Functional Analysis Of Bactericidal Protein Hcm1 And Sterol Carrier Protein GhSCP2D In Cotton

Cotton?Gossypium spp.?is an important economic crop and the largest source of textile fiber in the world.However,due to environmental degradation,cotton in the cotton-growing areas often suffers from drought,high salinity and low temperature,and other biotic stresses such as the Verticillium wilt,which is known as the cotton's cancer,and adversely affects the cotton growth and yield of cotton fiber.Upland cotton is one of the main cultivated cotton species planted in China accounting for more than 95%of the cotton production.However,cultivated cotton varieties of high-quality fiber,strong disease and stresses resistance,especially high reistance to Verticillium wilt,are rare,mainly because of lack of Upland cotton germplasms which are nature immune or highly resistant to Verticillium wilt.Therefore,through uncovering the molecular mechanism of cotton disease resistance using genetic engineering methods to foster excellent new varieties of cotton will be the main direction of cotton breeding in the future.Hcml is a novel protein which consists of three active domains including Harpinxooc protein,Cecropin A and Melittin.Harpinxooc protein could induce hypersensitive response in tobacco.Meanwhile,Cecropin A and Melittin can inhibit the growth of various pathogens in vitro.Through the transformation of epidermal cells via particle bombardment technology,we found that the Hcml protein was located in the membrane of plant cell,but not in the cell wall by treatment of 20%sucrose.To verify the function of Hcml,we obtained four homozygous transgenic lines through the over-expression vector,which was drived by the constitutive CaMV35S promoter were developed using pBI121,and used the method of Agrobacterium-mediated to transform Gossypium hirsutum WO.Southern and Wenstern blot analysis showed that,the four transgenic pure lines were transformed with one to three copies of Hcml protein respectively and they were able to translated into protein.These four transgenic lines have more stronger resistance of Verticillium wilt and Fusarium wilt in greenhouse and field indicating Hcml protein conferred plants a variety of diseases resistance.Furthermore,we found H2O2 content in the transgenic plants increased compared to the receptor W0,but the transcriptional expression of some resistance genes NPR1,PR1,HSR203J and HIN1 has not noticeably increased.After inoculation with Verticillium dahliae,the H2O2 content in the transgenic plants was rapidly increased and appeared two peaks within 0?12h,while in the receptor WO,the H2O2 content were increased slowly.We found that the transgenic plants appear necrotic cells?hypersensitive response?after the second peak and the expression of NPR1,PR1,HSR203J and HIN1 increased rapidly compared with the WO control.These results suggest that Harpinxooc protein of Hcml may make plants in a priming state.Once the plants infected by the pathogen,the Ttransgenic plants quick start plants allergic reactions and induce cell death to prevent further infection of pathogenic bacteria.In order to study the inhibition of Verticillium dahliae and Fusarium oxysporum,Hcm1 was expressed via the prokaryotic expression technology and cell-free elicitor preparations?CFEPs?of Hcm1 were extracted.We found CFEPs of Hcml could effectively inhibit the growth of Verticillium dahliae and fusarium oxysporum in vitro.Also,the crude protein extracted from the the Hcm1-transformed plants could inhibit the growth of these two pathogens.After transgenic plants and WO were inoculated with Verticillium dahliae V991 harboring GFP gene,we observed the weaker intensity of the fluorescence signal in the transgenic plants than in the WO receptor,indicating the content of Verticillium dahliae in transgenic plants is less than the receptor.This result was confirmed by qRT-PCR.These results suggest that Hcml protein can effectively inhibit the expansion of Verticillium dahliae in cotton.Cotton fibers are highly elongated single-cell trichomes that arose from the outer epidermis of the ovules.As the single-cell structure without the complication of cell division and multicellular development,cotton fiber was considered to be a unique experimental system for investigating the regulation of fiber related genes.The improvement of the cotton transformation efficiency and isolation of fiber-specific genes provided a platform for production of transgenic cotton and function analysis of cotton key genes.There is great significance for elucidating the function of fiber-specific genes,as is enable to promote the understanding of cotton fiber development mechanism and improve cotton quality by means of molecular breeding.We screened the differentially expressed genes among fiber initiation,elongation and secondary wall thickening in Upland cotton TM-1.28K₁78 ALL which was related to fiber development was screened out and it encodes a sterol carrier protein with high similarity to AtSCP2 in Arabidopsis by BLAST in NCBI.We