Gene Clone And Functional Analysis Of The Tetraspanin Gene MaPls1in Metarhizium Acridum

Entomopathogenic fungi are the largest category of entomopathogens. Differentfrom the bacteria and virus, entomopathogenic fungi can infect the insect by attachingto the insect cuticle under natural environment, which are efficient for insect withpiercing-sucking mouthparts. Therefore, entomopathogenic fungi have the mostpotential to kill insect. Although entomopathogenic fungi have characteristics ofnon-pollution, contacting and prevention of resurgence and so on, there are still somedisadwantages, for example, slow and unstable effeciency of insecticidal, whichseriously hinder their large-scale application. Therefore, to better understand thepathogenic machanism of entomopathogenic fungi will hep lay the theoretical basis forfor construction strains with high efficiency.Previous report showed that tetraspanins regulated cellular activities throughassociation with other components on membrane in most eukaryotes. The tetraspaninPls1controls an appressorial function required for host penetration in phytopathogenicfungi. However, the regulation mode of Pls1and signaling pathways it involves in are notclear. The same as most phytopathogenic fungi, entpmopathogenic fungi depend on aspecial infection structure, appressorium, to infect insect. In this study, a Pls1gene(MaPls1) was functionally characterized in the entomopathogenic fungus Metarhiziumacridum using gene deletion technique. Digital gene expression (DGE) was used toanalyze the pathways and downstream target genes regulated by MaPls1.The main results were as follows:1) Bioinformatics analysis of the MaPls1The cDNA of tetraspanin MaPls1was obtained from a full length cDNA library ofM. acridum germinating and differentiating on locust wing. The full-length cDNA ofMaPls1was1115bp with a200bp5’-untranslated region, an open reading frame (ORF) of675bp and a240bp3’-untranslated region. MaPls1genomic DNA had three exons (401bp,167bp and107bp) and two introns (90bp and71bp), encoding a protein of224aminoacid. Alignment of Pls1protein sequences revealed that the MaPls1shared conservedsecondary structures among phytopathogenic fungi. The length and position of fourtransmembrane domains in MaPls1was similar to MgPls1.2) Expression pattern and localization of the MaPls1Semiquantitative RT-PCR and quantitative real-time PCR (qRT-PCR) revealed strong expression of MaPls1in the mycelium and appressorium, but significant less expression inconidium and hyphal body. Consistently, confocal results showed that expression ofMaPls1-EGFP fusion proteins under the control of the native MaPls1promoter wasdetected only during formation of the mycelium and appressorium and accumulated on thecell membrane or in cytoplasm.3) MaPls1affects the germination on insect cuticleThe ΔMaPls1exhibited a significantly decreased germination on host insect matrix(wings or wing methanol and hexane extract) compared with WT. At extended growingtimes, the difference in germination between ΔMaPls1and WT diminished. However, whenexposed to non-host insect matrix (wing or wing methanol and hexane extract), ΔMaPls1showed germination rate similar to WT. Compared with germination on host insect matrix,both ΔMaPls1and WT had a significantly decreased germination on non-host insect wings(honey bee and cockroach), or hexane or methanol extracts of non-host insect wings at theearly times after inoculation. These results demonstrated that disruption of MaPls1led todelayed germination in M. acridum.4) MaPls1affects appressoria formation and turgorTo examine the role of MaPls1in regulating appressorium development, we conducteda time course of appressorium formation using WT, ΔMaPls1and a complemented strain onlocust wings. Results demonstrated that ΔMaPls1had delayed appressorium formation.Compared to WT and complemented strains, appressoria of the ΔMaPls1mutant were moreeasily collapsed when exposed to PEG-8000, indicating decreased appressorial turgorpressure in the ΔMaPls1mutant. The ΔMaPls1mutant contained fewer lipid droplets inboth the appressorium and the mycelium compared to WT and the complemented strainshown by nile red (NR) staining. This finding implied that MaPls1influenced the lipidstorage in M. acridum.5) MaPls1affects pathogenicityThe ΔMaPls1mutant had a significant reduction in virulence compared with WT, asassayed by infecting insect with a topical application of conidia on the pronotum. However,the ΔMaPls1mutant and WT had similar virulence (p>0.05) when conidia were directlyinjected into the insect hemocoel. In both bioassays, abundant conidia emerged on thecadavers died of either WT or complemented strain about10days after insect death.However, insect cadavers infected with ΔMaPls1mutant showed only a few conidia on thesurface.6) DGE analysis A total of226genes were identified to be differentially expressed between the WTand ΔMaPls1, including94up-regulated genes and132down-regulated genes in theΔMaPls1mutant. DGE revealed that genes involved in hydrolyzing host cuticle, proteintransportation, and cell wall synthesis and remodeling were dramaticallydown-regulated. MaPls1was involved in a wide crosstalk with other signal pathways,such as Ca2+/Calmodulin pathway, and GTPase and cAMP-PKA pathway.Taken together, these results demonstrated the important roles of MaPls1at theearly stage of infection-associated development by M. acridum.