| Fusarium head blight (FHB) is an economically devastating disease of cereal crops worldly. F. graminearum (teleomorph Gibberella zeae) is the major causal agent of FHB in China and other parts of the world. Infection of cereal crops with F. graminearum may lead to huge yield losses in epidemic years. More importantly, this disease mainly reduces grain quality and results in grain contaminated by trichothecene mycotoxins which pose a serious threat to human and animal health. Deoxynivalenol (DON) is the most abundant form of the trichoecenes found in infected grain and is an important virulence factor in wheat. Despite the serious damage caused by FHB, efficient strategies for the management of FHB are not available to date. Understanding the fundamental biology of this fungal pathogen can provide the basis for sustainable, long-term disease management. Direct replacement of a gene of interest with a selectable marker via homologous recombination is now relatively straight forward. Phospholipase C (PLC), an important phospholipid hydrolase, plays critical roles in a variety of cellular processes in eukaryotic cells. However, little is known about the roles of PLC on morphogenesis and pathogenesis in the Fusarium head blight (FHB) fungus Fusarium graminearum. We analyzed structure of FgPLCs and location of Fgplcs in F. graminearum by the method of bioinformatics. Based on the gene homologous combination and PEG-mediated gene transformation system theories, we produced deletion mutants of Fgplc1?Fgplc6 and compared their growth, development and pathogenicity with the wild-type strain and a complementation mutant of Fgplcl. We got results as follows:1. A whole-genome blast revealed that F. graminearum contains 6 orthologous of PLC. Based on the differences of the structures and the size of their ORF (open reading frame), six Fgplcs were named Fgplc1?Fgplc6. Protein domain analysis revealed that FgPLC1, containing not only X and Y domains, but also PH domain, EF hand domain, and C2 domain, resembles to mammal ?-PLC; FgPLC2-FgPLC5 contain same domains of X, Y and C2; FgPLC6 contains only the conserved domains of PLC.2. Deletion mutant of Fgplcl (Aplcl) showed slower growth rate (77.2% slower than the level of F. graminearum wild-type strain), shorter and tufted aerial hyphae, substantial hyperbtranching and lower hydrophobic property; and reduced conidiation (39.5% lower than that of the wild-type strain) and germination rate (38.4% lower than that of the wild-type strain at 24 h), smaller in size, and abnormal conidia with less or no membranes; and blocked sexual reproduction; and increased sensitivity to hyperosmotic stress factor KC1 or Sorbitol, decreased sensitivity to another hyperosmotic stress factor NaCl, decreased sensitivity to oxidative stress factor H2O2, decreased sensitivity to cell wall-damaging agents Congo red and CFW; and lower production of DON; and lower pathogenicity (96.00% lower than that of the wild-type strain). Furthermore, the functional complementation of Aplcl restored the characterizations of the wide type strain.3. Deletion mutants of Fgplc2-Fgplc6 genes (Aplc2-Aplc6) showed no visiable differences in hyphal growth, conidiation and perithecium formation compared with the wild-type strain. However, all of the five mutants showed substantial hyperbtranching and lower pathogenicity. Aplc6 showed increased sensitivity to KC1, Aplc4 showed decreased sensitivity to Sorbitol, Aplc2 and Aplc4 showed increased sensitivity to cell wall-damaging agent Congo red. All of the five Fgplcs were involved in DON biomass.Taken together, these results suggest that Fgplcl is involved in fungal growth, conidiation, perithecium development, stress responses, DON biomass and pathogenicity, Fgplc2-Fgplc6 conduct similar roles in the formation of hyphalbranches, stress responses, DON biomass and pathogenicities. |
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