Contribution Of Intracellular Trehalose To Beauveria Bassiana Thermotolerance And Characterization Of Enzyme-coding Genes To Regulate Trehalose Metabolism

Entomopathogenic fungi are widely applied in microbial control of arthropod pests, being well represented by the fungus Beauveria bassiana. Fungal cells (aerial conidia, blastospores or mycelia) are often formulated as active ingredients of mycoinsecticides and inevitably exposed to the stresses of high temperatures and solar UV irradiation, which are known to affect fungal viability and field performance. Fungal candidate strains with greater stress tolerance are likely to be more potential for successful use in pest control. Fungal trehalose is likely involved in crucial defense mechanisms that protect cells from damages by environmental stresses. However, neither trehalose accumulation nor trehalose metabolism in fungal cells has been related to the tolerance of the fungal biocontrol agents to multiple stresses. Thus, it is necessary to understand possible mechanism of trehalose metabolism in B. bassiana in response to multiple stresses.This study began from quantitative evaluation changes of intracellular trehalose and mannitol contents and enzymatic activities under thermal stress, resulting in a hypothesis that trehalose accumulation is likely involved in fungal thermotolerance. To confirm the hypothesis, the genes encoding B. bassiana neutral trehalase(BbNTH1) and trehalose-6-phosphate synthase (BbTPS1) were cloned and characterized. Crucial stress-responsive elements (STREs) to control BbNTHl expression under multiple stresses was identified by fusion of the reporter gene eGFP to its promoter. The biological function of BbTPS1 gene was revealed by suppressing its expression using antisense RNA-mediated gene silencing technique. The results are summarized as follows.Physiological implication of intracellular trehalose accumulation in response of B. bassiana to thermal stress. To explore possible role of intracellular trehalose accumulation in the tolerance of B. bassiana to summer-like thermal stress, three-day colonies of B. bassiana grown on the plates of a glucose-free medium at 25℃were separately exposed to 35,37.5 and 40℃for 1-18 h. Trehalose accumulation in the stressed mycelia increased from initial 4.2 to 88.3,74.7 and 65.5 mg/g biomass after 6-h exposure to 35,37.5 and 40℃respectively while intracellular mannitol level generally declined with more stressful temperatures and longer stress time. The stress-enhanced trehalose level was significantly correlated to the decrease of trehalase activity (r2=0.73) and the level of mannitol (r2=0.38), which was inversely correlated to the activity of mannitol dehydrogenase (r2=0.41) or mannitol 1-phosphate dehydrogenase (r2=0.30) under the stresses. All stressed cultures were successfully recovered at 25℃but their vigor depended on the stress intensity expressed as temperature, time length and the interaction of both (r2=0.98). The highest level of 6-h trehalose accumulation at 35℃was found enhancing the tolerance of the stressed cultures to the greater stress of 48℃for≤60 min. The results suggest that the trehalose accumulation result from metabolized mannitol and contribute to fungal thermotolerance. Trehalase also contributed to the thermotolerance by hydrolyzing accumulated trehalose under the conditions of thermal stress and recovery.Gene cloning and characterization of neutral trehalase (BbNTHl) from B. bassiana. A full-length 2387-bp fragment of neutral trehalase (BbNTH1) gene was cloned from B. bassiana, including a putative 2232-bp open reading frame with three 50-, 49- and 58-bp putative introns (Genbank accession number:EF122412). In sequence analysis online (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the dediced protein was featured with a cAMP dependent phosphorylation consensus site (RRGS) and a putative calcium binding site (DTDGNMQITIED). The gene was found encoding a 743-aa protein and expressed E. coli BL21 as the recombinant protein of 84.4 kDa in agreement with the predicted molecular weight. The purified BbNTHl showed high substrate specificity to trehalose (100%). Its maximal activity in trehalose hydrolysis was achieved at 55℃and pH 7.0. In the reaction at optimal 55℃and pH 7.0, the BbNTH1 activities (y) at 0.1-1.0 M trehalose (x) were well fitted to the non-linear Michaelis-Menten equation (r2=0.99), generating the parameters Km and Vmax (±SE) of 5.05±0.71 mM and 1290.98±22.73 U/mg protein. The catalytic efficiency (kcat/Km) for the hydrolysis of trehalose by BbNTH1 was estimated as 2.35×103 mM-1 s-1.Recognition of crucial stress-responsive elements to control BbNTHl expression in response to multiple stresses. To identify crucial stress-responsive elements (STREs) to control BbNTHl expression 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. As a result, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformant colonies controlled by the shortest fragment with last two STREs at -315 and -274 bp exhibited consistently brightest fluorescence under 7-day oxidative adaption of 0.3-1.2 mM menadione,6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40℃after 3-day growth at 25℃. 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.Gene cloning and functional analysis of trehalose-6-phosphate synthase (BbTPSl) from B. bassiana. A trehalose-6-phosphate synthase gene, BbTPS1, was cloned from B. bassiana. The function of BbTPSl was elucidated by suppressing its expression using the method of antisense RNA-mediated gene silencing. Two antisense-RNA plasmids vectoring both the transcriptional element of BbTPS1 hairpin inverted repeat fragments and the gene bar were constructed and then inserted into the genomic DNA of the wild-type strain Bb2860. Expression of BbTPS1 in selected transformants was measured by real time PCR. Transformants A2 and A4 with the suppressed BbTPS1 expression of 51.9% and 77.5% were used in the following experiments to analyze trehalose metabolsin in conidial germination and the role of the gene in response to various stresses. Conidial germination rates of A2 and A4 were significantly lower than that of the wild strain in nutrition-free saline (F2,6=42.5, P<0.01). The silenced expression of BbTPS1 increased significantly the sensitivity of germinating conidia to the 30-min thermal stress of 45℃(F2,6=29.2, P<0.01). The accumulation of trehalose was largely reduced in A2 and A4 mycelia compared to the wild-type strain during thermal stress at 35℃. The colony growths of both transformants and the wild-type strain also showed different responses to the oxidative stress of 1-3 mM menadione (F2,24=17.2, P<0.01) and the thermal stress of 48℃(F2,30=52.7, P<0.01). Our results highlight the important role of BbTPS1 gene in B. bassiana tolerance to the stresses.In summary, we developed an efficient assay system that enables to accurately quantify changes of intracellular trehalose and mannitol contents and enzymes activities under thermal stress and found that trehalose accumulation takes important part in fungal thermotolerance. The genes encoding B. bassaina neutral trehalase (BbNTH1) and trehalose-6-phosphate synthase(BbTPS1) were characterized for the first time. Two STREs in the downstream 560-bp region of the BbNTH1 promoter were found crucial to regulating not only constitutive but stress-inducible expression of BbNTH1 in response to multiple stresses. Significant contributions of BbTPSl to conidial germination and tolerance to thermal and oxidative stresses were confirmed by means of antisense RNA-mediated gene silencing. These results provide new insights into trehalose involvement in the stress tolerance of B. bassiana and suggest means to improving ecological fitness or field persistence of fungal biocontrol agents.