Npc1 Is Involved in Sterol Trafficking in the Filamentous Fungus Fusarium
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Fungal Genetics and Biology 48 (2011) 725–730 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Npc1 is involved in sterol trafficking in the filamentous fungus Fusarium graminearum ⇑ Andrew Breakspear a, Matias Pasquali b, Karen Broz c, Yanhong Dong a, H. Corby Kistler a,c, a Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108, USA b Centre de Recherche Public-Gabriel Lippmann, Département Environnement et Agro-biotechnologies – 41, rue du Brill, L-4422 Belvaux, Luxembourg c USDA ARS Cereal Disease Laboratory, 1551 Lindig Street, St. Paul, MN 55108, USA article info abstract Article history: The ortholog of the human gene NPC1 was identified in the plant pathogenic, filamentous fungus Fusar- Received 7 January 2011 ium graminearum by shared amino acid sequence, protein domain structure and cellular localization of Accepted 1 March 2011 the mature fungal protein. The Fusarium Npc1 gene shares 34% amino acid sequence identity and 51% Available online 11 March 2011 similarity to the human gene, has similar domain structure and is constitutively expressed, although up-regulated in ungerminated macroconidia and ascospores. GFP-tagged Npc1p localizes to the fungal Keywords: vacuolar membrane. Cultures derived from a Dnpc1 mutant strain contain significantly more ergosterol Niemann–Pick type C disease than cultures of the wildtype. Staining with the fluorescent, sterol binding dye filipin, shows that ergos- Ergosterol terol accumulates in vacuoles of the Dnpc1 mutant but not the wildtype strain. The Dnpc1 mutant has a Azole antifungals Fungicides temperature dependent reduction in growth and greater sensitivity to the ergosterol synthesis inhibiting fungicide tebuconazole compared with the wildtype strain or the mutant complemented with wildtype Npc1. The mutant also is significantly reduced in pathogenicity to wheat. Our results are consistent with the interpretation that Npc1p is important for normal transport of ergosterol from the vacuole and is essential for proper membrane function under particular environmental conditions. Published by Elsevier Inc. 1. Introduction translocation of cholesterol from the endosome to the endoplasmic reticulum where the sterol may be utilized as a structural compo- The process of ergosterol biosynthesis has been exploited for nent of the cellular membranes (Kwon et al., 2009). It is currently the control of fungal diseases of both plants and animals. Allyla- unclear whether cholesterol binding and translocation may be the mine and azole antifungal compounds each inhibit different enzy- primary, or merely an ancillary, function of Npc1p or the major matic steps in ergosterol biosynthesis (Ghannoum and Rice, 1999). deficiency resulting in NPC. Despite the importance of these compounds and their action for The goal of this study was to determine if a homologous process control of fungal diseases, little is known about regulation of sterol of sterol trafficking exists in filamentous fungi and to discover the homeostasis and transport within cells of filamentous fungi. role of a putative fungal NPC1 ortholog in sterol homeostasis of the In human, the gene NPC1 appears to be required for proper lipid fungus. Because of the complex domain structure and multifunc- trafficking from the lysosome (Lloyd-Evans and Platt, 2010). Muta- tional biochemical properties of human NPC1, a microbial model tions in the human NPC1 gene result in a fatal autosomal recessive for protein structure and function would be advantageous. Addi- disorder known as Niemann–Pick type C disease (Munkacsi et al., tionally, because of the importance of ergosterol biosynthesis as 2009). Niemann–Pick type C disease (NPC) is associated with accu- a target for antifungal therapy of human and plant diseases, further mulation of cholesterol and other lipids derived from low-density knowledge of ergosterol utilization and movement is warranted. lipoprotein (LDL) particles in the late endosome. The cholesterol- binding, Npc1 protein (Npc1p) is located in the endosomal membrane and current models suggest that it may result in 2. Materials and methods 2.1. Mutant generation ⇑ Corresponding author. Address: USDA ARS Cereal Disease Laboratory, 1551 Lindig Street, University of Minnesota, St. Paul, MN 55108, USA. Fax: +1 651 649 The split marker recombination procedure (Catlett et al., 2003) 5054. was used for gene replacement mutation of Npc1 (FGSG_09254.3) E-mail addresses: [email protected] (A. Breakspear), matias.pasquali@ gmail.com (M. Pasquali), [email protected] (K. Broz), [email protected] in Fusarium graminearum strain PH-1 (NRRL 31084) (Supplemental (Y. Dong), [email protected] (H. Corby Kistler). Fig. 1). Mycelium was grown 7 days in Complete Medium (CM) 1087-1845/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.fgb.2011.03.001 726 A. Breakspear et al. / Fungal Genetics and Biology 48 (2011) 725–730 (Correll et al., 1987) and DNA was extracted according