A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis

Abstract

Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host.

Figure 1. U. maydis–induced tumor formation in maize and predicted structure of Srt1.

(A) Ear tumors of a maize plant infected with U. maydis that caused tumor induction. (B) Uninfected (middle) and U. maydis–infected, tumorous (left) maize kernels, plus a tumor section (right) showing layers of black fungal teliospores. (C) Putative topology of Srt1.

Figure 2. srt1 deletion does not affect U. maydis growth in axenic culture.

Growth of SG200Δsrt1 on glutamine minimal media containing the monosaccharides (A) glucose or (B) fructose or the disaccharides (C) sucrose or (D) maltose is not reduced compared to the SG200 wild-type strain. Cultures from liquid glutamine minimal medium (1% glucose) were spotted in a series of 10-fold dilutions on the media indicated.

Figure 3. Srt1-GFP is specifically expressed in planta.

(A) Expression profile (real-time PCR) of srt1 in SG200 grown in liquid media supplemented with different carbon sources (left) or on plant tissue at different time points after infection. Gene expression was normalized to the expression of the constitutively expressed genes actin and eIF2B. Changes in srt1 expression are displayed relative to the lowest expression value. (B) The SG200Δsrt1::srt1-GFP mutant shown to have a functional Srt1-GFP protein in Figure 3 was grown in minimal medium with 1% glucose. Cells were photographed in white light or under GFP excitation light (bottom). DIC, differential interference contrast microscopy. (C) SG200Δsrt1::srt1-GFP mutant photographed after growth in minimal medium with 1% sucrose. (D) In contrast to (B) and (C), hyphae of the SG200Δsrt1::srt1-GFP mutant show Srt1::GFP-derived fluorescence when monitored after infection of plant tissue (3 dpi). A DIC image (top) and two merged fluorescence images (blue indicates autofluorescence of plant cell walls; green, Srt1::GFP fluorescence of fungal hyphae) are shown. Arrows point towards clamp cells, which are formed by U. maydis only during in planta growth. Asterisks mark cell-to-cell penetration points. Bars represent 10 µm.

Figure 4. Srt1 is necessary for pathogenic development of U. maydis.

(A) Tumor development at 7 dpi on maize leaves infected with the wild-type (wt) strain SG200, with an SG200Δsrt1 deletion mutant, with a mutant strain that had its str1 gene replaced by an srt1-GFP fusion construct under the control of the srt1 promoter (SG200Δsrt1::srt1-GFP), with the Δsrt1 deletion mutant complemented with a copy of srt1 in the ip locus (SG200Δsrt1-srt1::ip), or with a mutant strain that had its srt1 gene replaced by the Arabidopsis AtSUC9 cDNA under the control of the srt1 promoter (SG200Δsrt1::AtSUC9). (B) Disease rating at 7 dpi of plants infected with the wild-type strain (SG200), with three independent SG200Δsrt1 mutants, with SG200Δsrt1::srt1-GFP, with three independently complemented SG200Δsrt1-srt1::ip strains, and with SG200Δsrt1::AtSUC9. Percentage and range of tumor formation of infected plants are color-coded (n  =  total number of plants analyzed). Error bars indicate the standard deviations of mean expression values.

Figure 5. Srt1-dependent ¹⁴C-sucrose uptake in S. cerevisiae.

(A) Uptake of ¹⁴C-sucrose by srt1-expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM ¹⁴C-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).

Figure 6. Transport characteristics of Srt1.

(A) Transport is activated in the presence of the metabolizable carbon source glucose (Glc). (B) The pH optimum for sucrose uptake by Srt1 is in the acidic pH range. (C) Sucrose uptake is sensitive to the protonophore CCCP, but not to the SH-group inhibitor PCMBS. w/o, without. (D) The plateau of sucrose accumulation in baker's yeast results from an equilibrium of influx and efflux. Black symbols show the uptake of ¹⁴C-labeled sucrose and the onset of an immediate efflux, after replacement of labeled extracellular sucrose by unlabeled sucrose (black arrow). The grey region at the bottom of the graph shows the amount of sucrose that was sufficient to reach a concentration equilibrium of ¹⁴C-sucrose between the medium and the cell interior. White symbols show the onset of an immediate influx of ¹⁴C-labeled sucrose in an identical experiment that was started with unlabeled sucrose. The white arrow indicates the replacement of unlabeled extracellular sucrose by ¹⁴C-labeled sucrose. One of three experiments with identical results is presented. Error bars in (A) to (C) represent standard error (n = 3).

Figure 7. Subcellular localization of Srt1 in S. cerevisiae.

A functional Srt1::GFP fusion protein localizes specifically to the plasma membranes of S. cerevisiae. The fusion construct was expressed under the control of the S. cerevisiae pma1 promoter. The left image was taken under GFP excitation light; the corresponding image under transmission light is shown on the right side. The scale bar represents 5 µm.

Figure 8. Model of the bidirectional competition for extracellular sucrose at the plant/fungus interface.

Plants are known to use apoplastic sucrose either via plasma membrane-localized sucrose transporters (SUC or SUT proteins) or due to the activity of extracellular invertases (INV) via membrane-localized hexose transporters (STP or MST proteins). Srt1, a high-affinity sucrose H⁺-symporter, localizes to the fungal plasma membrane, and with its high substrate specificity and extremely low K _M value, it enables the fungus to efficiently use sucrose from the plant/fungus interface.