BbCrpa,a P4-ATPase,Confers The Resistance Of Beauveria Bassiana To Cyclosporine A And Fk506 Via Vesicle-Mediated Detoxification

Natural active products produced by microorganisms are important sources of pharmaceuticals.Meanwhile,many of them are detrimental to agricultural production and human health.Therefore,revealing the mechanism of cellular detoxification of the toxins is of great significance for both plant disease control and human health protection.Type ? P-type ATPases(P4-ATPases)are implicated in the initiation of vesicle biogenesis by translocating specific phospholipid substrates through membranes.Accumulating evidences indicate that P4-ATPases are involved in a variety of physiological processes,including cell surface growth,organelles biogenesis,endocytosis,secretion,protein sorting,and protein transport and storage.Although it has been reported that P1B-ATPases have a role in detoxification of heavy metals,little is known about whether P4-ATPases participate in detoxification of toxic secondary metabolites.If so,what is the mechanism?Cyclosporine A(CsA),a neutral lipophilic cyclic polypeptide and FK506(tacrolimus),a macrolide lactone,are produced by Beauveria nivea(Tolypocladium inflatum)and Streptomyces tsukubaensis,respectively.The two microorganismal products display antifungal activity and both of them are well-known immunosuppressive drugs to prevent graft rejection in organ transplant patients and autoimmune-disease therapy.Unfortunately,both drugs also result in significant side effects,such as nephrotoxicity and hepatotoxicity.However,the mechanism underlying resistance to these drugs has remained unknown.The entomopathogenic fungus Beauveria bassiana is a widely used biocontrol agent against pests.This fungus is also an endophyte of plants.Thus,B.bassiana becomes an emerging model system to study fungal growth,stress response,pathogenesis,and interactions between fungi and insects/plants.Our previous studies have indicated that B.bassiana shows high resistance to CsA and FK506.However,the mechanism underlying the resistance of the drugs has been totally unknown.In order to dissect the mechanism underlying the resistance,Agrobacterium tumefaciens-mediated transformation was used to construct the T-DNA insertional mutagenesis library of the fungus.Mutants that became sensitive to CsA were identified.The predicted protein,named as BbCrpa,responding to the resistance to CsA belongs to P4-ATPase subfamily.BbCRPA gene-knockout strain(?BbCRPA)became sensitive to CsA and FK506,and the CsA-/FK506-resistance defect could be complemented by the expression of BbCRPA in the?BbCRPA background.Further studies indicated that BbCrpaconfers resistance of B.bassiana to CsA and FK506 via vesicle-mediated transport pathway that deliver the compounds into detoxifying vacuoles for storage or degradation;the last 11 aa residues of BbCrpaN-terminus contains vacuolar localization signal and the mutagenesis of tyrosine-1325(Y1325)in the C-terminus renders partial loss of detoxification function of BbCrpa.Yeast two-hybrid and co-immunoprecipitation assays showed that CsA/FK506 promotes the interaction between BbCrpaC-terminus and a t-SNARE(target soluble N-ethylmaleimide-sensitive factor attachment protein receptor)PepB^Pep12,suggesting that CsA/FK506 not only is the cargo of vesicle-mediated transport,but also might participate in targeting the cargo-bearing vesicle to the vacuole.In addition,the results showed that BbCrpais functionally different with Saccharomyces cerevisiae P4-ATPase Drs2p and BbCrpamight be a novel member of P4-ATPase family.The main results are as follows:1 BbCrpaconfers the resistance of the cell to CsA and FK506The random insertion(T-DNA)library of B.bassiana was constructed via Agrobacterium-mediated transformation method that used p K2-Bar::Gus as transformation vector.Two CsA-sensitivity mutants were identified from?20,000colonies.The two T-DNA insertions took place in the same gene which owns a 4080 