Abstract: 【Objective】Cucumber Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum, is an important disease in cucumber (Cucumis sativus L.) and can result in serious economic loss. The objective of this study is to investigate the functions of phospholipase C4 (PLC4) in F. oxysporum.【Method】Chromosomal location of the FoPLC4 and structure of FoPLC4 were analyzed by bioinformatics methods. Based on the F. oxysporum f. sp. lycopersici genome database, the FoPLC4 was cloned using specific primers. The gene replacement vector carrying hygromycin phosphotransferase (HPH) gene was constructed based on the gene homologous recombination, and then transformed into protoplasts with PEG-mediated gene transformation. The FoPLC4 deletion mutant (?FoPLC4) was generated through hygromycin B selection and molecular assay. Biological characters of the mutant on PDA (potato dextrose agar) medium and its pathogenicity on cucumber seedlings were determined after inoculation of radicles with conidia suspensions.【Result】The FoPLC4 located on chromosome 4, contains 3 282 bp with no intron and encodes a long polypeptide of 1 093 amino acids (aa). The protein encoded by FoPLC4 resembled mammalian PLCδ and possessed five domains including an EF-hand domain, a pleckstrin homology domain, a C2 domain, and two catalytic domains. Transformants selected on hygromycin B plates were screened by PCR and RT-PCR. Compared with the wild type (WT) strain, the ?FoPLC4 mutant showed a similar colony growth rate, but exhibited sparse and fluffy aerial mycelium on PDA medium. The conidiation of the ?FoPLC4 mutant reduced by 82.2%, the ?FoPLC4 simultaneously produced micro- and macro-conidia while the WT strain did only produce micro-conidia, and macro-conidia accounted for 12.1% of all spores in the ?FoPLC4. Pathogenicity assay of the ?FoPLC4 showed that wilting symptoms of cucumber seedlings significantly reduced. After 10 days of inoculation, disease index decreased by 53.3% on inoculated seedlings. Furthermore, the functional complementation of ?FoPLC4 restored the characteristics of the WT strain.【Conclusion】FoPLC4 contains 3 282 bp with no intron and encodes a protein of 1 093 aa. Compared with WT, ?FoPLC4 significantly exhibited the change of spore types, decreased the number of spores and pathogenicity in cucumber seedlings. The results indicate that FoPLC4 plays multiple roles in the regulation of sporulation and pathogenicity in F. oxysporum.

Key words: Fusarium oxysporum , FoPLC4 , sporulation , pathogenicity