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.

Keywords: DMI; PDR transporter; RNA-Seq; SDHI; dicarboximide; multidrug resistance.

Figure 1

Neighbour‐joining phylogenetic tree of amino acid sequences of pleiotropic drug resistance (PDR) transporters from the dataset of Hulvey et al. (2012), including five additional full‐length PDR transporters from Sclerotinia homoeocarpa (ShPDR1, ShPDR1 deletion, contigs 2174, 4985 and 6230), Penicillium digitatum PMR3 and PMR4, and Aspergillus kawachii  PDR transporter (Lamping et al., 2010). The scale bar equals the proportion of amino acid substitutions.

Figure 2

Relative expression (RE) of ShPDR1 in the initial panel of eight isolates from five sites in New England in the absence and presence of propiconazole (0.1 μg/mL), iprodione (1 μg/mL) and boscalid (10 μg/mL) for 1 h. All bars (white, constitutive RE; light grey, propiconazole‐induced RE; dark grey, iprodione‐induced RE; black, boscalid‐induced RE) represent mean RE values. Error bars indicate one standard error of the mean. Isolates with an ‘I’ indicate propiconazole insensitive and with an ‘S’ represent propiconazole sensitive.

Figure 3

Relative expression (RE) values for ShPDR1 in practical field‐resistant (PFR) and sensitive Sclerotinia homoeocarpa isolates from Hickory Ridge Country Club and Hartford Golf Club. All bars (grey, constitutive RE of PFR isolates from propiconazole applied plots; white, constitutive RE of sensitive isolates from control plots) represent mean RE values. Error bars indicate one standard error of the mean.

Figure 4

Relative expression (RE) of ShPDR1 and ShatrD in two insensitive Sclerotinia homoeocarpa isolates (HRI11 and HFI40) in the absence and presence of propiconazole (0.1, 1 and 5 μg/mL) for 1 h. All bars (grey, RE of ShPDR1; white, RE of ShatrD) represent mean RE values. Error bars indicate one standard error of the mean. Significant differences between RE values of ShPDR1 and ShatrD are indicated: n.s., not significant; ***, significant at P < 0.0001.

Figure 5

Relationship between log₁₀ relative expression (RE) values of ShPDR1 and in vitro sensitivities to propiconazole (A), iprodione (B) or boscalid (C) (as measured by EC₅₀ for propiconazole and iprodione and EC₉₅ for boscalid) for 24 isolates from New England. The notation of isolates was according to Hulvey et al. (2012) [triangles, practical field‐resistant (PFR) isolates from Hartford Golf Club; diamonds, PFR isolates from Hickory Ridge Country Club; squares, insensitive isolates from four sites; black dots, sensitive isolates from five sites]. Each coloured shape indicates the same isolates for (A), (B) and (C).

Figure 6

Complementation of Saccharomyces cerevisiae strain AD1‐8 with ShPDR1 from Sclerotinia homoeocarpa. Sensitivity assays of the transformants were conducted on bacto‐yeast nitrogen base (YNB) agar medium lacking uracil, containing 2% galactose and amended with increasing concentrations of propiconazole, iprodione and boscalid fungicides.