ABA-Induced Sugar Transporter TaSTP6 Promotes Wheat Susceptibility to Stripe Rust^,

Upregulation of the wheat sugar transporter gene TaSTP6 promotes host susceptibility to stripe rust possibly due to increased hexose concentration.

Abstract

Biotrophic pathogens, such as wheat rust fungi, survive on nutrients derived from host cells. Sugar appears to be the major carbon source transferred from host cells to various fungal pathogens; however, the molecular mechanism by which host sugar transporters are manipulated by fungal pathogens for nutrient uptake is poorly understood. TaSTP6, a sugar transporter protein in wheat (Triticum aestivum), was previously shown to exhibit enhanced expression in leaves upon infection by Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. In this study, we found that Pst infection caused increased accumulation of abscisic acid (ABA) and that application of exogenous ABA significantly enhanced TaSTP6 expression. Moreover, knockdown of TaSTP6 expression by barley stripe mosaic virus-induced gene silencing reduced wheat susceptibility to the Pst pathotype CYR31, suggesting that TaSTP6 expression upregulation contributes to Pst host sugar acquisition. Consistent with this, TaSTP6 overexpression in Arabidopsis (Arabidopsis thaliana) promoted plant susceptibility to powdery mildew and led to increased Glc accumulation in the leaves. Functional complementation assays in Saccharomyces cerevisiae showed that TaSTP6 has broad substrate specificity, indicating that TaSTP6 is an active sugar transporter. Subcellular localization analysis indicated that TaSTP6 localizes to the plasma membrane. Yeast two-hybrid and bimolecular fluorescence complementation experiments revealed that TaSTP6 undergoes oligomerization. Taken together, our results suggest that Pst stimulates ABA biosynthesis in host cells and thereby upregulates TaSTP6 expression, which increases sugar supply and promotes fungal infection.

Wheat (Triticum aestivum) is one of the major food crops worldwide. With an increasing global population, the demand for wheat is rising. However, wheat is susceptible to a range of abiotic and biotic stresses, which pose varying degrees of threats to yield. Over the years, scientists have been searching for new agronomic traits in wheat to adapt to the stresses as well as to improve yield (Wellings, 2011). Stripe rust is caused by Puccinia striiformis f. sp. tritici (Pst), which is one of the most destructive fungal diseases in wheat (Hovmøller et al., 2010). The disease may cause significant yield loss and thus lead to food shortages and rising food prices. Pst is an obligate biotrophic fungus that acquires nutrients through specialized feeding structures called “haustoria.” Haustoria are fungal organs specific to biotrophic pathogens that form intimate interactions with the host cell to absorb nutrients (Sutton et al., 1999; Voegele and Mendgen, 2003). Control over nutrient flux from the plant to the pathogen is a potential novel and effective strategy to control a variety of plant pathogens. However, little is known about how infected host cells control the release of nutrients to their pathogenic boarders.

Pathogens, irrespective of their lifestyle, develop at the cost of nutrients released and generated by the host plant during plant-pathogen interactions. It is assumed that upon infection of above-ground plant parts, classical source tissue turns into sink tissue (Doidy et al., 2012; Bezrutczyk et al., 2018). Earlier studies of nutrient transfer from the host to the fungus were carried out on powdery mildew (Sutton et al., 1999; Hall and Williams, 2000). It was found that Glc is the primary carbon and energy source diverted to the fungal mycelium (Sutton et al., 1999; Hall and Williams, 2000). Later, work on other biotrophic pathogens followed. UfHXT1, a high-affinity hexose/proton symporter from Uromyces fabae, was found to be exclusively localized to the haustorial plasma membrane and play a pivotal role in carbon transfer to the fungus from the extrahaustorial matrix (Voegele et al., 2001). In addition, plant invertase activity was found to be increased after pathogen inoculation (Wirsel et al., 2001; Voegele et al., 2006), which might be attributed to the lack of Suc transporters in some biotrophic fungi (Spanu et al., 2010; Duplessis et al., 2011). Suc therefore has to be cleaved into Glc and Fru, which are subsequently taken up by the pathogen. Meanwhile, increased invertase activity can also alter the extracellular apoplastic hexose/Suc ratio and elicit a hexose-mediated defense response (Proels and Hückelhoven, 2014).

Sugar transport and especially partitioning across the plant plasma membrane by transporters is one of the most important processes for plant development and plant responses to biotic and abiotic factors (Lalonde et al., 2004; Lemoine et al., 2013). The main sugar transporter proteins in plants comprise both Suc transporters (SUTs/SUCs) and monosaccharide transporters. Both of the above-mentioned transporters belong to the major facilitator superfamily and are predicted to share a similar structure, with 12 putative transmembrane domains (TMDs) connected by hydrophilic loops, and to function as H⁺/sugar symporters (Doidy et al., 2012). Recently, a new class of sugar transporters termed “SWEETs” has been identified (Xuan et al., 2013). SWEETs are heptahelical proteins carrying a tandem repeat of 3-TMD separated by a single TMD (Chen et al., 2015b). These two families of transporters seem to be involved in balancing plant monosaccharide influx and efflux across the plasma membrane and between organelles within the cell. The plant MST family is fairly large, comprising 53 members in Arabidopsis and 65 in rice (Oryza sativa; Doidy et al., 2012). MSTs can be distinguished based on their substrate specificity (sugar transport proteins [STPs], polyol/monosaccharide transporters, inositol transporters; Büttner and Sauer, 2000; Büttner, 2010). STP members have been identified in different organisms (plants, animals, bacteria, archaea, and fungi) based on their capacity to catalyze the uptake of hexoses from the apoplastic space into the cell (Williams et al., 2000). So far, all of the characterized STPs are plasma membrane-localized H⁺/hexose symporters and show broad substrate specificity with the exception of AtSTP9, a Glc-specific transporter (Schneidereit et al., 2003), and AtSTP14, a Gal-specific transporter (Poschet et al., 2010).

Pathogen infection triggers alteration of sugar transport in host plants for improving pathogen access to nutrients. Sugar allocation at the plant-pathogen interfaces is mediated by sugar transporters, whose regulation patterns determine the outcome of the interaction (Lemoine et al., 2013). For example, AtSTP4 is induced upon infection by Erysiphe cichoracearum and correlated with Glc uptake in host tissue (Fotopoulos et al., 2003). The expression of the hexose transporter VvHT5 (AtSTP13 ortholog) is regulated by abscisic acid (ABA) during the transition from source to sink in response to infection by powdery or downy mildew (Hayes et al., 2010). Xanthomonas bacteria secrete transcription activator-like effectors that induce the transcription of SWEET transporters for sugar efflux into the apoplastic space (outside the symplast), where the bacteria acquire carbohydrates for energy and carbon (Chen, 2014). Plants also regulate sugar transporters to redistribute sugars away from the infection site, removing the pathogens’ energy source and limiting their proliferation. For example, transcript levels of AtSTP13 increased in Arabidopsis infected with Pseudomonas syringae pv tomato DC3000 (Nørholm et al., 2006). Another study revealed that AtSTP13 is activated by phosphorylation in the Arabidopsis-DC3000 interactions, which intensifies its hexose uptake activity to compete with bacteria for apoplastic monosaccharide (Yamada et al., 2016). Similarly, overexpression of AtSTP13 in Arabidopsis improved the capacity to take up Glc and conferred enhanced resistance against Botrytis cinerea (Lemonnier et al., 2014). In wheat, Lr67res (a natural mutation of TaSTP13), which impairs its hexose transport activity, provides partial resistance to all three wheat rust pathogen species (Pst and Puccinia triticina) and powdery mildew (Moore et al., 2015).

