The Functions Of β-tubulin And MaPpt1 Genes In Metarhizium Acridum

Widely publicized environmental concerns and health risks related to use of synthetic chemical insecticides have stimulated efforts to develop biological-control agents as alternatives or supplements to these chemicals to protect enviroment at present.Virtually all insect orders are susceptible to fungal diseases.So fungal diseases in insects are common and widespread and often decimate insect populations in spectacular epizootics.Consequently,fungal mycoinsecticides to substitute for synthetic chemical insecticides are important for protecting the environments and providing healthy food.It is an important way that entomopathogenic fungi have long been importantly studied for exploitation in biological control and integrated pest management strategies to protect the environments.In the life cycle of entomopathogenic fungi,the process of infection involves attachment of the fungal conidia to the insect cuticle,attended by the formation of germ tubes that differentiate into penetration structures termed appressoria.Penetration of the insect cuticle by appressoria involves the secretion of an array of cuticle degrading enzymes and mechanical pressure,the latter requiring cellular turgor pressure at the tip of the appressoria.Penetrating hyphae undergo a dimorphic transition as they enter the hemocoel,producing freely floating in vivo cells termed hyphal bodies.Entomopathogenic fungi penetrate the cuticle and produce conidium again when they consumed available nutrients of insects.Then,it has next new life cycle.Plant pathogenic fungi and entomopathogenic fungi have a similar infection process.A phytopathogen avirulence gene is a gene corresponding to a plant resistance gene in plant pathogenic fungi,including cytoskeleton and Ser/Thr protein phosphatase,and so on.The functions of entomopathogenic fungi genes corresponding to plant pathogenic fungi avirulence genes,such as β-tubulin and serine/threonine protein phosphatase Ppt1,are little known in entomopathogenic fungi.It is very important to study their functions for understanding the pathogenic mechanism of entomopathogenic fungi and the development of fungal biopesticides.β-tubulin is an elementary subunit of microtubules that form the cytoskeleton,participating in a wide range of cellular processes.In this paper,the effects of β-tubulin gene on cell morphology,sporulation,stress tolerance and virulence of Metarhizium acrium were studied by gene knockout and recovery.Knockout of β-tubulin impaired proper nuclear segregation,lipid droplet transport,and deposition of chitin to the cell wall.M.acridum β-tubulin mutants displayed wavy hyphal growth and densely packed,wrinkled colonies.Phialides reduction and conidial yield decreased significantly.Compared with WT,Δβ-tubulin had a significant reduction in virulence on the ocust Infection structures have bifurcated germ tubes;Appressoria formation rate decreased significantly,appressoria were aberrant in morphology and displayed decreased turgor pressure in Δβ-tubulin.The ability of the Metarhizium acridum β-tubulin mutant to proliferate in the insect hemolymph both in vitro and in vivo was also significantly reduced.Δβ-tubulin has no significant differences compared with WT in response to UV and heat treatment.So,β-tubulin has important functions for hyphal growth,sporulation and virulence,and not affects resistance characteristics.Ppt1(yeast)/PP5(humans)contains a unique tetratricopeptide repeat(TPR)domain and has been implicated in signal transduction mediated growth and differentiation,DNA damage/repair,cell cycle progression,and heat shock responses.Little,however,is well known concerning the functions of Ppt1/PP5 in filamentous fungi.Targeted gene deletion of the single Ppt1/PP5 gene in the insect pathogenic fungus,Metarhizium acridum,resulted in obviously changes in the colony morphology and conidiation type(from normal conidiation to microcyle conidiation)versus wild type strains.MaPpt1 mutants were unaffected in heat shock response or insect virulence,but it has higher UV-B tolerance than WT(wild type).Experiments containing an eGFP-tagged MaPpt1 fusion protein revealed dispersed cytoplasmic localization in spores,but enrichment at the septa in growing hyphae.11 potential target proteins were identified via an examination of the altered phosphoproteome of MaPpt1 versus wild type strains.DGE profiling(ΔMaPpt1 vs WT)data revealed 1846 possible regulative genes.The results of the two methods co-revealed that MaPpt1 involves fatty acid metabolism,DNA rapier,cell cycle,and glycolosis/gluconeogenesis signaling networks.The study provides a scientific basis for sporulation regulation and UV tolerance of filamentous fungi such as Metarhizium acridum.