Cloning And Functional Analysis Of Clk1, A Mitogen-activated Protein Kinase Gene Curvularia Lunata

Curvularia leaf spot of maize, caused by Curvularia lunata (Wakker) Boed., has become one of the most devastating fungal diseases in the most of maize-producing areas in China. Understanding molecular mechanisms of pathogenicity of the pathogen could lead to control the disease continuously and effectively. The mitogen-activated protein kinase (MAPK) cascade system, a model known to be involved in multiple signals transduction responsible for the filamentous pathogen development and pathogenicity to host plants play a crucial role in triggering pathogenic development, for instance, Pmk1, a MAPK gene isolated from Magnaporthe grisea, is well-conserved and essential for pathogenicity in many plant pathogens.Thus in this study, we focused Pmk1 cloning and function analysis and consequently reveal its role in C. lunata.development and pathogenicity .We cloned a MAPK gene that was highly homologous to Pmk1 (designated as Clk1 as to C.lunata) and present in the form of a single copy in genome. The gene was ensured as a 1271bp-DNA sequence and with four introns. Its predicted protein contained 352 amino acids. In order to understand the function of Clk1 in C. lunata, development and pathogenicity to maize plants, knockout of the targeted the gene was conducted. Two Clk1 deletion mutants were obtained. Both of them produced more aerial hyphae, lower vegetative growth rate in early development stage, but kept the same or even higher rate in elder stage compared to wild type. Moreover, the deletion mutants were more susceptible to low temperature, and lost their ability to produce typical conidia and infect intact maize leaf even though they could some extent colonize detached leave through artificial wounds. The significant reduction was found in the activity of cell wall degrading enzymes(PG, PMG, Cx) and toxin to destruct leaf , but not in pigment production. Another interesting finding was that deletion mutants could yield a structure like chlamydospores which might be associated with the reduced pathogenicity of deletion mutant.?These results indicated that Clk1 was essential for regulating infection processes in C. lunata, and this MAPK pathway may be widely conserved in fungal pathogens for pathogenicity.