ABA-Induced Sugar Transporter TaSTP6 Promotes WheatSusceptibility to Stripe Rust1[OPEN]

Baoyu Huai,a Qian Yang,a Yingrui Qian,b Wenhao Qian,a Zhensheng Kang,a and Jie Liub,2,3

aState Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&FUniversity, Yangling 712100, Shaanxi, ChinabState Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&FUniversity, Yangling 712100, Shaanxi, China

ORCID ID: 0000-0003-2278-1146 (J.L.).

Biotrophic pathogens, such as wheat rust fungi, survive on nutrients derived from host cells. Sugar appears to be the majorcarbon source transferred from host cells to various fungal pathogens; however, the molecular mechanism by which host sugartransporters are manipulated by fungal pathogens for nutrient uptake is poorly understood. TaSTP6, a sugar transporter proteinin wheat (Triticum aestivum), was previously shown to exhibit enhanced expression in leaves upon infection by Pucciniastriiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. In this study, we found that Pst infection caused increasedaccumulation 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 wheatsusceptibility to the Pst pathotype CYR31, suggesting that TaSTP6 expression upregulation contributes to Pst host sugaracquisition. Consistent with this, TaSTP6 overexpression in Arabidopsis (Arabidopsis thaliana) promoted plant susceptibility topowdery mildew and led to increased Glc accumulation in the leaves. Functional complementation assays in Saccharomycescerevisiae showed that TaSTP6 has broad substrate specificity, indicating that TaSTP6 is an active sugar transporter. Subcellularlocalization analysis indicated that TaSTP6 localizes to the plasma membrane. Yeast two-hybrid and bimolecular fluorescencecomplementation experiments revealed that TaSTP6 undergoes oligomerization. Taken together, our results suggest that Pststimulates ABA biosynthesis in host cells and thereby upregulates TaSTP6 expression, which increases sugar supply andpromotes fungal infection.

Wheat (Triticum aestivum) is one of the major foodcrops worldwide. With an increasing global popu-lation, the demand for wheat is rising. However,wheat is susceptible to a range of abiotic and bioticstresses, which pose varying degrees of threats toyield. Over the years, scientists have been searchingfor new agronomic traits in wheat to adapt to thestresses 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 dis-ease may cause significant yield loss and thus lead tofood shortages and rising food prices. Pst is an obli-gate biotrophic fungus that acquires nutrientsthrough specialized feeding structures called “haus-toria.” Haustoria are fungal organs specific to bio-trophic pathogens that form intimate interactionswith the host cell to absorb nutrients (Sutton et al.,1999; Voegele and Mendgen, 2003). Control overnutrient flux from the plant to the pathogen is a po-tential novel and effective strategy to control a vari-ety of plant pathogens. However, little is knownabout how infected host cells control the release ofnutrients to their pathogenic boarders.

Pathogens, irrespective of their lifestyle, develop atthe cost of nutrients released and generated by the hostplant during plant-pathogen interactions. It is assumedthat upon infection of above-ground plant parts, clas-sical source tissue turns into sink tissue (Doidy et al.,2012; Bezrutczyk et al., 2018). Earlier studies of nutrienttransfer from the host to the fungus were carried out onpowdery mildew (Sutton et al., 1999; Hall andWilliams, 2000). It was found that Glc is the primarycarbon and energy source diverted to the fungal my-celium (Sutton et al., 1999; Hall and Williams, 2000).Later, work on other biotrophic pathogens followed.UfHXT1, a high-affinity hexose/proton symporter

1This work was supported by the National Key Research and De-velopment Program of China (grant no. 2016YFD0100602) and theFundamental Research Funds for the Central Universities (grant no.2452019184).

2Author for contact: liujie2003@hotmail.com.3Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is: JieLiu (liujie2003@hotmail.com).

B.Y.H., Z.S.K., and J.L. conceived and designed the experiments;B.Y.H., Q.Y., Y.R.Q., and W.H.Q. performed the experiments; B.Y.H.and J.L. analyzed the data and wrote the article; all authors discussedthe results and commented on the article.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.00632

1328 Plant Physiology�, November 2019, Vol. 181, pp. 1328–1343, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved.

from Uromyces fabae, was found to be exclusively lo-calized to the haustorial plasma membrane and playa pivotal role in carbon transfer to the fungus from theextrahaustorial matrix (Voegele et al., 2001). In ad-dition, plant invertase activity was found to be in-creased after pathogen inoculation (Wirsel et al.,2001; Voegele et al., 2006), which might be attrib-uted to the lack of Suc transporters in some biotrophicfungi (Spanu et al., 2010; Duplessis et al., 2011). Suctherefore has to be cleaved into Glc and Fru, whichare subsequently taken up by the pathogen. Mean-while, increased invertase activity can also alter theextracellular apoplastic hexose/Suc ratio and elicita hexose-mediated defense response (Proels andHückelhoven, 2014).Sugar transport and especially partitioning across

