Intraspecific Variation In Tolerance Of Metarhizium Anisopliae To Upper Thermal Limits Of Summer And Characterization Of A New Conidial Wall Protein By Gene Cloning And Functional Analysis

As active ingredients of mycoinsecticides,aerial conidia of the entomopathogenic fungus, Metarhizium anisopliae,are readily produced on solid substrates such as small grains and formulated for wide application to insect control.The tolerance of aerial conidia of a fungal candidate to the upper limits of summer temperatures as one of importance environmental stresses is crucial to the field persistency and efficancy of its formulations.Proper quantification of conidial thermotolerance relies upon an efficient system of bioassay that generates biologically robust data for analysis.Such a system makes it possible to screen out desired candidates from a large collection of fungal stains.On the other hand,hydrophobic feautre of aerial conidia is known relating to their attachment to pest integument,a crucial step for the whole process of fungal infection to host insects,and is considered in association with the structure of cell wall that involves specific receptor-ligand and non-specific hydrophobic or electrostatic mechanisms.Cell wall protects the cell from environmental stress and defines an interface of fungus-insect interaction.Cell wall proteins(CWP) include multi-purpose hydrophobins or hydrophobin-like proteins that take parts in morphogenesis, hydrophobicity,adhesion,pathogenicity,antigenicity and defense for host immune reaction.This study sought to elucidate the variation of conidial thermotolerance of 18 M. anisopliae strains(with different host and geographic origins) under the thermal stress of 48℃and to characterize distinguished types under the stresses of 38-45℃by developing a quantitative assay system.A diversity of SDS-insoluble,formic-acid-extractable proteins from the conidial walls of the 18 isolates was examined.From those,a new 9.9-kDa conidial wall protein(CWP10) was characterized by gene cloning,protein expression and immunogold localization.The results are summarized as follows:Intraspecific variation of M.anisopliae tolerance to the upper thermal limits of summer.To develop an efficient assay system,aerial conidia(produced on steamed rice) of eight M.anisopliae(Ma) isolates,four M.anisopliae var.anisopliae(Maan) and six M. anisopliae var.acridum(Maac) with different host and geographic origins were wet-stressed at 48℃for≤180 min and used to initiate 14-day colony growths at 10-35℃.Survival indices (relative to unstressed conidia) of each isolate examined at 15-min intervals were fitted very well to a decline model(r²â‰¥0.975),yielding median lethal times(LT₅₀s) of 14.3-150.3 min as thermotolerant indices for the tested isolates at 48℃.Seven grasshopper-derived isolates from Africa had the mean LT₅₀ of 110(73-150) min but did not grow at 10 or 15℃.Five non-grasshopper isolates grew at 10-35℃but had much lower mean LT₅₀ of 16(10-26) min. Three isolates with typically low(Typeâ… ),medium(Typeâ…¡) or high(Typeâ…¢) tolerance to 48℃were separately assayed for up to 4-day tolerance of their conidia to the wet-stresses at 38,40,42 and 45℃,respectively.The resultant LT₅₀s decreased to 20,53 and 167 min at 48℃from 507,1612,and 8256 min at 38℃for typesâ… ,â…¡andâ…¢,respectively.For the distinguished types,the logarithms of the LT₅₀s were significantly correlated to the temperatures of 38-48℃with an inverse linearity(0.975≤r²â‰¤0.999).The method developed to quantify fungal thermotolerance would be useful for screening of fungal candidates for improved pest control in summer.Extraction and analysis of M.anisopliae conidial wall protines.Conidial wall proteins(CWP) were separately extracted from the aerial conidia of the 18 M.anisopliae isolates using formic acid.The overall mean(±SD) CWP contents(μg/mg conidia) differed significantly among the extracts of the tested isolates(F_17,36=221.8,P＜0.01),ranging from minimal 3.4±0.5(Maac 6421) to maximal 50.2±1.2(Ma 2421).On average,CWP proteins were richer in Ma(28.4±12.3) or Maan isolates(27.3±15.1) than Maac ones(14.7±8.4).The CWP contents also differed within each group(Ma:F_7,16=188.5,P＜0.01;Maac:F_5,12=129.8, P＜0.01;Maan:F_3,8=584.7,P＜0.01).At least nine bands(9.0-18.4 kDa) were distinguished from the SDS- PAGE profiles of the conidial extracts of the tested isolates except Maac 6421, which had no visible band perhaps due to its lowest content.A band of ca.9.9 kDa was shared by several isolates and thus selected as a target CWP(named CWP10) for the following study.Gene cloing and functional analysis of a new conidial wall protein(CWP10) from M. anisopliae.The target protein,CWP10,purified from the formic-acid extract of the aerial conidia of the isolate Maan 4132 was characterized as a new non-hydrophobic,non-glycosylated protein by gene cloning and protein expression and found existing in seven of the 18 M.anisopliae isolates examined.This 120-aa protein was encoded by a 363-bp, single-copy gene with three introns and up- and downstream sequences and proven more abundant in the intine of conidial wall than in the exine and cytoplasm via immunogold localization.Despite non-hydrophobic nature,the CWP10 included a 32-aa signal peptide with a central hydrophobic region of ca.10 residues within the N-terminus,which is terminated with four polar amino acids(QYYT).The cloned gene encoding the protein was successfully transformed into a wild strain of Beauveria bassiana in which the same protein was absent.The protein was expressed well in aerial conidia of B.bassiana,less in submerged conidia but absent in blastospores.Consequently,aerial conidia of the transformant showed significant increases of their hydrophobicity(10.5%) and adhesion to the hydrophobic substratum of polystyrene(55.2%) than the wild strain counterparts.However,the expressed protein had no significant effect on conidial tolerance to either a wet stress of 48℃or an iradiation of the weighted 365-nm UV-A dose of 7.0 J/cm² or of the weighted 312-nm UV-B dose of 0.7 J/cm².This is the first report on a non-hydrophobic CWP enhancing conidial hydrophobicity and adhesion to the hydrophobic substratum.In summary,the whole study resulted in the development of an efficient assay system that enables to accurately quantify fungal thermotolerance and would facilitate the selection of desired candidates for use in microbial control of insect pests.From the diverse CWP components of M.anisopliae,CWP10 was recognized and characterized as a new non-hydrophobic, non-glycosylated protein that has proven contributing significantly to the hydrophobicity of aerial conidia and their adhesion to hydrophobic surface.The results provide new insights into the importance of conidial wall proteins in fungal biology and suggest a promising approach to improving ecological fitness or field persistence of the fungal biocontrol agents.