our data suggest that pseudomonads pursue different media-dependent strategies that inhibit fungal growth. Metabolites such as phenazines are able to completely inhibit fungal surface growth in the presence of glucose, whereas GacA/GacS controlled inhibitors provide the same fungal growth effect on pectin/amino acid agar. [ABSTRACT FROM AUTHOR]

Verticillium wilt causes severe yield losses in a broad range of economically important crops worldwide. As many soil fumigants have a severe environmental impact, new biocontrol strategies are needed. Members of the genus Bacillus are known as plant growth-promoting bacteria (PGPB) as well as biocontrol agents of pests and diseases. In this study, we isolated 267 Bacillus strains from root-associated soil of field-grown tomato plants. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum. In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities. This indicates that chitinases contribute to antifungal activities. The present study identified B. thuringiensis isolates from tomato roots which exhibited in vitro antifungal activity against Verticillium species. [ABSTRACT FROM AUTHOR]