A Putative MAPK Kinase Kinase GeneSsos4is Involved in Mycelial Growth, Virulence, Osmotic Adaptation, and Sensitivity to Fludioxonil and is Essential for SsHog1 Phosphorylation inSclerotinia sclerotiorum

Abstract

The high osmolarity glycerol (HOG) pathway, comprising a two-component system and the Hog1 MAPK cascade, plays a pivotal role in eukaryotic organisms. Previous studies suggested that the biological functions of some key genes in the HOG pathway varied in filamentous fungi. In this study, we characterized a putative mitogen activated kinase (MAPK) kinase kinase gene Ssos4 in S. sclerotiorum, which encoded a phosphotransferase in the MAPK cascade. Compared to the wild-type progenitor HA61, the deletion mutant ∆Ssos4-63 exhibited impaired mycelial growth, sclerotia formation, increased hyphal branches and decreased virulence. The deficiencies of the deletion mutant ∆Ssos4-63 were recovered when the full-length Ssos4 gene was complemented. Deletion of Ssos4 increased the sensitivity to osmotic stresses, cell wall agents and the resistance to fludioxonil and dimethachlon. Intracellular glycerol accumulation was not induced in the deletion mutant ∆Ssos4-63 when treated with fludioxonil and NaCl and the phosphorylation of SsHog1 was also cancelled by the deletion of Ssos4. Consistent with the glycerol accumulation, increased expression levels of SsglpA and Ssfps1, controlling glycerol synthesis and close of glycerol channel under hyperosmotic stress respectively, were detected in the wild type stain HA61 but not in the deletion mutant ∆Ssos4-63. Moreover, the relative expression level of Sshog1 significantly decreased, whereas the expression level of Ssos5 increased in the deletion mutant ∆Ssos4-63. These results indicated that SsOs4 played important roles in mycelial growth and differentiation, sclerotia formation, virulence, hyperosmotic adaptation, fungicide sensitivity and the phosphorylation of SsHog1 in S. sclerotiorum.

A MAP kinase cascade broadly regulates development and virulence ofSclerotinia sclerotiorumand can be targeted by HIGS for disease control

“…For example, knocking out Sak1 in the B. cinerea high osmolarity pathway abolished compound appressoria formation required for host entry (Segmüller et al, 2007). Similarly in S. sclerotiorum , disruption of the osmolarity responsive MAPKKK Ssos4 led to reduced sclerotia formation and virulence (Li et al, 2021). Deletion of the cell wall integrity MAPK cascade components not only led to decreased cell wall integrity, but also decreased sclerotia formation and virulence (Bashi et al, 2016; Cong et al, 2022).…”

“…Deletion of the cell wall integrity MAPK cascade components not only led to decreased cell wall integrity, but also decreased sclerotia formation and virulence (Bashi et al, 2016; Cong et al, 2022). In contrast, depending on the dysregulated pathway, S. sclerotiorum exhibited differing mycelial morphologies, suggesting that downstream components of the pheromone response, high osmolarity and cell wall integrity MAPK pathways likely function independently with some overlap (Li et al, 2021; Cong et al, 2022). However, the details of crosstalk among the three MAPK signaling pathways in regulating S. sclerotiorum development and virulence require further careful analysis.…”

“…The disruption of the cell wall integrity MAPK pathway components, including Pkc1, Bck1, Mkk1, and Smk3, not only led to decreased cell wall integrity, but also decreased sclerotia formation and virulence (Bashi et al, 2016; Cong et al, 2022). Likewise, deletion of the MAPKKK gene Ssos4 , a component of the high osmolarity MAPK pathway associated with mitigating hyperosmotic stressors, led to reduced sclerotia formation and virulence (Li et al, 2021). SsSte12, the homolog of S. cerevisiae pheromone responsive transcription factor Ste12 activated by MAPK Kss1/Fus3, was studied through RNA interference (RNAi)-mediated gene knockdown in S. sclerotiorum .…”

Characterization of SsHog1 and Shk1 Using Efficient Gene Knockout Systems through Repeated Protoplasting and CRISPR/Cas9 Ribonucleoprotein Approaches in Sclerotinia sclerotiorum

“…Thus, the histidine kinase deletion mutants showed increased sensitivity to osmotic and oxidative stresses. , The Hog1, which is the last kinase of the HOG pathway, is regulated by the histidine kinase and is associated with a stress response, morphology (e.g., sclerotia formation or hyphal growth), and virulence in Fusarium graminearum and Verticillium dahliae. , Based on the role of two-component histidine kinases, , it was suggested that Shk1 controls both SsOs4-SsOs5-SsHog1 cascade and SsSkn7 signaling pathways, which are involved in osmolar adaptation and fludioxonil sensitivity in S. sclerotiorum . The Ssos4 gene, which is the upstream kinase in the Hog1 MAPK pathway, is involved in mycelial growth, reduced virulence, hyperosmotic adaptation, and resistance to phenylpyrrole and dicarboximide fungicides .…”

“…sclerotiorum . The Ssos4 gene, which is the upstream kinase in the Hog1 MAPK pathway, is involved in mycelial growth, reduced virulence, hyperosmotic adaptation, and resistance to phenylpyrrole and dicarboximide fungicides . However, the function of S.…”