Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses.

A neutral trehalase (NTH1) of fungal entomopathogen Beauveria bassiana was characterized for the first time as a 743-aa enzyme (84.4 kDa). To identify crucial stress-responsive elements (STREs) to control the expression of the NTH-coding gene (BbNTH1) in response to different stresses, the full-length promoter (-2713 bp) upstream of its open reading frame and three upstream-truncated fragments (-1912, -1060 and -560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. Consequently, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformants controlled by the shortest fragment with last two STREs at -315 and -274 bp exhibited consistently brightest fluorescence in mycelia under 3-h oxidative adaption of 0.3-1.2mM menadione, and in colonies under 6-day osmotic stress of 0.5-1M NaCl and thermal stress of 15-540 min at 40°C after 3-day growth at 25°C. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.