Functional Analysis Of Sclerotial Development Associated Protein SS-SL2in Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a notorious fungal pathogen with worldwide distribution. This fungus may infect many important crops, such as oilseed rape, sunflower, soybean, and cause Sclerotinia diseases. Sclerotia are not only the important tissue for breeding and over summer and winter to S. sclerotiorum, but also the important source of primarily infection for Sclerotinia diseases. Repress the formation of sclerotia may interrupt the disease cycle induced by S. sclerotiorum and research on the molecular mechanism involved sclerotial development may provide some clues to control the Sclerotinia diseases.Lack of efficient and easy transformation system is a great obstacle for studying the gene function in S. sclerotiorum. In this study, an Agrobacterium-medated transformation system was established for S. sclerotiorum by using the hyphae as receptor. A. tumefaciens cells containing pTFSS and fresh S. sclerotiorum mycelial plugs were co-cultured with acetovanillone for2days. Then the mycelial plugs were removed and the A. tumefaciens cells and S. sclerotiorum hypha were transferred into PDA medium containing30μg/ml of hygromycin B. The transformants emerged on the selective plates after3days. This method was simple and had a stable efficiency of transform. On average,1-3transformants can be obtained in each plate. This system provided an effective platform for gene functional study of involved in S. sclerotiorum. This research also showed that the HPH gene in transformants inherited steadily in the asexual stage of S. sclerotiorum, but it lost in the sexual progenies.To study the molecular mechanism involved in sclerotial development, a gene expression pattern during the stage of hyphal growth and sclerotial development was contrasted with Illumina-Solexa in our laboratory. According to the gene expression pattern, a gene named Ss-Sl2(SSIG₀5917, XM₀01592945) had higher expression level during sclerotial development, with the relative expression during sclerotial development400-fold greater than that at the hyphal growth stage. Thus, Ss-Sl2is supposedly to have a close relation with sclerotial development. Our results showed that Ss-Sl2protein has352aa residues and contains a signal peptide and two PAN modules. Its homologies appeared only in the some ascomycotina fungi and all of those are of unknown function. At the stage of vegetative hyphal growth, only a few gold particles could be observed dispersed in the cell walls and septa of hyphae. During the stage of sclerotial development, the transcript level of Ss-Sl2rised rapidly and a large number of Ss-S12protein were secreted and located in the thicken cell wall of sclerotia. The expression level of Ss-Sl2is down-regulated by cAMP level but has no relation with the medium pH. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2silenced strains were not significantly different from the wild type strain. Ss-Sl2silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanized to become mature sclerotia. Hyphae in these interwoven bodies were thin-walled, arranged loosely. These evidence showed that Ss-S12play an important role in sclerotial development of S. sclerotiorum.Identification and functional study on proteins which interacted with Ss-S12may provide clues to understand its mechanism involved in controlling sclerotial development and cell integrity. Our results showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hexl) and elongation factor1alpha (EF-la) could interact with the Ss-S12directly, and EF-1a was found to have only a weak interaction with Ss-S12. GAPDH-knockdown strains did not produce typical sclerotia, but only forming interwoven hyphal masses. The phenotype was similar to that of Ss-Sl2silenced transformants. Hexl-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2silenced strains. These evidences indicated that the Ss-S12may play the role in sclerotial development and the mantainence of cell integrity through its interaction with GAPDH and Hexl.The research profoundly analysed the function and mechanism of Ss-S12protein in S. sclerotiorum, which not only provides the theory basis for revealing the molecular mechanism of sclerotial development, but also offers important clues to control Sclerotnia disease. Besides, this research also provides important references for studying the function of the homologies of Ss-S12in some other fungi.