the plant plasma membrane by transporters is one ofthe most important processes for plant developmentand plant responses to biotic and abiotic factors(Lalonde et al., 2004; Lemoine et al., 2013). The mainsugar transporter proteins in plants comprise bothSuc transporters (SUTs/SUCs) and monosaccharidetransporters. Both of the above-mentioned trans-porters belong to the major facilitator superfamilyand are predicted to share a similar structure, with 12putative transmembrane domains (TMDs) connectedby hydrophilic loops, and to function as H1/sugarsymporters (Doidy et al., 2012). Recently, a new classof sugar transporters termed “SWEETs” has beenidentified (Xuan et al., 2013). SWEETs are heptahel-ical proteins carrying a tandem repeat of 3-TMDseparated by a single TMD (Chen et al., 2015b). Thesetwo families of transporters seem to be involved inbalancing plant monosaccharide influx and effluxacross the plasma membrane and between organelleswithin 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 dis-tinguished based on their substrate specificity (sugartransport proteins [STPs], polyol/monosaccharidetransporters, inositol transporters; Büttner andSauer, 2000; Büttner, 2010). STP members have beenidentified in different organisms (plants, animals,bacteria, archaea, and fungi) based on their capacityto catalyze the uptake of hexoses from the apoplasticspace into the cell (Williams et al., 2000). So far, all ofthe characterized STPs are plasma membrane-localized H1/hexose symporters and show broadsubstrate specificity with the exception of AtSTP9, aGlc-specific transporter (Schneidereit et al., 2003),and AtSTP14, a Gal-specific transporter (Poschetet al., 2010).Pathogen infection triggers alteration of sugar

transport in host plants for improving pathogen accessto nutrients. Sugar allocation at the plant-pathogeninterfaces is mediated by sugar transporters, whoseregulation patterns determine the outcome of the in-teraction (Lemoine et al., 2013). For example,AtSTP4 isinduced upon infection by Erysiphe cichoracearum andcorrelated with Glc uptake in host tissue (Fotopoulos

et al., 2003). The expression of the hexose transporterVvHT5 (AtSTP13 ortholog) is regulated by abscisicacid (ABA) during the transition from source to sink inresponse to infection by powdery or downy mildew(Hayes et al., 2010). Xanthomonas bacteria secretetranscription activator-like effectors that induce thetranscription of SWEET transporters for sugar effluxinto the apoplastic space (outside the symplast), wherethe bacteria acquire carbohydrates for energy andcarbon (Chen, 2014). Plants also regulate sugar trans-porters to redistribute sugars away from the infectionsite, removing the pathogens’ energy source and lim-iting their proliferation. For example, transcript levelsof AtSTP13 increased in Arabidopsis infected withPseudomonas syringae pv tomato DC3000 (Nørholmet al., 2006). Another study revealed that AtSTP13is activated by phosphorylation in the Arabidopsis-DC3000 interactions, which intensifies its hexose up-take activity to compete with bacteria for apoplasticmonosaccharide (Yamada et al., 2016). Similarly, over-expression of AtSTP13 in Arabidopsis improved thecapacity to take up Glc and conferred enhanced resis-tance against Botrytis cinerea (Lemonnier et al., 2014). Inwheat, Lr67res (a natural mutation of TaSTP13), whichimpairs its hexose transport activity, provides partialresistance to all three wheat rust pathogen species (Pstand Puccinia triticina) and powdery mildew (Mooreet al., 2015).Previous studies have reported that Suc accumula-

tion in Pst-challenged wheat leaves is significantly in-creased, accompanied by hexose accumulation due tosimultaneously enhanced invertase activity (Changet al., 2013; Liu et al., 2015). Nevertheless, few studiesare available on hexose transport in Pst-infected wheatplants. TaSTP6, a wheat sugar transporter gene thatresponds to stripe rust infection but appears insensitiveto other plant pathogen invasion, was isolated based ontranscriptome 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 wheatleaves either inoculated with Pst or treated with ABA.TaSTP6 promoter::b-glucuronidase fusions in trans-genic Arabidopsis were activated by ABA treatment.We found that Pst infection also resulted in increasedaccumulation of ABA. TaSTP6 expression knockdownenhanced wheat resistance to Pst. In addition, over-expression of TaSTP6 promoted Arabidopsis suscep-tibility to powdery mildew and resulted in increasedGlc accumulation in the leaves. Transient expressionanalysis in Nicotiana benthamiana leaves and wheatleaf protoplasts showed that TaSTP6 is localizedto the plasma membrane. Heterologous expressionin Saccharomyces cerevisiae revealed that TaSTP6 is ahexose/H1 symporter. Yeast two hybrid (Y2H) andbimolecular fluorescence complementation (BiFC)validated oligomerization of TaSTP6. Our results in-dicate that TaSTP6, as an ABA-inducible gene,

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increases wheat susceptibility to Pst possibly by reg-ulating the transition from source to sink tissue.

