AbstractWe deleted the FgStuA gene in Fusarium graminearum and demonstrate its involvement in spore development, pathogenicity and secondary metabolism. The FgStuA protein is a member of the APSES family which regulate morphogenesis and virulence in ascomycetes. FgStuA is closely related to FoStuA in F. oxysporum and stuA in Aspergillus, but unlike FoStuA mutants, the FgStuA mutants were greatly reduced in pathogenicity on wheat and colonization of apple slices. The mutant produces no detectable trichothecenes on the wheat cultivar Bobwhite, and <1% levels of wildtype in culture. Trichothecenes have shown to be pathogenicity factors in wheat, and this could be the reason for loss of ability to produce disease in the FgStuA mutants. The mutant was also greatly decreased in sporulation and produced no perithecia in culture. Thus both sporulation and trichothecene synthesis may be regulated under the control of FgStuA.

Pathogenicity and trichothecene production of wildtype PH-1 and ΔFgStuA mutant.A. Wheat heads inoculated with wildtype PH-1 (center) show tissue bleaching and deformed awns. No symptoms are seen in mock inoculated plants (right) or ones inoculated with ΔFgStuA (left). Deoxynivalenol (DON) concentrations (ppm) of inoculated spikelets are indicated (nd = not detected). B. Production of 15 ADON in culture. The ΔFgStuA mutant produced <1% the level of wildtype (WT). C. Expression heat map of trichothecene genes 72h after wheat infection. None of the trichothecene genes are expressed in ΔFgStuA, while most are highly expressed in WT. Grey signal = not detected.

Erik Lysøe1, Matias Pasquali2, Andrew Breakspear2, Sonja S. Klemsdal1, and H. Corby Kistler2,3

The transcription factor FgStuA influences spore development, pathogenicity and secondary metabolism in Fusarium

graminearum

1Bioforsk - Norwegian Institute of Agricultural and Environmental Research, Ås, Norway; 2Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108, USA; 3USDA ARS Cereal Disease Laboratory, University of Minnesota, St. Paul, MN 55108, USA

ΔFgStuA Wildtype Mock

nd 296 nd DON concentration (ppm) in wheat

FgStuA is a transcription factor and a predicted APSES helix loop helix DNA binding protein. A. The ΔFgStuA mutant has a greatly reduced sporulation rate and altered spore morphology. The mutant lacks conidiophores and phialides and produces spores directly from hyphea. This way of spore production seems slow and inefficient. On the spore-producing medium CMC for 62h, only 100 macroconidia could be found in ΔFgStuA, in contrast to 1x106 in the wildtype. The mutant macroconidia also germinate at a slower rate than wildype, about 4h later at 25C in liquid complete medium (CM). B. The chitin binding stain calcofluor shows diminished affinity for spores and germlings of the ΔFgStuA mutant compared to wildtype, and that septa are lacking from mutant macroconidia. C. The expression heat map shows relative expression levels of chitin-related genes in wildtype PH-1 and ΔFgStuA during three different experiments, on spore-producing CMC medium, on a secondary metabolite producing medium (SecMet) and during the infection process of wheat. Grey signal = not detected. The spore characteristics correlate well with reduced transcript levels in the mutant for genes related to chitin synthesis, especially on CMC and during wheat infection.

The red pigment aurofusarin. A. Production of the red pigment aurofusarin is highly reduced in the ΔFgStuA mutant on different growth media, such as V8 and PDA. B. Expression heat map of aurofusarin genes in wildtype PH-1 and ΔFgStuA during three different experiments. The lack of pigmentation in the mutant was reflected in the spore-producing CMC medium, where 17 adjoining probesets, including the known aurofusarin genes, where highly down-regulated or not detected in ΔFgStuA. Grey signal = not detected.

Summary - ΔFgStuA mutant characteristics:

1. Pathogenicity - Non-pathogenic on wheat - Reduced colonization of apple slices 2. Sporulation - Reduced sporulation rate, no perithecia - Lacks conidiophores and phialides - Altered spore morhology 3. Secondary metabolism - No trichothecene production in wheat - Very low 15ADON production in culture - Reduced pigmentation

AcknowledgementsWe thank Karen Hilburn (USDA-ARS Cereal Disease Laboratory) for outstanding technical assistance. This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant 2004-35604-14327 and the BILAT project 173277 supported by the Research Council of Norway.

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