Frank J. Maier

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence

SUMMARY Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.

PTK1, a Mitogen-Activated-Protein Kinase Gene, Is Required for Conidiation, Appressorium Formation, and Pathogenicity of Pyrenophora teres on Barley

Mitogen-activated protein kinases (MAPKs) are a group of protein kinases that execute a wide variety of roles in cellular signal transduction pathways such as osmoregulation, cell wall biosynthesis, growth, and differentiation. A polymerase chain reaction (PCR) with degenerate primers based on conserved regions of known MAPKs was used to clone the MAPK gene PTK1 from the leaf pathogen Pyrenophora teres (anamorph Drechslera teres), the causal agent of net blotch of barley (Hordeum vulgare L.). The predicted amino acid sequence shows high homology with MAPKs from other phytopathogenic fungi. The gene is present in the genome as a single copy. PTK1 is expressed during in vitro growth on complete medium, under conidiation-inducing conditions and during infection of barley leaves, as shown by reverse transcription-PCR studies. In order to assess the role of PTK1 in the life cycle of P. teres, targeted gene disruption was conducted. Mutants carrying an interrupted copy of the gene were deficient in conidiation, did not form appressoria on glass surfaces or on barley leaves, lost their ability to infect barley leaves, and could not colonize host tissues following artificial wounding.

Mutagenesis via Insertional - or Restriction Enzyme-Mediated - Integration (REMI) as a Tool to Tag Pathogenicity Related Genes in Plant Pathogenic Fungi

Abstract Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.

Developing Kernel and Rachis Node Induce the Trichothecene Pathway of Fusarium graminearum During Wheat Head Infection

The fungal pathogen Fusarium graminearum is the most common agent of Fusarium head blight (FHB) in small grain cereals and cob rot of maize. The threat posed by this fungus is due to a decrease in yield and, additionally, mycotoxin contamination of the harvested cereals. Among the mycotoxins, trichothecenes influence virulence of F. graminearum in a highly complex manner that is strongly host- as well as chemotype-specific. The factors inducing mycotoxin production during plant infection are still unknown. To evaluate the induction of the trichothecene pathway, the green fluorescence protein (GFP) gene was fused to the promoter of the TRI5 gene coding for the trichodiene synthase and integrated into the genome by homologous integration. The resulting mutant contains a fully functional TRI5 gene ensuring virulence on wheat and exhibits GFP driven by the endogenous TRI5 promoter. We are now able to monitor the induction of trichothecenes under real-time conditions. To localize the fungus in the plant tissue, the dsRed gene was integrated under constitutive control of the glycerol-3-phosphate dehydrogenase (gpdA) promoter. We are now able to show that, first, induction of GFP as well as trichothecene production in the reporter strain reflects TRI5 induction and trichothecene production in the wild type; second, expression of TRI5 is inducible during growth in culture; and, third, trichothecene production is not uniformly induced during the onset of infection but is tissue specific during fungal infection of wheat.

Phylogeny and evolution of mating-type genes from Pyrenophora teres, the causal agent of barley “net blotch” disease

The main aim of this study was to test the patterns of sequence divergence and haplotype structure at the MAT locus of Pyrenophora teres, the causal agent of barley ‘net blotch’ disease. P. teres is a heterothallic ascomycete that co-occurs in two symptomatological forms, the net form (NF) and the spot form (SF). The mating-type genes MAT1-1-1 and MAT1-2-1 were sequenced from 22 NF isolates (12 MAT1-1-1 and 10 MAT1-2-1 sequences) and 17 SF isolates (10 MAT1-1-1 and seven MAT1-2-1 sequences) collected from Sardinian barley landrace populations and worldwide. On the basis of a parsimony network analysis, the two forms of P. teres are phylogenetically separated. More than 85% of the total nucleotide variation was found between formae speciales. The two forms do not share any polymorphisms. Six diagnostic nucleotide polymorphisms were found in the MAT1-1-1 intron (1) and in the MAT1-1-1 (3) and MAT1-2-1 (2) exons. Three diagnostic non-synonymous mutations were found, one in MAT1-1-1 and two in MAT1-2-1. For comparison with P. teres sequence data, the mating-type genes from Pyrenophora graminea were also isolated and sequenced. Divergence between P. graminea and P. teres is of a similar magnitude to that between NF and SF of P. teres. The MAT genes of P. graminea were closer to those of SF than to NF, with the MAT1-2-1 SF peptide not different from the MAT1-2-1 peptide of P. graminea. Overall, these data suggest long genetic isolation between the two forms of P. teres and that hybridization is rare or absent under field conditions, with each form having some particular niche specialization. This indicates that research on resistance to P. teres should consider the two forms separately, as different species.

Preventing Fusarium Head Blight of Wheat and Cob Rot of Maize by Inhibition of Fungal Deoxyhypusine Synthase

Upon posttranslational activation, the eukaryotic initiation factor-5A (eIF-5A) transports a subset of mRNAs out of the nucleus to the ribosomes for translation. Activation of the protein is an evolutionary highly conserved process that is unique to eIF-5A, the conversion of a lysine to a hypusine. Instrumental for the synthesis of hypusine is the first of two enzymatic reactions mediated by deoxyhypusine synthase (DHS). We show that DHS of wheat and the pathogenic fungus Fusarium graminearum, which causes one of the most destructive crop diseases worldwide, are transcriptionally upregulated during their pathogenic interaction. Although DHS of wheat, fungus, and human can be equally inhibited by the inhibitor CNI-1493 in vitro, application during infection of wheat and maize flowers results in strong inhibition of the pathogen without interference with kernel development. Our studies provide a novel strategy to selectively inhibit fungal growth without affecting plant growth. We identified fungal DHS as a target for the development of new inhibitors, for which CNI-1493 may serve as a lead substance.