bp ORF and encodes a predicted protein with 1359 aa.Cluster analysis indicated that the protein belongs to Type IV P-type ATPases(P4-ATPases)and thus named as BbCrpa(cyclosporine A resistance P4-ATPase).It has 10 transmembrane domains and a cytosolic 268 aa(1-268 aa)N-terminus and an 86 aa(1274-1359 aa)C-terminus.The closest homologue in yeast P4-ATPases to BbCrpais Drs2p that shows 60%idenlity with BbCrpa.Susceptibility testing showed that BbCRPA gene-knockout strain(?BbCRPA)became sensitive to CsA and FK506,while the complemented strain(?BbCRPA::BbCRPA)showed resistance to the drugs similarly to the wild-type.Moreover,ectopic expression of BbCRPA in Verticillium dahliae could increase the resistance of the mutant to CsA.In contrast to BbCrpa,however,ectopic expression yeast Drs2p encoding gene DRS2 in?BbCRPA could not complement the CsA-/FK506-resistance defect,suggesting that BbCrpais functionally different with Drs2p in terms of toxin resistance.2 CsA/FK506 is transported to vacuoles through TGN-EE-LE vesicle transport pathway mediated by BbCrpaCsA and FK506 were labeled with 5-Carboxyfluorescein(5-FAM)to monitor the distribution of the drugs in the cells of the wild-type and?BbCRPA.Confocal microscopy observation showed that in the wild-type cells,CsA-5-FAM was colocalized with TGN[trans-Golgi-network,labeled by mRFP-fused PH^OSBP(the pleckstrin homology domain of the human oxysterol binding protein)],vesicles(labeled by fluorescent dye FM4-64),EEs(early endosomes,labeled by small GTPase Rab5),and LEs(late endosomes,labeled by small GTPase Rab7).Finally,the fluorescent labeled CsA was gathered in vacuoles.However,compared to the wild-type cells,CsA-5-FAM was hardly observed in vacuoles of?BbCRPA cells.Similar phenomenon was observed in the FK506-5-FAM treated?BbCRPA cells.These data indicated that CsA/FK506 is transported into vacuoles through“TGN-EE-LE-Vacuole”vesicle transport pathway which was mediated by BbCrpa.3 Subcellular localization of BbCrpaBbCrpawas fused with e GFP(enhanced green fluorescent protein)or mRFP(monomeric form of the red fluorescent protein)to monitor its subcellular localization.Confocal microscopy observation showed that e GFP::BbCrpaappeared in the apical plasma membrane and cytosolic structures in germinating conidia.In addition,the e GFP signal emerged in the subapical region termed Spitzenk(?)rper in germ tubes.In mature hypha,BbCrpalocalized primarily to TGN,then to secretory vesicles,EEs,LEs,and finally converged into vacuoles.Time-lapse dual-label microscopy combining either e GFP::BbCrpaand FM4-64 or e GFP::BbCrpaand mRFP::PH^OSBP indicated the dynamic trafficking through the pathway from the TGN to vesicle-early/late endosome-vacuole.What's more,the localization of BbCrpawas tightly associated with that of CsA or FK506 signal,and both BbCrpaand CsA/FK506 signal were finally converged into vacuoles.These results suggested that the vesicle,the formation of which is initiated by BbCrpa,could recognize CsA and FK506 cargos at TGN and then transport them into vacuoles for detoxification.In contrast to e GFP::BbCrpa,almost no e GFP::Drs2p singal was perceived in B.bassiana vacuoles.Combining with CsA-/FK506-detoxification defect of Drs2p,the data,confirmed that CsA/FK506 was transported into vacuoles for detoxification again.4 Contributions of BbCrpaN-and C-terminal residues to CsA/FK506detoxification4.1 Terminal graft with that from BbCrpaconfers the detoxification function to Drs2pOur previous studies have established that deletion of BbCrpaN-or C-terminus resulted in the defect of CsA resistance of B.bassiana.To further dissect the important roles of BbCrpaN-and C-terminus in drug detoxification,the N-and C-terminus of Drs2p were substituted with that from BbCrpa.The resulting genes were then expressed in?BbCRPA cell.The replacement with either N-or C-terminus along was unable to endow B.bassiana with the CsA-/FK506-resistance.However,the simultaneous substitution of both of BbCrpaN-and