The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual crop losses. The characteristic wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels, which undergo fluctuations in osmolarity. To gain insights into the mechanisms that confer the organisms' pathogenicity and enable them to proliferate in the unique ecological niche of the plant vascular system, we sequenced the genomes of V. dahliae and V. albo-atrum and compared them to each other, and to the genome of Fusarium oxysporum, another fungal wilt pathogen. Our analyses identified a set of proteins that are shared among all three wilt pathogens, and present in few other fungal species. One of these is a homolog of a bacterial glucosyltransferase that synthesizes virulence-related osmoregulated periplasmic glucans in bacteria. Pathogenicity tests of the corresponding V. dahliae glucosyltransferase gene deletion mutants indicate that the gene is required for full virulence in the Australian tobacco species Nicotiana benthamiana. Compared to other fungi, the two sequenced Verticillium genomes encode more pectin-degrading enzymes and other carbohydrate-active enzymes, suggesting an extraordinary capacity to degrade plant pectin barricades. The high level of synteny between the two Verticillium assemblies highlighted four flexible genomic islands in V. dahliae that are enriched for transposable elements, and contain duplicated genes and genes that are important in signaling/transcriptional regulation and iron/lipid metabolism. Coupled with an enhanced capacity to degrade plant materials, these genomic islands may contribute to the expanded genetic diversity and virulence of V. dahliae, the primary causal agent of Verticillium wilts. Significantly, our study reveals insights into the genetic mechanisms of niche adaptation of fungal wilt pathogens, advances our understanding of the evolution and development of their pathogenesis, and sheds light on potential avenues for the development of novel disease management strategies to combat destructive wilt diseases. Vascular wilts are chronic and very often severe plant diseases that cause billions of dollars in annual crop losses. The characteristic wilt symptom is a result of water blockage caused by the colonization and proliferation of pathogenic microbes in the plant xylem, a water-conducting system. We sequenced genomes of two Verticillium wilt pathogens and compared them to the genome of another wilt fungus, Fusarium oxysporum. The shared genomic features among these three wilt fungi suggest the acquisition of homologs of a bacterial glucosyltransferase, involved in adaptation to osmotic stress, through horizontal transfer. Analyses of glucosyltransferase gene deletion mutants in Verticillium dahliae revealed decreased virulence in the host plant Nicotiana benthamiana. Compared to other fungi, both Verticillium genomes encode more plant cell wall degrading enzymes, including those that are able to degrade cell walls of live plants. Between the two closely related Verticillium genomes, we discovered flexible genomic islands in the primary causal agent of Verticillium wilts, Verticillium dahliae. Coupled with the impressive arsenal of plant cell wall-degrading enzymes, these flexible genomic islands may have contributed to expanding genetic diversity for this organism to invade more plant hosts. In summary, our study reveals insights into the evolution and niche adaptation of fungal wilt pathogens and sheds light on the development of novel disease management strategies for combating the destructive wilt diseases.

Mycoviruses from plant pathogens can induce hypovirulence (reduced virulence) in their host fungi and have gained considerable attention as potential biocontrol tools. An increasing number of mycoviruses that induce fungal hypovirulence, from a wide variety of taxonomic groups, are currently being reported. Successful application of these viruses in disease management is greatly dependent on their ability to spread in the natural populations of the pathogen. Mycoviruses generally lack extracellular routes of transmission. Hyphal anastomosis is the main route of horizontal mycovirus transmission to other isolates, and conidia of vertical transmission to the progeny. Transmission efficiencies are influenced by both the fungal host and the infecting virus. Interestingly, artificial transfection methods have shown that potential biocontrol mycoviruses often have the ability to infect a variety of fungi. This expands their possible use to the control of pathogens others than those where they were identified. Mycovirus research is also focused on gaining insights into their complex molecular biology and the molecular bases of fungus−virus interactions. This knowledge could be exploited to manipulate the mycovirus and/or the host and generate combinations with enhanced properties in biological control. Finally, when exploring the use of mycoviruses in field conditions, the pathogen life style and the characteristics of the disease and crops affected will deeply impact the specific challenges to overcome, and the development of biocontrol formulations and delivery methods.

Background: The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to 15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae. Results: To shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 50.58 million reads). Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations. Conclusion: Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae. The identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development.

Fungi are mostly sessile organisms, and thus have evolved ways to cope with environmental changes. Many fungi produce ‘dormant’ structures, which all

Summary: Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast‐like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high‐affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site‐directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.