Previous studies have reported that Suc accumulation in Pst-challenged wheat leaves is significantly increased, accompanied by hexose accumulation due to simultaneously enhanced invertase activity (Chang et al., 2013; Liu et al., 2015). Nevertheless, few studies are available on hexose transport in Pst-infected wheat plants. TaSTP6, a wheat sugar transporter gene that responds to stripe rust infection but appears insensitive to other plant pathogen invasion, was isolated based on transcriptome analysis of Pst-infected wheat leaves (Hao et al., 2016) and an open-access exp-VIP platform (Borrill et al., 2016).

In this study, we further investigated the role of TaSTP6 in Pst nutrient acquisition and host infection. Upregulation of TaSTP6 transcripts occurred in wheat leaves either inoculated with Pst or treated with ABA. TaSTP6 promoter::β-glucuronidase fusions in transgenic Arabidopsis were activated by ABA treatment. We found that Pst infection also resulted in increased accumulation of ABA. TaSTP6 expression knockdown enhanced wheat resistance to Pst. In addition, overexpression of TaSTP6 promoted Arabidopsis susceptibility to powdery mildew and resulted in increased Glc accumulation in the leaves. Transient expression analysis in Nicotiana benthamiana leaves and wheat leaf protoplasts showed that TaSTP6 is localized to the plasma membrane. Heterologous expression in Saccharomyces cerevisiae revealed that TaSTP6 is a hexose/H⁺ symporter. Yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) validated oligomerization of TaSTP6. Our results indicate that TaSTP6, as an ABA-inducible gene, increases wheat susceptibility to Pst possibly by regulating the transition from source to sink tissue.

RESULTS

Identification and Sequence Analysis of TaSTP6

Complementary DNA (cDNA) containing an open reading frame (ORF) orthologous to the rice STP family member OsSTP6 (Supplemental Fig. S1), designated TaSTP6, was amplified by reverse transcription PCR (RT-PCR). The sequence of TaSTP6 was further used to query the Chinese Spring (CS) genome sequence. BLAST (http://plants.ensembl.org/Multi/Tools/Blast) results showed that there were three TaSTP6 copies located on chromosomes 2A, 2B, and 2D in the wheat genome. The encoding sequences of the three copies only differ in 39 nucleotides and share a sequence identity of 99.18% (Supplemental Fig. S2). Accordingly, the three proteins encoded by them share 99.56% sequence identity (Supplemental Fig. S3), indicating that these three copies may possess identical biological functions.

The ORF of TaSTP6 with 1,581 nucleotide residues is predicted to encode a 526-amino acid peptide with a pI of 9.26 and a calculated molecular mass of 57.5 kD. The membrane spanning model analysis indicates that TaSTP6 contains 12 predicted TMDs (Supplemental Fig. S4). The phylogenetic relationships of TaSTP6 with orthologous proteins from various species showed that TaSTP6 exhibits a high degree of conservation with the STP sequences from monocotyledons (Supplemental Fig. S5). These results indicate that TaSTP6 may encode a sugar transporter protein.

Upregulation of TaSTP6 Is Possibly Mediated by ABA during Pst Infection of Wheat

To confirm the expression pattern of TaSTP6, the promoter sequences of all three TaSTP6 copies were aligned and analyzed using the tools “PlantCARE” (Lescot et al., 2002) and “PLACE” (Higo et al., 1999). The results showed that they share high sequence identity and contain similar cis-regulatory elements, such as ABRE, CGTCA-motif, CE3, LTR, and WUN-motif (Supplemental Table S1), indicating that these three TaSTP6 copies possibly exhibit similar expression patterns. A pair of specific primers for RT quantitative-(q)PCR was designed and used to simultaneously assay the expression level of the three TaSTP6 copies (Supplemental Fig. S2).

During Pst infection of wheat, the expression profile of TaSTP6 was confirmed by RT-qPCR. The transcript levels of TaSTP6 were increased at 12- and 24-h post inoculation (hpi) in wheat leaves challenged with the Pst virulent pathotype CYR31. The highest TaSTP6 transcript levels were ∼16-fold at 24 hpi (Fig. 1A).

Figure 1.

Expression of TaSTP6 was upregulated by Pst infection and exogenous ABA treatment. A and B, TaSTP6 expression changes in response to Pst infection (A) and exogenous ABA treatment (B). Expression levels were normalized to TaEF-1α. The relative expression of TaSTP6 was calculated using the comparative threshold method (2^-ΔΔCt). Untreated leaves act as the control. C, ABA-induced TaSTP6 promoter activity in transgenic Arabidopsis. Transgenic Arabidopsis plants containing pCB308-TaSTP6np were sprayed with a solution containing 1 mm of ABA and 0.05% (v/v) TWEEN 20 or with 0.05% (v/v) TWEEN 20 alone, and sampled at 0, 2, 6, 12, 24, and 48 hpt for GUS-activity. D, Pst infection increases endogenous ABA in wheat leaves. Wheat leaves inoculated with Pst CYR31 were sampled at 8 and 10 hpi for HPLC-MS/MS analysis. Error bars represent variations among three independent replicates. Single (P < 0.05) and double asterisks (P < 0.01) indicate a significant difference from the untreated control according to Student’s t test. FW, fresh weight; MU, 4-methylumbelliferone.

The responses of TaSTP6 to various environmental stresses were also assayed. Wheat seedlings were treated with low temperature, wounding, NaCl, and polyethylene glycol (PEG)6000. The results showed that the expression level of TaSTP6 was increased at 2-h post treatment (hpt) and then decreased under low-temperature treatment (Supplemental Fig. S6). Wound treatment significantly induced transcripts of TaSTP6 at 2 hpt (Supplemental Fig. S6); transcript abundance of TaSTP6 decreased from 6 to 24 hpt and increased at 48 hpt (Supplemental Fig. S6). The expression of TaSTP6 was upregulated at 48 h after PEG6000 treatment, and it was 3-fold higher than that of the control at this time (Supplemental Fig. S6). Under NaCl treatment, no obvious changes were observed in the transcript level of TaSTP6 (Supplemental Fig. S6).

Three distinct combinations (ABRE–ABRE, CE3–CE3, and CE3–ABRE) may form as functional ABA-responsive complexes (ABRCs; Gómez-Porras et al., 2007) in the TaSTP6 promoter region (Supplemental Fig. S7A). Having identified the presence of a cluster of ABRCs in the TaSTP6 promoter region, we investigated TaSTP6 transcript levels in wheat leaves after treatment with exogenous ABA application. As shown in Figure 1B, after ABA treatment, the TaSTP6 transcript level was significantly upregulated with a peak at 12 hpt, whereas TaSTP6 expression appeared to be less sensitive to methyl jasmonate (MeJA) compared with ABA, although several MeJA-responsive elements are contained in the TaSTP6 promoter region (Supplemental Table S1).

To further analyze the involvement of the predicted motifs in ABA responsiveness, the construct pCB308-TaSTP6np was generated and transformed into Arabidopsis. The presence of the construct in Arabidopsis lines in the T2 generation was confirmed by PCR (Supplemental Fig. S7B). β-glucuronidase activity was measured in leaves of the TaSTP6np-6 line after ABA treatment. Our results show that ABA treatment caused an increase in reporter activity at 2 and 6 hpt (Fig. 1C). No significant changes occurred in the control (Fig. 1C). The GUS activity response to ABA supports the hypothesis that ABA-induced TaSTP6 expression is mediated via the ABRCs.

Because the transcript abundance of TaSTP6 was increased in wheat leaves treated with Pst and ABA, the endogenous ABA content in Pst-infected wheat leaves caused concern. HPLC analyses showed that the ABA concentration in Pst-infected wheat leaves was significantly upregulated at 8 and 10 hpi compared with the uninfected control (Fig. 1D). These results, presented in Figure 1, suggested that upregulation of TaSTP6 was possibly mediated by ABA in wheat leaves inoculated with Pst.