found six sterol carrier protein in the genome sequence of Gossypium hirsutum and four of them were cloned and named as GhSCPID,GhSCP2D?28K₁78_ALL?,GhSCP3A and GhSCP3D,respectively.It was predicted that GhSCP2D,GhSCP3A and GhSCP3D contain the peroxisomal targeting signal 1?PTS1?which encoded by its C-terminal fragment while GhSCPID was unable to predict signal peptide due to the mutation of stop codon.To examine the subcellular localization,a green fluorescence protein?GFP?was fused with a linker to its C-terminus?GhSCP:GFP?,under the control of the cauliflower mosaic virus?CaMV?35S promoter,was transiently expressed in TM-1 protoplasts.Laser confocal imaging revealed that the GhSCP1D fusion protein was targeted to chloroplast.GhSCP2D,GhSCP3A and GhSCP3D fusion protein was targeted to peroxisome.RNA-seq data analysis and quantitative reverse transcription?qRT?-PCR results showed that GhSCP2D specifically expressed in cotton fiber and highly expressed in the fibers at 5 DPA.GhSCPID,GhSCP3A and GhSCP3D predominantly expressed in the cotyledons.The expression of these four genes was induced by sucrose,especially GhSCP2D gene.To further study the roles of GhSCP2D in cotton fiber development,the antisense construct of GhSCP2D driven by the constitutive CaMV35S promoter were developed using pBI121 as the fragment.Then,the plant-expressed vectors were transformed into the Upland cotton WO by the Agrobacterium-mediated transformation.All the individuals of TO,T1,T2 and T3 transgenic plants were detected by the PCR analysis using the NPT? and promoter-gene specific primers.Three transgenic pure lines were obtained after three rounds selections.The expression level of GhSCP2D in ODPA?ovules with fibers?,and 5DPA,10DPA,15DPA?fibers?of transgenic pure lines and WO was investigated by quantitative reverse transcription?qRT?-PCR.The expression level of GhSCP2D was significantly reduced than that of the wild-type plants.The fiber length of the three transgenic lines was much shorter compared with WO and the fiber strength and fineness of three transgenic lines slightly decreased.According to previous studies,we speculate that AtSCP2 may play a role in the regulation of sucrose transport in Arabidopsis.So we measured the sucrose content of 5 DPA fibers and found sucrose content of three transgenic lines was significantly lower than that in the WO.These results indicated that inhibiting the expression of GhSCP2D may result in sucrose transport disruption.As it has been known,sucrose was imported into fiber cell through symplast pathway?plasmodesmata?or apoplastic pathway which was assisted by sucrose transporters.Sucrose transporters were divided into sucrose transporter protein and SWEETs proteins.RNA-seq data analysis and quantitative reverse transcription?qRT?-PCR showed that Sucrose transporters were nearly no expression in 5DPA fibers.Therefore,GhSCP2D may regulate sucrose transport of symplast pathway.The expression of sucrose transporters significantly increased in the transgenic plants compared to WO.These results further illustrated that GhSCP2D regulate sucrose transport of symplast pathway rather than apoplastic pathway.Based on these results we speculated that apoplastic pathways can compensate symplast pathways when sucrose transport of symplast pathways blocked.Sucrose transport of Symplastic pathway depends on the plasmodesmata permeability.Plasmodesmata permeability regulated by H2O2 and callose.H2O2 could increase the plasmodesmata permeability by increasing the number of inter-branch and twin plasmodesmata or decrease plasmodesmata permeability by inducing callose deposition in plasmodesmata.Correspondingly,the H2O2 content in the fibers at 5DPA of transgenic plants significantly increased compared with receptor WO,indicating that GhSCP2D may affect sucrose transport by regulating callose deposition in plasmodesmata.GhMYB9 encodes a R2R3 MYB transcription factor in upland cotton genome.Our studies showed that GhMYB9 predominantly expressed in the flowers and fibers of cotton.To gain a better understanding of its regulatory mechanism,we isolated the 5'-flanking region of GhMYB9,which was 1,487 bp in length.Cis-acting element prediction showed that this region contained the basic structure of the core promoter elements?TATA-box,CAAT-box?and transcription start site?TSS?.Other motif such as defense and stress responsiveness?TC-rich repeats?,the anaerobic induction?ARE?and MYB binding sites involved in drought-inducibility?MBS?were also found.Histochemical assay showed that the GhMYB9 promoter governed the beta-glucuronidase?GUS?expression mainly in the seed,fibers and flowers of transgenic cotton.Also the activity of the promoter was induced by auxin in the fibers of transgenic cotton.This was consistent with its transcript abundance in different tissues.Further deletion analysis confirmed that the promoter region from-1,231 to-860 was required for auxin response.Our findings provide a useful reference for understanding the transcriptional regulation mechanism of GhMYB9 gene.