The Wor1-like Protein Fgp1 Regulates Pathogenicity, Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

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

The Nuclear Protein Sge1 of Fusarium oxysporum Is Required for Parasitic Growth

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth

Investigating Agrobacterium-Mediated Transformation of Verticillium albo-atrum on Plant Surfaces

Background: Agrobacterium tumefaciens has long been known to transform plant tissue in nature as part of its infection process. This natural mechanism has been utilised over the last few decades in laboratories world wide to genetically manipulate many species of plants. More recently this technology has been successfully applied to non-plant organisms in the laboratory, including fungi, where the plant wound hormone acetosyringone, an inducer of transformation, is supplied exogenously. In the natural environment it is possible that Agrobacterium and fungi may encounter each other at plant wound sites, where acetosyringone would be present, raising the possibility of natural gene transfer from bacterium to fungus. Methodology/Principal Findings: We investigate this hypothesis through the development of experiments designed to replicate such a situation at a plant wound site. A. tumefaciens harbouring the plasmid pCAMDsRed was co-cultivated with the common plant pathogenic fungus Verticillium albo-atrum on a range of wounded plant tissues. Fungal transformants were obtained from co-cultivation on a range of plant tissue types, demonstrating that plant tissue provides sufficient vir gene inducers to allow A. tumefaciens to transform fungi in planta. Conclusions/Significance: This work raises interesting questions about whether A. tumefaciens may be able to transform organisms other than plants in nature, or indeed should be considered during GM risk assessments, with furthe

Distinct colonization patterns and cDNA-AFLP transcriptome profiles in compatible and incompatible interactions between melon and different races of Fusarium oxysporum f. sp. melonis

Background: Fusarium oxysporum f. sp. melonis Snyd. & Hans. (FOM) causes Fusarium wilt, the most important infectious disease of melon (Cucumis melo L.). The four known races of this pathogen can be distinguished only by infection on appropriate cultivars. No molecular tools are available that can discriminate among the races, and the molecular basis of compatibility and disease progression are poorly understood. Resistance to races 1 and 2 is controlled by a single dominant gene, whereas only partial polygenic resistance to race 1,2 has been described. We carried out a large-scale cDNA-AFLP analysis to identify host genes potentially related to resistance and susceptibility as well as fungal genes associated with the infection process. At the same time, a systematic reisolation procedure on infected stems allowed us to monitor fungal colonization in compatible and incompatible host-pathogen combinations. Results: Melon plants (cv. Charentais Fom-2), which are susceptible to race 1,2 and resistant to race 1, were artificially infected with a race 1 strain of FOM or one of two race 1,2 w strains. Host colonization of stems was assessed at 1, 2, 4, 8, 14, 16, 18 and 21 days post inoculation (dpi), and the fungus was reisolated from infected plants. Markedly different colonization patterns were observed in compatible and incompatible host-pathogen combinations. Five time points from the symptomless early stage (2 dpi) to obvious wilting symptoms (21 dpi) were considered for cDNA-AFLP analysis. After successful sequencing of 627 transcript-derived fragments (TDFs) differentially expressed in infected plants, homology searching retrieved 305 melon transcripts, 195 FOM transcripts expressed in planta and 127 orphan TDFs. RNA samples from FOM colonies of the three strains grown in vitro were also included in the analysis to facilitate the detection of in planta-specific transcripts and to identify TDFs differentially expressed among races/strains. Conclusion: Our data suggest that resistance against FOM in melon involves only limited transcriptional changes, and that wilting symptoms could derive, at least partially, from an active plant response. We discuss the pathogen-derived transcripts expressed in planta during the infection process and potentially related to virulence functions, as well as transcripts that are differentially expressed between the two FOM races grown in vitro. These transcripts provide candidate sequences that can be further tested for their ability to distinguish between races. Sequence data from this article have been deposited in GenBank, Accession Numbers: HO867279-HO867981

Regulation of proteinaceous effector expression in phytopathogenic fungi

Effectors are molecules used by microbial pathogens to facilitate infection via effector-triggered susceptibility or tissue necrosis in their host. Much research has been focussed on the identification and elucidating the function of fungal effectors during plant pathogenesis. By comparison, knowledge of how phytopathogenic fungi regulate the expression of effector genes has been lagging. Several recent studies have illustrated the role of various transcription factors, chromosome-based control, effector epistasis, and mobilisation of endosomes within the fungal hyphae in regulating effector expression and virulence on the host plant. Improved knowledge of effector regulation is likely to assist in improving novel crop protection strategies

Aspergillus as a multi-purpose cell factory: current status and perspectives

Aspergilli have a long history in biotechnology as expression platforms for the production of food ingredients, pharmaceuticals and enzymes. The achievements made during the last years, however, have the potential to revolutionize Aspergillus biotechnology and to assure Aspergillus a dominant place among microbial cell factories. This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products. New trends and concepts related to Aspergillus genomics and systems biology will be discussed as well as the challenges that have to be met to integrate omics data with metabolic engineering attempts

A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. The life cycle of V. dahliae includes three vegetative phases: parasitic, saprophytic and dormant. The dormant microsclerotia are the primary infectious propagules, which germinate when they are stimulated by root exudates. In this study, we report the first application of Agrobacterium tumefaciens-mediated transformation (ATMT) for construction of insertional mutants from a virulent defoliating isolate of V. dahliae (V592). Changes in morphology, especially a lack of melanized microsclerotia or pigmentation traits, were observed in mutants. Together with the established laboratory unimpaired root dip-inoculation approach, we found insertional mutants to be affected in their pathogenicities in cotton. One of the genes tagged in a pathogenicity mutant encoded a glutamic acid-rich protein (VdGARP1), which shared no significant similarity to any known annotated gene. The vdgarp1 mutant showed vigorous mycelium growth with a significant delay in melanized microsclerotial formation. The expression of VdGARP1 in the wild type V529 was organ-specific and differentially regulated by different stress agencies and conditions, in addition to being stimulated by cotton root extract in liquid culture medium. Under extreme infertile nutrient conditions, VdGARP1 was not necessary for melanized microsclerotial formation. Taken together, our data suggest that VdGARP1 plays an important role in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. Overall, our findings indicate that insertional mutagenesis by ATMT is a valuable tool for the genome-wide analysis of gene function and identification of pathogenicity genes in this important cotton pathogen

Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f. sp. cepae

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus Verticillium dahliae by Agrobacterium tumefaciens-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacteriumtumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae