Ds transposition mediated by transient transposase expression in Heiracium aurantiacum

The feasibility of using transient transposase expression to mobilize Ds elements for gene tagging in Hieracium aurantiacum was evaluated. A T-DNA construct carrying the Ac transposase gene and either a visible marker gene (uidA) or the conditionally-lethal marker gene (codA) was transferred to H. aurantiacum leaf discs (previously transformed with a Ds element) by co-cultivation with Agrobacterium tumefaciens. Shoots were regenerated directly from the co-cultivated leaf discs under selection for antibiotic resistance resulting from Ds excision. Most regenerants carried unique transposition events. Of 84 regenerated plants, twenty one (25%) did not express the marker gene and the DNA coding sequence of the transposase could not be detected in seven (8.3%). Potential advantages of this method over conventional gene-tagging methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; and the transient nature of transposase expression should facilitate the stabilisation of the transposed element.     

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola

Trichoderma species conidiate in response to blue light, however, unlike in the blue-light model fungus Neurospora crassa, conidiation in Trichoderma spp. has been considered to be non-circadian. In this study we uncovered evidence for circadian conidiation in Trichoderma pleuroticola and identified orthologues of the key N. crassa clock components, wc-1 (blr-1) and frq.     

Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)

 An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis. Received: 12 October 1998 / Revision received: 17 May 1999 Accepted: 14 June 1999     

Ambient pH intrinsically influences Trichoderma conidiation and colony morphology

Conidiation in Trichoderma has been demonstrated to be favoured by a low ambient pH and more recently PacC (Pac1) mediated pH-regulation has been implicated in the control of conidiation. In this study, ambient pH effects on conidiation were investigated in three isolates (Trichoderma hamatum, Trichoderma atroviride and Trichoderma pleuroticola) exposed to a single blue-light burst or to mycelial injury. Disks of conidiation were observed for T. atroviride in response to a single light exposure, which clearly demonstrates that all cells are potentially competent for photoconidiation. Previous studies have suggested T. hamatum does not conidiate in response to mycelial injury, however, in this study a clear injury response was observed from pH 2.8 to 3.2. T. pleuroticola displayed three distinct pH-dependent colony morphologies from pH 2.8 to 5.2. Conidiation was strictly low pH-dependent on buffered media and observed at all pH values on unbuffered media. The dependence of the conidial phenotype on the buffering state of the medium rather than the pH per se, was unexpected as it has been suggested that conidiation is PacC regulated. Conversely, excretion of an anthraquinone was strictly pH-dependent regardless of the buffering state. These studies highlight the complexity of ambient pH effects on Trichoderma spp. and demonstrate a need to widen the scope of research to multiple species.     

Agrobacterium-mediated transformation of Sclerotinia sclerotiorum

Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.     

Approaches to functional genomics in filamentous fungi

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.     

Isolate-specific conidiation in Trichoderma in response to different nitrogen sources

A characteristic feature of Trichoderma is the production of concentric rings of conidia in response to alternating light/dark conditions and a single ring of conidia in response to a single burst of light. In this study, conidiation was investigated in four biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens) and one isolate from the mushroom pathogen species, T. pleuroticola. All five isolates produced concentric conidial rings under alternating light/dark conditions on potato-dextrose agar (PDA), however, in response to a 15min burst of blue light, only T. asperellum and T. virens produced a clearly defined conidial ring. Both T. pleuroticola and T. hamatum photoconidiated in a disk-like fashion and T.?atroviride produced a broken ring with a partially filled in appearance. In the presence of primary nitrogen, T. asperellum and T. pleuroticola conidiated in a disk, whereas, when grown in the presence of secondary nitrogen, a ring of conidia was produced. Primary nitrogen promoted photoconidiation and competency to conidiate in response to light appeared dependent on the nitrogen catabolite repression state of the cell. Mycelial injury was also investigated in the same five isolates of Trichoderma on PDA and under different nitrogen statuses. For the first time, we report that conidiation in response to injury is differentially regulated in different isolates/species of Trichoderma.