Clonging And Functional Analysis Of CLM1 In Curvularia Lunata

Curvularia lunata (Wakker) Boedijn, a causal agent of Curvularia leaf spot of maize, is distributed widely in maize growing regions in China. Currently, the study on the molecular mechanisms of pathogenicity of this disease is still in initial stage. Therefore, the research on its field is beneficial to the further understanding of the pathogenesis of C. lunata and guiding the prevention and control of the disease. In the past decade, many data have shown that mitogen-activated protein kinases (MAPKs), involved in multiple signals transduction, were relevant to pathogenicity in various pathogenic fungi. Thus in this study, we focus on cloning and analyzing Clm1 to reveal its role in C. lunata.The whole sequence and flanking regions of Clm1 is amplified by Inverse PCR and RACE. Clm1 contains a 1.609kb DNA sequence with a 1.251kb coding region interrupted six introns (Genbank: HQ851366). It encodes a MAPK, which is homologous to Slt2 in Saccharomyces cerevisiase related to cell wall integrity. C. lunata strain CX-3 is transformed to enhanced green fluorescent protein (eGFP) using plasmid pCAMBIA1300, which contains the eGFP gene fused to the 5' noncoding region of Clm1. The results indicate that the upstream segment of Clm1 has promoter like activity, and further confirm the reliability of the ORF of Clm1. In order to understand its function in C. lunata, disruption of the targeted gene is conducted by ATMT. Based on the comparison between Clm1 deletion mutant and wild type strain, it is indicated that the production of conidiospores is reduced in Clm1 mutants, in which the percentage of deformed spore is increased. The pigment of Clm1 disruption is also altered. On the other hand, semiquantitative RT-PCR analysis is shown that Clm1 is associated with the expression of Brn1 in a certain extent. In the characteristic of cell wall integrity, the outer wall of deletion mutants is basically disappeared. In addition, Clm1 has some effects on the activity of cellulase, production of toxin compound and expression of toxin synthesis related gene Clt1. The infection assays are also revealed that the virulence of Clm1 deletion mutants is reduced. In short, the results above illustrate that Clm1 is closely related to developmental traits associated with pathogenicity and pathogenic biochemical factors in C. lunata, and further confirm that MAPK signaling transduction pathway plays an important role in the pathogenicity in pathogenic fungi.