Functional Analysis Of A Beauveria Bassiana Antifungal Peptide BbAFP1 And Its Application In Increasing Verticillium Wilt Resistance In Cotton

Cotton Verticillium wilt caused by Verticillium dahliae Kleb.,a typical soil-borne vascular disease,seriously threatens cotton yield and fiber quality and is called as“the cancer of cotton”.Presently,there are no fungicides available to cure plants once they are infected,and there are few germplasms of upland cotton that are resistant to V.dahliae.Transgenic engineering is a potential way to increase cotton disease resistance.However,the lack of high-efficiency resistance genes for genetic engineering hinders the development transgenic cotton with high Verticillium wilt resistance.The entomogenous fungus Beauveria bassiana has been widely used as a fungal insecticide against agriculrutal and forest pests.In addition to killing insects,B.bassiana can also interact with plants and inhibit the occurrence of fungal diseases.Therefore,it has been gradually becoming a good source to explore plant disease-resistance genes.In our previous studies,an antifungal peptide gene was cloned from B.bassiana and named BbAFP1.Its encoding product had a significant inhibitory effect on a wide range of phytopathogenic fungi including V.dahliae.However,to date,BbAFP1's action mechanism,biological function in B.bassiana and resistance to Verticillium wilt in cotton are still not clear.To address the above problems,we firstly analyzed the binding ability of BbAFP1 toward fungal cell wall and the effects on target fungal cell membranes potential changes,reactive oxygen species(ROS)production and cell membrane disruption,and elucidated the action mechanism.Secondly,we observed the expression and localization of BbAFP1 in B.bassiana during fungal growth and development processes.Functions of BbAFP1 in B.bassiana against environmental fungi was evaluated by constructing knocking out(BbAFP1)and overexpressing strains(BbAFP1^OE)of BbAFP1 and co-culturing them with other fungi.Finally,to increase the antifungal ability of BbAFP1,several binding domains,which can bind to various targets in fungal cell membrane or wall,were fused to the C-terminus of BbAFP1,respectively.The fusion antifungal peptide genes as well as wild type BbAFP1 were expressed in cotton and tobacco,and transgenic plants with higher disease resistance were obtained.The main results are as follow:1.The protein characteristics of BbAFP1Antifungal activity analysis showed BbAFP1 significantly inhibited the conidia germination and hyphal gowth of Fusarium oxysporum,Magnaporthe orzyae,V.dahliae,Alternaria solani,Fusarium graminearum,Botryis cinerea,Alternaria brassicae,however,had no inhibitory effect on bacteria and yeast.The minimum inhibitory concentrations(MIC)of BbAFP1 against phytopathogenic fungi ranged from5 to 15?M,and F.oxysporum had the lowest MIC(5?M)among them.Thermostability analysis showed that the antifungal activity of BbAFP1 was not affected by treatment at 100?for up to 2 h,and with only slight decreases(20-30%)after treatment for 4-6 h,indicating BbAFP1 has superior thermostability.The activity analysis further revealed BbAFP1 exhibits higher antifungal activity under acidic conditions.2.The action mechanism of BbAFP1In order to probe the potential action mechanisms,the purified BbAFP1 was labeled with FITC(Fluorescein isothiocyanate).Binding experiments demonstrated that BbAFP1 could bind to chitin in the cell wall of phytopathogenic fungi.In order to analyse the key amino acids that may be involved in chitin binding and the effect of chitin binding on antifungal activity,a series of site directed mutants of BbAFP1,including Y^37A,F^50A,F^59A,Y^74A,Y^79A,and Y^37AF^50Adouble mutant were obtained.Binding and antifungal activity assay showed that the binding ability of mutant BbAFP1^F50Atowards powdered chitin and fungal cell wall was obviously reduced;and the inhibition activity of BbAFP1^F50Aagainst A.brassicae was also decreased obviously,with MIC against sensitive fungi increased?3-5-fold compared to wild-type protein.The above results indicated that a phenylalanine residue(F⁵⁰)plays an important role in chitin binding and antifungal activity of BbAFP1.The effect of BbAFP1 on fungal cell membrane potential was analyzed using the voltage-sensitive dye Di SC3(5).BbAFP1 treatment resulted in the rapid depolarized of the A.brassicae cell membrane potential within 2 minutes.The production of reactive oxygen species in A.brassicae treated with BbAFP1 was detected by H₂DCFDA dye.The result showed that a rapid burst of ROS production was observed within 5 min after addition of BbAFP1 to cells.Membrane disruption was visualized by staining of BbAFP1^FITCtreated A.brassicae cells with propidium iodide(PI).These results suggested that BbAFP1 enriches onto the surface of pathogenic fungi with its chitin binding ability,then causes the membrane potential change,elicits an ROS burst,and ultimately disrupts the cell membrane.3.The biological function of BbAFP1 in B.bassianaBbAFP1 possesses a secretory signal peptide in the N-terminus,therefore,we speculated the antifungal peptide may be induced by environmental fungi and secreted to inhibit growth of competing fungi.However,the fluorescence analysis of P_BbAFP1::e GFP strain showed the expression of BbAFP1 was not induced by phytopathogenic fungi,but was specifically expressed in mature aerial conidia.In order to examine the localization of BbAFP1 in B.bassiana,a fusion protein BbAFP1::e GFP under the control of its native promoter was constructed and transformed into B.bassiana.The results showed that BbAFP1 was not secreted