The Frq-Frh Complex Light-Dependently Delays Sfl1-Induced Microsclerotia Formation in Verticillium dahliae.

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.

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.

Tomato Xylem Sap Hydrophobins Vdh4 and Vdh5 Are Important for Late Stages of Verticillium dahliae Plant Infection.

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.

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.

Verticillium longisporum Elicits Media-Dependent Secretome Responses With Capacity to Distinguish Between Plant-Related Environments.

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.

Verticillium dahliae transcription factors Som1 and Vta3 control microsclerotia formation and sequential steps of plant root penetration and colonisation to induce disease.

Verticillium dahliae nuclear transcription factors Som1 and Vta3 can rescue adhesion in a FLO8-deficient Saccharomyces cerevisiae strain. Som1 and Vta3 induce the expression of the yeast FLO1 and FLO11 genes encoding adhesins. Som1 and Vta3 are sequentially required for root penetration and colonisation of the plant host by V. dahliae. The SOM1 and VTA3 genes were deleted and their functions in fungus-induced plant pathogenesis were studied using genetic, cell biology, proteomic and plant pathogenicity experiments. Som1 supports fungal adhesion and root penetration and is required earlier than Vta3 in the colonisation of plant root surfaces and tomato plant infection. Som1 controls septa positioning and the size of vacuoles, and subsequently hyphal development including aerial hyphae formation and normal hyphal branching. Som1 and Vta3 control conidiation, microsclerotia formation, and antagonise in oxidative stress responses. The molecular function of Som1 is conserved between the plant pathogen V. dahliae and the opportunistic human pathogen Aspergillus fumigatus. Som1 controls genes for initial steps of plant root penetration, adhesion, oxidative stress response and VTA3 expression to allow subsequent root colonisation. Both Som1 and Vta3 regulate developmental genetic networks required for conidiation, microsclerotia formation and pathogenicity of V. dahliae.