RESULTS

Identification and Sequence Analysis of TaSTP6

Complementary DNA (cDNA) containing an openreading frame (ORF) orthologous to the rice STP familymember OsSTP6 (Supplemental Fig. S1), designatedTaSTP6, was amplified by reverse transcription PCR(RT-PCR). The sequence of TaSTP6 was further used toquery the Chinese Spring (CS) genome sequence. BLAST(http://plants.ensembl.org/Multi/Tools/Blast) resultsshowed that there were three TaSTP6 copies located onchromosomes 2A, 2B, and 2D in the wheat genome. Theencoding sequences of the three copies only differ in 39nucleotides and share a sequence identity of 99.18%(Supplemental Fig. S2). Accordingly, the three pro-teins encoded by them share 99.56% sequence identity(Supplemental Fig. S3), indicating that these threecopies may possess identical biological functions.

The ORF of TaSTP6 with 1,581 nucleotide residues ispredicted to encode a 526-amino acid peptide with a pIof 9.26 and a calculated molecular mass of 57.5 kD. Themembrane spanning model analysis indicates thatTaSTP6 contains 12 predicted TMDs (SupplementalFig. S4). The phylogenetic relationships of TaSTP6 withorthologous proteins from various species showed thatTaSTP6 exhibits a high degree of conservation with theSTP sequences from monocotyledons (Supplemental

Fig. S5). These results indicate that TaSTP6may encodea sugar transporter protein.

Upregulation of TaSTP6 Is Possibly Mediated by ABAduring Pst Infection of Wheat

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

During Pst infection of wheat, the expression profileof TaSTP6 was confirmed by RT-qPCR. The transcriptlevels of TaSTP6 were increased at 12- and 24-h postinoculation (hpi) in wheat leaves challenged with thePst virulent pathotype CYR31. The highest TaSTP6transcript levels were ;16-fold at 24 hpi (Fig. 1A).

The responses of TaSTP6 to various environmentalstresses were also assayed. Wheat seedlings weretreated with low temperature, wounding, NaCl, andpolyethylene glycol (PEG)6000. The results showedthat the expression level of TaSTP6 was increased at2-h post treatment (hpt) and then decreased under

Figure 1. Expression of TaSTP6 was upregulated by Pst infection and exogenous ABA treatment. A and B, TaSTP6 expressionchanges in response to Pst infection (A) and exogenous ABA treatment (B). Expression levels were normalized to TaEF-1a. Therelative expression of TaSTP6 was calculated using the comparative threshold method (2-DDCt). Untreated leaves act as thecontrol. C, ABA-induced TaSTP6 promoter activity in transgenic Arabidopsis. Transgenic Arabidopsis plants containing pCB308-TaSTP6npwere sprayedwith a solution containing 1mM of ABA and 0.05% (v/v) TWEEN20 orwith 0.05% (v/v) TWEEN20 alone,and sampled at 0, 2, 6, 12, 24, and 48 hpt for GUS-activity. D, Pst infection increases endogenous ABA in wheat leaves. Wheatleaves inoculated with Pst CYR31 were sampled at 8 and 10 hpi for HPLC-MS/MS analysis. Error bars represent variations amongthree independent replicates. Single (P, 0.05) and double asterisks (P, 0.01) indicate a significant difference from the untreatedcontrol according to Student’s t test. FW, fresh weight; MU, 4-methylumbelliferone.

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low-temperature treatment (Supplemental Fig. S6).Wound treatment significantly induced transcripts ofTaSTP6 at 2 hpt (Supplemental Fig. S6); transcript a-bundance of TaSTP6 decreased from 6 to 24 hpt andincreased at 48 hpt (Supplemental Fig. S6). The expres-sion of TaSTP6 was upregulated at 48 h after PEG6000treatment, and it was 3-fold higher than that of thecontrol at this time (Supplemental Fig. S6). Under NaCltreatment, no obvious changes were observed in thetranscript 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 (SupplementalFig. S7A). Having identified the presence of a cluster ofABRCs in the TaSTP6 promoter region, we investigatedTaSTP6 transcript levels in wheat leaves after treatmentwith exogenous ABA application. As shown inFigure 1B, after ABA treatment, the TaSTP6 transcriptlevelwas significantly upregulatedwith a peak at 12 hpt,whereas TaSTP6 expression appeared to be less sensitiveto methyl jasmonate (MeJA) compared with ABA, al-though several MeJA-responsive elements are containedin the TaSTP6 promoter region (Supplemental Table S1).To further analyze the involvement of the pre-

