Siegfried Salomon

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.

Enhanced mycotoxin production of a lipase-deficient Fusarium graminearum mutant correlates to toxin-related gene expression

Fusarium graminearum causes important diseases of small-grain cereals and maize and produces several mycotoxins. Among them, deoxynivalenol (DON) and zearalenone (ZEA) can accumulate in feedstuffs and foods to health-threatening levels. Although DON is important for fungal virulence in wheat, disease severity in the field does not correlate with mycotoxin concentrations. We compared gene expression and mycotoxin production of lipase-deficient mutants (Δfgl1), strongly reduced in virulence, and the respective wild-type isolate. Δfgl1 mutants exhibited up-regulated DON production during wheat head infection. On isolated wheat kernels, DON was only produced in low quantities, but higher in wild-type than in Δfgl1 mutants. In contrast, neither wild-type nor Δfgl1 mutants produced ZEA during wheat head infection. However, ZEA was clearly detectable on wheat kernels. Here, Δfgl1 mutants revealed a dramatically enhanced ZEA production. We could correlate the altered amounts of DON and ZEA directly with the expression of the toxin-related genes Tri5 for DON and PKS4 and PKS13 for ZEA. Based on Tri5 expression and the infection pattern of the wild-type and Δfgl1 mutants, we suggest that the transition zone of rachilla and rachis is important in the induction of DON synthesis. Gene expression studies indicate an involvement of the lipase FGL1 in regulation of 8 PKS genes and ZEA production.

Enzymatic properties and expression patterns of five extracellular lipases of Fusarium graminearum in vitro.

Fusarium graminearum is a filamentous fungus that causes devastating diseases on plants of economic importance including maize, wheat, and barley. F. graminearum is able to utilize triglycerides as a carbon source during growth. Extracellular lipases are the preferred enzymes to catalyze the hydrolysis of fats and oils. Lipases are ubiquitous enzymes of considerable physiological significance and industrial potential. Previously, FGL1 was the first described F. graminearum extracellular lipase associated with virulence. We report the biochemical characterization of FGL1 and four new secreted lipases of F. graminearum. The lipase genes of F. graminearum wild-type strain 8/1 were sequenced, cloned and over-expressed in Pichia pastoris. We show that the lipases have their temperature optimum between 30 and 40°C and a pH optimum of ∼7. A broad range of lipase substrates, from C4 to C18 p-nitrophenyl esters, were hydrolyzed efficiently by the lipases, indicating the true lipolytic activity of the enzymes. Expression patterns of these lipases were also analyzed by semiquantitative RT-PCR in F. graminearum cultured in water supplemented with 2% (v/v) wheat germ oil at 28°C. Transcripts of all examined lipases are detectable and the genes are regulated differently under these culture conditions. Our data indicated that F. graminearum possesses a ubiquitous source of secreted lipases, which could be used for industrial intentions. We also provided the foundation of lipase expression in vitro, which is necessary for further characterization.

The secreted lipase FGL1 is sufficient to restore the initial infection step to the apathogenic Fusarium graminearum MAP kinase disruption mutant Δgpmk1

Mitogen activated protein (MAP) kinases are key components in signalling networks. In Fusarium graminearum, one of the most devastating fungal plant pathogens, two MAP kinases are known to be involved in pathogenicity, Mgv1 and the Gibberella pathogenicity MAP kinase Gpmk1. Δgpmk1 mutants with a disrupted GPMK1 gene are unable to infect wheat spikelets. They exhibit altered secretion of several extracellular enzymes. Among those, the lipase FGL1 is known to be a major virulence factor of F. graminearum. FGL1 gene expression was decreased in Δgpmk1 strains during wheat head infection. To uncouple FGL1 expression from Gpmk1 activity, we generated Δgpmk1 strains that constitutively express FGL1 under control of the Cochliobolus heterostrophus glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter. The level of extracellular lipolytic activity of these strains in culture was comparable to the wild-type. These mutants showed fully restored conidiation and partial complementation of defects in development that were reported from the Δgpmk1 mutant. They also partially complemented the apathogenic disease phenotype of the Δgpmk1 mutants causing lesions in directly inoculated wheat spikelets. But in contrast to wild-type, their growth was restricted to directly inoculated spikelets. A barrier-like formation was observed at the rachis node. Based on these results, we could show that the lipase FGL1 is necessary but not sufficient to restore complete pathogenicity to the apathogenic Δgpmk1 mutant. Hence, we hypothesize that the MAP kinase Gpmk1 is involved in the regulation of additional factors required for complete virulence of F. graminearum.

Downregulation of Glucan Synthase-Like (TaGSL) genes in wheat leads to inhibition of transgenic plant regeneration

RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants.