Functional characterization of sterol regulatory element transcription factors in the biocontrol fungus Clonostachys rosea

Sammanfattning: Phytopathogens account for a large portion of the yield loss in the world today. Developing sustainable strategies to limit their effect for increasing food safety and security is one of important challenge for agriculture and horticulture production systems. Using biological control is one of the cornerstones in Integrated Pest Management (IPM), in which multiple control strategies are used and with the aim to reduce pesticide use. The biological control agent Clonostachys rosea IK726 is a fungal antagonist of many important phytopathogens. The ability of C. rosea IK726 to tolerate relatively higher dose of fungicide makes it possible to use it in IPM strategies that includes both BCA and fungicides. To develop a successful IPM strategy, the underlying mechanisms of fungicide tolerance in C. rosea need to be further investigated. The role of Sterol Regulatory Element Binding Proteins (SREBP’s) in azole tolerance, pathogenesis and hypoxia have recently been studied in numerous fungi and provided further knowledge of the sterol regulatory pathway in fungi. The aim of this study was to identify proteins involved in sterol regulatory pathway and characterize the biological function of SREBPs in C. rosea with emphasis on their role in fungicide tolerance, hypoxic resilience, antagonism and biocontrol. Blast search against C. rosea genome identified two genes coding for SREBP (named SRE1 and SRE2), one for INSIG (insulin induced-genes) and two for SCAP (SREBP cleavage-activating protein). The result was validated by conserved domain analysis and phylogenetic analysis. Functional characterization of SRE1 and SRE2 in C. rosea was performed by generating gene deletion mutants of sre1 and sre2. Gene deletion of sre1 (Δsre1) resulted in mutants with reduced growth rate (p=0,000331, p= 0,000030, p= 0,011457) on the medium supplemented with 1/60 recommended dosage of proline and (p=0,004100, p=0,000233, p= 0,000169) 4 mM cobalt chloride (hypoxia-mimetic agent) compared to that of the WT, suggesting an increased sensitivity of Δsre1 to prothioconazole and hypoxia. However, deletion of sre2 showed no significant difference in growth rate under the same conditions. Interestingly, the strains Δsre1_1 (p=0,045334056) and Δsre2_55 (p=0,008146712) increased shoot length significantly compared to WT and the overgrowth of Δsre1_1 (p= 0,042264) and Δsre1_15 (p= 0,020591) was significantly different to WT during dual cultivation. SRE1 and SRE2 likely influence the hyphal structure and pigmentation since this was a consistent observed phenotype within the gene deletion strains.