dicted motifs in ABA responsiveness, the constructpCB308-TaSTP6np was generated and transformed

into Arabidopsis. The presence of the construct inArabidopsis lines in the T2 generation was confirmedby PCR (Supplemental Fig. S7B). b-glucuronidase ac-tivity was measured in leaves of the TaSTP6np-6 lineafter ABA treatment. Our results show that ABAtreatment caused an increase in reporter activity at 2and 6 hpt (Fig. 1C). No significant changes occurred inthe control (Fig. 1C). The GUS activity response to ABAsupports the hypothesis that ABA-induced TaSTP6expression is mediated via the ABRCs.Because the transcript abundance of TaSTP6 was in-

creased in wheat leaves treated with Pst and ABA, theendogenous ABA content in Pst-infected wheat leavescaused concern. HPLC analyses showed that the ABAconcentration in Pst-infected wheat leaves was signifi-cantly upregulated at 8 and 10 hpi compared with theuninfected control (Fig. 1D). These results, presented inFigure 1, suggested that upregulation of TaSTP6 waspossibly mediated by ABA in wheat leaves inoculatedwith Pst.

Knockdown of TaSTP6 Reduces Wheat Susceptibilityto Pst

To determine a possible role of TaSTP6 during thewheat response to Pst, the barley stripe mosaic virus

Figure 2. Functional analysis of TaSTP6 during the wheat-Pst interaction using the BSMV-VIGS system. A, Mild chlorotic mosaicsymptomswere observed on the leaves of wheat seedlings at 9 dpi with BSMV:g, BSMV:TaSTP6-as1, and BSMV:TaSTP6-as2, andphotobleaching was evident on the fourth leaves of wheat plants infected by BSMV:TaPDS (a positive control treatment). Wheatleaves inoculated with FES buffer (MOCK). B, Disease phenotypes of the fourth leaves preinoculated with BSMV constructs andthen challenged with Pst CYR31. C, Fungal biomass measurements of total DNA extracted from BSMV-treated wheat leavesinfected byCYR31 at 14 dpi based on qPCR. Ratio of total fungalDNA to total wheat DNAwas assessed by normalizing the data tothewheat gene TaEF-1a and the Pst gene PstEF1. D, Silencing efficiency of TaSTP6 in the fourth leaves of the control (BSMV:g) andTaSTP6-silenced (BSMV:TaSTP6-as1 and BSMV:TaSTP6-as2) plants. Leaves for RT-qPCR were sampled at 0, 24, 48, 72, and120 hpi. Data were normalized to the TaEF-1a expression level. The results were obtained from three biological independentreplicates. Values represent the means 6 SE of three independent samples. Asterisks indicate a significant difference (P , 0.05)according to Student’s t test.

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(BSMV)-virus induced gene silencing (VIGS) systemwas used to knock down the expression of TaSTP6.For co-silencing of the three copies of TaSTP6, twohighly conserved regions in the ORF of TaSTP6 werechosen and amplified (Supplemental Fig. S2). Noevident defects occurred in wheat growth and mildchlorotic mosaic symptoms were displayed onBSMV-treated wheat seedlings (Fig. 2A). To visualizethe functionality of the VIGS system, BSMV:TaPDS(Holzberg et al., 2002) was used as a positive control.Thereafter, we used Pst CYR31 to inoculate theBSMV-treated wheat seedlings. A reduced diseasephenotype was observed in TaSTP6-knockdownplants at 14 d post inoculation (dpi; Fig. 2B). Fur-thermore, plants treated with BSMV:TaSTP6-as1 orBSMV:TaSTP6-as2 showed a similar phenotype(Fig. 2B).

To test whether the disease phenotype was corre-lated with fungal growth in the host tissue, fungalbiomass measurements were conducted on infected

leaves. Total genomic DNA was extracted fromwheat leaves inoculated with Pst, and the rela-tive levels of TaEF-1a and PstEF1 (Yin et al., 2009)were quantified using qPCR to generate standardcurves (Supplemental Fig. S8). The fungal biomasswas significantly decreased by;29% in BSMV:TaSTP6-as1 leaves and by 18% in BSMV:TaSTP6-as2 leavesat 14 dpi compared to control plants (Fig. 2C).This is consistent with the reduced diseasephenotype.

RT-qPCR determined that endogenous TaSTP6transcription was successfully silenced in the fourthleaves of BSMV-VIGS plants. The TaSTP6 transcriptin BSMV:TaSTP6-as1-inoculated or BSMV:TaSTP6-as2-inoculated leaves was reduced by;50% to 75% at0, 24, 48, 72, and 120 hpi with CYR31 compared withBSMV:g (control) leaves (Fig. 2D). Taken together,our results demonstrate that the expression ofTaSTP6 was substantially knocked down in ourexperiments.

