Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.     

The role of NADPH oxidase and MAP kinase phosphatase in UV-B-dependent gene expression in Arabidopsis

Plant responses to supplementary UV-B irradiation have been reported to include formation of reactive oxygen species (ROS), hydrogen peroxide, in particular, and regulation by mitogen-activated protein kinase (MAPK) cascades which in turn are fine-tuned by MAPK phosphatases (MKPs). Here we present direct genetic evidence for the involvement of plasma membrane NADPH oxidase, a source of superoxide and hydrogen peroxide in the apoplasts, in UV-B signalling in Arabidopsis thaliana, by analysis of gene expression of the UV-B molecular markers in NADPH oxidase (atrbohD, F and DF) and MAP kinase phosphatase 1 (MKP1) knockout mutants (mkp1). Whereas the NADPH oxidase mutants were affected in UV-B-dependent CHS, PYROA and MEB5.2 gene expression, the mkp1 mutant was affected in the general expression pattern of the pathogenesis-related (PR) and PDF1.2 genes. The results indicate involvement of MKP1 in repressive action on gene expression of more general stress response pathways, similar to those activated by pathogen attack, while NADPH oxidase is involved in quantitative (rather than absolute) regulation of more UV-B-specific genes. The expressions of the molecular markers in the knockout mutant mkp1 and in its complemented lines (lines 6 and 10) were similar, as opposed to the responses of the corresponding wild-type Wassilewskija-4 (Ws-4). Lines 6 and 10 showed much higher MKP1 mRNA than Ws-4 but did not complement the mutant. This suggests a complex dependency of the MAPK phosporylation level of the PR and PDF1.2 genes. Both NADPH oxidase mutants and the mkp1 mutant phenotypically responded to UV-B by growth retardation.     

Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele

When confronted with a marked increase in external osmolarity, budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth. One input that stimulates the HOG pathway requires the integral membrane protein and putative osmosensor Sho1, which recruits and enables activation of the MAPK kinase kinase Ste11. In mutants that lack the downstream MAPK kinase (pbs2Delta) or the MAPK (hog1Delta) of the HOG pathway, Ste11 activated by hyperosmotic stress is able to inappropriately stimulate the pheromone response pathway. This loss of signaling specificity is known as cross talk. To determine whether it is the Hog1 polypeptide per se or its kinase activity that is necessary to prevent cross talk, we constructed a fully functional analog-sensitive allele of HOG1 to permit acute inhibition of this enzyme without other detectable perturbations of the cell. We found that the catalytic activity of Hog1 is required continuously to prevent cross talk between the HOG pathway and both the pheromone response and invasive growth pathways. Moreover, contrary to previous reports, we found that the kinase activity of Hog1 is necessary for its stress-induced nuclear import. Finally, our results demonstrate a role for active Hog1 in maintaining signaling specificity under conditions of persistently high external osmolarity.     

球孢白僵菌Hog1 MAPK同源基因BbHog1的克隆及特征分析

根据几种丝状真菌Hog1 MAPK的保守氨基酸序列设计简并引物,从昆虫病原真菌球孢白僵菌中扩增出MAPK同源基因的部分片段,然后利用YADE法延伸该片段的上、下游邻接序列,获得MAPK编码基因的全长序列,命名为BbHog1。序列分析表明,该基因编码358个氨基酸的多肽,推测分子量为40.99kDa,等电点为5.49。BbHog1含有MAPK保守的蛋白激酶激活域(TGY),序列与粗糙脉孢霉os-2(AF297032)、烟曲霉OSM1(XM_747571)、隐球酵母HOG1(AF243531)和酿酒酵母Hog1(Z73285)等Hog1 MAPK高度同源,相似性分别为94%、89%、83%和80%。系统聚类结果表明,BbHog1与酵母Hog1 MAPK同源。Southern杂交表明,BbHog1在球孢白僵菌基因组中以单拷贝形式存在。Northern分析表明,BbHog1在高渗、亚高温和营养胁迫等条件下的表达明显升高。由此推测,BbHog1基因可能与球孢白僵菌对逆境胁迫的适应性调节密切相关。     

The Saccharomyces cerevisiae Sln1p-Ssk1p two-component system mediates response to oxidative stress and in an oxidant-specific fashion

Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism. In addition, environmental gamma and UV radiation, as well as several chemicals also generate reactive oxygen species, which induce oxidative stress. Thus oxidative stress constitutes a major threat to organisms living in aerobic environments. Oxidative stress induces the expression of several genes in yeast Saccharomyces cerevisiae. However, the primary sensor(s) that trigger the response is unknown. This study demonstrates that primary sensors of osmotic stress, the Sln1p-Ssk1p two-component proteins, are involved in sensing oxidative stress specifically induced by hydrogen peroxide and diamide, but not by other oxidants used in the study. Wild type and sln1-ssk1 mutant were treated with hydrogen peroxide, diamide, menadione, UV, and gamma-radiation. Results show that sln1-ssk1 mutant is only sensitive to hydrogen peroxide and diamide but not to other oxidants. S. cerevisiae contains an additional cell surface osmosensor, Sho1p, that targets the osmotic signal to Hog1p. Data is presented that shows Sho1 and Hog1 proteins are also involved in signaling oxidant-specific cellular damage. Furthermore, it is demonstrated that expression of the mammalian homolog of Hog1p provides protection from oxidative stress induced by hydrogen peroxide. These results suggest that Sln1p-Ssk1p and Sho1p signal transduction pathways participate in oxidative stress response. However, this response to oxidative stress is limited to specific oxidants.     

Checkpoints in a yeast differentiation pathway coordinate signaling during hyperosmotic stress

All eukaryotes have the ability to detect and respond to environmental and hormonal signals. In many cases these signals evoke cellular changes that are incompatible and must therefore be orchestrated by the responding cell. In the yeast Saccharomyces cerevisiae, hyperosmotic stress and mating pheromones initiate signaling cascades that each terminate with a MAP kinase, Hog1 and Fus3, respectively. Despite sharing components, these pathways are initiated by distinct inputs and produce distinct cellular behaviors. To understand how these responses are coordinated, we monitored the pheromone response during hyperosmotic conditions. We show that hyperosmotic stress limits pheromone signaling in at least three ways. First, stress delays the expression of pheromone-induced genes. Second, stress promotes the phosphorylation of a protein kinase, Rck2, and thereby inhibits pheromone-induced protein translation. Third, stress promotes the phosphorylation of a shared pathway component, Ste50, and thereby dampens pheromone-induced MAPK activation. Whereas all three mechanisms are dependent on an increase in osmolarity, only the phosphorylation events require Hog1. These findings reveal how an environmental stress signal is able to postpone responsiveness to a competing differentiation signal, by acting on multiple pathway components, in a coordinated manner.     

Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway

Dominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER-associated degradation. Accumulation of misfolded proteins in the ER activates an ER-to-nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1-D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen-activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress-dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.     

A two‐component histidine kinase Shk1 controls stress response,sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high‐osmolarity glycerol mitogen‐activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H₂O₂‐induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real‐time polymerase chain reaction (qRT‐PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21‐activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild‐type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two‐component HKs in Sclerotinia.     

Combination of two activating mutations in one HOG1 gene forms hyperactive enzymes that induce growth arrest

Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis. Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway. Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically. We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties. These alleles, HOG1(D170A,F318L) and HOG1(D170A,F318S), induced a severe growth inhibition and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes. Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176. Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death. This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect.     

KdgR, an IClR family transcriptional regulator, inhibits virulence mainly by repression of hrp genes in Xanthomonas oryzae pv. oryzae

KdgR has been reported to negatively regulate the genes involved in degradation and metabolization of pectic acid and other extracellular enzymes in soft-rotting Erwinia spp. through direct binding to their promoters. The possible involvement of a KdgR orthologue in virulence by affecting the expression of extracellular enzymes in Xanthomonas oryzae pv. oryzae, the causal agent of rice blight disease, was examined by comparing virulence and regulation of extracellular enzymes between the wild type (WT) and a strain carrying a mutation in putative kdgR (ΔXoo0310 mutant). This putative kdgR mutant of X. oryzae pv. oryzae showed increased pathogenicity on rice without affecting the regulation of extracellular enzymes, such as amylase, cellulase, xylanase, and protease. However, the mutant carrying a mutation in an ortholog of xpsL, which encodes the functional secretion machinery for the extracellular enzymes, showed a dramatic decrease in pathogenicity on rice. Both mutants of kdgR and of xpsL orthologs showed higher expression of two major hrp regulatory genes, hrpG and hrpX, and the genes in the hrp operons when grown in hrp-inducing medium. Thus, both genes were shown to be involved in repression of hrp genes. The kdgR ortholog was thought to suppress virulence mainly by repressing the expression of hrp genes without affecting the expression of extracellular enzymes, unlike findings for the kdgR gene in soft-rotting Erwinia spp. On the other hand, xpsL was confirmed to be involved in virulence by promoting the secretion of extracellular enzymes in spite of repressing the expression of the hrp genes.     