Knockdown of TaSTP6 Reduces Wheat Susceptibility to Pst

To determine a possible role of TaSTP6 during the wheat response to Pst, the barley stripe mosaic virus (BSMV)-virus induced gene silencing (VIGS) system was used to knock down the expression of TaSTP6. For co-silencing of the three copies of TaSTP6, two highly conserved regions in the ORF of TaSTP6 were chosen and amplified (Supplemental Fig. S2). No evident defects occurred in wheat growth and mild chlorotic mosaic symptoms were displayed on BSMV-treated wheat seedlings (Fig. 2A). To visualize the functionality of the VIGS system, BSMV:TaPDS (Holzberg et al., 2002) was used as a positive control. Thereafter, we used Pst CYR31 to inoculate the BSMV-treated wheat seedlings. A reduced disease phenotype was observed in TaSTP6-knockdown plants at 14 d post inoculation (dpi; Fig. 2B). Furthermore, plants treated with BSMV:TaSTP6-as1 or BSMV:TaSTP6-as2 showed a similar phenotype (Fig. 2B).

Figure 2.

Functional analysis of TaSTP6 during the wheat-Pst interaction using the BSMV-VIGS system. A, Mild chlorotic mosaic symptoms were observed on the leaves of wheat seedlings at 9 dpi with BSMV:γ, BSMV:TaSTP6-as1, and BSMV:TaSTP6-as2, and photobleaching was evident on the fourth leaves of wheat plants infected by BSMV:TaPDS (a positive control treatment). Wheat leaves inoculated with FES buffer (MOCK). B, Disease phenotypes of the fourth leaves preinoculated with BSMV constructs and then challenged with Pst CYR31. C, Fungal biomass measurements of total DNA extracted from BSMV-treated wheat leaves infected by CYR31 at 14 dpi based on qPCR. Ratio of total fungal DNA to total wheat DNA was assessed by normalizing the data to the wheat gene TaEF-1α and the Pst gene PstEF1. D, Silencing efficiency of TaSTP6 in the fourth leaves of the control (BSMV:γ) and TaSTP6-silenced (BSMV:TaSTP6-as1 and BSMV:TaSTP6-as2) plants. Leaves for RT-qPCR were sampled at 0, 24, 48, 72, and 120 hpi. Data were normalized to the TaEF-1α expression level. The results were obtained from three biological independent replicates. Values represent the means ± se of three independent samples. Asterisks indicate a significant difference (P < 0.05) according to Student’s t test.

To test whether the disease phenotype was correlated with fungal growth in the host tissue, fungal biomass measurements were conducted on infected leaves. Total genomic DNA was extracted from wheat leaves inoculated with Pst, and the relative levels of TaEF-1α and PstEF1 (Yin et al., 2009) were quantified using qPCR to generate standard curves (Supplemental Fig. S8). The fungal biomass was significantly decreased by ∼29% in BSMV:TaSTP6-as1 leaves and by 18% in BSMV:TaSTP6-as2 leaves at 14 dpi compared to control plants (Fig. 2C). This is consistent with the reduced disease phenotype.

RT-qPCR determined that endogenous TaSTP6 transcription was successfully silenced in the fourth leaves of BSMV-VIGS plants. The TaSTP6 transcript in BSMV:TaSTP6-as1-inoculated or BSMV:TaSTP6-as2-inoculated leaves was reduced by ∼50% to 75% at 0, 24, 48, 72, and 120 hpi with CYR31 compared with BSMV:γ (control) leaves (Fig. 2D). Taken together, our results demonstrate that the expression of TaSTP6 was substantially knocked down in our experiments.

Histological Observations of Fungal Growth in TaSTP6-Knockdown Plants

To quantify the reduced disease phenotype in TaSTP6-knockdown lines after infection with CYR31, the infected leaves were studied using histological observations. At 24 hpi, no significant histological differences were observed in TaSTP6-knockdown plants. In BSMV:TaSTP6-as1 and BSMV:TaSTP6-as2-inoculated leaves, the number of hyphal branches, haustorial mother cells, haustoria, and overall hyphal length were decreased at 48 hpi (Fig. 3, A–C, J, and K). In addition, colonization and formation of secondary hyphae were strongly restricted in the TaSTP6-knockdown seedlings compared with the plants inoculated with BSMV:γ (control) at 48, 72, and 120 hpi (Fig. 3, D–I, and L; Supplemental Fig. S9, A and B). These results indicate that knockdown of TaSTP6 results in restricted fungal development in the wheat-Pst interaction.

Figure 3.

Histological observation of fungal growth in wheat leaves infected by BSMV:γ and recombinant BSMV after inoculation with Pst CYR31. A to I, Fungal structures were stained with wheat germ agglutinin (WGA). A to I, Growth of Pst in wheat leaves inoculated with BSMV:γ, BSMV:TaSTP6-as1, or BSMV:TaSTP6-as2 at 48 hpi (A to C), 72 hpi (D to F), and 120 hpi (G to I) was observed under a fluorescence microscope. J, Average number of hyphal branches (HB), haustorial mother cells (HMC), and haustoria (H) of Pst in each infection site were counted at 48 hpi. K, Significant decrease in the length of infection hyphae (IH) in TaSTP6-silenced plants at 48 hpi. The length of IH was measured from the substomatal vesicle (SV) to the apex of the longest infection hyphae. L, TaSTP6-silenced plants show a significant decrease in infection unit area at 120 hpi. Values represent the mean ± se of three independent samples (50 infection sites each time). Asterisks indicate a significant difference (^*P < 0.05, ^**P < 0.01) from BSMV:γ inoculated plants using Student’s t test and one-way ANOVA. (A to C) Scale bar = 20 μm; (D to F) scale bar = 50 μm; (G to I) scale bar = 100 μm.

Overexpression of TaSTP6 Promotes Arabidopsis Susceptibility to Powdery Mildew

Furthermore, to elucidate the possible function of TaSTP6 during the wheat-Pst interaction, we introduced a TaSTP6 overexpression construct into Arabidopsis and generated several transgenic TaSTP6 overexpression (TaSTP6-OE) lines. Two lines (TaSTP6-OE1 and TaSTP6-OE2) showing distinct TaSTP6 mRNA accumulation in leaves (Supplemental Fig. S10) were inoculated with powdery mildew (Li et al., 2019), which resulted in an enhanced susceptibility phenotype (Fig. 4A). Consistent with the disease phenotype, TaSTP6-OE plants had more conidiophores per colony than wild-type Col-0 plants during the early infection stage at 4 dpi when the fungus began asexual reproduction (Fig. 4, B and C). Sugar concentration in leaves from these two transgenic lines was then analyzed using HPLC. As shown in Figure 4D, the Glc content was significantly increased in the transgenic lines compared with the control plants, whereas no obvious difference was observed for other type of sugar, indicating that TaSTP6 is an active sugar transporter.

Figure 4.

Overexpression of TaSTP6 promotes Arabidopsis susceptibility to powdery mildew. A, Representative images of Arabidopsis leaves of indicated genotypes infected with tobacco powdery mildew at 12 dpi. TaSTP6 overexpression lines (TaSTP6-OE1 and -OE2) were more susceptible than wild type. B, Representative microscopic images of single colonies of powdery mildew on leaves of indicated genotypes at 4 dpi. Fungal structures were stained by trypan blue. Scale bars = 200 μm. C, Total number of conidiophores per colony on leaves of indicated genotypes at 4 dpi. The bar-chart shows combined data from three independent experiments (at least 30 colonies were counted for each genotype per experiment). D, Glc concentrations in the leaves of TaSTP6 overexpression lines and wild-type plants. Values are the means ± se of three replicates. Double asterisks indicate a significant difference (P < 0.01) from wild-type plants according to the Student’s t test and one-way ANOVA. FW, fresh weight; WT, wild type.

