BbAPL, A Function-Unknown Gene Involved In Aerial Hyphae Formation Of Beauveria Bassiana

Entomopathogenic fungi are important natural control agents for many insects. Compared with other pathogenic microbes,insect-pathogenic fungi infect their hosts by penetrating the host cuticle directly and have a significant superiority for practical microbial control of sucking-insects.However,the efficiency of entomopahtogenic fungi is easy to be affected by environmental factors,and inconsistent results limit their widespread application of fungal insecticides in integrated pest management(IPM). Understanding the mechanism of fungal pathogenesis and tolerance to adverse condition will promote the commercial development of mycoinsecticides.Beauveria bassiana is one of the most important entomopathogenic fungi.To identify genes involved in pathogenicity and development of B.bassiana,T-DNA insertion mutant collections were constructed by Agrobacterium tumefaciens-mediated transformation.A deficient mutant in colony morphology and conidiation,namely 212 was isolated from the collections.A function-unknown gene was isolated from mutant 212 and its function was investigated by gene disruption through homologous recombination.The mainly results are as follows:1 The identification and cloning of T-DNA insertional mutant1.1 Mutant identification and characterizationTo identify genes involved in pathogenicity and development of B.bassiana, T-DNA insertion mutant collections had been generated by Agrobacterium tumefaciens-mediated transformation.From the collections,a deficient mutant 212 in colony morphology and conidiation was isolated.Southern blotting result indicated that T-DNA was a single copy in mutant 212 genome.In order to identify the disrupted gene in 212,DNA flanking the T-DNA insertion was isolated using YADE method followed by sequencing.Homology searches were carded out on NCBI web site.The results suggested that T-DNA was inserted into the encoding region for a hypothetical gene in mutant 212.1.2 Isolation and sequence ananlysis of BbAPLThe genomic DNA and cDNA sequences of a function-unkown gene were cloned by YADE and 3'RACE methods,respectively.This gene consisted of 1489 bp and contained a 58-bp intron.Its ORF encoded a protein of 476 aminoes with a Wr of 49.8 kDa and pI of 7.14.The deduced amino sequence contained a conserved acid phosphatase domain.Thus,this gene was named BbAPL(Acid Phosphatase-Like in_B. bassiana).There was no any function-known gene significantly similar to BbAPL.The result of southern blotting showed that BbAPL was single copy in the genome of B. bassiana.2 Expression and localization of BbAPL2.1 Expression and localization of BbAPL in B.bassianaTo detect BbAPL localization in B.bassiana,BbAPL::eGFP gene fusion was produced by introducing the eGFP to the C-terminus of the BbAPL ORF and controlled by native promoter of BbAPL.The gene fusion was transformed into wild-type strain Bb0062,and transformants containing BbAPL::eGFP were selected.Fluorescence could be easily detected in conidia and hyphae.The result was consistant with RT-PCR which showed that BbAPL transcripted persistently in aerial hyphae(4 days and 8 days) and conidia(14 days).Fluorescence could be detected in aerial hyphae but not in sustrate hyphae.BbAPL::eGFP mainly accumulated in cytoplasm and on septum.2.2 Phosphatase activity detectionBbAPL was successfully expressed in P pastoris and the activity was detected with different substrates.BbAPL was active on monoester pNPP(p-nitrophenyl phosphate) but not diester bis-(ρ-nitrophenyl).Although,BbAPL shared a similar conserved domain(RHG)with phytase from other filamentous fungi,it was not active on phytic acid.And the optimal pH was 6.0.3 Function analysis of BbAPLIn order to investigate the function of BbAPL in B.bassiana,this gene was disrupted by homologous recombination and four BbAPL-disrupted transformants(RC2, RC53,RC59 and RC63)were identified by PCR and southern blotting.3.1 BbAPL did not affect the tolerance of to adverse conditions.The growth of BbAPL-disrupted mutants had no significant difference with that of wild type strain at adverse conditions(high temperature or osmotic stress).And the conidia tolerance to ultraviolet and high temperature was also not changed.These results indicated BbAPL was not essential for the tolerance to adverse condition.3.2 Disruption of BbAPL caused thin colony and decreased biomass and conidial yield. The colony of each BbAPL-disrupted strain on Czapek medium was thinner than that of wildtype strain.But the colonies of all strains had no significant difference on SDAY medium.The biomass of each transformant in liquid Czapek medium was determined. Results showed that the biomass of BbAPL-disrupted strains obviously decreased and was only 33%of that of wild type strain.Meanwhile,conidial yield of each BbAPL-disrupted stain was also significantly decreased and attained 45%of that of wild type strain.The decrease in conidial yield possiblly resulted from the thinner colony and the reduced biomass.3.3 BbAPL was required for the formation and development of aerial hyphae.Aerial hyphae in BbAPL-disrupted tansformants were significantly thinner than that in wild type strain.Further investigation suggested that the development of aerial hyphae was inhibited due to the disruption of BbAPL.The penetration and formation of aerial hyphae of BbAPL-disrupted stains was lagged compared with that of wild type strain.3.4 Disruption of BbAPL decreased hydrophobicity of aerial hyphae and conidia, as well as the adhesion to hydrophobic surface.In filamentous fungi,hydrophobicity of the colony surface correlated with formation of aerial hyphae.To investigate this,hydrophobicity of each strain was detected.Surface hydrophobicity was significantly declined in BbAPL-disrupted strains. A phase(water/hexadecane)distribution test showed that hydrophobicity of conidia was also decreased by 16%-20%compared with that of wild type strain.The attachment to hydrophobic surface was partially affected,and adhesion of aerial conidia and hyphae to hydrophobic surface declined by 8%and 13%,respectively.3.5 Anastomosis formation between aerial hyphae in BbAPL-disrupted transformants.In colony of BbAPL-disrupted strain,aerial hyphae were combined through short hyphal bridge,but it was hardly founded in that of wildtype strain.We suggested that the decreased hydrophobicity of aerial hyphae was one of the reasons of anastomosis formation.3.6 Growth of BbAPL-disrupted strain was sensitive to high concentration of abiophosphate. The growth of BbAPL-disrupted strains was different on Czapek contained various concentrations of abiophosphate(K₂HPO₄).The growth of aerial mycelium in BbAPL-disrupted strains was inhibited on high concentration abiophosphate medium compared with that of wildtype strain.But on low concentration of abiophosphate,the growth of aerial mycelium in BbAPL-disrupted strains has no significantly difference with that of wildtype strain.This indicated that BbAPL correlated with utilization of abiophosphate.3.7 BbAPL was not essential for the virulence against aphid.Bioassay result showed that the accumulative mortalities had no significant difference after inoculation with conidia suspension(1×10⁸ conidia/ml)for 168 h.But, if aphids were washed by spraying of 0.05%Tween-80 after inoculation with conidia suspension,the accumulative morality(59%-63%)caused by BbAPL-disrupted strains was lower than that(70%)by wild type strain.It could be accounted for the effect of BbAPL on attachment to hydrophobicity surface.