Figure 3. Histological observation of fungal growth inwheat leaves infected by BSMV:g and recombinant BSMVafter inoculationwith Pst CYR31. A to I, Fungal structures were stained with wheat germ agglutinin (WGA). A to I, Growth of Pst in wheat leavesinoculated with BSMV:g, BSMV:TaSTP6-as1, or BSMV:TaSTP6-as2 at 48 hpi (A to C), 72 hpi (D to F), and 120 hpi (G to I) wasobserved under a fluorescence microscope. J, Average number of hyphal branches (HB), haustorial mother cells (HMC), andhaustoria (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 longestinfection hyphae. L, TaSTP6-silenced plants show a significant decrease in infection unit area at 120 hpi. Values represent themean 6 SE of three independent samples (50 infection sites each time). Asterisks indicate a significant difference (*P , 0.05,**P , 0.01) from BSMV:g inoculated plants using Student’s t test and one-way ANOVA. (A to C) Scale bar 5 20 mm; (D to F)scale bar 5 50 mm; (G to I) scale bar 5 100 mm.

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Histological Observations of Fungal Growth inTaSTP6-Knockdown Plants

To quantify the reduced disease phenotype inTaSTP6-knockdown lines after infection with CYR31,the infected leaves were studied using histological ob-servations. At 24 hpi, no significant histological differ-ences were observed in TaSTP6-knockdown plants. InBSMV:TaSTP6-as1 and BSMV:TaSTP6-as2-inoculatedleaves, the number of hyphal branches, haustorialmother cells, haustoria, and overall hyphal length weredecreased at 48 hpi (Fig. 3, A–C, J, and K). In addition,colonization and formation of secondary hyphae werestrongly restricted in the TaSTP6-knockdown seedlingscompared with the plants inoculated with BSMV:g(control) at 48, 72, and 120 hpi (Fig. 3, D–I, and L;Supplemental Fig. S9, A and B). These results indicatethat knockdown of TaSTP6 results in restricted fungaldevelopment in the wheat-Pst interaction.

Overexpression of TaSTP6 Promotes ArabidopsisSusceptibility to Powdery Mildew

Furthermore, to elucidate the possible function ofTaSTP6 during the wheat-Pst interaction, we introduced

a TaSTP6 overexpression construct into Arabidopsis andgenerated several transgenic TaSTP6 overexpression(TaSTP6-OE) lines. Two lines (TaSTP6-OE1 and TaSTP6-OE2) showing distinct TaSTP6 mRNA accumulation inleaves (Supplemental Fig. S10) were inoculated withpowdery mildew (Li et al., 2019), which resulted in anenhanced susceptibility phenotype (Fig. 4A). Consistentwith the disease phenotype, TaSTP6-OEplants hadmoreconidiophores per colony than wild-type Col-0 plantsduring the early infection stage at 4 dpi when the fungusbegan asexual reproduction (Fig. 4, B and C). Sugarconcentration in leaves from these two transgenic lineswas then analyzed using HPLC. As shown in Figure 4D,the Glc content was significantly increased in the trans-genic lines comparedwith the control plants, whereas noobvious difference was observed for other type of sugar,indicating that TaSTP6 is an active sugar transporter.

TaSTP6 Is Localized to the Plasma Membrane

In plants, STPs have been shown to function asplasma membrane proteins (Büttner, 2010). To confirmthe TaSTP6 subcellular localization, transient expres-sion of a TaSTP6-GFP construct inN. benthamiana leaves

Figure 4. Overexpression of TaSTP6promotes Arabidopsis susceptibility topowdery mildew. A, Representativeimages of Arabidopsis leaves of indi-cated genotypes infected with tobaccopowdery mildew at 12 dpi. TaSTP6overexpression lines (TaSTP6-OE1 and-OE2) were more susceptible than wildtype. B, Representative microscopicimages of single colonies of powderymildew on leaves of indicated geno-types at 4 dpi. Fungal structures werestained by trypan blue. Scale bars 5200 mm. C, Total number of con-idiophores per colony on leaves ofindicated genotypes at 4 dpi. The bar-chart shows combined data from threeindependent experiments (at least 30colonies were counted for each gen-otype per experiment). D, Glc con-centrations in the leaves of TaSTP6overexpression lines and wild-typeplants. Values are the means 6 SE ofthree replicates. Double asterisks indi-cate a significant difference (P , 0.01)from wild-type plants according to theStudent’s t test and one-way ANOVA.FW, fresh weight; WT, wild type.