TaSTP6 Is Localized to the Plasma Membrane

In plants, STPs have been shown to function as plasma membrane proteins (Büttner, 2010). To confirm the TaSTP6 subcellular localization, transient expression of a TaSTP6-GFP construct in N. benthamiana leaves and wheat leaf protoplasts was carried out. Similar results were observed in these two systems. Free GFP, as a control, was detected throughout the cytosol and nucleus (Fig. 5). Transient expression of TaSTP6-GFP in N. benthamiana leaves showed that GFP fluorescence colocalized with the fluorescence of n-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM4-64; Thermo Fisher Scientific), a membrane-selective fluorescent dye (Fig. 5A). In wheat leaf protoplasts, chlorophyll autofluorescence (red) was found inside the ring of GFP fluorescence (green) emitted by the TaSTP6-GFP fusion protein (Fig. 5B). These results indicate that TaSTP6 is localized to the plasma membrane.

Figure 5.

Subcellular localization of TaSTP6 in N. benthamiana leaves and wheat leaf protoplasts. A and B, Subcellular localization of free GFP (as control) and TaSTP6-GFP with transient expression under the control of the 35S promoter in cells of N. benthamiana leaves (A, scale bars = 10 μm) and wheat leaf protoplasts (B, scale bars = 5 μm). FM4-64 was used to stain the plasma membrane. GFP fluorescence is in green. Red fluorescence indicates FM4-64 labeling the plasma membrane or chlorophyll auto-fluorescence. Merged GFP/FM4-64 (A) and GFP/chlorophyll (B) images are shown. Bright-field images show the equivalent field observed under white light. All of the signals were monitored by confocal microscopy. Comparable expression and localization patterns were observed in three independent biological replicates.

Functional Characterization of TaSTP6 by Heterologous Expression in Yeast

To identify the function of TaSTP6, several recombinant plasmids were transformed into the hexose transport-defunct S. cerevisiae mutant EBY.VW4000 (Wieczorke et al., 1999). Yeast cells carrying pDR195-GFP or pDR195-TaSTP6-GFP were monitored by confocal microscopy. We found that TaSTP6 was also localized to the plasma membrane of yeast (Fig. 6A). Thus, TaSTP6 exhibits the same subcellular localization in S. cerevisiae and wheat and it is possible that the transport properties of TaSTP6 can be measured in S. cerevisiae.

Figure 6.

Expression of TaSTP6 in S. cerevisiae. A, Localization of GFP and TaSTP6-GFP in the EBY.VW4000 strain. Bright-field and GFP fluorescence images were taken by confocal microscopy and merged. Scale bars = 2 μm. B, Growth of EBY.VW4000 carrying either the vector pDR195 or plasmid pDR195-TaSTP6 on maltose (control), and the monosaccharides Glc, Fru, and Man 2% (w/v) each. These experiments were repeated three times (two different transformants each time), with similar results.

We then tested the mutant carrying the empty pDR195 vector (Rentsch et al., 1995) or the recombinant plasmid pDR195-TaSTP6 for growth on synthetic dropout (SD)-Ura medium supplemented with different carbon sources. The results show that the complemented strain grew normally on media with Glc, Fru, or Man as the sole carbon source (Fig. 6B). These results suggest that TaSTP6 is a typical monosaccharide transporter.

To determine the kinetic parameters of TaSTP6, [¹⁴C]Glc uptake assays were performed. Our results show that the complemented strain was able to take up [¹⁴C]Glc with a K_m of 49.8 ± 4.75 μm and a V_max of 290 ± 15 pmol mg⁻¹ min⁻¹ (Fig. 7B), whereas yeast cells carrying the empty vector did not take up Glc (Fig. 7A). The pH optimum for TaSTP6 was determined to be ∼5.5 (Fig. 7C).

Figure 7.

Characterization of TaSTP6 transport activity in S. cerevisiae. A, Uptake of [¹⁴C]Glc into the yeast strain EBY.VW4000 transformed with pDR195-TaSTP6 (black circle) or pDR195 alone (white triangle) per milligram fresh weight at an initial outside concentration of 100 μm of Glc at pH 5.5. B, Concentration-dependent [¹⁴C]Glc uptake. The Lineweaver-Burk plot of a typical K_m determination is presented. The estimated K_m is 49.8 ± 4.75 μM. The V_max was determined to be 290 ± 15 pmol mg⁻¹ min⁻¹ cells. C, Relative uptake rate of [¹⁴C]Glc into EBY.VW4000 transformed with pDR195-TaSTP6 at different pH values at an initial outside concentration of 100 μm of Glc. The time interval for [¹⁴C]Glc uptake was 5 min. D, Determination of the substrate specificity and sensitivity to uncouplers of TaSTP6. Carrier [¹⁴C]Glc concentration was 100 μm (without competitor as one internal control, indicated by Control). Competing sugars were added 30 s before the addition of labeled [¹⁴C]Glc at a concentration of 10 mm (10 mm of Glc as another control, indicated by Glc). Transport activities of TaSTP6 for other sugars were determined by competitive inhibition of [¹⁴C]Glc (100 μM) uptake in the presence of nonradioactive sugars in 100-fold excess. CCCP and p-(chloromercuri) benzene sulfonic acid (PCMBS) were added to a final concentration of 50 μM. Values are presented as units relative to the values from the internal control taken as 100%. Data represent means and ses (se) of three independent biological replicates. ^*P < 0.05, ^**P < 0.01 based on the Student’s t test and one-way ANOVA. FW, fresh weight.

To test the substrate specificity of TaSTP6, competition experiments were performed. Transport of [¹⁴C]Glc was measured in the presence of nonradioactive sugars supplied at 100-fold excess. It was directly shown that the uptake of [¹⁴C]Glc was strongly suppressed by Glc and Man (Fig. 7D), whereas Fru and Gal did not interfere with Glc uptake (Fig. 7D), although the complemented strain grew on SD containing Fru as the sole carbon source (Fig. 6B). It is inferred that TaSTP6 has a higher affinity for Glc than Fru. Also, pentose, Ara, and Xyl reduced Glc uptake (Fig. 7D), indicating that they might be additional substrates of TaSTP6. The proton uncoupler carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) significantly decreased Glc uptake (Fig. 7D), demonstrating that TaSTP6 transport activity is driven by a proton gradient across the plasma membrane as shown previously for other STPs (Büttner, 2010). In common with the other plant STPs, no inhibition was observed with the SH group inhibitor p-(chloromercuri) benzene sulfonic acid (Fig. 7D), a transmembrane transport inhibitor directly blocking the transporter from binding substrate (M’Batchi et al., 1986). Taken together, these results suggest that TaSTP6 is an energy-dependent symporter with broad substrate specificity and a preference for Glc.

TaSTP6 Forms Oligomers

Many STPs have been shown to exist as oligomers (Hebert and Carruthers, 1992; Xuan et al., 2013). This led us to investigate whether TaSTP6 can form oligomers using the split ubiquitin system. TaSTP6 fused with C-terminal half of ubiquitin (Cub) served as bait, and TaSTP6 fused with the N-terminal half of ubiquitin (NubG) was used as prey. Homo-oligomerizations were assessed by yeast growth (containing bait and prey plasmids) on SD media (-Trp, -Leu, -Ade, and -His) containing X-Gal. As shown in Figure 8A, the cells cotransformed with TaSTP6-NubG and TaSTP6-Cub grew on the above-mentioned medium, indicating that TaSTP6 is capable of forming a homooligomer in yeast.