球孢白僵菌Hog1 MAPK同源基因BbHog1的克隆及特征分析

根据几种丝状真菌Hog1 MAPK的保守氨基酸序列设计简并引物,从昆虫病原真菌球孢白僵菌中扩增出MAPK同源基因的部分片段,然后利用YADE法延伸该片段的上、下游邻接序列,获得MAPK编码基因的全长序列,命名为BbHog1。序列分析表明,该基因编码358个氨基酸的多肽,推测分子量为40.99kDa,等电点为5.49。BbHog1含有MAPK保守的蛋白激酶激活域(TGY),序列与粗糙脉孢霉os-2(AF297032)、烟曲霉OSM1(XM_747571)、隐球酵母HOG1(AF243531)和酿酒酵母Hog1(Z73285)等Hog1 MAPK高度同源,相似性分别为94%、89%、83%和80%。系统聚类结果表明,BbHog1与酵母Hog1 MAPK同源。Southern杂交表明,BbHog1在球孢白僵菌基因组中以单拷贝形式存在。Northern分析表明,BbHog1在高渗、亚高温和营养胁迫等条件下的表达明显升高。由此推测,BbHog1基因可能与球孢白僵菌对逆境胁迫的适应性调节密切相关。     

球孢白僵菌Hog1 MAPK同源基因BbHog1的克隆及特征分析

根据几种丝状真菌Hog1 MAPK的保守氨基酸序列设计简并引物,从昆虫病原真菌球孢白僵菌中扩增出MAPK同源基因的部分片段,然后利用YADE法延伸该片段的上、下游邻接序列,获得MAPK编码基因的全长序列,命名为BbHog1。序列分析表明,该基因编码358个氨基酸的多肽,推测分子量为40.99kDa,等电点为5.49。BbHog1含有MAPK保守的蛋白激酶激活域(TGY),序列与粗糙脉孢霉os-2(AF297032)、烟曲霉OSM1(XM_747571)、隐球酵母HOG1(AF243531)和酿酒酵母Hog1(Z73285)等Hog1 MAPK高度同源,相似性分别为94%、89%、83%和80%。系统聚类结果表明,BbHog1与酵母Hog1 MAPK同源。Southern杂交表明,BbHog1在球孢白僵菌基因组中以单拷贝形式存在。Northern分析表明,BbHog1在高渗、亚高温和营养胁迫等条件下的表达明显升高。由此推测,BbHog1基因可能与球孢白僵菌对逆境胁迫的适应性调节密切相关。     

Evolution of osmosensing signal transduction in Metazoa: stress-activated protein kinases p38 and JNK

Sponges (Porifera) represent the most basal branch of the Metazoa alive today. We show that two central stress-activated protein kinases involved in the osmosensing pathway, p38 mitogen-activated protein kinase (MAPK) and JNK, can complement for the ancestral MAPK Hog1 in the yeast Saccharomyces cerevisiae. S. cerevisiae mutants lacking Hog1 (hog1-Delta 1) have been complemented with the sponge SDJNK and SDp38 genes. Western blotting has revealed that, after transformation, the hog1-Delta 1+ SDJNK(sense) and hog1-Delta 1+ SDp38(sense) clones express the sponge proteins. Functional studies have demonstrated that the complemented clones grow under hyperosmotic conditions (0.6 M NaCl). Furthermore, the expressed sponge kinases undergo phosphorylation in S. cerevisiae at 0.6 M NaCl. This report documents that the metazoan signal transduction kinases, p38 and JNK, which were originally derived from an common ancestor with yeast HOG1, have retained their function after their specification.