Fungal fludioxonil sensitivity is diminished by a constitutively active form of the group III histidine kinase

Abstract

a b s t r a c tThe fungicide fludioxonil is used to control plant-pathogenic fungi by causing improper activation of the Hog1-type MAPK. However, the appearance of fludioxonil resistant mutants, mostly caused by mutations in the group III histidine kinases, poses a serious problem. Moreover, such mutations cause also hyperosmotic sensitivity and the underlying mechanism has been elusive for a long time. Using Saccharomyces cerevisiae as an experimental host, we show that those phenotypes are conferred by a constitutively active form of the group III histidine kinase. Our results explain the different reasons for fludioxonil resistance conferred by its deletion and missense mutation.

Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p

“…AMY1000 was constructed from S. cerevisiae strain R1158 (MATa his3-1 leu2-0 met15-0 URA3::CMV-tTA) (20) by inserting tetO7-TATA in the promoter region of the SLN1 gene and deleting the STE11 gene with a hygromycin-resistant gene cassette. S. cerevisiae strain YSH2472 (MATa leu2-3,112 trp1-1 his3-11,15 ade2-1 can1-100 sln1::LEU2 ssk1::kanMX ura3-1::YIpSSRE-lacZ) was used as host for ␤-galactosidase reporter assay (17).…”

“…Earlier studies showed that the toxicity of fludioxonil against S. cerevisiae cells expressing group III HHK including DhNik1p is based on activation of the Hog1p MAPK (17). Therefore, we determined the level of phosphorylated Hog1p in S. cerevisiae strain AMY1000 expressing linker deletion mutants by immunoblotting after exposing the cells to fludioxonil.…”

“…Fungicidal action of fludioxonil involves inhibition of group III HHK resulting in constitutive activation of the HOG pathway (6). Our earlier work showed that heterologous expression of DhNik1p confers sensitivity to fludioxonil in S. cerevisiae and fludioxonil inhibits the activity of DhNik1p in vivo (17). Therefore, we studied whether the HAMP-like linkers had any role in modulating the fludioxonil sensitivity conferred by DhNik1p.…”

“…We next monitored the in vivo effect conferred by fludioxonil on the histidine kinase activity of linker mutants using an SSRElacZ reporter system (17). In this system, the SSRE-lacZ reporter activity depends on phospho-relay from DhNik1p to cerevisiae strain AMY1000 carrying vector pRS423 was diluted to ϳ0.05 A 600 and incubated with or without doxycycline.…”

“…Deletion analysis suggested that the HAMP domains are functionally distinct. Although the fourth domain is dispensable for histidine kinase activity of DhNik1p, it regulates the osmosensing function as well as the fludioxonil sensitivity mediated by the molecule (9,17). In DhNik1p, the HAMP domains are interspersed with linker sequences.…”