Figure 8.

Homooligomerization of TaSTP6. A, Split ubiquitin assay for homo-oligomerization of TaSTP6. Interactions of a TaSTP6-Cub fusion with a TaSTP6-Nub fusion and a wild-type variant of NubI (positive control) or a mutant variant of negative control (NubG) were tested. APP and Fe65 were used as another positive control. Cells of yeast strain NMY51 harboring the indicated plasmid combinations were grown on SD medium (containing 20 μg/mL X-gal and 70 mm of 3-AT). Positive interaction was visualized by analysis of reporters (i.e. His auxotrophy and β-galactosidase activity) expression in drop assays. For yeast transformants, four serial 1:10 dilutions are shown for each combination. L, Leu; T, Trp; A, Ade; H, His. B, In vivo BiFC analysis of TaSTP6 homo-oligomerization. Yn:TaSGT1 + TaRAR1:Yc is shown, see upper (positive control); Yn:TaSTP6 + TaSTP6:Yc is shown in the middle; and Yn:TaSTP6+Yc is shown in the bottom. Agrobacterium-mediated transient expression of indicated constructs in N. benthamiana leaves. Bright-field (BF) and YFP fluorescence (in green) images were taken by confocal microscopy and merged. Scale bars = 10 μm. All of the assays were repeated independently at least three times with comparable results.

To further validate the oligomerization of TaSTP6 in plants, BiFC assay in transiently transformed N. benthamiana leaves was carried out. The BiFC results showed that strong yellow fluorescence signals were obtained when agrobacteria carrying Yn:TaSTP6 and TaSTP6:Yc were coinfiltrated (Fig. 8B), which was similar to the positive control (Yn:TaSGT1 and TaRAR1:Yc; Fig. 8B). However, with the coexpression of Yn:TaSTP6 and the empty vector Yc in N. benthamiana leaves, no fluorescence was observed (Fig. 8B). This supports our finding that TaSTP6 is capable of forming homooligomers.

DISCUSSION

Plants convert CO₂ into sugar by photosynthesis, and a broad spectrum of plant-interacting microbes has evolved sophisticated strategies to enhance their access to these sugars (Voegele and Mendgen, 2011). Uptake and exchange, as well as competition for sugars at the plant-pathogen interface, are mediated by sugar transporters, and the regulation of these sugar transporters is integral to the outcome of a plant-pathogen interaction (Doidy et al., 2012). Pst, as an obligate biotrophic fungus, must acquire nutrients from host cells by haustoria. However, few studies have focused on sugar partitioning in the wheat-Pst interaction. In this study, TaSTP6, a wheat STP gene, was cloned and its transcript profiles were measured. Additionally, the TaSTP6 functional properties were determined via the BSMV-VIGS system, complementation in yeast, Y2H, and BiFC. Our results indicate that TaSTP6 induced by ABA contributes to wheat susceptibility to Pst possibly by modulating the source-sink transition.

Pst-induced TaSTP6 Upregulation Is Possibly Mediated by ABA

Numerous studies have found that pathogen infection may result in sugar redistribution within the host tissue (Lemoine et al., 2013; Bezrutczyk et al., 2018). This transition is primarily triggered by pathogen-regulated plant sugar transporters. For example, the transcript levels of AtSTP4 were significantly increased in Arabidopsis leaves inoculated with Fusarium oxysporum or E. cichoracearum (Truernit et al., 1996; Fotopoulos et al., 2003), whereas P. syringae and B. cinerea infection resulted in upregulation of AtSTP13 in Arabidopsis (Lemonnier et al., 2014; Yamada et al., 2016). In this study, the expression of TaSTP6 was upregulated in Pst-infected wheat leaves. Thus, TaSTP6 appears to be involved in carbohydrate transport and partitioning in infected tissues. In addition, it has been reported that the expression of other sugar transporter genes is induced in rust-infected wheat plants. For example, the expression level of TaSTP13 is increased during leaf rust infection of wheat (Savadi et al., 2017). Five SWEET family members are induced in wheat leaves infected by stem rust (Gao et al., 2018). Therefore, we speculate that these sugar transporters could participate in sugar distribution in the interaction between wheat and rust fungi.

The regulation mode of sugar transporters varies in different pathosystems. For example, the rice homologs OsSWEET11 and OsSWEET14 are specifically exploited by bacterial pathogens for nutritional gain by means of enhancing their transcription through direct binding of a bacterial effector to the promoters of these SWEET transporter genes (Chen et al., 2010). In addition, ABA has been shown to play a central role in the regulation of VvHT5 expression in response to infection by biotrophic pathogens (Hayes et al., 2010). In this study, TaSTP6 was found to be strongly induced by ABA. Interestingly, our data also show that Pst infection can lead to an increased level of endogenous ABA in wheat. Thus, it is reasonable to hypothesize that Pst-induced transcriptional activation of TaSTP6 might be mediated by ABA (Fig. 9). Previous studies found that ABA levels also increased in wheat leaves infected by stem rust fungi (Chigrin et al., 1981). Therefore, we speculate that it is possibly a kind of conserved regulatory pattern through which rust fungi manipulate host plants for invasion and nutrient acquisition by altering the ABA content of wheat leaves.

Figure 9.

Possible model depicting the role of TaSTP6 in the wheat-Pst interaction. During Pst infection of wheat, ABA levels are increased and the expression of TaSTP6 might be upregulated by an ABA-responsive transcription factor. Increased TaSTP6 expression results in enhanced import of apoplastic hexoses into Pst-infected wheat cells. Accumulation of cytoplasmic hexose promotes infection by Pst due to a sufficient carbon supply for absorption by haustoria. Expression of TaSTP6 is maintained at a low level in the uninoculated wheat leaves. HMC, haustorial mother cells; EHM, extrahaustorial matrix; TF, transcription factor.

TaSTP6 Contributes to Wheat Susceptibility to Stripe Rust Possibly by Promoting the Source-Sink Transition

Numerous studies have found that sugar transporters play a central role in the host-pathogen interaction. For example, AtSTP13 confers enhanced Arabidopsis resistance to Botrytis and Pseudomonas by reducing the apoplastic sugar content (Lemonnier et al., 2014; Yamada et al., 2016). Moore et al. (2015) found that inactivation of TaSTP13 increased wheat resistance to all three rust and powdery mildew fungi. To determine the role of TaSTP6 in the wheat response to Pst infection, the BSMV-VIGS system was used. The reduced disease symptoms and restricted hyphal growth in the TaSTP6-knockdown wheat plants infected by CYR31 suggest that silencing of the TaSTP6 gene could partially decrease host susceptibility to virulent Pst, whereas overexpressing TaSTP6 enhanced Arabidopsis susceptibility to powdery mildew. In addition, similar to previous results reported by Cheng et al. (2018) and Wang et al. (2019), overexpression of TaSTP6 in Arabidopsis increased Glc accumulation in the leaves. STPs have been characterized as H⁺/hexose symporters that transport hexose from the apoplast to the cytoplasm (Doidy et al., 2012). Thus, it is reasonable to assume that cytoplasmic Glc concentration was increased in the leaves of TaSTP6 overexpression lines. Because biotrophic fungi such as rust fungi or powdery mildew acquire nutrients from the cytoplasm of the host cell by haustoria (Voegele and Mendgen, 2003), enhanced Arabidopsis susceptibility to powdery mildew is possibly due to the role of TaSTP6 in the source-sink transition. Accordingly, we inferred that suppression of TaSTP6 expression could partially restrict sugar transport from the apoplast to Pst-invaded host cells, resulting in a reduction in the number of uredia. Previous studies have found that pathogens can alter sugar transport in hosts to enhance their access to carbohydrates. For example, Xanthomonas species induce the expression of SWEET family members to release sugar into the apoplastic space (bacterial colonization sites) by delivering transcription activator-like effector proteins (Chen, 2014). Powdery mildew infection led to the upregulation of VvHT5 expression to increase cytoplasmic hexose accumulation in the infected cells (Hayes et al., 2010). Therefore, it is inferred that TaSTP6 is induced by Pst to import more sugar into haustoria-invaded host cells for the supply of carbon sources (Fig. 9).

