Identification Of The Genes Regulated By BbMPK1 MAPK Involved In The Penetration Of Insect Cuticle In Beauveria Bassiana

Entomopathogenic fungi are important insect mortality agents that have been intensively investigated for application in pest insect control.However,little is known about the molecular basis of pathogenesis which limits the widespread use of fungal agents.And thus,an understanding of the molecular mechanism of fungal pathogeniciy is essencial for comencial development and improvement of these biocontrol fungi.Mitogen-activated protein kinases（MAPKs）,a family of serine-threonine protein kinases,are widespread in eukaryotic cells and play crucial roles in transduction of a variety of extracellular signals and regulation of various development and differentiation processes.Recent studies showed that MAPK pathways are involved in the development,differentiation,virulence and adaptability to the adverse environment of fungi,demonstrating that MAPK cascades are important clues to understand the molecular mechanism of pathogenesis,development and differentiation of fungi.In our previous study,a MAPK-encoding gene BbMPK1 was isolated from an important insect fungal pathogen,Beauveria bassiana,and shown to encode a functional homolog of yeast FUS3/KSS1.A BbMPK1 null mutation was generated in B.bassiana by targeted gene replacement,and the resulting mutants showed defects in penetration into insect cuticle and in appressorium formation.And thus,it is very useful to detect the difference of gene expression between the mutant with the wild-type strain during penetration and appressorium formation for elucidating the molecular mechanism involved in penetration and appressorium formation of the fungal pathogen.In order to understand the mechanism of infection of B.bassiana,we isolated genes involved penetration and appressorium by the suppression subtractive hybridization（SSH） method using the wild type strain and the BbMPK1 disruption mutant.1 Isolation of genes regulated by BbMPK1 involved in penetration in B.bassianaWhen B.bassiana wild-type strain Bb0062 penetrated and invaded the cuticle of the 3^rd instar larvae of Pieris rapae 36 h after inoculation,RNA were extracted from cuticle of the inoculated insect and used to synthesize cDNAs which was as the tester of suppression subtractive hybridization（SSH）.The driver cDNA was synthesized using RNA isolated from the insect cuticle inoculated by the BbMPK1 disruption mutant△BbMPK12167 as same as the wild-type strain.The SSH library was constructed and the genes possibly regulated by BbMPK1 was screened and sequenced.Forty clones differently expressed between the wild-type and the mutant strains were screened using the PCR-Select Differential Screening method and sequenced.The results indicated that the 40 clones encode 13 different amino sequences.Surprisingly,the 13 putative amino sequences showed no homology to any fungal genes.Among these sequences,four were highly homologous to the proteins related to the insect immunization response（insect serpin 1,lectin 4,lebocin 4 and chemosensory protein CSP2）,and five were homologous insect cuticle protein（cuticle protein, cuticle protein 3,cuticular protein CPR67,cuticular protein CPR103 and cuticular protein CPFL4B）. Other 4 putative amino sequences show homology to insect olfactory protein,Nicotiana tabacum cytochrome P450 like_TB,Pisum sativum senescence-associated protein and a insect protein of unknow function,respectively.These resuld suggested that the isolated genes in this study possibly are involved in the insect immunity response to the fungal pathogen.2 Isolation of genes regulated by BbMPK1 related to appressorium formation in B.bassianaIt was demonstrated previously that disruption of BbMPK1 in B.bassiana resulted in a defect in appressorium differentiation.Here,we induced appressorium formation of B.bassiana on cicada hind wings.RNA was extracted and used to synthesize cDNA which was as the tester of the suppression subtractive hybridization（SSH）.The driver cDNA was synthesized using RNA isolated from mutant induced on the cicada hind wings as same as the wild-type strain.The SSH library was constructed and the genes possibly regulated by BbMPK1 was screened and sequenced..One hundred and ninety five clones differently expressed between the wild-type strain and the△BbMPK1 mutant was screened from 1047 clones using the PCR-Select Differential Screening method and sequenced.Analysis of sequence showed that these 195 clones encode 75 different amino sequences.Thirteen putative amino acid sequences showed homology with fungal mitochondrial protein,hydrophobin 3 precursor,translation elongation factor 1 alpha,aldehyde dehydrogenase,hydrophobin,copper-zinc superoxide dismutase,involucrin repeat protein, non-histone chromosomal protein 6,chromatin assembly factor 1 subunit A,sugar transporter, ADP-RIBOSYLATION FACTOR,histone H3,and ATP-dependent RNA helicase,respectively. Twenty-six putative proteins were homogous with hypothetical proteins of some filamentous fungi such as Magnaporthe grisea,Neurospora crassa,Aspergillus nige,Gibberella zeae,Aspergillus terreus,Chaetomium globosum,and so on.Tweenty two putative amino sequences showed homology with some proteins or hypothetical proteins of bacterium,protozoon and vegetoanimal. Other 14 sequences showed no significant similarity with any protein in GenBank.