into the environment as expected,but localized on the cell wall of conidia.When P_BbAFP1::BbAFP1::e GFP conidia were treated by mild sonication,the GFP signal was lost in conidia and was recovered in the cell-free supernatant.Fluorescence localization observation of P_BbAFP1::BbAFP1::e GFP strain during germination showed that the GFP signal remained associated with the conidia up to 6 h post-inoculation,after which the signal was gradually lost from the cells;at 12 h post-inoculation,very weak signal could be detected on the cells.Western blot and ELISA detection confirmed BbAFP1 was released into the solution.To further probe the biological functions of specific expression in mature conidia and binding to cell wall of BbAFP1,targeted gene knockout and overexpression strains were constructed.The above strains were co-cultured with A.brassicae or Trichoderma sp.to analyze the inhibitory effect against competing fungi.B.bassiana strains expressing BbAFP1,including wild type and overexpression strains,inhibited growth of co-cultured fungi,whereas targeted gene deletion of BbAFP1 significantly decreased this inhibitory effect.These results suggested that BbAFP1 was specifically expressed in mature aerial conidia of B.bassiana and could been preloaded in the cell wall.When encountering the appropriate environment,the preloaded BbAFP1 was released into the environment,and then inhibited the growth of competing fungi,to ensure that B.bassiana could obtain enough nutrition and space to complete the growth and development.4.The targeted improvement of BbAFP1Previous studies have found that overexpression of BbAFP1 in tomatoes enhanced their disease resistance.To further increase the antifungal ability of BbAFP1,several fusion peptides with different tags that can bind to various targets in fungal cells,including the Ch BD binds chitin,the Sp BD binds sphingolipid,the Er BD binds ergosterol were designed.We preliminarily evaluated the disease resistance of fusion antifungal peptides in plant by agrobacterium-mediated transient expression system.Transient expression in cotton showed that cotyledons expressing BbAFP1::Er BD exhibited the highest resistance level against V.dahliae,and the disease indexs of BbAFP1::Er BD,BbAFP1::Sp BD,BbAFP1::Ch BD were 27.0,32.5 and 46.8,respectively.Transient expression in cotton and tobacco further showed that fusion peptide BbAFP1::Er BD exhibited higher disease resistance than Er BD or BbAFP1alone toward V.dahliae and B.cinerea.5.Improving the disease resistance of cotton and tobacco by BbAFP1::Er BDTo evaluate the potential of BbAFP1::Er BD in genetic engineering of disease-resistance plant,we introduced BbAFP1::Er BD and BbAFP1 into cotton and tobacco,and obtained the transgenic plant lines.We analyzed the disease resistance of transgenic plants in different generations by various inoculation methods.The results showed that:1)After inoculation V.dahliae onto detached leaves(T₀),the lesion areas on BbAFP1::Er BD transgenic leaves were significantly smaller than those on BbAFP1-expressing cotton leaves.2)Disease resistance of T₂generation transgenic cotton lines(BbAFP1::Er BD-61 and BbAFP1-35)was evaluated by inoculating the fungal pathogens V.dahliae and B.cinerea onto leaves.The results showed that BbAFP1::Er BD conferred higher resistance towards both phytopathogenic fungi in cotton.3)Disease resistance of T₂generation BbAFP1-35-and BbAFP1::Er BD-61-expressing plants was further analyzed using the root dipping method.Severe symptoms of Verticillium wilt appeared on wild-type plants,while no or only slight symptoms appeared in BbAFP1-35 or BbAFP1::Er BD-61 transgenic cotton plants,with the latter showing a better level of resistance.4)After inoculation V.dahliae onto T₀generation transgenic detached leaves,and the disease indexes of BbAFP1::Er BD transgenic lines were significantly lower than that of BbAFP1.All of the results indicated that ergosterol binding domain could further improve the disease resistance of BbAFP1 towards phytopathogenic fungi.6.The action mechanism of BbAFP1::Er BDGene expression detection showed BbAFP1 and BbAFP1::Er BD has no significant effects on the expression of endogenous PR genes in cotton with or without V.dahliae inoculation.The location of BbAFP1::Er BD towards V.dahliae cells was analyzed by immunofluorescence method using total proteins extracted from plant roots.After treating V.dahliae cells for 1 h,the fluorescent signal for BbAFP1 was found to mainly localize in the cell surface of V.dahliae,while the signal for BbAFP1::Er BD was concentrated on some specific patches in the cell envelope and appeared inside the fungal cells.Propidium iodide(PI)staining verified that more V.dahliae conidia were disrupted after treatment with the total protein from BbAFP1::Er BD transgenic cotton compared to BbAFP1,indicating BbAFP1::Er BD could disrupt the cell membrane more quickly.In addition,fungal growth in transgenic plants was observed by inoculated a GFP-expressing V.dahliae strain V991-GFP.At 6 d post inoculation,a large number of fluorescent hyphae were found in wild-type cotton root,however,the amount of hyphae were decreased significantly in BbAFP1-35 or BbAFP1::Er BD-61 transgenic cotton,with the latter showing less hyphae,indicating BbAFP1::Er BD had stronger ability to inhibit the colonization process of V.dahliae.In conclusion,our results elucidated the protein characteristics,action mechanism and biological function of BbAFP1,enriching the understanding of antimicrobial peptides.In addition,this study provides a new strategy and new gene for genetic engineering of Verticillium wilt resistance.