Molecular Mechanism Involved In The Cell-Wall Protein Related Thermotolerance Of Beauveria Bassiana And Applied To Genetic Improvement Of The Fungal Thermotolerance

Entomopathogenic hyphomycetes are important fungal biocontrol agents against insect pests, being well represented by the fungal species Beauveria bassiana. It is well known that the shelf life and field persistence of a fungal formulation, including intact cells (e.g., conidia, balstospores and/or mycelia) as inocula or active ingredients, are affected conspicuously by environmental temperature. Thus, it is necessary to understand possible mechanisms involved in the thermotolerance of the fungal bioconctrol agents and to explore technical approaches to improving ecological fitness of fungal formulations to crop environments for better control of insect pests.This study began from quantitative evaluation of conidial thermotolerance of selected fungal biocontrol agents. The contents of hydrophobin-like or formic-acid-extractable (FAE) proteins on conidial wall were found significantly relating to the conidial thermotolerance, resulting in a hypothesis that the cell-wall FAE proteins are likely involved in conidial thermotolerance. To confirm the hypothesis, the full length sequence of the gene encoding amino acid sequence of a 15.0-kDa FAE protein, which was most influential on the thermotolerance of B. bassiana conidia, was cloned and characterized as a new gene. The suppression of this gene in B. bassiana by an antisense RNA technique was found causing significant decrease of the conidial thermotolerance using a new transformation system based on B. bassiana blastospores. The new system was also applied to separate integration of two foreign genes, functionally resisting o environmental stress, into B. bassiana, generating transformants which produced significantly more thermotolerable conidia. Overall, the study developed a new technical platform for analysis of gene functions and genetic improvement of B. bassiana and other fungal biocontrol agents. Detailed results are summarized as follows.Conidial thermotolerance versus FAE proteins. The assay protocol to evaluate quantitatively conidial thermotolerance was developed by exposing fungal conidia to the thermal stress of 48℃(in water bath) for certain periods of time. The ratio of conidial viability (measured as germination rate) after a given period of the thermal stress to that of blank control (unstressed conidia) was defined as survival index I_s. When the conidia of 11 isolates of B. bassiana and Paecilomyces fumosoroseus with different host and geographic origins were separately exposed to 48℃for 15-150 min, the survival indices of all over the time of exposure (t) fit very well to the logistic equation I_s=1/[1+exp(a+bt)] (r²>0.97, P<0.01). For a given isolate, the fitted equation was used to estimate median lethal time, LT₅₀, of its conidia under the thermal stress. This estimate was a charateristic index for the conidial thermotolerance of each isolate. As a result, the LT₅₀s of six B. bassiana isolates were averaged as 40.7 (10.1-61.9) min whereas those of five P. fumosoroseus isolates were on average only 4.6 (2.8-6.2) min.For a given isolate, conidial thermotolerance was significantly affected by medium components and culture conditions. Given peptone as mere nitrogen source, 2-4% glucose was optimal for the expression of conidial thermotolerance, followed by 2% sucrose and starch. When NH₄NO₃ was used as mere nitrogen source, 4% glucose was optimal, followed by 1% starch and sucrose. Optimized culture conditions for best expression of the thermotolerance of B. bassiana conidia were 25℃and pH 5-6, which were nearly identical to those for optimal growth of the same fungal species. The conidial thermotolerance was enhanced when Mn²⁺ was added to SDAY at the rate of <50μg/mL, altered slightly when Zn²⁺ was added at 50-200μg/mL, but decreased remarkably when Fe³⁺ or Cu²⁺ were added even at a very low rate. The conidial thermotolerance decreased as more ions accumulated in conidia.The contents of FAE proteins on conidial wall differed significantly between the tested fungal species or among the different isolates of each species. The contents were also affected by medium components and culture conditions. When peptone was used as mere nitrogen source for growth of a B. bassiana isolate, 2.0% glucose or sucrose was most favorable to accumulation of FAE proteins but starch affected the content at lesser degree. Given NH₄NO₃ as mere nitrogen source, the different maximal contents of FAE proteins were achieved at 2% glucose, 2% sucrose and 1% starch, respectively. Metal ions decreased the FAE protein content at 0-50μg/mL, slightly affected the content at 50-200μg/mL, but increased significantly the content at≥200μg/mL.SDS-PAGE profiles of the FAE proteins were consistent for each fungal species. Three FAE proteins with molecular weights of 12.0, 15.0 and 17.5 kDa were identified from the extracts of B. bassiana conidia and designated as BCW12, BCW15 and BCW17 respectively. The proteins BCW15 and BCW17 were constitutively expressed in the conidia of both B. bassiana and P. fumosoroseus. However, expression of the protein BCW12 in B. bassiana only was affected by medium components and culture conditions. When peptone was used as mere nitrogen source, BCW12 was expressed only at lower concentrations of glucose or sucrose but not affected by starch. Given NH₄NO₃ as mere nitrogen source, expression of BCW12 was affected by neither carbon type nor concentration. The BCW12 was expressed at all tested concentrations of Fe³⁺ but suppressed by Mn²⁺, Cu²⁺ and Zn²⁺. Other factors including pH, water availability and incubation temperature were found not affecting the expression of the FAE protein.The LT₅₀s (y) of all the isolates of B. bassiana and e P. fumosoroseus were significantly correlated with the contents (x) of their FAE proteins [y=exp(0.762+0.137x), r²=0.81; F=37.5, P<0.01]. For the isolate B. bassiana 2860, the survival indices of the conidia stressed at 48℃for 45 or 30 min were also correlated well with the contents of the FAE proteins from conidia produced on different carbon- and nitrogen-based substrates. With these results in consideration, a hypothesis is proposed that at least some of the conidial FAE proteins are possibly involved into the conidial thermotolerance. Among the recognized FAE proteins, the constitutively expressed BCW15 and BCW17 were of greater significance than the conditionally expressed BCW12. Cloning and characterization of the FAE protein BCW15. The thermotolerance-relating FAE protein BCW15 was purified by means of selective resolution in 30% acetonitrile. The protein was separated by SDS-PAGE and transferred onto PVDF membrane for sequencing the amino acids of its N-terminus. The resultant N-terminus was SFPGGQFIIRN . Based on this M-terminal sequence, the degenerated primer was designed for amplifying the coding sequence of the gene BCW15 by 3'-RACE. As a result, the partial upstraem sequence of the gene BCW15 was cloned using YADE protocol. Further BLAST analysis in NCBI indicates that BCW15 was the first discovered full-length gene encoding a cell-wall protein in B. bassiana. The new gene consisted of a 393-bp ORF encoding 131 amino acids and no signal peptide sequence. The 3'-RACE products included two fragments of～500 and 600 bp. No difference was found between the two coding sequences except the untranslated regions of their 3'-terminuses. The whole promoter of the gene BCW15 was not cloned, but its partial upstream sequence was rich in nucleotides A and T. The gene BCW15 was characterized with two copies in genomic DNA of B. bassiana and with no intron in the genomic sequences.A common, conserved sequence of amino acids (HNDRVVGAWDQDVKIV) was found from the BCW15 sequences of the six B. bassiana isolates with different host and geographic origins. Based on divergence in amino acid sequences of the protein BCW15, the two groups BCW15a and BCW15b were classified. This suggests a possible use of BCW15 as a new molecular marker for the phylogeny of B. bassiana.Construction of blastospore-based transformation system. To confirm the function of the protein BCW15 and to improve conidial thermotolerance of a fungal candidate, a new transformation system was developed to ease genetic manipulation based on B. bassiana blastospores. The concentration of 200μg/mL phosphinothricin added to basal medium (Czapek's medium) was used as selection pressure for putative transformants. The plasmid pABeG vectoring the phosphinothricin resistance gene bar and the green fluorescence protein gene egfp was constructed to test the efficiency of the blastospore-based transformation system. The B. bassiana blastospores produced in a nitrogen-limited liquid medium became competent to receive foreign gene(s) after they were treated with 0.1 mol/L LiAc. The constructed plasmid pABeG was successfully inserted into the blastospores via the mediation of polyethylene glycol (PEG). As a result, the transformation frequency of 24±4.58 transformants/μg plasmid DNA (n=3) was achieved. All randomly taken transformants were found constitutively expressing the green fluorescence protein in either fungal growth in vitro or infection in vivo. Subsequently, the Poly-A-trap vectors of the gene bar were constructed as pUCBAR-TF and pUCBAR-TR. The pUCBAR-TF was successfully inserted into B. bassiana genomic DNA with the technique of restriction enzyme mediated integration (REMI). This is the first application of the Poly A technique to labeling a functional gene in fungal biocontrol agents.The blastospore-based transformation system developed to ease genetic manipulation of filamentous fungi was featured with greater convenience and higher efficiency. A combination of the novel transformation system with the gene trap technique would greatly facilitate identification of functional genes and genetic improvement of the fungal agents.Confirmation of the biological function of BCW15. The function of the FAE protein BCW15 was determined by suppressing its expression using an antisense technique. To achieve this goal, a plasmid vectoring both the transcriptional element of BCW15 antisense RNA and the gene bar was constructed as pAN52-antiBCW15-Bar. This ansense vector was then inserted into the genomic DNA of the B. bassiana 2860 blastospores using the REMI technique. The results were exciting. First, the conidial FAE contents of five randomly taken transformants (A1-A5) were significantly lower than those of the wild strain (CK) and the transformant (TCK) integrated with pAN52-Bar only (F_6,14=107.2, P<0.01). Based on SDS-PAGE analysis, expression of the protein BCW15 was entirely suppressed in all the five transformants whereas expressions of two other FAE proteins, BCW12 and BCW17, were not affected at all. Second, the conidial survival indices of all the five