Mechanism On The Loss Of Function Of P4-ATPase VdDrs2 In Reducing Of Verticillium Dahliae Pathogenicity

Verticillium dahliae is one of the most notoriously devastating phytopathogenic fungi,which causes Verticillium wilt disease in a wide range of hosts including many commercial crops and trees,including cotton,resulting huge economic losses worldwide.Due to frequent mutation,complex pathogenic mechanisms,and the lack of effective germplasm resources for disease resistance in upland cotton,the progress of cotton breeding against Verticillium wilt is slow.As the result,the cotton area suffered from Verticillium wilt increases year by year.Therefore,Verticillium wilt is called the"cancer"of cotton.Studying the pathogenic mechanism of V.dahliae and its interaction with the host can provide new ideas and new strategies for cotton breeding against Verticillium wilt,which has important theoretical and practical significance.P4-ATPases are ATP-driven flippase enzymes that facilitate the initiation and formation of vesicles by transporting phospholipids to asymmetric membrane distribution（from the outside of the cytoplasm or the inside of the organelle to the cytoplasmic side）.The P4-ATPases associated vesicle trafficking is involved in various biological processes.Previous studies have shown that P4-ATPases-involved vesicle trafficking is associated with the pathogenicity of human and plant pathogenic fungi.However,how do P4-ATPases influence the pathogenicity of pathogens?The cellular processes and molecular mechanisms are far from understood.Verticillium toxins are considered as important virulence factors for V.dahliae.Yet,whether is the vesicle transport involved in toxin transport and whether P4-ATPases are involved in the interaction between V.dahliae and host are also not clear.To answer these questions,we cloned and sequenced four P4-ATPase genes from V.dahliae,and investigated the effects of gene knockout or interference on the pathogenicity and antioxidant response of the P4-ATPases.It was found that the pathogenicity ofΔVdDrs2 decreased most significantly compared to wild-type strain V991,and theΔVdDrs2 was extremely sensitive to H₂O₂.Thus,the investigation of mechanism of VdDrs2 in the pathogenicity and anti-oxidative stress was carried out.The main results are as follows:1 Knockout of VdDrs2 leads to the greatest decrease in the pathogenicity of V.dahliaeFour candidate P4-ATPase genes（VdDrs2,VdDnf1,VdNeo1 and VdP4-4）were obtained by BLAST search against V.dahliae genome using the amino acid sequences of Saccharomyces cerevisiae P4-ATPases.VdDrs2,VdNeo1 and VdP4-4 were successfully knocked out in V.dahliae V991 by homologous recombination strategy.Due to the failure of the gene knock-out of VdDnf1,the expression of the gene was down-regulated by RNA interference technology.Infected cotton plants with the mutant strain and wild-type strain V991,it was shown that the pathogenicity of four P4-ATPases mutants to cotton was obviously reduced.Among them,ΔVdDrs2 showed the greatest decrease in pathogenicity.2 Knockout of VdDrs2 leads to V.dahliae extremely sensitive to H₂O₂The wild-type strain V991 and mutant strains were treated with different stress agents（MND,H₂O₂,Congred,CFW and KCl）.ΔVdDrs2 strain became extremely sensitive to H₂O₂ and did not grow at all on PDA plates containing H₂O₂.The growth of other mutants was not significantly different with the wild-type strain V991.3 Knockout of VdDrs2 delays V.dahliae infection,microsclerotia and melanin formation3.1 Knockout of VdDrs2 delays the infection of V.dahliae to cottonThe wild-type strain V991,ΔVdDrs2 and the complemented strain（Com）were inoculated into cotton by root-unwounded and root-wounded method,respectively.The symptom severity in the cotton caused byΔVdDrs2 was significantly reduced.Using root-unwounded method,the disease index ofΔVdDrs2 infected cotton was 9.2%,while that of wild-type strain V991 and of Com was 84.2%and 76.2%,respectively.For root-wounded method,the disease index of the cotton infected withΔVdDrs2 was90.0%,not