title: A pleiotropic drug resistance transporter is involved in reduced sensitivity to multiple fungicide classes in Sclerotinia homoeocarpa (F.T. Bennett).
abstract: Dollar spot, caused by Sclerotinia homoeocarpa, is a prevalent turfgrass disease, and the fungus exhibits widespread fungicide resistance in North America. In a previous study, an ABC-G transporter, ShatrD, was associated with practical field resistance to demethylation inhibitor (DMI) fungicides. Mining of ABC-G transporters, also known as pleiotropic drug resistance (PDR) transporters, from RNA-Seq data gave an assortment of transcripts, several with high sequence similarity to functionally characterized transporters from Botrytis cinerea, and others with closest blastx hits from Aspergillus and Monilinia. In addition to ShatrD, another PDR transporter showed significant over-expression in replicated RNA-Seq data, and in a collection of field-resistant isolates, as measured by quantitative polymerase chain reaction. These isolates also showed reduced sensitivity to unrelated fungicide classes. Using a yeast complementation system, we sought to test the hypothesis that this PDR transporter effluxes DMI as well as chemically unrelated fungicides. The transporter (ShPDR1) was cloned into the Gal1 expression vector and transformed into a yeast PDR transporter deletion mutant, AD12345678. Complementation assays indicated that ShPDR1 complemented the mutant in the presence of propiconazole (DMI), iprodione (dicarboximide) and boscalid (SDHI, succinate dehydrogenase inhibitor). Our results indicate that the over-expression of ShPDR1 is correlated with practical field resistance to DMI fungicides and reduced sensitivity to dicarboximide and SDHI fungicides. These findings highlight the potential for the eventual development of a multidrug resistance phenotype in this pathogen. In addition, this study presents a pipeline for the discovery and validation of fungicide resistance genes using de novo next-generation sequencing and molecular biology techniques in an unsequenced plant pathogenic fungus.
authors.affiliation: Stockbridge School of Agriculture, University of Massachusetts, 230 Stockbridge Rd., Amherst, MA, 01003-9320, USA.
authors.forename: Hyunkyu
authors.initials: H
authors.lastname: Sang
authors.forename: Jon
authors.initials: J
authors.lastname: Hulvey
authors.forename: James T
authors.initials: JT
authors.lastname: Popko
authors.forename: John
authors.initials: J
authors.lastname: Lopes
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Swaminathan
authors.forename: Taehyun
authors.initials: T
authors.lastname: Chang
authors.forename: Geunhwa
authors.initials: G
authors.lastname: Jung
mesh_terms: D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D001203:Ascomycota / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001713:Biphenyl Compounds / Q000494:pharmacology; D060467:Disease Resistance / Q000187:drug effects; D025141:Drug Resistance, Fungal / Q000187:drug effects*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005659:Fungicides, Industrial / Q000494:pharmacology*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005816:Genetic Complementation Test; D006827:Hydantoins / Q000494:pharmacology; D016014:Linear Models; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D010802:Phylogeny; D010935:Plant Diseases / Q000276:immunology / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D017423:Sequence Analysis, RNA; D059467:Transcriptome / Q000235:genetics; D014230:Triazoles / Q000494:pharmacology
chemical_list: D001713:Biphenyl Compounds; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D006827:Hydantoins; D026901:Membrane Transport Proteins; D014230:Triazoles; C045950:propiconazole; D009536:Niacinamide; C550088:2-chloro-N-(4-chlorobiphenyl-2-yl)nicotinamide; D000620:Aminoimidazole Carboxamide; C033148:iprodione
keywords: DMI; PDR transporter; RNA-Seq; SDHI; dicarboximide; multidrug resistance