A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis methods
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Srt1 Is an Energy-Dependent, Sucrose-Specific Transporter of the Fungal Plasma Membrane
(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substrates added at 100-fold molar excess. w/o, without. (C) Michaelis-Menten kinetics of sucrose uptake rates (pH 5.0) indicate a K M of 26±4.3 µM (standard error [SE]). Error bars represent SE ( n = 3).
The Arabidopsis Sucrose Transporter AtSUC9 Can Functionally Replace Srt1
To validate that sucrose uptake is the primary function of Srt1 during biotrophic growth, we tested whether another transporter with a well-characterized sucrose uptake activity can functionally replace Srt1. We selected the sucrose transporter AtSUC9 from Arabidopsis thaliana. This plant transporter is plasma membrane localized, transports sucrose and maltose, and is sensitive to CCCP and PCMBS. Moreover, AtSUC9 has a K M -sucrose of 0.5 mM, which is quite low for a plant sucrose transporter but still 20-fold higher than the K M -sucrose of Srt1 ( ). In strain SG200Δ srt1:: AtSUC9, the AtSUC9 cDNA was inserted into the srt1 locus.
Srt1 Enables U. maydis to Feed on Apoplastic Sucrose without Extracellular Hydrolysis
Under growth chamber conditions, an U. maydis mutant that had its srt1 gene replaced by an srt1 promoter/ AtSUC9 cDNA fusion showed wild-type virulence ( ). With a K M -sucrose of 0.5 mM, AtSUC9 has a lower substrate affinity than Srt1, but still one of the lowest K M -sucrose values determined for plant sucrose transporters. In contrast, the K M -sucrose of ZmSUT1, the sucrose transporter responsible for phloem loading in maize and, thus, the competing transporter at the U. maydis /maize interface, varies from 3.7 mM at pH 5.6 to 12.4 mM at pH 6.5. These different K M values may explain the successful replacement of Srt1 by AtSUC9. Nevertheless, it could well be that SG200Δ srt1:: AtSUC9 would show reduced virulence in the field, where growth conditions are more competitive.
Materials and Methods
Escherichia coli strain TOP10 (Invitrogen) was used for cloning purposes. For plant infections, U. maydis cells were grown at 28°C in YEPSL. For RNA extraction, U. maydis was grown in glutamine minimal medium, which is based an the minimal medium described by Holliday with 30 mM l -glutamine as nitrogen source. Plant infections with U. maydis were performed as described. The U. maydis strain used in this study is SG200, a haploid, solopathogenic strain that can infect maize plants without a mating partner. S. cerevisiae strains used for analyses of Srt1 were EBY.VW4000 ( MATa; leu2-3,112; ura3-52; trp1-289; his3-Δ1; MAL2-8c; SUC2; Δhxt1-17; Δgal2; Δstl1; Δagt1; Δmph2; Δmph3), SEY2102 ( MATα; ura3-52; leu2-3,112; his4-519; suc2-Δ9; gal2), D458-1B ( MATα; leu2; itr1; ino1), and DBY2617 ( MATa; his4-539; lys2-801; ura3-52; suc2-43...
Transport Studies with Radiolabeled Substrates
S. cerevisiae cells were grown to an absorbance at 600 nm (A 600 nm ) of 1.0, harvested, washed twice with water, and resuspended in buffer to an A 600 nm of 10.0. If not otherwise indicated, uptake experiments were performed in 50 mM Na-phosphate buffer (pH 5.0) with an initial substrate concentration of 1 mM 14 C-labeled sucrose (or another 14 C-labeled or 3 H-labeled substrate). Cells were shaken in a rotary shaker at 29°C, and transport tests were started by adding labeled substrate. Samples were withdrawn at given intervals, filtered on nitrocellulose filters (0.8-µm pore size), and washed with an excess of distilled H 2 O. Incorporation of radioactivity was determined by scintillation counting. Competition analyses were performed with 0.1 mM 14 C-sucrose in the presence of 10 mM competitor (100-fold excess). For analyses of the energy dependence of sucrose transport, d...
Transport Studies with Radiolabeled Substrates
For influx/efflux analyses in the plateau of sucrose accumulation ( ), identical amounts of S. cerevisiae cells were incubated in two flasks with either 100 µM 14 C-labeled sucrose or with unlabeled sucrose, and sucrose uptake was determined in the flask with the labeled substrate. When the plateau was reached (after 35 min), the cells were quickly pelleted and washed in Na-phosphate buffer (pH. 5.0). Cells from the unlabeled flask were then resuspended to the initial volume with 100 µM 14 C-sucrose, cells from the labeled flask with 100 µM unlabeled sucrose, and uptake experiments were continued.
Quantitative Real-Time PCR Analysis
To analyze srt1 expression on different carbon sources, SG200 was grown in glutamine minimal media supplemented with the indicated amount of the respective carbon source to an optical density at 600 nm (OD 600 ) of 1.0 for 6 h. Precultures were grown overnight in glutamine minimal medium containing 1% of glucose. RNA samples were frozen in liquid nitrogen for two independently conducted replicates.
Supporting Information
14 C-maltose is not a substrate for Srt1. Uptake of 14 C-maltose (closed circles) was determined in parallel with the uptake of 14 C-sucrose (open circles) in the same srt1 -expressing S. cerevisiae cells that had been used to determine transport in. The extracellular pH was 5.0, substrate concentration was 1 mM. Although 14 C-maltose transport was analyzed for much longer than the transport of 14 C-sucrose (see also ), no significant import of 14 C-maltose into srt1 -expressing cells could be observed. Error bars represent standard error ( n = 3).