significantly different with that of cotton inoculated with V991（96.7%）and Com（92.5%）.This indicates that VdDrs2 plays an important role in the infection of V.dahliae during penetrating cotton cells.eGFP gene was transformed into wild-type strain（eGFP-Vd991）andΔVdDrs2stain（eGFP-ΔVdDrs2）,respectively.The spores of eGFP-Vd991 and eGFP-ΔVdDrs2were inoculated with cotton roots.Fluorescence signal was observed in the spores on the surface of roots treated with eGFP-Vd991 and eGFP-ΔVdDrs2 spores,indicating that the spores are able to attach to the surface of cotton roots.Then,eGFP-labeled hyphae emerged in eGFP-V991-infected cotton root tissue and the hyphae grew up along vascular bundles.However,no obvious eGFP signal was observed in eGFP-ΔVdDrs2-infected cotton roots.The observation results further verified that the deletion of VdDrs2 impaired the penetration ability of the pathogen.3.2 Knockout of VdDrs2 decreases the sporulation,delays conidia germination and impairs the microsclerotia formation of V.dahliaeCompared with the wild-type strain V991 under the same culture conditions,colony ofΔVdDrs2 was significantly smaller,the sporulation yield decreased,and the spore germination delayed.The growth rate ofΔVdDrs2 was significantly lower than that of V991 under different carbon and nitrogen sources,indicating that the disruption of VdDrs2 delayed the growth of V.dahliae.Microsclerotia are the main dormant structure of V.dahliae in soil and the primary infection source of Verticillium wilt.Disruption of VdDrs2 severely decreased melanin deposition and disrupted microsclerotia formation of V.dahliae.4 VdDrs2 is localized to plasma membrane,vacuoles,and trans-Golgi networkIntroducing the eGFP::VdDrs2 fusion protein gene intoΔVdDrs2 strain could restore the mutant pathogenicity to cotton,indicating that the fusion of eGFP did not affect the function of VdDrs2.Laser confocal observation found that VdDrs2 was localized to the cytoplasmic membrane and vacuoles of V.dahliae.The trans-Golgi network（TGN）marker protein PH^OSBP fused with mRFP was co-expressed with eGFP::VdDrs2.The observations showed that eGFP::VdDrs2 co-localized with mRFP::PH^OSBP in aggregate structures.The above results indicate that VdDrs2 localizes to the plasma membrane,vacuole and TGN.The eGFP::VdDrs2 expressing strain was treated with vesicle transport inhibitors BFA,AG18 and Con A,respectively.Laser scanning confocal microscopy showed that AG18 and Con A had no obvious effect on the subcellular localization of eGFP::VdDrs2,while BFA,an inhibitor of Golgi-associated vesicle trafficking,led to the appearance of eGFP::VdDrs2 fluorescent punctate structures,indicating that VdDrs2 was involved in TGN-related vesicle trafficking.5 Loss of function of VdDrs2 decreases the secretion of Verticillium toxinsThe toxic severity of the leaves treatedΔVdDrs2 crude toxin was significantly lower than that of V991.Metabolites in the fermentation broth of V991 andΔVdDrs2were detected by HPLC.The metabolites in the fermentation broth ofΔVdDrs2 strain were significantly less than those of wild-type strain V991.To confirm the decrease of Verticillium toxins inΔVdDrs2 strain.Tow known Verticillium toxins,SFA（Sulfacetamide,SFA）and FB1（Fumonisin B1,FB1）,were used as reference to detect the content of these two toxins.As assumed,the contents of SFA and FB1 in theΔVdDrs2 strain were significantly lower than those in V991.The results above indicated that VdDrs2 was involved in the secretion of Verticillium toxins.6 Loss of function of VdDrs2 reduces the secretion of the peroxiredoxin VdPrxB that contributes to scavenging of H₂O₂6.1 Loss of function of VdDrs2 reduces H₂O₂scavenging ability in V.dahliaeThe accumulation of H₂O₂ inΔVdDrs2-infected cotton leaves was lower than that in wild-type strain V991-and the complemented strain（Com）-infected leaves,suggesting that the deletion of VdDrs2 attenuated the ability to scavenge ROS.Comparing the expression of VdDrs2 before and after H₂O₂ treatment,it was found that the expression of this gene was elevated by