Abstract
BackgroundMetarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Unfortunately, its worldwide commercial use has been restricted by a short shelf life and inconsistencies in virulence. In our previous study, a gene (GenBank accession number EFZ01626) was found to be significantly upregulated in heat-treated conidia. In the present study, this gene was characterized via gene disruption and complementation strategies.ResultsThe gene (amplified by rapid amplification of cDNA ends PCR) was 1219 bp long and contained an open reading frame (ORF) of 777 bp. It encoded a protein of 234 amino acid residues with a 26-residue signal peptide. Bioinformatics analyses did not identify conserved functional domains; therefore, it was assumed to be a secreted virulence-associated protein according to its signal peptide and bioassay results. We found that the conidial germination rate of the ΔMrSVP mutant fungi dramatically decreased after heat shock treatment in a thermotolerance test. In addition, transcription levels of all tested heat shock–related genes were significantly lower in the mutant than in the wild type. We also demonstrated that the mean lethal time to death (LT50) of ΔMrSVP significantly increased relative to the wild type in insect bioassays (both topical inoculation and injection) involving Galleria mellonella. Moreover, similar rates of appressorium formation between ΔMrSVP and the wild type—and the significantly different expression of virulence-related genes such as acid trehalase and sucrose nonfermenting protein kinase in the haemocoel after injection—revealed that MrSVP is required for virulence in the insect haemocoel.ConclusionsOverall, our data suggest that the Mrsvp gene contributes to thermotolerance and virulence of M. robertsii. Furthermore, this gene is deeply involved in the mycosis of insect cadavers and in immune escape rather than insect cuticle penetration during infection.
Highlights
Metarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides
Identification of the Mrsvp gene and sequence analysis The sequencing results on the 3′- and 5′-rapid amplification of cDNA ends (RACE)-amplified fragments revealed that the cDNA sequence is 1219 bp long (GenBank accession number EFZ01626)
The open reading frame consists of 777 nucleotides, and the 5′ untranslated region (UTR) and 3′-UTR are 278 and 164 bp, respectively
Summary
Metarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Proteins secreted during the insect cuticle penetration stage, e.g., protease, chitinase, lipase, esterase, and other cuticle-degrading enzymes, have been widely studied [6]. Metarhizium propagates in the nutrient-rich insect haemocoel via immune evasion and adaptation to osmotic stress [9], which is involved in expression of the secreted proteins. The species can express the MOS1 receptor protein to adapt to the high osmotic pressure of hemolymph. After that, it can grow by means of secreted acid phosphatase and trehalase to degrade organic phosphorus and trehalose, respectively, in the haemocoel of insects [11]. The fungi eventually secrete a large number of secondary metabolites to interfere with, inhibit, or counter the host immune response and to kill the insect
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