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(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substrates added at 100-fold molar excess. w/o, without. (C) Michaelis-Menten kinetics of sucrose uptake rates (pH 5.0) indicate a K M of 26±4.3 µM (standard error [SE]). Error bars represent SE ( n = 3).
To validate that sucrose uptake is the primary function of Srt1 during biotrophic growth, we tested whether another transporter with a well-characterized sucrose uptake activity can functionally replace Srt1. We selected the sucrose transporter AtSUC9 from Arabidopsis thaliana. This plant transporter is plasma membrane localized, transports sucrose and maltose, and is sensitive to CCCP and PCMBS. Moreover, AtSUC9 has a K M -sucrose of 0.5 mM, which is quite low for a plant sucrose transporter but still 20-fold higher than the K M -sucrose of Srt1 ( ). In strain SG200Δ srt1:: AtSUC9, the AtSUC9 cDNA was inserted into the srt1 locus.
Under growth chamber conditions, an U. maydis mutant that had its srt1 gene replaced by an srt1 promoter/ AtSUC9 cDNA fusion showed wild-type virulence ( ). With a K M -sucrose of 0.5 mM, AtSUC9 has a lower substrate affinity than Srt1, but still one of the lowest K M -sucrose values determined for plant sucrose transporters. In contrast, the K M -sucrose of ZmSUT1, the sucrose transporter responsible for phloem loading in maize and, thus, the competing transporter at the U. maydis /maize interface, varies from 3.7 mM at pH 5.6 to 12.4 mM at pH 6.5. These different K M values may explain the successful replacement of Srt1 by AtSUC9. Nevertheless, it could well be that SG200Δ srt1:: AtSUC9 would show reduced virulence in the field, where growth conditions are more competitive.
Escherichia coli strain TOP10 (Invitrogen) was used for cloning purposes. For plant infections, U. maydis cells were grown at 28°C in YEPSL. For RNA extraction, U. maydis was grown in glutamine minimal medium, which is based an the minimal medium described by Holliday with 30 mM l -glutamine as nitrogen source. Plant infections with U. maydis were performed as described. The U. maydis strain used in this study is SG200, a haploid, solopathogenic strain that can infect maize plants without a mating partner. S. cerevisiae strains used for analyses of Srt1 were EBY.VW4000 ( MATa; leu2-3,112; ura3-52; trp1-289; his3-Δ1; MAL2-8c; SUC2; Δhxt1-17; Δgal2; Δstl1; Δagt1; Δmph2; Δmph3), SEY2102 ( MATα; ura3-52; leu2-3,112; his4-519; suc2-Δ9; gal2), D458-1B ( MATα; leu2; itr1; ino1), and DBY2617 ( MATa; his4-539; lys2-801; ura3-52; suc2-438). Cells were grown in minimal medium (0.67% yeast nitrogen base without amino acids plus required amino acids depending on the strain) containing 2% maltose (EBY.VW4000) or glucose (all other strains) at 29°C.
S. cerevisiae cells were grown to an absorbance at 600 nm (A 600 nm ) of 1.0, harvested, washed twice with water, and resuspended in buffer to an A 600 nm of 10.0. If not otherwise indicated, uptake experiments were performed in 50 mM Na-phosphate buffer (pH 5.0) with an initial substrate concentration of 1 mM 14 C-labeled sucrose (or another 14 C-labeled or 3 H-labeled substrate). Cells were shaken in a rotary shaker at 29°C, and transport tests were started by adding labeled substrate. Samples were withdrawn at given intervals, filtered on nitrocellulose filters (0.8-µm pore size), and washed with an excess of distilled H 2 O. Incorporation of radioactivity was determined by scintillation counting. Competition analyses were performed with 0.1 mM 14 C-sucrose in the presence of 10 mM competitor (100-fold excess). For analyses of the energy dependence of sucrose transport, d -glucose was added to the yeast cells 2 min before the start of the experiment to a final concentration of 10 mM. For inhibitor analyses, CCCP (carbonylcyanide m -chlorophenylhydrazone) or PCMBS ( p -chloromercuribencene sulfonate) were used at final concentrations of 50 µM.
For influx/efflux analyses in the plateau of sucrose accumulation ( ), identical amounts of S. cerevisiae cells were incubated in two flasks with either 100 µM 14 C-labeled sucrose or with unlabeled sucrose, and sucrose uptake was determined in the flask with the labeled substrate. When the plateau was reached (after 35 min), the cells were quickly pelleted and washed in Na-phosphate buffer (pH. 5.0). Cells from the unlabeled flask were then resuspended to the initial volume with 100 µM 14 C-sucrose, cells from the labeled flask with 100 µM unlabeled sucrose, and uptake experiments were continued.
To analyze srt1 expression on different carbon sources, SG200 was grown in glutamine minimal media supplemented with the indicated amount of the respective carbon source to an optical density at 600 nm (OD 600 ) of 1.0 for 6 h. Precultures were grown overnight in glutamine minimal medium containing 1% of glucose. RNA samples were frozen in liquid nitrogen for two independently conducted replicates.
14 C-maltose is not a substrate for Srt1. Uptake of 14 C-maltose (closed circles) was determined in parallel with the uptake of 14 C-sucrose (open circles) in the same srt1 -expressing S. cerevisiae cells that had been used to determine transport in. The extracellular pH was 5.0, substrate concentration was 1 mM. Although 14 C-maltose transport was analyzed for much longer than the transport of 14 C-sucrose (see also ), no significant import of 14 C-maltose into srt1 -expressing cells could be observed. Error bars represent standard error ( n = 3).
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