transformants after 30-min exposure to the thermal stress at 48℃were in the range of 0.38-0.55, which was significantly smaller lower (F_6,14=20.6, P<0.01) than those of the strains CK (0.66) and TCK (0.68). There was no significant difference in the survival indices between the CK and TCK strains. Third, the conidia of the strains CK and TCK and of the antisense transformant A1 with an intermediate survival index after 30 min stressed at 48℃were further exposed to the thermal stress until their viability declined to zero, resulting in excellent fit of all the time-dependent declining trends to the logistic equation (r²â‰¥0.98, P<0.01). As a result, the LT₅₀s for the conidial thermotolerance of the strains CK, TCK and Al were estimated as 41.1, 40.4 and 31.9 min, respectively. The LT₅₀ of the antisense transformant was 8.5-9.2 min shorter than those of CK and TCK. Conclusively, the BCW15 was an FAE protein significantly contributing to the conidial thermotolerance but had no detectable effect on normal growth and conidiation of the fungal agent tested. It has also proved that the ansense technique was very useful for analyzing the function of the fungal gene.Expression and function of metallothionein in transformed B. bassiana. A foreign gene MT encoding the metallothionein of Neurospora crassa was integrated into the wild strain B. bassiana 2860 to evaluate its possible effect on conidial thermotolerance. This started from constructing the binary plasmid pAN52-MT-Bar vectoring the two genes MT and bar, followed by inserting the plasmid into B. bassiana blatospores using the REMI method. As a result, the mycelia of five randomly taken transformants (M1-M5) has significantly higher contents of the thio-group proteins than those of the strains CK and TCK (F_6,14=14.52, P<0.01). Western blotting analysis indicates that the N. crassa metallothionein was well expressed in the mycelia of the three transformants with high contents of the thio-group proteins. Moreover, the conidial survival indices of the three transformants after 30-min exposure to 48℃was averaged as 0.74 (0.71- 0.76), being significantly greater (F_4,10=8.4, P<0.05) than that of CK (0.66) or TCK (0.68). The LT₅₀ of the transformant M4 (whose conidia had a survival index close to the average after 30-min exposure to 48℃) was estimated as 45.8 min in a prolonged exposure of its conidia to the same thermal stress time by fitting the declining trend to the logistic equation (r²=0.98, P<0.01). This estimate was enhanced by 4.7 and 5.4 min compared to those of CK (41.1 min) and TCK (40.4 min) respectively. Thus, the conidial thermotolerance was improved by integration of the N. crassa metallothionein into the wild strain of B. bassiana.Expression and function of thioretoxin in transformed B. bassiana. Another foreign gene thxA encoding Escherichia coli thiretoxin was also introduced into the B. bassiana strain to evaluate its possible effect on conidial thermotolerance. Similarly, the binary plasmid pAN52-THX-Bar vectoring the genes thxA and bar was constructed at first and then transformed into B. bassiana blatospores using the REMI technique. As a result, specific activities of thredoxin in the mycelia of five randomly taken transformants (T1-T5) were significantly higher (F_6,14=142.9, P<0.01) than those of the strains CK and TCK, which were close to zero. Western blotting analysis demonstrates that the E. coli thiredoxin was well expressed in the mycelia of the three thxA-inserted transformants showing high specific activities of thredoxin. After 30-min exposure to the thermal stress of 48℃, the conidia of the three transformants had a mean survival index of 0.78 (0.76- 0.81) significantly greater (F_4,10=17.1,P<0.01) than CK (0.66) and TCK (0.68). The LT₅₀ of the transformant T1 (whose conidia had a survival index close to the mean after 30-min exposure to 48℃) was estimated as 49.2 min in a prolonged exposure of its conidia to 48℃by fitting the declining trend to the logistic equation (r²=0.98, P<0.01). This LT50 was increased by 8.1 and 8.8 min compared to those of CK (41.1) and TCK (40.4), respectively. Thus, integration of the E. coli thioredoxin into B. bassiana enhanced the conidial thermotolerance more than integration of the N. crassa metallothionein.In summary, the whole study has resulted in establishment of a quantitative method for evaluating conidial thermotolerance and modification of the protocol for extracting FAE proteins from conidia. Recognition of thermotolerance-relating FAE proteins by correlation analysis led to a new hypothesis that the FAE proteins take important parts in the conidial thermotolerance. A gene encoding the conidial wall protein BCW15 was cloned and characterized as new and its significant contribution to conidial thermotolerance was first confirmed by means of the antisense RNA technique and the newly developed, blastospore- based transformation system. Genetically engineered strains with enhanced thermotolerance of their conidia to the thermal stress were obtained by integration of E. coli thioredoxin or N. crassa metallothionein into B. bassiana. The results provide new insights into stress biology of B. bassiana and suggest a promising approach to improving ecological fitness or field persistence of fungal formulations. That is genetic manipulation of the fungal candidates by exploiting stress resistant or tolerant function genes and the transformation system based on fungal blastospores.