Gene Cloning, Identification And Functional Analysis Of Superoxide Dismutases (SODs) In Beauveria Bassiana

As a classic entomopathogenic hyphomycete, Beauveria bassiana has been widely applied in microbial control of arthropod pests due to easy production, formulation and environmental safety. The virulence and field persistence of a fungal formulation are known to be affected by multiple stresses. Of those, oxidative stress is most important because the burst of intracellular reactive oxygen species (ROS) can be triggered both by outdoor stresses, such as high temperature, solar UV irradiation and chemical pesticides often encountered in summer, and fungal interaction with host pests in the course of infection. Thus, exploring the underlying protective mechanism of the fungal pathogen against oxidative stress and manipulating antioxidant genes for genetic improvement of fungal candidate strains will greatly facilitate the development of high-quality mycoinsecticides. Superoxide dismutases (SOD) are very important antioxidant enzymes in eukaryotic cells and constitute a primary source of cellular defenses against ROS damages. However, little is known about SOD genes of fungal entomopathogens. Thus, this study sought to elucidate the structure, subcellular localization and function of possible SOD members in Beauvria bassiana and explore the feasibility of exploiting their genes for improving fungal strains. The results are summarized below.Gene cloning of B. bassiana Cu/Zn-SOD (BbSodl) and its active expression without chaperon. A Cu/Zn-SOD was characterized for the first time from Beauveria bassiana by gene cloning, heterogeneous expression and function analysis. This 154-aa SOD (BbSodl) was deduced from a 465-bp gene cloned, showing 49-96% sequence identity to Cu/Zn-SODs known from other 57 fungi. BbSodl and its form engineered with two site-directed mutations P143S and P145L (BbSodl-Mut) or fused to copper chaperon Lys7 (BbSodl-Lys7) were expressed well in Escherichia coli. Crude extracts and purified BbSodl-Mut from cell cultures exhibited much higher antioxidation activities than the counterparts of BbSodl-Lys7 whereas BbSodl showed no substantial activity. The engineered enzymes were best induced by overnight incubation at 20℃in Luria-Bertani medium including 2.5 mM Cu2+,0.5 mM Zn2+ and 0.5 mM IPTG (isopropyl-d-thiogalactopyranoside) after 5-h growth at 37℃. Our results highlight alternative means to producing highly active fungal Cu/Zn-SOD in E. coli by making use of the two site-directed mutations without chaperon.Gene cloning and characterization of a cytosolic Mn-SOD (BbSod2) in B. bassiana and its contribution to the fungal virulence and stress tolerance. A new Mn-SOD (BbSod2) was characterized from B. bassiana. This 209-aa enzyme showed no more than 71% sequence identity to other 74 fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn2+, suppressed by Cu2+ and Zn2+ but inhibited by Fe3+. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain resulted in 4-10-fold increases in SOD activity. Compared to the wild-type strain, a transformant with maximal SOD activity (1157.9±74.7 U/mg protein) was 93% and 61% more tolerant to superoxide generating menadione in colony growth (EC50:2.41±0.03 versus 1.25±0.01 mM) and conidial germination (EC50:0.89±0.06 versus 0.55±0.07 mM), respectively, and 23% more tolerant to UV-B irradiation (LD50:0.49±0.02 versus 0.39±0.01 J/cm2). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT50:4.5 (4.2-4.8) versus 5.7 (5.2-6.4) days]. Our study highlights for the first time that the Mn2+-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.Gene cloning and subcellular localization of a mitochondrial Mn-SOD (BbSod3) in B. bassiana. A new mitochondrial Mn-SOD (BbSod3) was identified from B. bassiana. The SOD-coding gene BbSod3 was 879 bp in length, including two introns and three exons. The deduced protein consisted of 230 amino acid residues with the predicted molecular weight of 24.7 kDa and the isoelectronic point of 7.6. In online database search, the deduced BbSod3 showed 36-93% sequence identity to 75 Mn-SODs known from other fungi and was most close to the mitochondrial Mn-SOD of Cordyceps militaris. BbSod3 was found sharing all conserved amino acid residues with the Mn-SOD family. The first 35 amino acid residues of BbSod3 were predicted as a putative mitochondrium-targeting signal peptide with an export probability of 0.9987. This was further confirmed using transgenic strains expressing a fusion of the signal peptide and enhanced green fluorescent protein (eGFP). The eGFP was well expressed in the intracellular reticular components of transgenic B. bassiana strains and also well stained as red fluorescence with mitochondrium specific probe MitoTracker Red.Interaction of cytosolic and mitochondrial Mn-SOD genes in B. bassiana. Based on SOD reactions to specific inhibitors and Western blots, BbSod2 and BbSod3 were identified as two main SOD enzymes expressed in 3-day colony mycelia of B. bassiana. To reveal the interaction of the two enzymes, three RNAi plasmids containing a transcriptional unit for hairpin RNA expression were constructed for silencing the expression of the gene BbSod2, BbSod3 or both. As a result, Mn-SOD expression at both mRNA and SOD activity levels were significantly reduced in all RNAi transformants. Of those, three representative transformants with lowest SOD activity for each of the RANi reactions (C2 for BbSod2, Ml for BbSod3 and CM3 for both) were selected for stress tolerance assays in paralleled with the wild-type strain. Their tolerance to menadione (ROS-generating compound) was significantly reduced in both colony growth(F3,32=3,659, P<0.01) and conidial germination (F3,11=132, P<0.01). The conidial tolerance of C2, M1 and CM3 to the oxidative stress was reduced by 38,52 and 66%, respectively. The silenced gene expression also increased the conidial susceptibility to UV-A irradiation by 32,18 and 22%(LD50:15.83±0.66,17.97±0.41 and 16.96±0.40 J/cm2 for C2, M1 and CM3 versus 21.81±0.60 J/cm2 for the wild-type strain). However, no significant effect on conidial tolerance to thermal or osmotic stress and UV-B irradiation was found in the three transformants.In summary, the whole study resulted in the characterization of the three new SOD genes in B. bassiana and will facilitate the understanding of the SOD family in the fungal entomopathogen. The successful high-activity expression of the fungal CuZn-SOD in E. coli cell by making use of the two site-directed mutations without the use of a chaperon suggests alternative means to exploring eukaryotic Cu/Zn-SOD. Significant contributions of the cytosolic Mn-SOD to the B. bassiana tolerance to oxidative stress and UV-B irradiation highlight the feasibility of improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes.