Functional Analysis Of White Collar-1 Fawc1 And Vivid Favvd In Fusarium Asiaticum

The Fusarium graminearum species complex is the most common causative agent of Fusarium head blight (FHB) in cereals all over the world. In addition to direct yield losses, the mycotoxins produced by F. graminearums in host plants, such as trichothecenes (DON) and zearalenone, are threatening human and animal health. F. graminearum clade consists of nine phylogenetical subgroups, most of which cannot be distinguished from each other by morphological characters. Fusarium.asiaticum, which is one of the nine phylogenetical subgroups, is the main pathogen of FHB in Asia region. Our lab firstly reported that F. asiaticum is an emerging pathogen jeopardizing postharvest asparagus spears, and is highly pathogenic in broad range of fruits and vegetables, indicating that F. asiaticum could do potential harm to the postharvest fruits and vegetables.Our previous work showed that light can adjust the pathogenicity of pathogenic fungi. In order to further clarify the influence of light on the F. asiaticum, the function of blue light receptor genes of Fawcl and Favvd were studied by the methods of molecular genetics. The results were as follows:1. By comparing the colony morphology, growth rate, conidiation of F. asiaticum as influenced with different light, we identified blue light and wihte light can inhibit the growth of F. asiaticum. Additionally, three blue light receptor genes, Fawcl, Fawc2 and Favvd, were confirmed to be induced by illumination, and Fawcl and Favvd could be induced by light most greatly.2. Analysis of homologous evolutionary relationships shows that FaWCl has a common ancestor with other WC-1 family proteins; FaWCl and FaVVD are highly conservative by the analysis in their respective functional domains of amino acid sequence; and FaWC1、FaVVD have a LOV domain as a light receptor. Through further comparing the conserved functional domain of WC-1 proteins in 25 species fungi, the functional domains possess certain difference, which may be the reason for the difference function in the different fungi.3. Fawcl and Favvd were knocked out by means of homologous recombination strategy with PEG-mediated protoplast transformation, and 51 tand 83 transformants were obtained, respectively. There are 6 knock-out mutant strains of Fawc 1 were identified by DNA level identification from 51 transformants and 9 knock-out mutant strains of Favvdwere identified by the same way from 83 transformants.Three strains randomly selected form the knock-out mutant strainsrespectively, which were identified by the PCR analysis of RNA transcript and Southern bloting.Finally,3 Fawcl deletion mutants named △ Fawcl-1、△Fawc1-24、△ Fawc1-48 and 3 Favvd deletion mutants named △ Favvd-1、△ Favvd-50、△ Favvd-83 were used for further study.4. By comparing analysis with the mutants and wild type, the results were as follows:the AFawcl mutants produced abnormal perithecia and more conidia, moreover, the mutants displayed significantly reduced pathogenicity; interestingly, apart from stronger pathogenicity, there were no difference between wildtype and AFawcl mutantsin morphology. Therefore, the White callar 1- and VIVID-like proteins of F. asiaticum FaWCl and FaVVD adversely regulate the development, secondary metabolism and pathogenicity.5. To further investigate the reason of weakened pathogenicity in △Fawcl but strengthened one in △Favvd knockout mutants, the pathogenic process of the pathogens were analyzed, including the adhesion of pathogens, penetration of hyphae, the secretion of cell wall degradation enzymes, DON toxin and ROS generation. The absence of Fawcl led to greatly reduced hydrophobicity and penetration of hyphae. Comparing with △Fawc1 mutants, the △ Favvd mutants showed totally opposite in corresponding aspects. Thus we proved that Fawcl and Favvd participate in pathogenic process, and Favvd may be the negative regulatory factors to Fawcl.In conclusion, we identified the function of Fawcl and Favvd in controlling development, secondary metabolism, sexual and asexual, and pathogenicity in Fusarium asiaticum. This study provides the reference to control F. asiaticum in postharvest fruits and vegetables.