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and wheat leaf protoplasts was carried out. Similarresults were observed in these two systems. FreeGFP, as a control, was detected throughout the cy-tosol and nucleus (Fig. 5). Transient expression ofTaSTP6-GFP in N. benthamiana leaves showed thatGFP fluorescence colocalized with the fluorescence ofN-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl) pyridinium dibromide (FM4-64;Thermo Fisher Scientific), a membrane-selective fluo-rescent dye (Fig. 5A). In wheat leaf protoplasts, chlo-rophyll autofluorescence (red) was found inside thering of GFPfluorescence (green) emitted by the TaSTP6-GFP fusion protein (Fig. 5B). These results indicate thatTaSTP6 is localized to the plasma membrane.

Functional Characterization of TaSTP6 by HeterologousExpression in Yeast

To identify the function of TaSTP6, several recombi-nant plasmids were transformed into the hexosetransport-defunct S. cerevisiae mutant EBY.VW4000(Wieczorke et al., 1999). Yeast cells carrying pDR195-GFP or pDR195-TaSTP6-GFP were monitored by con-focal microscopy. We found that TaSTP6 was alsolocalized to the plasma membrane of yeast (Fig. 6A).Thus, TaSTP6 exhibits the same subcellular localizationin S. cerevisiae and wheat and it is possible that thetransport properties of TaSTP6 can be measured inS. cerevisiae.

We then tested the mutant carrying the emptypDR195 vector (Rentsch et al., 1995) or the recombi-nant plasmid pDR195-TaSTP6 for growth on syntheticdropout (SD)-Ura medium supplemented with differentcarbon sources. The results show that the complementedstrain grew normally on media with Glc, Fru, or Man asthe sole carbon source (Fig. 6B). These results suggestthat TaSTP6 is a typical monosaccharide transporter.

To determine the kinetic parameters of TaSTP6,[14C]Glc uptake assays were performed. Our resultsshow that the complemented strain was able to take up[14C]Glc with a Km of 49.8 6 4.75 mM and a Vmax of290 6 15 pmol mg21 min21 (Fig. 7B), whereas yeastcells carrying the empty vector did not take up Glc(Fig. 7A). The pH optimum for TaSTP6 was deter-mined to be ;5.5 (Fig. 7C).

To test the substrate specificity of TaSTP6, competi-tion experiments were performed. Transport of [14C]Glc was measured in the presence of nonradioactivesugars supplied at 100-fold excess. It was directlyshown that the uptake of [14C]Glc was strongly sup-pressed by Glc andMan (Fig. 7D), whereas Fru and Galdid not interfere with Glc uptake (Fig. 7D), although thecomplemented strain grew on SD containing Fru as thesole carbon source (Fig. 6B). It is inferred that TaSTP6 hasa higher affinity for Glc than Fru. Also, pentose, Ara, andXyl reduced Glc uptake (Fig. 7D), indicating that theymight be additional substrates of TaSTP6. The protonuncoupler carbonyl cyanide m-chlorophenyl-hydrazone(CCCP) significantly decreased Glc uptake (Fig. 7D),

Figure 5. Subcellular localization of TaSTP6 inN. benthamiana leaves and wheat leaf protoplasts.A and B, Subcellular localization of free GFP (ascontrol) and TaSTP6-GFP with transient expres-sion under the control of the 35S promoter in cellsofN. benthamiana leaves (A, scale bars5 10 mm)and wheat leaf protoplasts (B, scale bars5 5 mm).FM4-64 was used to stain the plasma membrane.GFP fluorescence is in green. Red fluorescenceindicates FM4-64 labeling the plasma membraneor chlorophyll auto-fluorescence. Merged GFP/FM4-64 (A) and GFP/chlorophyll (B) images areshown. Bright-field images show the equivalentfield observed under white light. All of the sig-nals were monitored by confocal microscopy.Comparable expression and localization pat-terns were observed in three independent bio-logical replicates.

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demonstrating that TaSTP6 transport activity is driven bya proton gradient across the plasma membrane as shownpreviously for other STPs (Büttner, 2010). In commonwith the other plant STPs, no inhibition was observedwith the SH group inhibitor P-(chloromercuri) benzenesulfonic acid (Fig. 7D), a transmembrane transport in-hibitor directly blocking the transporter from bindingsubstrate (M’Batchi et al., 1986). Taken together, theseresults suggest that TaSTP6 is an energy-dependentsymporter with broad substrate specificity and a prefer-ence for Glc.