Biological Characteristics of TaSTP6

The STPs, responsible for uptake of hexoses from the apoplastic space across the plasma membrane, are encoded by 14 highly homologous genes in Arabidopsis (Büttner, 2010). Each AtSTP is a monosaccharide/H⁺ symporter localized to the plasma membrane and has a broad substrate spectrum, except for AtSTP9 and AtSTP14 (Schneidereit et al., 2003; Büttner, 2010). The TaSTP6 protein sequence was found to share high homology with earlier reported plasma membrane H⁺-monosaccharide symporters, indicating that TaSTP6 potentially encodes a plasma membrane-localized sugar transporter. To confirm this speculation, a TaSTP6-GFP fusion was expressed in N. benthamiana epidermal cells and wheat mesophyll protoplasts, and clear plasma membrane localization was shown. The exclusive localization of the STPs is different from that of the SWEET family members. Some SWEET family members have been found in different subcellular compartments. For example, AtSWEET2, AtSWEET16, and AtSWEET17 have been reported to localize to the vacuolar membrane (Chen et al., 2015a).

Compared with other STPs, TaSTP6 was identified as a broad-spectrum monosaccharide transporter that could accept Glc, Fru, and Man as a substrate. In addition, TaSTP6 exhibits a preference for Glc. Its K_m value for Glc is comparable to the values measured for most other plant STPs characterized so far (Lecourieux et al., 2014; Rottmann et al., 2018) with the exception of AtSTP3, which has a lower affinity for Glc (Büttner and Sauer, 2000). TaSTP6 also mediates the uptake of Xyl like some other STPs, such as AtSTP1, VvHT1, and OsMST5 (Ngampanya et al., 2003; Büttner, 2010; Lecourieux et al., 2014). The observed inhibitory effect of CCCP indicates that TaSTP6 uses the energy of the proton gradient across the plasma membrane and functions as a proton symporter like all of the other STPs. The above-mentioned results suggest that TaSTP6 is an energy-dependent hexose/H⁺ symporter with high affinity for Glc. These characterized properties correlate with TaSTP6-mediated sugar transport in the wheat-Pst interaction.

Oligomerization is thought to play a role in the regulation of sugar transport properties or it may contribute to protein stability. For example, mammalian sugar transporters, part of the same superfamily as plant sugar transporters, exhibit regulatory effects within a complex of dimers (Hebert and Carruthers, 1992; Zottola et al., 1995; Hamill et al., 1999). In plants, the first indication that Suc transporters exist in oligomeric complexes was derived from gel filtration experiments of plasma membrane fractions (Li et al., 1991). Subsequently, it was confirmed that sugar transporters function by forming homodimers (Xuan et al., 2013; Moore et al., 2015). In this study, BiFC and Y2H experiments indicated that TaSTP6 is capable of forming oligomeric structures, which might be of functional significance for the regulation of its transport properties. Recently, similar findings regarding oligomerization of sugar transporters have been reported. For example, LR67res (a TaSTP13 allele) in wheat can form heterodimers with functional transporters (TaSTP13), which results in decreased Glc uptake (Moore et al., 2015).

CONCLUSION

This study reveals a potential role of TaSTP6 in the wheat-Pst interaction. During Pst infection of wheat, ABA levels increase and the upregulated expression of TaSTP6, possibly through the action of an ABA-responsive transcription factor, results in enhanced import of apoplastic hexoses into Pst-infected cells. Accumulation of cytoplasmic hexose promotes infection of Pst due to sufficient carbon supply by the absorption of haustoria, as shown in Figure 9. Nevertheless, the specific transcription factor regulating the expression of TaSTP6 and the exact functional mechanism of TaSTP6 during the wheat-Pst interaction still needs further exploration.

MATERIALS AND METHODS

Plant Materials, Pathogen Infection, and Chemical Treatments

Nicotiana benthamiana and Arabidopsis (Columbia-0 background) were used in this study. Plants were grown as described in Liu et al. (2015). Powdery mildew (Golovinomyces cichoracearum) SICAU1 was maintained on Nicotiana tabacum leaves at 23°C (16-h light, 8-h dark) in a growth room. Wheat (Triticum aestivum) seedlings of ‘Suwon 11’ and the Pst virulent pathotype CYR31 was used in the wheat–Pst interaction study.

For the cold treatment, 14-d–old wheat seedlings were incubated at 4°C. Wound treatment was performed by cutting the wheat leaves using a pair of sterilized scissors. For the drought and salt stress treatments, the roots of wheat seedlings were immersed in 20% (w/v) PEG6000 and 200 mm of NaCl, respectively. For the chemical treatments, 14-d–old seedling surfaces were sprayed with 1 mm of ABA or 1 mm of MeJA in 0.05% (v/v) TWEEN 20. Control leaves were sprayed with 0.05% (v/v) TWEEN 20 only. Leaves were collected at 0, 2, 6, 12, 24, and 48 hpt.

All of the sampled leaves were stored at −80°C before RNA extraction. For different treatments, three independent biological replications were performed.

Genomic DNA and Total RNA Extraction, and RT-qPCR Analysis

Genomic DNA was extracted by the cetyltrimethylammonium bromide method (Porebski et al., 1997). Total RNA from wheat was extracted using a Quick RNA isolation Kit (Huayueyang Biotechnology) according to the manufacturer’s instructions. The potential genomic DNA was digested with DNase I. First-strand cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and oligo(dT)₁₈ primer.

Gene expression was carried out by RT-qPCR using the SYBR Green method on a CFX Connect Real-Time PCR Detection System (Bio-Rad). TaEF-1α was selected as the endogenous reference for normalization. RT-qPCR performed in a 25-μL reaction mixture containing UltraSYBR Mixture (CWBIO), 10 pmol each of the forward and reverse gene-specific primers (Supplemental Table S2), and 2 μl of diluted cDNA (1:20). RT-qPCR analysis represented data from three biological repeats, with each group containing three technical repeats. RT-qPCR data were analyzed with the comparative 2^-ΔΔCt method (Pfaffl, 2001).

Cloning of TaSTP6 and Sequence Analysis

The ORF of TaSTP6 was amplified via PCR using the specific primers (Supplemental Table S1) designed based on transcriptome sequencing of wheat seedlings infected by CYR31 (Hao et al., 2016). The obtained fragment was aligned with the T. aestivum ‘CS’ genome using data of the International Wheat Genome Sequencing Consortium (https://urgi.versailles.inra.fr/blast) and portal “Ensembl Plants” (http://plants.ensembl.org/Multi/Tools/Blast), and predicted chromosomal locations and related sequences were obtained from this website. The promoter region sequences of TaSTP6 were then isolated and putative cis-acting regulatory elements were analyzed by the databases “PlantCARE” (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and “PLACE” (https://www.dna.affrc.go.jp/PLACE/?action=newplace).

The amino acid sequence of TaSTP6 was analyzed with the “ProtParam” tool of “ExPASy” (http://www.expasy.org) to identify physicochemical properties. Multiple sequence alignments were carried out using the software “DNAMAN6.0” (Lynnon Biosoft) and “ClustalW2.0” (Chenna et al., 2003), and polygenetic relationships were inferred with the neighbor-joining method using the software “MEGA 5.0” (Tamura et al., 2011). The transmembrane region of TaSTP6 was then predicted using the “TMHMM Server v. 2.0” (http://www.cbs.dtu.dk/services/TMHMM/). The subsequent topologies of TaSTP6 were visualized according to these predictions using the software “Protter” (Omasits et al., 2014).

