The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits.

Hexose kinases play a central role in the initiation of sugar metabolism of living organisms and have also been implicated in carbon catabolite repression in yeasts and plants. In this study, the genes encoding glucokinase (Glk1) and hexokinase (Hxk1) from the plant-pathogenic ascomycete Botrytis cinerea were isolated and functionally characterized. Glk1-deficient mutants were indistinguishable from the wild-type in all growth parameters tested. In contrast, Deltahxk1 mutants lacking Hxk1 showed a pleiotropic growth defect. On artificial media, vegetative growth was retarded, and conidia formation strongly reduced. No or only marginal growth of Deltahxk1 mutants was observed when fructose, galactose, sucrose or sorbitol were used as carbon sources, and fructose inhibited growth of the mutant in the presence of other carbon sources. B. cinerea mutants containing hxk1 alleles with point mutations leading to enzymically inactive enzymes showed phenotypes similar to the Deltahxk1 disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Deltahxk1 mutants was dependent on the plant tissue: on leaves, lesion formation was only slightly retarded compared to the wild-type, whereas only small lesions were formed on apples, strawberries and tomatoes. The low virulence of Deltahxk1 mutants on fruits was correlated with their high contents of sugars, in particular fructose. Heterologous expression of Hxk1 and Glk1 in yeast allowed their enzymic characterization, revealing kinetic properties similar to other fungal hexokinases and glucokinases. Both Deltaglk1 and Deltahxk1 mutants showed normal glucose repression of secreted lipase 1 activity, indicating that, in contrast to yeast, B. cinerea hexose kinases are not involved in carbon catabolite repression.