Role Of A Conidia Cell Surface Protein,eukaryotic Aspartic Protease,in Development And Virulence In The Insect Fungal Pathogen,Beauveria Bassiana

Aspartic proteases?APs?are a class of proteolytic enzymes with aspartic acid residues as catalytic active sites,which are widely found in animals,plants,fungi and viruses.It is demonstrated that APs are involved in the digestion of animals and development of plants,parasitism and virulence in fungi and bacteria.However,roles of APs are unclear in development and virulence in fungal species.Our group isolated a cell surface protein with locus tag of BBA₀4972,a from the insect fungal pathogen,Beauveria bassiana conidia when identified cell surface proteins from different morphological cells,conidia,blastospores and hyphal bodies,which is annotated as eukaryotic aspartyl protease,designed CsAP1.In this thesis,CsAP1 was characterized in the development and virulence of B.bassiana by combination of gene expression analysis,gene disruption,overexpression and reverse complementation strategies.The main findings are as follows.1.Sequence analysis and expression profiles of CsAP1Sequence analysis indicated that the coding region of CsAP1 with 1939 bp contained an intron and encoded a 621-amino-acid polypeptide with a theoretical molecular weight of 64.815 kDa and a calculated pI of 4.39.Bioinformatic analysis revealed that CsAP1belonged to the eukaryotic aspartyl protease family,which contained two aspartic acid residues and two characteristic catalytic fingerprints"DTGS and DTGT".The phylogenetic analysis suggested that the CsAP1 of B.bassiana clustered in group with APs of Sclerotinia sclerotiorum.Gene expression analysis showed that CsAP1 was highly expressed in conidia,but repressed in the hyphal bodies,and induced by insect nutrition.The CsAP1::eGFP was constructed under the constitutive promoter P_gpdA and introduced into B.bassiana to investigate subcellular localization of the protein.The results showed eGFP fluorescence distributed on the cell surface,however,no eGFP signal was detected in other mophological cells,suggesting that the fusion protein might be secreted.These data demonstrated that CsAP1 is a conidia surface protein,which might be involved in utilization of insect nutrient.2.CsAP1 is involved in fungal development and cell surface featureTo reveal role of CsAP1 in B.bassiana,CsAP1 disruption mutant??CsAP1?,overexpression?OE-CsAP1?and reverse complementation?Comp?strains were generated.Disruption of CsAP1 did not obvious changes in colony growth,but resulted in a significant reduction in conidiation.?CsAP1 displayed a distinct decrease in conidia yield either on basic medium or rich medium,with 26.7%⁷6.7%decrease as compared to the wild type strain?WT?.No obvious difference was detected in conidia yield between Comp and WT strains.The results suggested that CsAP1 is involved in conidiation.Cell surface assays indicated that disruption of the gene led to a significant decrease in conidia hydrophobicity?²2.93%?,but dramatic increase was examined in OE-CsAP1conidia?⁸8%?,compared to WT conidia.Adhesion of OE-CsAP1 conidia on the hydrophobic matrix was increased by 33.18%³8.32%.On the hydrophilic matrix,adhesion of OE-CsAP1 conidia decreased by 38.88%⁴3.18%and?CsAP1 conidia increased by 23.67%as compared to those of WT.However,no obvious difference was probed in adhesion either on the hydrophobic matrix or the hydrophobic matrix between Comp and WT conidia.It was noted that adhesion of OE-CsAP1 conidia on weakly polar matrix was significantly lower?44.48%⁶8.26%?than those of WT conidia.Whereas,disruption of CsAP1 did not cause any obvious changes in ability of conidia to weakly polar substrate.The results suggested that CsAP1 was involved in formation of the cell surface feature.3.Overexpression of CsAP1 results in an increase in production of secreted proteaseProduction of secreted proteases was assayed on either skim milk powder or casein-contained agar plates.Halo-to-colony diameter ratios were 0.69/0.21 or 0.23/0 for OE-CsAP1 and the wild-type strains respectively.The results showed that overexpression of the gene resulted in a significant increase in activities of extracellular proteases and Whereas,no obvious difference in extracellular activities between the gene disruption mutant and WT strains.4.CsAP1 is involved in the utilization of insect nutrition in B.bassianaTo examine role of CsAP1 in utilization of insect nutrient,conidia were inoculated on silkworm cuticle or haemolymph–contained agar plates.The?CsAP1 displayed sparse aerial hyphae in growing colony on the insect nutrient-contained agar,compared to compact colony for WT.Obvious decrease in biomass accumulation was also detected for the?CsAP1.Whereas,it is surprised that colony growth and biomass accumulation of OE-CsAP1 was similar to those of WT strain.The data demonstrated that CsAP1 was involved in the utilization of insect nutrition.5.CsAP1 is a virulence factor of B.bassianaThe third-instar larvae of the Galleria mellonella were used for bioassays by inoculation of fungal conidia with topical application and injection.The results showed that?CsAP1 strain displayed as slight decrease in virulence in both of bioassays and OE-CsAP1 strain increase.While,no difference was examined in virulence between Comp and WT strains.The biomass of hyphal bodies show a decrease?27.21%⁵0.7%?in proliferation of hyphal bodies was determined in the?CsAP1 mutant as compared WT while the quantity of hyphal bodies OE-CsAP1 was increased by 7.6%⁵9.5%.The results suggested that CsAP1 was involved in the utilization of insect nutrition and contributes fungal virulence in B.bassiana..