Development of Cordyceps javanica BE01 with enhanced virulence against Hyphantria cunea using polyethylene glycol-mediated protoplast transformation

Abstract

Cordyceps javanica has promising application prospects as an entomopathogenic fungus with a wide range of hosts. To enhance the virulence of C. javanica, a polyethylene glycol (PEG)-mediated protoplast genetic transformation system was constructed. Strains overexpressing the subtilisin-like protease genes CJPRB and CJPRB1 and the tripeptidyl peptidase gene CJCLN2-1 were constructed with this system, and the effects of these strains on Hyphantria cunea were tested. The aminoglycoside G418 was used at 800 μg ml−1 to screen the transformants. C. javanica hyphae were degraded with an enzyme mixture to obtain protoplasts at 1.31 × 107 protoplasts ml−1. The transformation of 2 μg of DNA into 1,000 protoplasts was achieved with 20% PEG2000, and after 6 h of recovery, the transformation efficiency was 12.33 ± 1.42 transformants μg−1 plasmid. The LT50 values of CJPRB, CJPRB1, and CJCLN2-1-overexpressing C. javanica strains were 1.32-fold, 2.21-fold, and 2.14-fold higher than that of the wild-type (WT) strain, respectively. The three overexpression strains showed no significant differences from the WT strain in terms of colony growth, conidial yield, and conidial germination rate. However, the infection rate of the CJPRB1 strain was faster than that of the WT strain, with infection occurring within 4–5 days. The CJCLN2-1 strain had a significantly higher mortality rate than the WT strain within 4–10 days after infection. A C. javanica genetic transformation system was successfully constructed for the first time, and an overexpression strain exhibited enhanced virulence to H. cunea compared with the WT strain.

Role of the Subtilisin-like Serine Protease CJPRB from Cordyceps javanica in Eliciting an Immune Response in Hyphantria cunea

“…The P. pastoris expression vectors pPICZαA and pPICZαA-GFP and the expression vector PYF11-CJPRB [ 9 ] (CJPRB GenBank number: OM468894) were stored in the Pathology Laboratory of Nanjing Forestry University.…”

“…RNA extraction from larvae of H. cunea and cDNA synthesis were performed according to Wang’s method [ 9 ]. The RT–qPCR system and conditions were as described by Wang [ 9 ]. The primers for the defense-related genes are shown in Table 1 .…”

“…Cordyceps species are a group of globally distributed entomopathogenic fungi that are capable of infecting more than 40 species of insects in 8 orders, exhibiting a broad spectrum of insecticidal activity [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. Among Cordyceps spp., Cordyceps javanica is capable of infecting insects of 12 genera in 3 orders [ 9 ]. The mortality rate of Hyphantria cunea larvae infected by the strain C. javanica BE01 isolated by us previously was over 85% at 1 × 10 8 conidia/mL, indicating that this strain has good application prospects [ 10 ].…”

“…Our previous results showed that C. javanica BE01 overexpressing the subtilisin-like serine protease CJPRB could accelerate H. cunea death [ 9 ]. Therefore, to further understand the role of the CJPRB protein in the pathogenesis of BE01, in this study, we investigated the changes in protective enzymes and the expression of immune defense-related genes in the larvae of H. cunea after infection and after feeding and injection with the CJPRB protein.…”