supplementation of H₂O₂.The expression profile of antioxidant stress-related genes inΔVdDrs2 and wild-type V991 was tested before and after H₂O₂ treatment.With presence of H₂O₂,knockout of VdDrs2 resulted in the decreased expression of VdCat1,VdCat3,VdCatA,VdCatE,VdCatF,VdSod1,VdSod3 and VdPrxB inΔVdDrs2 relative to V991.6.2 Loss of function of VdDrs2 decreases the secretion of ROS scavenger peroxiredoxin VdPrxBComparing the secreted proteins produced by wild-type strain V991 withΔVdDrs2strain under H₂O₂ treatment and no treatment conditions,a peroxiredoxin VdPrxB that had been shown a key role in ROS detoxification was identified in the reduced secreted proteins caused by the deletion of VdDrs2.A fusion protein of eGFP::VdPrxB was expressed in V991 andΔVdDrs2,and the intracellular and extracellular secreted protein levels of VdPrxB were detected.The results showed that the level of intracellular VdPrxB in theΔVdDrs2 was always lower than that in the wild-type strain V991,regardless of whether H₂O₂ was added or not.In the absence of H₂O₂,the level of intracellular VdPrxB secreted byΔVdDrs2 was 36.5%lower than that by the wild-type strain V991.With presence of H₂O₂,it was 49.4%lower than the control.For the extracellular proteins,the relative amount of VdPrxB secreted into the extracellular space was 84.6%lower than that of the wild-type strain V991 without the addition of H₂O₂,and 81.1%lower when H₂O₂ was added.It can be seen that loss of VdDrs2 had a much greater effect on reducing extracellular VdPrxB than intracellular proteins,indicating that the deletion of VdDrs2 mainly inhibited the secretion of VdPrxB into extracellular.6.3 Loss of function of VdPrxB makes V.dahliae very sensitive to H₂O₂,and significantly reduces its pathogenicitySimilar to theΔVdDrs2,knockout of VdPrxB in V.dahliae also led the fungus very sensitive to H₂O₂.Moreover,the pathogenicity ofΔVdPrxB to cotton was significantly decreased than wild-type strain V991.6.4 Subcellular localization of VdPrxBThe fluorescent protein gene eGFP was fused with VdPrxB,and the fusion gene that was expressed in V991 andΔVdPrxB respectively.The results of H₂O₂treatment showed that the expression of eGFP::VdPrxB inΔVdPrxB could rescued the resistance of the mutant to H₂O₂,indicating that the fusion of eGFP did not affect the function of VdPrxB.Confocal laser microscope observation of VdPrxB localization results showed that VdPrxB is distributed in the cytoplasm and also has a signal in the tonoplast.6.5 Loss of function of VdDrs2 alters the subcellular localization of VdPrxBeGFP::VdPrxB cassette was expressed in V991（eGFP::VdPrxB-V991）andΔVdDrs2（eGFP::VdPrxB-ΔVdDrs2）,respectively,and the effect ofΔVdDrs2 on the protein localization of VdPrxB was observed.Knockout of VdDrs2 decreased VdPrxB signaling in the tonoplast and cytoplasm,accompanied by an increase in VdPrxB signaling in the vacuole.6.6 VdDrs2 colocalized with VdPrxB under H₂O₂ stress conditionVdPrxB::mRFP cassette and eGFP::VdDrs2 cassette were co-expressed in V991.The localization of VdPrxB::mRFP and eGFP::VdDrs2 were observed under H₂O₂treatment and no treatment condition,respectively.The results showed that VdDrs2 was not colocalized with VdPrxB in the absence of H₂O₂ treatment,while VdDrs2 was colocalized with VdPrxB in a spherical structure in the presence of H₂O₂.Taken together,the role of P4-ATPase VdDrs2 in pathogenicity of V.dahliae is proposed as follows:VdDrs2 mediated vesicle transport is involved in the secretion of Verticillium toxins and peroxiredoxin VdPrxB.Knockout of VdDrs2 reduces Verticillium toxin secretion and thus impairs the pathogenicity of V.dahliae.Meanwhile,the decrease of VdPrxB secretion increases the sensitive of V.dahliae to H₂O₂,which makes the pathogen vulnerable to the stress of ROS generated by the host during the infection.Therefore,increasing of ROS generation of hosts,while interfering with the synthesis mycotoxins and the secretion of toxins and scavengers of the pathogen,should be an effective strategy for the Verticillium wilt disease control.