Functional Analyses Of Two Mannitol Dehvdrogenases And Five Catalases In Beauveria Bassiana And Construction Of Engineered Strain With High Per Os Virulence To Caterpillars

As a classic fungal entomopathogen, Beauveria bassiana has been developed into dozens of mycoinsecticides and widely applied in insect pest control. The fungal application is often restricted due to the defect of slow action and the sensitivity to the stresses of high temperature and solar UV irradiation often encountered in summer. Thus, there is an urgent need for exploring possible mechanisms involved in the defence of the fungal pathogen against multiple stresses and improving genetically fungal virulence to target pests.Mannitol, a small sugar polyol, is widely distributed in fungi and can be served as carbohydrate resources and involved in crucial defense mechanisms that protect cells from damages by environmental stresses. Catalases (CAT) are important antioxidant enzymes in eukaryotic cells and play important roles in protecting cells from the damage of intracellular reactive oxygen species (ROS). However, the regulation of mannitol metabolism and the function of catalases in entomopathogenic fungi are not well understood at present. On the other hand, integrating Bacillus thuringiensis (Bt) vegetative insecticidal protein Vip3Aal into B. bassiana under a constitutive promoter PgpdA from Aspergillus nidulans could increase fungal per os virulence to the neonates of Spodoptera litura but the amount of the target toxin expressed in fungal conidia was too small to kill older larvae. Thus, the present study sought to elucidate a possible mechanism involved in mannitol metabolism and explore the functions of CAT members in B. bassiana in response to multiple stresses by means of gene knockouts and phenotypic analyses. Moreover, a fragment found from the upstream regions of B. bassiana Class I hydrophobin gene (hydl) was used a novel, strong promoter to drive high yield expression of the midgut toxin Vip3Aal in transgenic B. bassiana, yielding an engineered strain highly insecticidal to all instars of S. litura larvae. The results are summarized below.Characterization of B. bassiana mannitol1-phosphate dehydrogense (BbMPD). A full-length1334-bp fragment of mannitol1-phosphate dehydrogenase gene (BbMPD) was cloned from B. bassiana2860(Bb2860herein), including a putative1176-bp open reading frame (ORF) with a158-bp intron. The gene was found encoding a391amino acid protein and showing56-74%sequence identity to MPDs known from other17fungi. The deduced protein was featured with a putative NADH cofactor binding site (GAGRIG) at the N-terminal. The gene was successfully expressed in E. coli BL21and the recombinant protein (84.4kDa) was well in agreement with the predicted molecular weight. The purified BbMPD showed high substrate specificity to fructose6-phosphote (Fru6P). Its maximal activity in Fru6P reduction was achieved at30℃and pH7.0. In the reaction at optimal 30℃and pH7.0, the BbMPD activities (y) at0.05-5mM Fru6P (x) were well fitted to non-linear Michaelis-Menten equation (r2=0.99), generating the parameters Km and Vmax (±SE) of0.89±0.05mM and913.5114.9U/mg proteins. The catalytic efficiency (kcat/Km) for the reduction of Fru6P by BbMPD was estimated as1.31×104mM-1s-1.Gene cloning of B. bassiana mannitol dehydrogense (BbMTD). A full-length864-bp fragment of mannitol dehydrogenase gene(BbMTD) was cloned from Bb2860. Three potential stress-responsive elements (AGGGG) and five elements meeting the consensus sequence (SYGGRG) for binding CREA, a broad domain regulator for carbon catabolism repression in Aspergillus, were lcoated in its5'flanking regions. The864-bp ORF with a63-bp intron encoded for a266amino acid protein with the predicted molecular weight of28.2kDa and the isoelectric point of6.6. The deduced protein, featured with a putative NADPH cofactor binding site (GPKGIG) at N-terminal, was found sharing high sequence identity to MTDs known from other fungi.Expression patterns of BbMPD and BbMTD. The wild-type strain Bb2860was grown on the plates of Saubouraud dextrose agar plus1%yeast extract (SDAY) for7days at25℃to monitor the transcription levels of the two genes in fungal colonies. As a result, the mRNA level of BbMTD increased steadily during the first four-day growth, followed by a20-fold increase on day5relative to day2. Unlike BbMTD, the transcription level of BbMPD fluctuated in a narrow range during the7-day growth and peaked on day4. Under the osmotic stress of1M NaCl) or the oxidative stress of50mM H2O2, the expression levels of both genes increased with stress time. Three-hour stress resulted in an increase of5(NaCl) or11.4(H2O2) fold in BbMTD expression and of10.9(NaCl) and74.9(H2O2) fold in BbMPD expression, respectively. In contrast, both BbMPD and BbMTD expression levels were suppressed by50and63%, respectively, after3-h heat stress at35℃. The results suggest that both genes take important parts in B. bassiana response to multiple stresses.Functional analyses of BbMPD and BbMTD. Two gene disruption mutants, ABbMPD and ABbMTD, were constructed via Agrobacterium tumefaciens-mediated transformation. Enzymatic activity assays and polyol content analysis showed that both BbMPD and BbMTD were crucial enzymes for mannitol metabolism. The BbMPD disruption resulted in almost all loss of MPD activity to reduce fructose-6-phosphate while the BbMTD knockout led to～80%loss of MTD activity compared with the wild-type strain. The two mutants and Bb2860grown on SDAY plates or incubated in Saubourau dextroe broth (SDB) differed significantly in intracellular mannitol content. The mannitol contents in mycelia and conidia decreased68and83%for△BbMPD, and16and38%for△BbMTD, respectively, compared with the Bb2860counterparts. The mannitol contents of△BbMPD and ΔBbMTD were enhanced respectively by3.0and1.7fold after the SDB-cultured mycelia were transferred to fresh SDB containing1M NaCl for24-h osmotic stress or by40.7and62.3%after exposure to35℃. Accompanied by the decreases of mannitol contents, trehalose contents increased in ΔBbMPD and ΔBbMTD cells under normal or stressed conditions.The conidial yield of ΔBbMPD on SDAY was significantly reduced by25-27%compared with similar yields of Bb2860and ΔBbMTD. The two mutants showed some differences in colony growth and spore germination on minimal medium with different carbon sources. ΔBbMPD was similar to Bb2860in colony size irrespective of a carbon source but fructose, glucose and mannitol delayed50%spore germination (GT50) for3.0,3.1and6.6h, respectively. In contrast, ΔBbMTD grew much more slowly than Bb2860and its GT50was delayed for7.1h by mannitol but not affected by fructose and glucose. Moreover, ΔBbMPD and ΔBbMTD conidia were38and18%less tolerant to the oxidative stress of H2O2,39and16%less tolerant to the stress of UV-B irradiation, and22and11%less tolerant to the wet-heat stress of45℃, respectively. However, both gene knockout mutants were not affected in conidial tolerance to osmotic stress and and virulence to the apterous adults of green peach aphid Myzus persicae.Gene cloning, sequence analysis and expression patterns of B. bassiana catalases. Five catalase (CAT) genes were cloned for the first time from B. bassina, including BbcatA, BbcatB, BbcatP, BbcatC and BbcatD. These genes were found encoding three monofunctional catalases (BbcatA, BbcatB, and BbcatC) and two biofunctional catalase-peroxidases (BbcatP and BbcatD). Of those, BbcatC was a small subunit catalase found in B. bassiana and other four were all large subunit catalases. Phylogenetic analysis indicated that five catalases were classified to different groups. BbcatA, a Clade A catalase, is mainly involved in spore-specific processes, such as germination. Its transcription level increased during conidiation but significantly decreased during spore germination and hyphal growth. In SDB cultures including50mM H2O2for3-h oxidative stress, the transcription level of BbcatA was enhanced by4.5fold. BbcatB and BbcatD were Clade B catalases due to the presence of a secretive signal peptide of19amino acids at the N-terminal. The BbcatB gene transcribed increasingly during colony growth, resulting in a peak of30.2fold increase on day7relative to day2. The transcript level of BbcatD was relatively low during the first5-day growth and drastically increased53.1and158.3fold on days6and7, respectively. BbcatC and BbcatP were Clade P members (peroxisomal