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

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Abstract
A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive pro- moter 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 Aspergil- lus nidulans gpdA gene encoding glyceraldehyde-3- phosphate dehydrogenase was used as a constitutive pro- moter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indi- cated 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 sac- charose triggered the repression, whereas mannitol, xy- lose, 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 pycnidio- spores fluoresced. The use of GFP transformants also al- lowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the bio-