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Exploring Infection of Wheat and Carbohydrate Metabolism in Mycosphaerella graminicola Transformants with Differentially Regulated Green Fluorescent Protein Expression

February 2001 , Volume 14 , Number  2
Pages  156 - 163

Eric A. Rohel , 1 Andrew C. Payne , 2 Bart A. Fraaije , 2 and Derek W. Hollomon 2

1INRA Centre de Recherches Agronomiques de Rennes, UMR BiO3P, Domaine de la Motte, B.P. 35327, 35653 Le Rheu Cedex, France; 2IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, BS41 9AF Bristol, U.K.

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Accepted 12 September 2000.

A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.

Additional keywords: plant-pathogenic fungus, septoria blotch, Septoria tritici, UB7.

© 2001 The American Phytopathological Society