catalases) due to their peroxisome-targeting signal (PTS). BbcatC transcribed increasingly during colony growth while BbcatP transcibed at a level much lower than other four genes during7-day colony growth. The transcriptional levels of both genes were induced by H2O2added to SDB cultures for3-h oxidative stress, which, for example, increased BbcatP transcription by13.5fold. Our study revealed that the B. bassiana catalases may take different parts in regulating fungal response to oxidative stress.Functional analyses of five B. bassiana catalases. The disruption mutants of five B. bassiana CAT genes, i.e., ΔBbcatA, ΔBbcatB, ΔBbcatC, ABbcatD and AbbcatP, were constructed by means of A. tumefaciens-mediated transformation. Enzymatic activity assays indicated that BbcatB, BbcatP and BbcatD primarily contributed to the CAT activities of the fungal species under normal conditions. In the profiles of non-denaturing polyacrylamide gels stained with ferricyanide, however, only two CAT-active bands were found in the wild-type strain grown under normal conditions. The upper and lower bands corresponded to BbcatB and BbcatP because they were absent in the ΔBbcatB and ΔbbcatP profiles, respectively.ΔBbcatC and ΔBbcatP were very sensitive to the oxidative stress of50mM H2O2with their colony growth being greatly suppressed (e.g., nearly100%for AbbcatP). However, other three mutants showed the same response to the oxidative stress as the wild-type strain. All mutant and wild-type strains displayed no significant difference in colony growth on SDAY plates under the stress of1M NaCl,1μg/ml carbendazim or4mM menadione or incubated at33℃. Exceptionally, the colony growth of ABbcatB was suppressed on menadione-inclusive plates. Moreover, conidial tolerance to H2O2was reduced by42.6,57.0and34.5%for ΔBbcatA, ΔBbcatC and AbbcatP, respectively. The conidia of ΔBbcatB and ABbcatD were52.4and47.6%less tolelrance to UV-B irradiation than the wild-type strain, respectively. Conidial tolerance to the heat stress of45℃was similar for all wild-type and mutant strains except AbbcatA, whose median lethal time (LT50) was reduced by40%.ΔBbcatA, ΔBbcatP and ΔBbcatD displated significantly reduced virulence to the second-instar larvae of S. litura compared to the wild-type strain. Their LT50estimates were delayed for1.4,1.8and0.9days, respectively. The same estimates of ABbcatB, ABbcatC and the wild-type strain fell in the narrow range of5.0-5.1days. Thus, the three catalases are potential virulence factors in B. bassiana.Great elevation of fungal per os virulence to caterpillars by high yield expression of a midgut-acting toxin in transgenic conidia under a novel promoter. Beuaveria bassiana infects insect pests normally through host cuticle but hardly causes per os infection. We obtained a great progress in high yield expression of Bacillus thuringensis Vip3Aal, a midgut-acting toxin, in transgenic conidia for enhanced fungal virulence to caterpillars through spore ingestion. The toxin production relied on a homologous promoter (Phydl-t1) found to drive the toxin-coding gene expression specifically during conidiation. The gene transcription level in the4-day colonies of all examined transformants was3.4-112fold higher than that in a transgenic strain (BbV28) under the control of PgpdA, a heterologous promoter widely used for gene expression in fungi. The best transformant BbHV8produced9.8-fold-higher Vip3Aal yield in ripe conidia than BbV28. The release of active Vip3Aal into larval midguts from BbHV8conidia ingested within24h was4.4-fold more than that from BbV28. In the bioassays of spore suspensions sprayed onto S. litura larvae (for normal infection) and lotus leaf discs (for feeding), the LC50S of BbHV8against instars II and III on days2-5and3-8were reduced by11-24and8-80fold (declining with time), respectively, compared with the BbV28estimates. BbHV8killed90%instars II-V larvae within3-8days under the economic sprays of678-1389conidia/mm2whereas BbV28and wild-type strain could not kill the older larvae effectively. Conclusively, the high toxin yield in conidia enhanced greatly the toxin release into midguts, the fungal per os virulence to caterpillars and the potential of BbHV8for pest control.