C-terminus to Drs2p led to a small but observable increase of resistance against the drugs.These dada demonstrated that both N-and C-terminus were indispensable for the CsA and FK506 detoxification.4.2 Last 11 aa residues of BbCrpaN-terminus contains vacuolar localization signalFused the last 11 aa residues(B^N258-268,FASFLPKFLFE)with mRFP,the protein could colocalize with e GFP::BbCrpain vacuoles,indicating that the last 11 aa residues of BbCrpaN-terminus contains vacuolar localization signal.However,further deletion beyond 258(B^N259-268)caused a loss of the vacuolar targeting.In addition,when the K264(Lys)was replaced by Ala(A)in the last 11 aa residues in BbCrpaC-terminus,the mutated residues lost the vacuolar guiding function,suggesting the requirement of the hypothetical mono-ubiquitination in vacuolar localization associated the last 11 aa residues of BbCrpaN-terminus.4.3 Site-directed mutagenesis of tyrosine-1325(Y1325)in the C-terminus decreases detoxification activity of BbCrpaThe results from our previous studies have indicated that tyrosine-1325(Y1325)plays important role in the BbCrpa-mediated detoxification.In this study,when Y1325was replaced by Ala(A),the substitution led to a significant decrease in CsA/FK506-resistance compared to the wild-type.However,the mutant Y1325A showed the resistance to CsA/FK506 higher than?BbCRPA,suggesting that the functional redundancy might exist between Y1325 and Y1341 and/or Y1350 in the1325-1359 aa region of BbCrpaC-terminus.To test this hypothesis,double and triple substitution mutations of the three Tyr residues were generated.The double substitutional transformant Y1325A-Y1341A or Y1325A-Y1350A,and triple substitutional transformant Y1325A-Y1341A-Y1350A exhibited a significant decrease in CsA/FK506 resistance similar to?BbCRPA.However,the double substitution mutation of Y1341A-Y1350A showed the resistance to the drugs similar to Y1325A.These data indicated that Y1325 has an important role in CsA/FK506 resistance,which is functionally redundant with Y1341-Y1350.5 BbCrpaC-terminus interacts with PepB^Pep12 and CsA/FK506promotes the interactionTo identify the potential protein(s)that interact(s)with BbCrpaand participate(s)in CsA/FK506 transport pathway in B.bassiana,yeast two-hybrid assay was performed using full length BbCrpaas a bait.A predicted protein which might interact with BbCrpawas isolated.This protein contains syntaxin-2(SN)and t-SNARE coiled-coil(CC)conservative domains and a single transmembrane domain and shows 53.5%identity to Aspergillus nidulans t-SNARE PepA^Pep12,and hence is named as PepB^Pep12.Down-regulation of PEPB using RNA interference decreased the resistance to CsA and the gene expression was induced by CsA/FK506,suggesting that PepB^Pep12 is also involved in the CsA/FK506 detoxification associated with BbCrpa.PepB^Pep12 was labeled by e GFP or mRFP to monitor its subcellular localization.Confocal microscopy observation showed that PepB^Pep12 localizes to TGN,EEs,and LEs.Besides,PepB^Pep12could colocalize with BbCrpa.Co-immunoprecipitation(Co-IP)assay confirmed the interaction of BbCrpaC-terminus and PepB^Pep12.Importantly,CsA/FK506 could promote the interaction.Yeast two-hybrid assay revealed that the CC domain of PepB^Pep12 was responsible for the interaction between BbCrpaC-terminus and PepB^Pep12.These data suggested that CsA/FK506 not only serves as the cargo of the vesicle transport but also participates in guiding the vesicle to the destination membrane.In conclusion,this study revealed the molecular mechanism of the intrinsic resistance possessed by the entomopathogenic fungus B.bassiana against CsA and FK506 and demonstrated a new function of P4-ATPases in cell detoxification which will enrich our knowledge of the proteins.Furtheremore,this vesicle-mediated cell detoxification strategy provides a new approach in plant disease control,food safety management,and human health protection,and thus will produce tremendous economic and social benefits.