Cloning and Heterologous Expression of Serine Protease SL41 Related to Biocontrol in Trichoderma harzianum

Abstract

Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases are involved in fungal growth and have been related to biocontrol processes. To assess the functional role of serine proteases from Trichoderma harzianum T88, an effective biocontrol agent, on inhibition of phytopathogenic fungi, a gene (SL41) encoding a serine protease was isolated by 5′ and 3′ RACE (rapid amplification of cDNA ends). Northern blot analysis indicated that SL41 was induced in response to cell walls of different fungi. This protease gene was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. After induction, the enzyme activity was culminated (16.2 units ml^-1) at 60 h of cultivation. The optimal enzyme reaction temperature was 40°C and optimal pH was 10.5. Northern blot analysis indicated that the amount of the transcripts increased with the culture time in agreement with the measured enzyme activity. Antifungal activity of serine protease against five phytopathogens was investigated in vitro. It can inhibit the mycelial growth of phytopathogenic fungi and exerted broad spectrum antifungal activity against phytopathogenic fungi. This is the first time that the different regulation of serine protease in T. harzianum response to five phytopathogenic fungi was shown, the protease was functionally expressed in a heterologous host, and its antagonistic activity was evaluated in vitro.