Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.

Vegetative fusion is essential for the development of an interconnected colony in many filamentous fungi. In the ascomycete fungus Neurospora crassa, vegetative fusion occurs between germinated conidia (germlings) via specialized structures termed "conidial anastomosis tubes" (CATs) and between hyphae within a mature colony. In N. crassa, both CAT and hyphal fusion are under the regulation of a conserved MAP kinase cascade (NRC1, MEK2, and MAK2). Here we show that the predicted downstream target of the MAK2 kinase pathway, a Ste12-like transcription factor known as PP1, regulates elements required for CAT and hyphal fusion. The PP1 regulatory network was revealed by expression profiling of wild type and the Dpp-1 mutant during conidial germination and colony establishment. To identify targets required for cell fusion more specifically, expression-profiling differences were assessed via inhibition of MAK2 kinase activity during chemotropic interactions and cell fusion. These approaches led to the identification of new targets of the cell fusion pathway that, when mutated, showed alterations in chemotropic signaling and cell fusion. In particular, conidial germlings carrying a deletion of NCU04732 (Dham-11) failed to show chemotropic interactions and cell fusion. However, signaling (as shown by oscillation of MAK2 and SO to CAT tips), chemotropism, and cell fusion were restored in Dham-11 germlings when matched with wild-type partner germlings. These data reveal novel insights into the complex process of self-signaling, germling fusion, and colony establishment in filamentous fungi. CELL fusion between genetically identical cells is important in development (for example, myoblast fusion during muscle formation) and occurs in many multicellular organisms from simple ascomycete fungi to mammals (Chen et al. 2007;Aguilar et al. 2013). Cell fusion between genetically identical cells can be mediated by cells that have differentiated, but in some cases, also between cells in an identical developmental state, for example, cell fusion between germinating asexual spores (conidia) of filamentous fungi (Pandey et al. 2004;Roca et al. 2005a;Read et al. 2010). In filamentous fungi, these fusions are integral to the formation of an interconnected hyphal network, which mediates genetic mixing and the sharing of resources (Simonin et al. 2012;Roper et al. 2013). How this process is initiated and maintained and what proteins are involved are still mostly unknown.In filamentous ascomycete fungi, a conserved MAP kinase pathway that is involved in pheromone response and mating in Saccharomyces cerevisiae (Ste11, Ste7, and Fus3) (Bardwell 2005) is required for cell fusion and heterokaryon formation during vegetative growth (Hou et al. 2002;Wei et al. 2003;Pandey et al. 2004;Fu et al. 2011;Jun et al. 2011;Dettmann et al. 2012). This conserved pathway also plays a role in sexual development and secondary metabolism and is required for the virulence of both plant and animal fungal pathogens (Roman et al. 2007;Rispail and Di Piet...

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein.