Integration of dsRNA against host immune response genes augments the virulence of transgenic Metarhizium robertsii strains in insect pest species

Abstract

The slow lethality of fungal biopesticides to insects restrains their widespread application as a strategy of pest control. In this study, unary, binary and ternary transgenic Metarhizium robertsii were created by integrating genes that encode the scorpion neurotoxin BjaIT, the cuticle-degrading protease Pr1A, and a double-stranded RNA (dsRNA) that targets host gnbp3, individually or collectively under a constitutive promoter to enhance virulence. Compared with the parental wild type, all unary transgenic strains had increased virulence against four insect species, Tenebrio molitor, Locusta migratoria, Plutella xylostella and Galleria mellonella, whereas the binary transgenic strain expressing both pr1A and BjaIT had increased virulence to T. molitor and L. migratoria, with no change in virulence against P. xylostella and G. mellonella. Importantly, all ternary transgenic strains simultaneously expressing pr1A, BjaIT, and the dsRNA specific to host gnbp3 exhibited the highest increase in insect-specific virulence. This finding highlights a novel strategy for genetic engineering of dsRNAs that target genes associated with the host immune response alongside virulence genes to maximize fungal virulence and lethality against insect pests.

Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii

“…For the samples of M. robertsii during the infection progress, conidia were used for cuticle infection by dipping the fifth-instar Galleria mellonella L. (Lepidoptera: Pyralidae) larvae into a conidial suspension (1 × 10 7 conidia mL −1 ) for 10 s. Then, the infected larvae were maintained in a stable photoperiod with 16 h of light and 8 h of dark at 28 °C without feeding. Infected G. mellonella were collected at 24 h, 48 h, and 96 h. Quantitative RT-PCR was performed [ 20 ]. In brief, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction, and complementary DNA were synthesized with a gDNA remover kit (Toyobo, Osaka, Japan) as the manufacturer’s instructions.…”

A life-and-death struggle: interaction of insects with entomopathogenic fungi across various infection stages

“…Transferring an esterase gene (Mest1) from the generalist M. robertsii to the locust specialist M. acridum enabled the latter to expand its host range (116,196) Suppressing or evading host insect immunity through genetically engineering and RNAi Isaria fumosorosea I. fumosorosea with Toll-like receptor 7 targeted dsRNA is more virulent than wild fungus against white fly (Bemisia tabaci) (197,198) M. robertsii M. robertsii expressing dspr1A (cuticle-degrading protease Pr1A) and dsBjaIT (the scorpion neurotoxin) exhibited an increased virulence (199) M. anisopliae DsRNA-expressed M. anisopliae targeting Apolipophorin-D and Relish exhibited higher virulence (200,201) M remain elusive. Moreover, the interaction between host-pathogen transcends mere immunological interplay, which encompasses precontact communication and the dispersal of EPF post-infection.…”

A novel partitivirus orchestrates conidiation, stress response, pathogenicity, and secondary metabolism of the entomopathogenic fungus Metarhizium majus

“…The 3 × FLAG was fused in frame with the N-terminus of the MmPV1 ORFs 1 and 2 and the constructs were introduced into the binary plasmid pDHt-SK-bar-PgpdA to generate respectively pDHt-SK-bar-PgpdA-ORF1 and pDHt-SK-bar-PgpdA-ORF2, which were transferred into Agrobacterium tumefaciens strain EHA105. Then ORF1-and ORF2-expressing RCEF0577 stains were obtained via Agrobacterium tumefaciens-mediated transformation (ATMT) according to previous descriptions [63], and verified by RT-PCR, RT-qPCR and immunoblotting.…”