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

Abstract

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, Dhxk1 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 Dhxk1 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 Dhxk1 disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Dhxk1 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 Dhxk1 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 Dglk1 and Dhxk1 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. INTRODUCTIONHexose kinases are crucial enzymes for most living cells, by catalysing the intracellular trapping and feeding into metabolism of the hexoses glucose and fructose after their uptake. In addition, hexokinases have been shown in yeast, plants and mammals to participate in glucose signalling (De Winde et al., 1996;Moore et al., 2003;Efrat et al., 1994). Saccharomyces cerevisiae has three enzymes catalysing hexose phosphorylation, namely hexokinases (EC 2 . 7 . 1 . 1) Hxk1 and Hxk2, and glucokinase (EC 2 . 7 . 1 . 2) Glk1. Each of these enzymes can support growth on glucose, while either of the two hexokinases is required for growth on fructose (Lobo & Maitra, 1977;Gancedo et al., 1977). Hxk2, in addition, is mainly required for catabolite repression, which prevents the expression of genes involved in catabolism of less preferred carbon sources in the presence of a more favourable nutrient such as glucose (Entian, 1980). In the presence of glucose, Hxk2 interacts with the carbon catabolite repressor Mig1 and thereby is translocated into the nucleus. In the nucleus, the Hxk2-Mig1 complex seems to form a repressor complex that binds to the promoters of carbon-catabolite-repressible genes (Ahuatzi ...

Aspergillus fumigatus Catalytic Glucokinase and Hexokinase: Expression Analysis and Importance for Germination, Growth, and Conidiation

“…Therefore, the glkA phenotype was in agreement with previously reported observations of A. nidulans and B. cinerea (19,40). In contrast, the hxkA deletion strain showed pleiotropic growth phenotypes similar to those of B. cinerea but not A. nidulans (19,40). In general, conidium formation of the ⌬hxkA mutant was delayed on nearly all carbon sources tested, which was visualized by the delayed appearance of the gray-greenish color on top of the colonies.…”

“…However, with complete medium as well as all other sugars and nonfermentable carbon sources, no difference in colony formation was observed, implying that GlkA function was completely complemented by HxkA. Therefore, the glkA phenotype was in agreement with previously reported observations of A. nidulans and B. cinerea (19,40). In contrast, the hxkA deletion strain showed pleiotropic growth phenotypes similar to those of B. cinerea but not A. nidulans (19,40).…”

“…A previous investigation of glucokinase and hexokinase mutants from A. nidulans indicated that only the mutation of hexokinase, but not glucokinase, leads to a defect in sugar utilization, whereby the defect of the hexokinase mutant was restricted to an inability to grow on fructose (19). Interestingly, the respective mutants of Botrytis cinerea showed a similar unaltered phenotype for a glucokinase mutant, but a hexokinase mutant was severely affected on various sugars and also some gluconeogenic carbon sources such as acetate and glycerol (40). Since the mutation in the A. nidulans glkA4 mutant was not analyzed by sequencing, it remains unclear whether the mutated enzyme still retained some residual activity.…”

“…In particular, the role of GlkA was elusive, since the mutation of this gene in several other fungi, such as A. nidulans (19) or B. cinerea (40), revealed no visible phenotype. In terms of colony morphology on various carbon sources, our investigations confirmed these observations.…”

“…In contrast to investigations of A. nidulans, the deletion of the hexokinase in the fruit-pathogenic fungus Botrytis cinerea revealed a pleiotropic growth defect on various carbon sources, whereas a glucokinase mutant, in agreement with data for A. nidulans, displayed no altered phenotype. Furthermore, for B. cinerea, attempts to generate a double-deletion strain were unsuccessful, implying the synthetic essentiality of the two genes (40).…”