TaSTP6 Forms Oligomers

Many STPs have been shown to exist as oligomers(Hebert and Carruthers, 1992; Xuan et al., 2013). Thisled us to investigate whether TaSTP6 can form oligo-mers using the split ubiquitin system. TaSTP6 fusedwith 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-oligomerizationswere assessed by yeast growth (containing bait andprey plasmids) on SD media (-Trp, -Leu, -Ade, and-His) containing X-Gal. As shown in Figure 8A, the cellscotransformed with TaSTP6-NubG and TaSTP6-Cubgrew on the above-mentioned medium, indicatingthat TaSTP6 is capable of forming a homooligomerin yeast.To further validate the oligomerization of TaSTP6 in

plants, BiFC assay in transiently transformed N. ben-thamiana leaves was carried out. The BiFC resultsshowed that strong yellow fluorescence signals wereobtained when agrobacteria carrying Yn:TaSTP6 andTaSTP6:Yc were coinfiltrated (Fig. 8B), which wassimilar to the positive control (Yn:TaSGT1 and TaR-AR1:Yc; Fig. 8B). However, with the coexpression ofYn:TaSTP6 and the empty vector Yc in N. benthamianaleaves, no fluorescence was observed (Fig. 8B). Thissupports our finding that TaSTP6 is capable of forminghomooligomers.

DISCUSSION

Plants convert CO2 into sugar by photosynthesis, anda broad spectrum of plant-interacting microbes hasevolved sophisticated strategies to enhance their accessto these sugars (Voegele and Mendgen, 2011). Uptakeand exchange, as well as competition for sugars atthe plant-pathogen interface, are mediated by sugartransporters, and the regulation of these sugar trans-porters is integral to the outcome of a plant-pathogeninteraction (Doidy et al., 2012). Pst, as an obligate bio-trophic fungus, must acquire nutrients from host cellsby haustoria. However, few studies have focused onsugar partitioning in the wheat-Pst interaction. In thisstudy, TaSTP6, a wheat STP gene, was cloned and itstranscript profiles were measured. Additionally, theTaSTP6 functional properties were determined via theBSMV-VIGS system, complementation in yeast, Y2H,and BiFC. Our results indicate that TaSTP6 induced byABA contributes to wheat susceptibility to Pst possiblyby modulating the source-sink transition.

Pst-induced TaSTP6 Upregulation Is Possibly Mediatedby ABA

Numerous studies have found that pathogen infec-tion may result in sugar redistribution within the hosttissue (Lemoine et al., 2013; Bezrutczyk et al., 2018).This transition is primarily triggered by pathogen-regulated plant sugar transporters. For example, the

Figure 6. Expression ofTaSTP6 in S. cerevisiae. A, Localization ofGFPandTaSTP6-GFP in the EBY.VW4000 strain. Bright-field and GFP fluorescenceimages were taken by confocal microscopy and merged. Scale bars 52 mm. B, Growth of EBY.VW4000 carrying either the vector pDR195 orplasmid pDR195-TaSTP6 on maltose (control), and the monosaccharidesGlc, Fru, and Man 2% (w/v) each. These experiments were repeated threetimes (two different transformants each time), with similar results.

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transcript levels of AtSTP4were significantly increasedin Arabidopsis leaves inoculated with Fusarium oxy-sporum or E. cichoracearum (Truernit et al., 1996;Fotopoulos et al., 2003), whereas P. syringae and B. ci-nerea infection resulted in upregulation of AtSTP13 inArabidopsis (Lemonnier et al., 2014; Yamada et al.,2016). In this study, the expression of TaSTP6 wasupregulated in Pst-infected wheat leaves. Thus, TaSTP6appears to be involved in carbohydrate transport andpartitioning in infected tissues. In addition, it has beenreported that the expression of other sugar transportergenes is induced in rust-infected wheat plants. For ex-ample, the expression level of TaSTP13 is increasedduring leaf rust infection of wheat (Savadi et al., 2017).Five SWEET family members are induced in wheatleaves infected by stem rust (Gao et al., 2018). Therefore,we speculate that these sugar transporters could par-ticipate in sugar distribution in the interaction betweenwheat and rust fungi.

The regulation mode of sugar transporters varies indifferent pathosystems. For example, the rice homologsOsSWEET11 andOsSWEET14 are specifically exploitedby bacterial pathogens for nutritional gain by means ofenhancing their transcription through direct binding of

a bacterial effector to the promoters of these SWEETtransporter genes (Chen et al., 2010). In addition, ABAhas been shown to play a central role in the regulationof VvHT5 expression in response to infection by bio-trophic 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 canlead to an increased level of endogenous ABA in wheat.Thus, it is reasonable to hypothesize that Pst-inducedtranscriptional activation of TaSTP6might be mediatedby ABA (Fig. 9). Previous studies found that ABA levelsalso increased in wheat leaves infected by stem rustfungi (Chigrin et al., 1981). Therefore, we speculate thatit is possibly a kind of conserved regulatory patternthrough which rust fungi manipulate host plants forinvasion and nutrient acquisition by altering the ABAcontent of wheat leaves.

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

Numerous studies have found that sugar transportersplay a central role in the host-pathogen interaction.