Plasmid Construction

To further verify that TaSTP6 responds to ABA treatments, a 2,000-bp region upstream of TaSTP6-2D was amplified by PCR using genomic DNA as a template. The promoter fragments were cloned into the XbaI/BamHI restriction sites in plasmid pCB308 (Xiang et al., 1999), yielding construct pCB308-TaSTP6np.

To analyze the subcellular localization of TaSTP6 in plants, we generated TaSTP6-GFP fusion constructs by using the SalI/BamHI restriction sites of vector pTF486 (Yu et al., 2008), under the control of the Cauliflower mosaic virus 35S promoter.

To overexpress TaSTP6 in plants, the full-length TaSTP6 ORF was amplified and inserted into the Gateway vector pENTR/D-TOPO (Invitrogen). The full-length TaSTP6 ORF was then cloned into the expression vector pK7FWG2 (Karimi et al., 2007), resulting in construct pK7FWG2-TaSTP6.

To characterize the function of TaSTP6 by heterologous expression in yeast, the ORF of TaSTP6 was amplified with primers for insertion into the XhoI/BamHI sites in vector pDR195. Similarly, the GFP-fusion TaSTP6 and free GFP fragments were cloned using the recombinant binary vector pK7FWG2-TaSTP6 as a template and inserted into the same restriction sites (XhoI/BamHI) of pDR195 to produce the GFP fusion constructs pDR195-TaSTP6-GFP and pDR195-GFP, respectively, for expression in yeast.

To investigate oligomerization of TaSTP6, the TaSTP6 ORF was cloned into the SfiI restriction site of vectors pBT3-SUC-Cub and pPR3N-NubG (Stagljar et al., 1998) to construct pBT3-SUC -Cub-TaSTP6 and pPR3N-NubG-TaSTP6 recombinant plasmids. In addition, the coding regions of TaSTP6 were subcloned into vector pSPYNE(R)173 and pSPYCE(M; Waadt et al., 2008) with restriction sites BamHI and XhoI to generate the pSPYNE(R)173-TaSTP6 and pSPYCE(M)-TaSTP6 vectors, respectively.

The plasmids used for silencing TaSTP6 were constructed as described in Holzberg et al. (2002). Two cDNA fragments derived from the coding sequence (196 bp, nucleotides 1–196; 204 bp, nucleotides 1,178–1,373) based on results of a BLASTn (National Institutes of Health) search of the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov/) show the lowest sequence similarity with other wheat genes and the highest polymorphism within the STP family. Three copies shared 98.98% and 99.75% nucleotide identity, respectively, with each other for the two VIGS fragments. Consequently, two cDNA fragments were amplified by PCR to construct the recombination plasmids TaSTP6-as1 and TaSTP6-as2, respectively, in an antisense orientation.

TaSTP6-2D was used for vector construction and primers for all of the plasmid constructions are listed in Supplemental Table S2.

Arabidopsis Transformation and Inoculation

The plasmid pCB308-TaSTP6np and pK7FWG2-TaSTP6 were sequenced and transferred into Agrobacterium tumefaciens strain GV3101 and subsequently transformed into Arabidopsis using the floral dip method (Clough and Bent, 1998). Transgenic Arabidopsis were identified on half-strength Murashige and Skoog medium containing 10 mg/L of BASTA (Bayer) or 50 μg/mL of kanamycin. Selection-marker–resistant seedlings were checked and transferred into single pots filled with soil. Then, the seedlings were transferred to a growth chamber and they were allowed to grow for the next generation of seeds. Methods of inoculation with powdery mildew and conidiophore counting were the same as those described by Xiao et al. (2005).

GUS Activity Assay

For the measurement of GUS activity, 6-week–old transgenic lines were sprayed with 1 mm of ABA and 0.05% (v/v) TWEEN 20. Leaves were sampled just before ABA spraying to measure basal GUS activity, and at 2 6, 12, 24, and 48 hpt to determine ABA-induced GUS activity. For the parallel control, leaves were sprayed with 0.05% (v/v) TWEEN 20 only. Total proteins extraction and quantitative GUS assays were performed as described previously by Jefferson et al. (1987). Protein concentrations were determined as described by Bradford (1976). The standard curves were prepared with 4-methylumbelliferone. Fluorescence strength was measured on an Infinite 200 PRO Multimode Plate Reader (Tecan Life Sciences) with the excitation at 455 nm and emission at 365 nm. Three biological replicates were taken to measure the GUS activity.

Extraction and Analysis of ABA Accumulation

To identify the ABA level in wheat leaves, 14-d–old seedlings were infected by CYR31. Wheat leaves were harvested at 8 and 10 hpi. The levels of ABA were determined by the company Shanghai Applied Protein Technology. One-hundred milligrams of infected leaves was ground in a precooled mortar and extracted at 4°C for 12 h in 1 mL 1% (v/v) formic acid (FA) in acetonitrile (ACN) and water (1:1, v/v). One milliliter of 1% (v/v) ACN spiked with 200 ng of D6-ABA (OIChemIm) as an internal standard was added to each sample and then samples were homogenized and vortexed. After centrifugation at 14,000g for 10 min at 4°C, supernatants were transferred to fresh 2-mL Eppendorf tubes. The extractions were combined and dried under N₂ gas, then resuspended in 100 μL of 50% (v/v) ACN and subsequently filtered through a 0.22-μm Millipore filter. The supernatants were transferred to glass bottles and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Subsequently, 5 μL of supernatant was injected into an Acquity I-Class LC Ultra-Performance Liquid Chromatograph (Waters) and a 5500 QTrap Quadruple Tandem Mass Spectrometer (AB Sciex). HPLC analysis was performed using an Acquity UPLC BEH C18 (Waters) column (2.1 mm × 100 mm; 1.7 μm). The mobile phase A solvents consisted of ultrapure water/0.05% (v/v) FA, and the mobile phase B solvents consisted of 0.05% (v/v) FA in ACN. MS conditions were as follows: the pressure of the air curtain gas, ion Source gas1, and ion Source gas1 were 30, 45, and 45 psi, respectively; the ion spray voltage was 4,500 V; and the source temperature was 500°C. Three biological replications were performed.

BSMV-Mediated TaSTP6 Gene Silencing

Infectious BSMV RNAs were prepared as described by Wang et al. (2012). Four BSMV viruses (BSMV:γ, BSMV:TaPDS, BSMV:TaSTP6-as1, and BSMV:TaSTP6-as2) were inoculated individually into the second fully expanded leaves of wheat seedlings as described previously by Scofield et al. (2005). MOCK-treated seedlings were inoculated with 1 × FES buffer (0.1 m glycine pH 8.9, 0.06 m K ₂HPO₄, 1% sodium pyrophosphate, 1% celite, 1% bentonite) as negative controls. BSMV-treated wheat seedlings were placed in a growth chamber at 25°C. When the virus phenotype was observed and photographed (12 d after BSMV treatment), the fourth leaves were further treated with CYR31. The plants were then maintained at 16°C and the fourth leaves were sampled at 0, 24, 48, 72, and 120 hpi for histological observation and RNA isolation. We used RT-qPCR to confirm the silencing efficiency of TaSTP6 for each assay. At 14 dpi, when extensive fungal growth was visible in leaves, the infection phenotypes of Pst were examined and photographed. Three independent sets of inoculations were performed, which consisted of 50 seedlings inoculated for each BSMV virus.

