Construction Of Protoplast-Based Transformation System Of Fungal Biocontrol Agent Paecilomyces Fumosoroseus With Restriction Enzyme-Mediated Integration (REMI)

As an entomopathogenic hyphomycetous fungus to infect a wide range of hosts, Paecilomyces fumosoroseus is one of the most important fungal biocontrol agents against insect pests and has been developed into commercial formulations for use in microbial control. Since celluar inocular such as conidia, mycelia and blastospores are always active ingredients of the formulations, the tolerance or resistance of the fungal candidates to environmental stress, such as drought, heat, ultraviolet, fungicides and herbicides, to large extent determines their adaptation to field conditions and compatibility with chemical control of plant diseases and weeds. Thus, improvement of the fungal formulations largely relies upon a candidate strain that has desirable features not only in virulence and inocular production but also in stressed tolerance or resistance, which can be attained by means of genetic manipulation and molecular breeding. This study sought to develop a technical protocol for protoplast-based genetic transformation of P. fumosoroseus Pfr116, a fungal candidate highly virulent to sucking insects, via the method of restriction enzyme-mediated integration. (REMI), and to test the protocol by integrating a phosphinothricin resistance gene into the protoplasts of the fungal strain using a constructed plasmid vectoring the gene. The results are summarized as follows.Preparation of Pfrll6 protoplasts. Conidia of P. fumosoroseus Pfr116, a wild strain derived from the whitefly Bemisia tabaci, was suspended evenly in Sabouraud dextrose broth and shaken at the regime of 25℃ and 120 r/min for 24 h. Fresh broth inoculated with the liquid culture by 10% was shaken at the same regime for another 24 h. The resultant mycelia were soaked in 0.3% mercapto ethanol for 10 min and then transfen-ed to a reaction system including 0.6 mol/mL KCl as osmotic stabilizer and one of five different enzymes (2% snailase, 2% lysing enzyme, 0.5% snailase plus 1.5% lysing enzyme, 1.5% snailase plus 0.5% lysing enzyme, and 1% snailase plus 0.6% lysozyme plus 0.4% cellulose) for 2-h reaction at 28℃, shaken by 90 r/min. As a result, a maximal yield of 1.1×10~6 protoplasts per miligram mycelia was achieved in the reaction system including the miature of 0.5% snailase and 1.5% lysing enzyme. The regeneration rate of the prepared protoplasts was up to 25.7% on the plates of Czapek's medium including 0.8 mol/L sucrose as osmotic stabilizer for the protoplast regeneration.Generation of protoplast transformants and their molecular identification. In trials to screen molecular markers for transformation, a concentration of 0.3 mg/mL RPT was found inhibiting the protoplast regeneration of the wild fungal strain whereas hygormycin B did not show inhibitory effect even at the concentration of 0.4 mg/mL. Thus,pAn52-lN-5ar, a plasmid vectoring the PPT-resistant gene Bar, was constructed for integrating it into the genomic DNA of Pfrl 16 protoplasts with the REMI method. The plasmid was linearized by the restriction enzyme Hind III and a concentration of 107.5 fig/mL was reclaimed using a Cycle-Pure Kit. The linearized plasmid was integrated into the Pfrl 16 protoplasts in a 50-uL reaction system at the concentrations of 0, 10, 30 and 50 units Hind III, respectively. After 5-day cultivation on the plates of Czapek's medium containing 0.3 mg/mL PPT, the reaction systems at 10 and 30 units Hind III resulted in the efficiency of 12 transformants per ug plasmid DNA. After three consecutive 7-day cultures at 25°C on 24-well Czapek's plates exclusive of PPT, the resultant transformants were all capable of growing on Czapek's medium including 0.4 mg/mL PPT. The result suggests the presence of stable PPT-resistance in all the transformants.Genomic DNAs were separately extracted from eight transformants randomly taken and used as template DNA and 5ar-specific primers in the PCR and Southern Blotting analyses, respectively. As a consequence, the gene Bar was detected as a singular specific band in all the transformed genomes but absent in the genome of th wild strain Pfrl 16. This indicates that the gene Bar was successfully integrated into the chromosomal DNAs of the protoplast transformants.Growth and conidiation potential of the transformants. Among 47 transformants consecutively maintained on Czapek's plates, two had much more fluffy colonies but apparently lost conidiation capability. After 9-day incubation on the plates, these two transformants, 02C1 and 02D6, 1 had the yields of 1.2* 105 and 8.2x105 conidia per mm2, respectively. These were 100 and 14.6 fold lower than the yield of 1.2><107 conidia per mm2 produced by the Pfrl 16 wild type. Thus, the two transformants could be conidiation-depressive mutants.Based on the results presented above, the protoplast- and REMI-based method has been successfully applied to transformation of the wild strain Pfrl 16 and shown advantages of low copy, high transformation rate and stable heredity. The two conidiation-depressive mutants resulted from the transformation would be of potential use for cloning or analyzing functional genes relating to conidiation. Thus, method is useful for genetic manupulation of P. fumosoroseus and other fungal biocontrol agents.