Functional Analysis Of Genes In Sphingolipid Biosynthesis Pathway In Penicillium Digitatum

Sphingolipids are not only an important component of cell membrane, involve in the constitution of lipid rafts, and participate in substance absorption and transportation, the intermediates in sphingolipid pathway are also as signal molecules, participate in numerous cellular events, such as cell proliferation, ion homeostasis and response to stress. Recent studies show that sphingolipid pathway is involved in the regulation of pathogenesis of pathogenic fungi. Although sphingolipids are ubiquitous in animals, plants and fungi, but the molecular structures and synthesis pathway of fungal sphingolipids are different from that in plants and animals, the key enzymes involved in sphingolipid biosynthesis pathway of pathogenic fungi are supposed to be the ideal targets of novel antifungal drug or fungicides. Therefore, understanding the sphingolipd pathway and its regulation, characterizing the genes encoding the key enzymes in this pathway have theoretical and practical significance for the development of new fungicides. Penicillium digitatum, causing green mold of postharvest citrus, is one of the most destructive pathogens in citrus industry. Losses of citrus fruit from green mold are usually estimated to as high as90%of the decay gross loss of postharvest citrus. In this study, we indentified the sphingolipid biosynthesis pathway of P. digitatum based on its sequenced genome data; we established the method of high liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) to determine the main sphingolipds; we characterized the functions of two genes (PdMitl and PdGcsl) in sphingolipd pathway in P. digitatum. The main findings are as follows1Sphingolipid biosynthesis pathway of P. digitatum is not identical to that of S. cerevisiaeHomology search in genome sequence database of P. digitatum by using sequences of S. cerevisiae as references showed that most genes participated in sphingolipid biosynthesis of S. cerevisiae had their homologous sequences in the genome of P. digitatum. However, several significant differences were found between these two species. The orthologs of Tsc3p (temperature-sensitive suppressor of calcium sensitivity) was absent in P. digitatum genome, we presumed that only EKV07450and EKV05826, the orthologs of Lcbl and Lcb2encoding two subunits of serine palmitoyltransferase (SPT) in S. cerevisiae, participated the first step reaction of sphingolipd biosynthesis in P. digitatum. The ortholog of Iptlp (inositol phosphotransferase) was absent in P. digitatum, thus we presumed that the terminal product in sphingolipid pathway might not be M(IP)2C but MIPC in P. digitatum. P. digitatum had homologous genes that participated the complete GlcCer biosynthesis pathway and generated GlcCer (d18:1/18:0h) and GlcCer (d18:2/18:0h), however they were absent in S. cerevisiae2Sphingolipids participate in the hyphae polar growth of P. digitatumMyriocin, fumonisin B1(FB1) and aureobasidin A(AbA) were inhibitors targeting the sphingolipid biosynthesis pathway. The MICs of myriocin, FB1and AbA to the growth of P. digitatum were0.8,0.8and0.4μg/ml, respectively. On the media containing a half MIC concentrations of these inhibitors, the hyphal polar growth was arrested, and the germination tubers of conidia germinated on these inhibitor-contained media branched earlier than these on inhibitor-free media, suggesting that sphingolipids were required for the hyphal polar growth of P. digitatum.3Establishment of High liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) to identify sphingolipids in P. digitatumThis detection was operated in positive ionization mode by multiple reaction monitoring (MRM) technique using precursor ion scan and product ion scan. The retaining time of standards also provided references. Qualitative analysis indicated that P. digitatum has sphingosine (d18:1), phytosphingosine (t18:0), GlcCer (d18:2/18:0h) and GlcCer (d18:1/18:0h), their [M+H+]+were300,318,742and744.4PdMitl was required for hyphal growth, sporulation, spore germination, involved in the sensitivity to divalent cation and amphotericin BIn the plasma membrane, MIPC and other complex sphingolipids constitute the lipid rafts with sterols. The functions of the lipid raft were elaborated including material transport and endocytosis. An ortholog of Aspergillus fumigatus Mitl encoding mannose:inositolphosphorylceramide transferase was identified and named PdMitl in P. digitatum. To determine the functions of PdMitl, a gene-disruption mutant was generated by gene homologous recombination using an Agrobacterium tumefaciens-mediated transformation procedure. Compared to the wildtype strain PdKH8, the colony diameter of PdGcsl deletion mutant was decreased severely, the sporulation and the germination percent of conidia of PdMitl deletion mutant was also severely impaired. These phenotypes were repaired, when PdMitl was introduced into PdMitl deletion mutant. These data indicated that PdMitl was involved in hyphal growth, sporulation and spore germination in P. digitatum.The content of ergosterol in APdMitl was higher than that in wild type strain, and the sensitivity of APdMitl to amphotericin B was increased. The fusion of vacuole under high concentrations of Ca2+in APdMitl was blocked. APdMitl also showed hypersensitive to high concentrations of Ca2+and Mg2+. Under treatment with250mmol/1Ca2+for6h, the relative expression level of three Ca2+ATPase genes PMC1, PMR1and PMCA in wild type strain were significantly up regulated compared with untreated wildtype, but were not happened in PdMitl deletion mutant This result indicated that PdMitl is directly or indirectly involved in the expression regulation of these three Ca2+ATPase, thus determined the sensitivity of P. digitatum to Ca2+.5PdGcsl encoded GlcCer synthase and was required for hyphal growth, sporulation, spore germination and virulenceGlcCer (Glucosylceramide) is synthesized by uridine diphosphate-glucose: ceramide glucosyltransferase encoded by GlcCer synthase (Gcs) gene in Pichia pastoris and other fungi. An ortholog of Candida albicans and Cryptococcus neoformans Gcsl was identified in P. digitatum and named PdGcsl. PdGcsl had two nucleotide recognition domains NRD2L and NRD2S, and also had D1, D2, D3and Q/RXXRW motif. PdGcsl deletion mutant was generated using the same method above. GlcCer (d18:1/18:0h) and GlcCer (d18:2/18:0h) were absent in the PdGcsl deletion mutant using HPLC-MS/MS, demonstrated that PdGcsl was the real gene encoding GlcCer synthase, and participated the synthase of GlcCer. Compared to the wildtype strain PdKH8, the colony diameter of PdGcsl deletion mutant on PDA and C YA was decreased severely, the sporulation and the germination percent of conidia of PdGcsl deletion mutant was severely impaired with32%and44.7%reduction. These data indicated that PdGcsl was involved in hyphal growth, sporulation and spore germination in P. digitatum. Compared to the wildtype strain PdKH8, the virulence of PdGcsl deletion mutant on Ponkan(Citrus reticulata Blanco) was impaired, the symptom appeared delayed, the lesion size induced by PdGcsl deletion mutant was only half of that induced by the wild type strain. This result indicated GlcCer participated the pathogenesis of P. digitatum. GlcCer was the first small molecule required for pathogenicity in P. digitatum we have known so far.