Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.
Bottom Line: Further truncating P hyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in P hyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein).Therefore, P hyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of P gpdA, a promoter widely used for gene expression in fungi.Conclusively, P hyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.
Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.Show MeSH
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Mentions: In bioassays with normal conidial suspensions of BbHV8, BbV28 and Bb2860, standardized fungal sprays (2 × 107 conidia per spray) resulted in the mean (± SD) deposit of 391 (± 20) conidia per square millimetre onto both S. litura larvae (for cuticle infection) and lotus leaf discs (for their ingestion). In the probit analyses of time-mortality trends, the median lethal time (LT50) and associated 95% confidence interval (CI) against the second-instar larvae were estimated as 6.4 (6.0−6.9) days for Bb2860 (Pearson's χ2 = 6.02, d.f. = 5, P = 0.30), 5.4 (5.1−5.8) days for BbV28 (χ2 = 0.60, d.f. = 5, P = 0.99) and 2.5 (2.3−2.6) days for BbHV8 (χ2 = 0.40, d.f. = 3, P = 0.94), as shown in Fig. 3a. The same estimates of BbHV8 against instars III and IV were 4.7 (4.5−5.0) days (χ2 = 10.12, d.f. = 6, P = 0.12) and 7.2 (6.8−7.8) days (χ2 = 2.21, d.f. = 4, P = 0.70) respectively. In contrast, neither Bb2860 nor BbV28 had computable LT50 against later stage larvae because the two strains killed only 22% and 37% of third-instar larvae on day 8 and even fewer of fourth-instar larvae. Moreover, fungal outgrowths as a typical symptom of mycosis occurred heavily on all larvae that died of Bb2860 and sparsely on those that died of BbV28, but were absent on those killed by BbHV8 after 3−5 day incubation at saturated humidity (Fig. 3b). These data indicate that BbHV8 not only killed the younger larvae more rapidly than BbV28 and Bb2860 but also showed high killing activity on later stage larvae. The enhanced insecticidal activity of BbHV8 to S. litura larvae was likely attributable to the ingestion of more toxin molecules expressed in conidia.
Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.