RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae.

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.

Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens.

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.

Transposable elements in phytopathogenic Verticillium spp.: insights into genome evolution and inter- and intra-specific diversification.

BACKGROUND: Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. Whole genome comparative analysis of the recently sequenced strains VdLs.17 and VaMs.102 revealed that non-random insertions of transposable elements (TEs) have contributed to the generation of four lineage-specific (LS) regions in VdLs.17. RESULTS: We present here a detailed analysis of Class I retrotransposons and Class II "cut-and-paste" DNA elements detected in the sequenced Verticillium genomes. We report also of their distribution in other Vd and Va isolates from various geographic origins. In VdLs.17, we identified and characterized 56 complete retrotransposons of the Gypsy-, Copia- and LINE-like types, as well as 34 full-length elements of the "cut-and-paste" superfamilies Tc1/mariner, Activator and Mutator. While Copia and Tc1/mariner were present in multiple identical copies, Activator and Mutator sequences were highly divergent. Most elements comprised complete ORFs, had matching ESTs and showed active transcription in response to stress treatment. Noticeably, we found evidences of repeat-induced point mutation (RIP) only in some of the Gypsy retroelements. While Copia-, Gypsy- and Tc1/mariner-like transposons were prominent, a large variation in presence of the other types of mobile elements was detected in the other Verticillium spp. strains surveyed. In particular, neither complete nor defective "cut-and-paste" TEs were found in VaMs.102. CONCLUSIONS: Copia-, Gypsy- and Tc1/mariner-like transposons are the most wide-spread TEs in the phytopathogens V. dahliae and V. albo-atrum. In VdLs.17, we identified several retroelements and "cut-and-paste" transposons still potentially active. Some of these elements have undergone diversification and subsequent selective amplification after introgression into the fungal genome. Others, such as the ripped Copias, have been potentially acquired by horizontal transfer. The observed biased TE insertion in gene-rich regions within an individual genome (VdLs.17) and the "patchy" distribution among different strains point to the mobile elements as major generators of Verticillium intra- and inter-specific genomic variation.

Microsclerotia development in Verticillium dahliae: Regulation and differential expression of the hydrophobin gene VDH1.

The vascular wilt fungus Verticillium dahliae produces persistent resting structures, known as microsclerotia, which are important for this plant pathogen's long-term survival. Previously, we identified a hydrophobin gene (VDH1) that is necessary for microsclerotial production. The current study of VDH1's expression, and its regulation, was undertaken to provide insight into the largely uncharacterized molecular mechanisms relevant to microsclerotial development. Reporter gene analysis showed that VDH1 is specifically expressed in developing microsclerotia, as well as in hyphal fusions and conidiophores, suggesting that VDH1 mediates the development of microsclerotia from conidiophores and other hyphal structures. We report also on the effects of nutrient availability on the regulation of microsclerotial development in V. dahliae; the gene's activity appears to be regulated in response to carbon availability. Lastly, constitutive expression of VDH1 results in delayed disease symptom development, but has no noticeable effect on in vitro microsclerotial development.

Genomics spurs rapid advances in our understanding of the biology of vascular wilt pathogens in the genus Verticillium.

The availability of genomic sequences of several Verticillium species triggered an explosion of genome-scale investigations of mechanisms fundamental to the Verticillium life cycle and disease process. Comparative genomics studies have revealed evolutionary mechanisms, such as hybridization and interchromosomal rearrangements, that have shaped these genomes. Functional analyses of a diverse group of genes encoding virulence factors indicate that successful host xylem colonization relies on specific Verticillium responses to various stresses, including nutrient deficiency and host defense-derived oxidative stress. Regulatory pathways that control responses to changes in nutrient availability also appear to positively control resting structure development. Conversely, resting structure development seems to be repressed by pathways, such as those involving effector secretion, which promote responses to host defenses. The genomics-enabled functional characterization of responses to the challenges presented by the xylem environment, accompanied by identification of novel virulence factors, has rapidly expanded our understanding of niche adaptation in Verticillium species.

Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7.

The colonization process of Olea europaea by the defoliating pathotype of Verticillium dahliae, and the in planta interaction with the endophytic, biocontrol strain Pseudomonas fluorescens PICF7 were determined. Differential fluorescent protein tagging was used for the simultaneous visualization of P. fluorescens PICF7 and V. dahliae in olive tissues. Olive plants were bacterized with PICF7 and then transferred to V. dahliae-infested soil. Monitoring olive colonization events by V. dahliae and its interaction with PICF7 was conducted using a non-gnotobiotic system, confocal laser scanner microscopy and tissue vibratoming sections. A yellow fluorescently tagged V. dahliae derivative (VDAT-36I) was obtained by Agrobacterium tumefaciens-mediated transformation. Isolate VDAT-36I quickly colonized olive root surface, successfully invaded root cortex and vascular tissues via macro- and micro-breakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site, and once established on/in root tissues, hindered pathogen colonization. For the first time using this approach, the entire colonization process of a woody plant by V. dahliae is reported. Early and localized root surface and root endophytic colonization by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt epidemics in olive.

A hydrophobin gene, VDH1, is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae.

The wilt fungus Verticillium dahliae Kleb. produces desiccation- and cold-tolerant resting structures, known as microsclerotia, which are the primary source of disease inoculum in the field. In an exploration of the molecular mechanisms involved in the development of these important structures, we have identified in V. dahliae a differentially expressed, class II hydrophobin gene (VDH1). vdh1 mutants generated through targeted gene disruption show a severe reduction in microsclerotia production, indicating that the gene is important for this type of development. Although vdh1 mutants do produce normal conidiophores and spores, desiccation-tolerance of the spores is reduced. The VDH1 gene is not, however, needed for normal disease development in tomato. VDH1's functions are multi-faceted, and seem generally relevant to long-term survival in V. dahliae.

Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae.

Verticillium dahliae is an important soil-borne fungal pathogen that causes vascular wilt diseases in a large variety of important crop plants. Due to its persistence in the soil, control of Verticillium wilt relies heavily on soil fumigation. The global ban on methyl bromide, a highly effective soil fumigant, poses an urgent need to develop alternative control measures against Verticillium wilt; and these might be more forthcoming with a better understanding of the molecular and cellular mechanisms that underpin the pathogenicity of V. dahliae. In this study, we assessed the role in growth, development, and pathogenicity of VMK1, a gene encoding a mitogen-activated protein (MAP) kinase (hence, Verticillium MAP Kinase 1). Disruption of VMK1 via Agrobacterium tumefaciens-mediated transformation, in two V. dahliae isolates, one from lettuce and the other from tomato, resulted in severely reduced virulence in diverse host plants, suggesting that VMK1 is essential for pathogenicity and that the MAP kinase-mediated signaling pathway has a conserved role in fungal pathogenicity. The vmk1 mutants also exhibited reduced conidiation and microsclerotia formation, suggesting that the gene is important for multiple cellular processes.

Cloning and targeted disruption, via Agrobacterium tumefaciens-mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae.

A gene encoding a trypsin protease was isolated from a tomato isolate of Verticillium dahliae. The gene, designated VTP1, contains two introns and is predicted to encode a protein of 256 amino acids. The gene is present in V. dahliae isolates from different host plants and in V. albo-atrum; weakly hybridizing sequences are present in V. tricorpus. VTP1 cDNA sequences were identified in a sequence tag analysis of genes expressed under growth conditions that promote microsclerotia development. Replacement of the gene, by Agrobacterium tumefaciens-mediated transformation (ATMT), with a mutant allele construct did not noticeably alter either pathogenicity or growth in culture. Searches of expressed sequence tag databases showed that, in addition to the VTP1 gene, V. dahliae contains two genes encoding subtilisin-like proteases similar to those produced by pathogenic Aspergillus spp. This is the first description of the application of ATMT to the molecular analysis of phytopathogenic Verticillium spp.