The fungal biomass changes in the Pst-infected wheat leaves were analyzed as described by Chang et al. (2017). All of the primers used are listed in Supplemental Table S2.

Histological Observations of Fungal Growth

To identify the function of TaSTP6 in the wheat-Pst infections, the fungal development was observed by microscopy. The leaf fractions were fixed and cleared as described in Wang et al. (2007). Wheat germ agglutinin conjugated to Alexa Fluor 488 (Invitrogen) was used to stain the Pst infection structures as described in Ayliffe et al. (2011). The hyphal branches, haustorial mother cells, haustoria hyphal, length, and infection area were observed and measured using the software “cellSens Entry” (Olympus). Stained tissue was examined under blue-light excitation (excitation wavelength 450–480 nm, emission wavelength 515 nm) with a BX51 Microscope (Olympus). The final data of each index were the mean of at least 50 infection sites for each of the five randomly selected leaf segments per treatment.

Extraction and Determination of Water-Soluble Carbohydrates

The leaves of 4-week–old transgenic plants were weighed to obtain 2 g of fresh weight and ground in liquid nitrogen. Extraction of water-soluble carbohydrates and HPLC analysis were performed as described by Chang et al. (2013).

Subcellular Localization

To determine the membrane localization of TaSTP6, N. benthamiana leaves were infiltrated with GV3101 carrying pK7FWG2-TaSTP6. For plasma membrane staining by FM4-64 (Thermo Fisher Scientific; Fischer-Parton et al., 2000), leaves were immersed in a phosphate-buffered saline of 5 μm of FM4-64. Wheat leaf protoplast isolation and the transient expression of pTF486-TaSTP6 constructs were performed as described by Ahmed et al. (2017). Fluorescence signals were monitored 2 d later or 18-h post transformations using an FV1000 Confocal Laser Microscope (Olympus). All of the assays were performed in duplicate and repeated at least three times.

Functional Characterization of TaSTP6 by Heterologous Expression in Yeast

For the complementation and subcellular localization assays in Saccharomyces cerevisiae, pDR195, pDR195-TaSTP6, pDR195-GFP, and pDR195-TaSTP6-GFP were transformed into a hexose transporter-deficient yeast strain EBY.VW4000 (Wieczorke et al., 1999) using the lithium acetate method (Soni et al., 1993). The transformants were selected on sd medium supplemented with maltose as the sole carbon source without uracil at 30°C for 4 d, and transformants were confirmed via PCR. In addition, to determine the expression of TaSTP6 in S. cerevisiae, the yeast cells harboring pDR195-TaSTP6-GFP were observed by confocal microscopy. The transformants expressing the free GFP construct were used for the control. The positive transformants were grown on SD medium supplemented with 2% (w/v) maltose for 1 d. Dilutions of 10³, 10⁴, 10⁵, and 10⁶ cells mL⁻¹ in water were quantified with a hemocytometer, and 6 µL of cells was dropped on solid SD medium containing Glc, Fru, or Man as the unique carbon source. Three biological replicates were performed.

For the Glc uptake assays, the transformants preparation was performed as described by Cheng et al. (2015). Cells were then harvested by centrifugation, washed twice and resuspended in 25 mm of sodium phosphate buffer (pH 5.5) to an OD₆₀₀ of 10. Transport assays with transgenic yeast strains were performed as described by Sauer and Stadler (1993). Whatman syringe filter and Whatman glass microfiber filters, Grade GF/B (Sigma-Aldrich), were used. All of the assays were performed in triplicate and repeated at least twice.

Split-Ubiquitin Analysis

The split-ubiquitin system (Stagljar et al., 1998) was used to investigate polymerization of TaSTP6. To ensure correct expression and functionality of this system, the bait construct pBT3-SUC-Cub-TaSTP6 was cotransformed with the prey control vector, which express the wild-type N-terminal half of ubiquitin (NubI ; positive) portion or the NubG (negative) portion bearing an Ile to Gly mutation. Self-interaction of TaSTP6 was determined by cotransformation of pBT3-SUC-Cub-TaSTP6 and pPR3N-NubG-TaSTP6. The transformed S. cerevisiae NMY51 were cultured on solid SD-Leu-Trp (SD-LW) medium at 30°C for 3 d.

A single colony was cultured in liquid SD-LW medium for 36 h, and then serial 1:10 dilutions were dropped on either solid SD-Leu-Trp-His-Ade (SD-LWHA) or SD-LWHA containing X-Gal supplemented with 70 mm of 3-aminotriazole (a competitive inhibitor of the HIS3 gene product).

The vectors pTSU2-amyloid A4 precursor protein (APP) and amyloid beta A4 precursor protein-binding family B member 1 (pNubG-Fe65) were combined as the positive control. Interactions were tested by analysis of reporters (i.e. His auxotrophy and β-galactosidase activity). All of the assays were repeated at least three times.

BiFC Assay

nYFP and cYFP sequences were fused to the N-terminal and C-terminal sequences of TaSTP6 in vectors pSPYNE(R)173 and pSPYCE(M), respectively. Yn:TaSGT1 and TaRAR1:Yc were used as the positive control (Wang et al., 2015). The recombinant plasmids were transferred into GV3101 by electroporation and coinfiltrated into N. benthamiana leaves (Xuan et al., 2013). YFP fluorescence was detected by confocal microscopy, with an excitation laser at 488 nm. All of the assays were repeated independently at least three times with comparable results.

Statistical Analysis

Data analysis was performed using the software “SPSS v17.0” (IBM). Statistical analyses of independent experiments were reported as the mean ± se (se). Significance was determined by conducting Student’s t tests or one-way ANOVA followed by Least Significant Difference and Bonferroni tests.

Accession Numbers

Sequence data from this article can be found through the “Ensembl Plants” portal (http://plants.ensembl.org/Triticum_aestivum/Info/Index) and the National Center for Biotechnology database (http://www.ncbi.nlm.nih.gov/) with the following accession numbers: TaSTP6-2A (TraesCS2A02G205500), TaSTP6-2B (TraesCS2B02G232900), and TaSTP6-2D (TraesCS2D02G230100LC.1), TaEF-1α (Q03033), AtUBC21 (AT5G25760).

The accession numbers of protein sequences used for phylogenetic analysis are listed in Supplemental Table S3.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Phylogenetic analysis of TaSTP6 and the STPs from Arabidopsis and rice.

• Supplemental Figure S2. Multialignment of the encoding sequences of the three copies of TaSTP6; TaSTP6 cDNA sequence from T. aestivum ‘Suwon 11’ was amplified.

• Supplemental Figure S3. Multialignment of the deduced TaSTP6 proteins.

• Supplemental Figure S4. Schematic view of the structure of TaSTP6.

• Supplemental Figure S5. Phylogenetic analysis of TaSTP6 and selected homologous proteins from other plants.

• Supplemental Figure S6. Expression profiles of TaSTP6 in response to abiotic stress.

• Supplemental Figure S7. Analysis of the promoter region of TaSTP6 and PCR identification of transgenic Arabidopsis plants.

• Supplemental Figure S8. Standard curves generated for the absolute quantification of genomic DNA of Pst and wheat.

• Supplemental Figure S9. Quantitative assessment of histological observations of fungal development in the control (BSMV:γ) and TaSTP6-silenced (BSMV:TaSTP6-as1 and BSMV: TaSTP6-as2) wheat plants challenged with Pst CYR31.

• Supplemental Figure S10. RT-PCR analysis of TaSTP6 expression in TaSTP6-OE and wild-type leaves.

• Supplemental Table S1. Motifs detected in the TaSTP6 promoter sequence based on the analysis of PlantCARE and PLACE software.

• Supplemental Table S2. The primers and strains used in this study.

• Supplemental Table S3. The accession numbers of gene or protein sequences used in this study.