Expression Of An Insect Immunity Regulator Serpin In Entomopathogenic Fungus Beauveria Bassiana Leads To Increased Virulence

Entomopathogenic fungi are important natural regulators of insect populations and have been used as environmentally friendly alteratives to chemical pesticides in pest control.. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and higher environmental conditions. It can take between6to14d for the fungus to kill target insects, which imposes a significant limitation regarding the use of these fungus. Mycosis involves several steps that includes;(1) adhesion of spores (conidia) to the host cuticle,(2) germination on the insect surface and hyphal penetration of the exoskeleton into the hemocoel,(3) proliferation and immune evasion in the hemocoel, and (4) outward hyphal growth and sporulation on the host cadaver. In the course of infection, the insect will use their body wall and the immune system active defense, resist the infection of the host. As the fungal infection of host, the immune system could appears to be mediated via Toll-like receptor signaling pathway that results in hemocyte activation (phagocytosis, nodule formation, or encapsulation), synthesis of antimicrobial peptides, and activation of phenoloxidase (PO) and melanization pathways. Research shows that the Spaetzle/Toll/Cactus signaling cascade pathway in Drosophila has been shown to mediate responses to fungal infection, and Toll-mediated activation is controlled by serine protease inhibitors (Spn43Ac). Thus, we reasoned that expression of a serpin by a fungal pathogen during the invasion process might inhibit innate immune activation to increase the virulence of the fungus. In this study, a serpin Spn43Ac from D. melanogaster was expressed in B. bassiana and the virulence was evaluated. The main results are as follows:1. The construction of Bbsp:Spn43Ac expression vectorThe Drosophila Spn43Ac cDNA sequence was obtained by removed the intron of Spn43Ac gene using the PCR overlap methods. A secretion signal peptide (Bbsp) derived from the B. bassiana chitinase gene Bbchitl was added to the cloned Spn43Ac ORF, obtain fusion gene Bbsp:Spn43Ac. The fusion gene was introduced into B. bassiana and Spn43Ac expression strains were obtained by screening and real-time quantitative PCR.2. Expression of Bbsp:Spn43Ac in B. bassiana elevated virulenceTopical bioassays, which represent the natural route of infection, revealed enhanced virulence of the Bbsp:Spn43Ac strain. Bbsp:Spn43Ac strain has an LT50(3x107conidia/mL) of84h compared to110h for wild type strain against G. mellonella. Topical bioassays using M. persicae adults resulted in an LTso=98h for the Bbsp:Spn43Ac, and LTso=117h for the wild type (1x107conidia/mL). These data indicate16-24%decrease in the LT50for the Bbsp:Spn43Ac expressing strain as compared to the wild type parent. Expression of Bbsp:Spn43Ac also decreased LD50. Using G. mellonella, the LD50for Bbsp:Spn43Ac=2.0x107conidia/mL and for the wild type LD50=6.0x107conidia/mL, indicating a2-fold reduction in the number of spores required for the same level of mortality.3. Analysis the mechanism of virulence improvement in Bbsp:Spn43Ac strains1) Expression of Bbsp:Spn43Ac has no effect on the germination and growth of fungusThe germination and growth of fungus in host body wall is a key step when entomopathogenic fungi infect susceptible hosts, we analyzed the effects of Spn43Ac on this step. The results showed that expression of Bbsp:Spn43Ac in B. bassiana did not affect the germination and growth in the artificial medium and insect body wall (cicada wing), and Spn43Ac protein had no effect on the activity of cuticle degradation protease CDEP-1.2) Hyphal body development in infected G. mellonella larvaeAs fungal infection of the host, hyphal body could grow in insect hemolymph. Microscopic observation of hemolymph samples showed greater hyphal body proliferation in larvae injected with Bbsp:Spn43Ac than wild type. Quantification of hyphal body production over a time course post-injection (60-108h) indicated a2-4fold increase in hyphal bodies during the later stages of infection for the Bbsp:Spn43Ac strain compared to wild-type.3) Expression of Bbsp:Spn43Ac inhibited the PO activation of G. MellonellaPhenoloxidase (PO) cascade activation is one of the main reaction of insect immune system, we wonder if expression of Spn43Ac in B. bassiana can affect the activation of PO. Results showed that injection of Bbsp:Spn43Ac spores can inhibit phenoloxidase activity of haemolymph than injection of WT spores, significant differences (P<0.01) in PO activity were seen8h post-infection between Bbsp:Spn43Ac and WT strains.4) GST:Spn43Ac inhibit PO activation in vitroGST:Spn43Ac was heterologous expressed and purified from E. coli. Results showed a concentration dependent inhibition of chymotrypsin activation of PO activity, with a～74%decrease in PO activity seen in reactions containing6μM GST:Spn43Ac as compared to controls that included GST and inactivated GST:Spn43Ac. In vitro assay showed inhibition of phenoloxidase activation in the presence of Spn43Ac.This study showed that expression of host immune responses regulator Spn43Ac inhibited the activation of PO cascade and significantly improved the proliferation rate of fungi in the hosts, finally increased the strain virulence. This provides a new train of thought for using of genetic engineering to improve strains and screen of high virulence factors.