to a pub- (Goswami et al., 2006). For verification of NPC1 deletion and com- lished protocol (Pasquali et al., 2004). PCR was performed using plementation, 2 lg of genomic DNA was digested with BstXI and primers listed in Table S1. PCR products were purified with QIA- probed with an internal fragment of the NPC1 gene amplified with quick PCR Purification kit (Qiagen, Valencia, CA). Protoplast prepa- primers FGSG_09254_INTF and FGSG_09254_INTR. For verification ration and fungal transformation were performed as described of the hygromycin resistance (hph) gene in these strains, 1.5 lgof previously (Hou et al., 2002). Transformants were cultivated on DNA was digested with BsrGI and probed with the hph gene frag- V8 juice agar (Seong et al., 2009) with 250 lg/ml hygromycin B ment amplified from pCX62 with primers HYG/F and HYG/R. For (Calbiochem, La Jolla, CA). Single spore isolates were preliminarily confirmation of GFP tagging, 1.5 lg of genomic DNA was digested screened by PCR and two candidate mutants were retained and with BglII and probed with the eGFP gene fragment amplified from analyzed by Southern blot as described below. In addition, PCR pGFP::hph::loxP with primers GFPcodF and GFPcodR. The hph was used for verification of mutants with primers 9254-N1F and probe and internal NPC1 fragment probe were used to verify pres- 9254-N2R (Table S1), using a rapid microwave treatment of the ence of hph and NPC1, respectively. fungal samples for DNA extraction (Pasquali et al., 2010). One mu- tant was chosen for further study and designated PH-1 Dnpc1. 2.3. Pathogenicity testing To complement the Dnpc1 mutant, wildtype Npc1 was ampli- fied from strain PH-1 genomic DNA using primers FGSG_09254FP Wheat plant growth, pathogenicity tests and mycotoxin analy- and FGSG_09254RP (Table S1) that were modified with 50 phos- sis were as detailed previously (Goswami and Kistler, 2005) but phate to permit ligation into a dephosphorylated vector. The with the following modifications. Wheat plants cv. Norm were fer- resulting product was purified using a QIAquick PCR purification tilized with Nutricote 13-13-13 type 100 two weeks after planting kit (Qiagen) and pSM334 (Hou et al., 2002) was completely and with 20-20-20 fertilizer 4 weeks after planting. Inoculum was digested with EcoRV and dephosphorylated using Antarctic phos- prepared by growing cultures in 100 ml CMC for 4–5 days at 25 °C, phatase (New England BioLabs, Ipswich, MA) to prevent re-circu- 12 h fluorescent light, with shaking at 150 rpm. Conidia were col- larization. The linear vector and Npc1 fragment were then ligated lected by filtering cultures through one layer of Miracloth and cen- using T4 DNA ligase (New England BioLabs) at a 3:1 insert vector trifuging the filtrate for 10 min at 2500g. Supernatant was ratio and 1 ll of the ligation mixture was used to transform One- discarded and pelleted conidia washed twice with sterile water Shot TOP10 chemically competent cells (Invitrogen, Carlsbad, (centrifuge 10 min, 2500g). The concentration of conidia was ad- CA). Ten colonies were picked and subject to plasmid purification justed to 106 conidia per ml water. To aid in adhesion to the wheat using the QIAprep spin miniprep kit (Qiagen). Restriction digests spikelet, Triton X-100 was added to a final concentration of 0.1% (v/ confirmed the presence of the insert in all ten colonies, and one, v). Awns were removed from the central spikelet of wheat heads at designated pNPC1COMP was chosen to transform the Dnpc1 mu- anthesis, and the central spikelet inoculated with 104 conidia using tant. Protoplasting and transformation was carried out as previ- a micropipette. Inoculated plants were then placed in a dew cham- ously described (Hou et al., 2002) and yielded two transformants ber for 24 h. The door to the chamber was then opened for a half that were cultivated on V8 juice agar supplemented with 250 lg/ hour, closed and dew applied for another 24 h. Inoculated plants ml geneticin. One transformant was selected for further study were then transferred to a growth chamber (18 °C, 16 h day, and designated PH-1 Dnpc1::Npc1. 16 °C night measured at head height) for 14 days. For each strain, To generate Npc1p tagged with green fluorescent protein (GFP), ten wheat heads were inoculated per replication. Each strain had a fusion PCR protocol was used (Yang et al., 2004). Primers GFPF four replicates for a total of 40 heads per strain. Head blight symp- and GFPR (Table S1) were used to amplify a 2.4 kb region of toms were rated 14 days post inoculation (dpi) by counting the pGFP::hph::loxP (GenBank accession: FJ457011) (Honda and number of symptomatic spikelets per inoculated head (Proctor et Selker, 2009) containing the GFP and hygromycin resistance genes al., 1995). A total of 10 spikelets were evaluated per head, four (Supplemental Fig. 2). A 1 kb region of Npc1 immediately upstream spikelets below the inoculated central spikelet, the inoculated of the predicted stop codon was amplified with primers 09254LF1F spikelet, and five spikelets above.