טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHila Tal
SubjectSignal Transduction through the MAP Kinases of the Maize
Pathogen Cochliobolus: Activation and
Downstream Targets
DepartmentDepartment of Biology
Supervisor Full Professor Horwitz Benjamin
Full Thesis textFull thesis text - English Version


Abstract

Pathogenic fungi need to respond to environmental changes, especially to the presence of the host. Cochliobolus heterostrophus, the agent of Southern corn leaf blight, is a haploid ascomycete and a necrotrophic foliar pathogen. This pathogenic fungus, like other eukaryotes, is thought to use conserved signaling cascades like the MAPK pathway in order to respond to environmental changes. The pathogenicity MAP kinase Chk1 of Cochliobolus heterostrophus is required for formation of infection structures (appressoria), conidiation, melanin biosynthesis, virulence and female fertility. Phosphorylation and kinase activity of Chk1 are required for its function since mutants with non-phosphorylatable and kinase-negative Chk1 have the same phenotype as deletions. We followed CHK1 localization, quantity, amount of phosphorylation and expression of downstream genes during conidial germination on an inductive surface and in suspension. I observed that Chk1 protein is abundant in ungerminated conidia, accumulates in maturing appressoria and sometimes was found in appressorial nuclei when grown on an inductive surface, but was uniformly distributed in suspension-grown hyphae. Expression of Chk1-dependent genes was up-regulated on an inductive surface, but not in mycelium growing in suspension. Despite this evidence of Chk1 activity, its phosphorylation and total protein quantity were nearly the same in conidia germinating on an inductive surface and in suspension during the entire incubation period. Our results suggest that activation of Chk1, at least during appressoria formation, is manifested by its local accumulation followed by increased nuclear translocation, but not significant changes in phosphorylation. MAP kinase Mps1 of Cochliobolus heterostrophus is a homolog of the yeast cell-integrity MAPK Slt2, which functions to maintain the cell wall, and is essential for the formation of functional appressoria and for virulence. The targets of Mps1 in C. heterostrophus are unknown.  In this study we found that alkalinization of the environment causes Mps1 activation. By construction of an SSH library and dimethylation analysis between WT and mutant of this protein in alkali environment and in control I identified gene targets and candidate phospho-protein targets of Mps1. Most of the gene targets of Mps1 under alkaline stress belong to large group of cell wall proteins and secreted proteins, among them we found proteins that construct the cell wall like UDP-galactose-4-epimerase and fructose-6-phosphate amidotransferase, some allergens, chitin binding protein, adhesin protein and BYS1 domain protein. The phospho-protein targets that we found include proteins predicted to be involved in transcription and translation, for example eIF4B, and a CBF/NF-Y family transcription factor.