Mechanism Of Synthetic Antifungal Peptides Against Verticillium Dahliae And Its Application In Cotton Breeding

Cotton production occupies a central position in national economy of China contributed from agriculture sector. According to the data provided by the National Bureau of Statistics of China, the average yield (1229kg/ha) of cotton from2010is declined as compared to last five years. This reduction in the cotton yield is mainly due to the occurrence of a devasting pathogen Verticillium dahliae in cotton growing areas and caused a severe disease known as Verticillium wilt. For so long time, scientists are engaged for identifying resistant resources against this pathogen but could not find any one in commercially grown tetraploid cotton(Gossypium hirsutum). However biotechnology and genetic engineering as new emerging sciences set up new avenues for combating Verticillium wilt in cotton by transferring resistant genes from sources other than plants.Yangtze River and Yellow River are the two main cotton growing areas in China. About15different isolates of Verticillium dahliae, were collected from these two places and maintained in our laboratory for future work. The isolates were characterized into hyphae, sclerotia and intermediate-type on the basis of colony morphology. Further molecular identification of these isolates using four pairs of specific primers indicated that, except Fusarium oxysporum and Zhengzhou strain of Verticillium dahliae, all the other tested strains belong to Verticillium daliae including some virulent defoliating strains. To understand the interaction between cotton and Verticillium dahliae, seedling bioassays were performed using VD(Verticillium dahliae) toxin (protein-lipids-polysaccharide complexes, PLPs) and spore suspension following root dip method. The VD-toxin caused the cotton seedlings to wilt immediately while symptoms appearance with spore suspension was little delayed. Moreover, antioxidant activity in different organs of the same treatment was not the same.In addition to greenhouse screening, field screening against Verticillium dahliae was also during2007and2008conducted using16CMS-based hybrid cotton developed by our lab. In august2007, monthly average temperature was above30℃and no wilting was recorded. However at the end of august the high disease index was observed. Whereas in2008due to low temperature and frequent rainfall, which are suitable factors for the growth of Verticillium dahliae, the incidence of field disease reached at peak in August. Moreover at different time period, cotton materials exhibited different disease index for Verticillium wilt and according to their performance,16CMS-based hybrid cotton can be divided into four groups, highly resistant sea island cotton restorer line, highly susceptible chicken leaf recovery line and ’Xiangzamian2’while other materials were either tolerant to disease or between tolerant and susceptible. The correlation analysis results showed that the final yield and fiber quality performance of the CMS-based materials were significantly correlated with the disease severity. In order to utilize the phenomenon of heterosis, F1intraspecific hybrids of upland cotton and interspecific hybrids between sea and upland cotton were developed. Hybrids though exhibited some resistance but it was not as high as that of sea iseland cotton.From already available database having more than1500antimicrobial peptide sequences,600were identified to possess antifungal properties. Out of these, finally20different types of anti-fungal peptides were selected as a parent peptide and modified for high resistance against Verticillium dahliae and low toxicity to other organisms. However, their conservative antibacterial core domain (or amino acid residues) was retained. The interaction of these antimicrobial peptides with fungal, animal, bacterial cells and cotton protoplast was studied. Results revealed that these antimicrobial peptides efficiently inhibited the growth of Verticillium dahliae, low toxicity to animal red blood cells and cotton protoplast. The interaction between fungus isolate and antimicrobial peptide was highly significant and0-defensin BTD-S can inhibit five different isolates while hybrid peptide CM-S can inhibit four kinds of Verticillium dahliae. The minimum inhibitory concentration (MIC) for these two peptides was less than5μM and possessed stronger antimicrobial activity as compared to synthetic peptide D4E1(MIC=9.61μM). No hemolytic activities were observed to sheep red blood cells and cotton protoplast even at concentration level up to200μg/ml. These qualities make these two antimicrobial peptides more demanding for using them in developing wilt resistant cultivars.Circular dichroism (CD) spectra of BTD-S in the far UV wavelength displayed a characteristic positive peak is195nm and205nm negative peak, indicating a typical β-sheet conformation of BTD-S. Similarly a positive peak at190nm and the negative peak at222nm and208nm for CM-S indicated a a-helix conformation and match well with the prediction from bioinformatics softwares. The advanced structural analysis of antimicrobial peptides showed that the six cysteins amino acid residues within the molecule of the BTD-S formed three disulfide bonds (like a ladder) making it more stable with durable microbicidal properties. Wherease CM-S with a-helix structure having seven net positive charge and amphiphilic properties (hydrophobic surface) make it easier to interact with the membrane of Verticillium dahliae.Under the optical microscope, antimicrobial peptides concentration (20μg/ml) leads to the significant inhibition of conidial germination. Cell membrane permeation of fungal spores was also observed using fluorescence microscopy technique and severe cell injury was observed in response to treatment with antimicrobial peptides. This injury can be visualized from permeation of large molecules of propidium iodide (PI) into the fungus cells which then stained the nucleus and emitted red fluorescence after excitation. Similarly scanning electron microscopy (SEM) also revealed retarded growth of fungal spores with some extravasations on the cell surface and rupturing of the cell. Whereas flow cytometric analysis (FCA) showed that BTD-S treatment differentiated fungal cells into a new group of small sized cells (mini cells) and their number increased continuosly upon increasing the concentration of the BTD-S. These findings were inconsistent to that of optical microscopy, florescence microscopy and scanning electron microscopy. Therefore it can be concluded from this study that the BTD-S and CM-S interacted with pathogenic fungal cell membranes, changed the membrane permeability and thus disturbing the normal metabolism of the cells. Using bioinformatics tools, amino acid sequence of the BTD-S was translated into coding nucleotide sequence and then inserted into the prokaryotic expression vector pET32a to construct the recombinant expression vector of the BTD-pET32a. This recombinant vector was then transferred into Escherichia coli BL21(DE3) and after IPTG induction a significant overexpression of the target protein was detected. The study provided a comprehend mechanisim of antimicrobial peptide interaction with Verticillium dahliae and laid a basis for utilizing the coding sequences of these peptides to transfer into cotton for developing transgenic wilt resistant cultivars.