Figure 7. Characterization of TaSTP6 transport activity in S. cerevisiae. A, Uptake of [14C]Glc into the yeast strain EBY.VW4000transformed with pDR195-TaSTP6 (black circle) or pDR195 alone (white triangle) per milligram fresh weight at an initial outsideconcentration of 100mM of Glc at pH 5.5. B, Concentration-dependent [14C]Glc uptake. The Lineweaver-Burk plot of a typical Km

determination is presented. The estimated Km is 49.8 6 4.75 mM. The Vmax was determined to be 290 6 15 pmol mg21 min21

cells. C, Relative uptake rate of [14C]Glc into EBY.VW4000 transformed with pDR195-TaSTP6 at different pH values at an initialoutside concentration of 100 mM of Glc. The time interval for [14C]Glc uptake was 5 min. D, Determination of the substratespecificity and sensitivity to uncouplers of TaSTP6. Carrier [14C]Glc concentration was 100 mM (without competitor as one in-ternal control, indicated by Control). Competing sugars were added 30 s before the addition of labeled [14C]Glc at a concen-tration of 10 mM (10 mM of Glc as another control, indicated by Glc). Transport activities of TaSTP6 for other sugars weredetermined by competitive inhibition of [14C]Glc (100 mM) uptake in the presence of nonradioactive sugars in 100-fold excess.CCCPand P-(chloromercuri) benzene sulfonic acid (PCMBS) were added to a final concentration of 50 mM. Values are presentedas units relative to the values from the internal control taken as 100%. Data represent means and SEs (SE) of three independentbiological replicates. *P , 0.05, **P , 0.01 based on the Student’s t test and one-way ANOVA. FW, fresh weight.

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For example, AtSTP13 confers enhanced Arabidopsisresistance to Botrytis and Pseudomonas by reducing theapoplastic sugar content (Lemonnier et al., 2014;Yamada et al., 2016). Moore et al. (2015) found thatinactivation of TaSTP13 increased wheat resistance toall three rust and powdery mildew fungi. To determinethe role of TaSTP6 in the wheat response to Pst infec-tion, the BSMV-VIGS system was used. The reduceddisease symptoms and restricted hyphal growth in theTaSTP6-knockdown wheat plants infected by CYR31suggest that silencing of the TaSTP6 gene could par-tially decrease host susceptibility to virulent Pst,whereas overexpressing TaSTP6 enhanced Arabidopsissusceptibility to powdery mildew. In addition, similarto previous results reported by Cheng et al. (2018) andWang et al. (2019), overexpression of TaSTP6 in Ara-bidopsis increased Glc accumulation in the leaves. STPshave been characterized as H1/hexose symporters thattransport hexose from the apoplast to the cytoplasm

(Doidy et al., 2012). Thus, it is reasonable to assume thatcytoplasmic Glc concentration was increased in theleaves of TaSTP6 overexpression lines. Because bio-trophic fungi such as rust fungi or powdery mildewacquire nutrients from the cytoplasm of the host cell byhaustoria (Voegele and Mendgen, 2003), enhancedArabidopsis susceptibility to powdery mildew is pos-sibly due to the role of TaSTP6 in the source-sink tran-sition. Accordingly, we inferred that suppression ofTaSTP6 expression could partially restrict sugar trans-port from the apoplast to Pst-invaded host cells,resulting in a reduction in the number of uredia. Pre-vious studies have found that pathogens can alter sugartransport in hosts to enhance their access to carbohy-drates. For example, Xanthomonas species induce theexpression of SWEET family members to release sugarinto the apoplastic space (bacterial colonization sites)by delivering transcription activator-like effector pro-teins (Chen, 2014). Powdery mildew infection led to the

Figure 8. Homooligomerization ofTaSTP6. A, Split ubiquitin assay forhomo-oligomerization of TaSTP6. In-teractions of a TaSTP6-Cub fusion witha TaSTP6-Nub fusion and a wild-typevariant of NubI (positive control) or amutant variant of negative control(NubG) were tested. APP and Fe65were used as another positive control.Cells of yeast strain NMY51 harboringthe indicated plasmid combinationswere grown on SDmedium (containing20 mg/mL X-gal and 70 mM of 3-AT).Positive interaction was visualized byanalysis of reporters (i.e. His auxotro-phy and b-galactosidase activity) ex-pression in drop assays. For yeasttransformants, four serial 1:10 dilu-tions are shown for each combination.L, Leu; T, Trp; A, Ade; H, His. B, Invivo BiFC analysis of TaSTP6 homo-oligomerization. Yn:TaSGT1 1 TaR-AR1:Yc is shown, see upper (positivecontrol); Yn:TaSTP6 1 TaSTP6:Ycis shown in the middle; and Yn:TaST-P61Yc is shown in the bottom.Agrobacterium-mediated transient ex-pression of indicated constructs inN. benthamiana leaves. Bright-field(BF) and YFP fluorescence (in green)images were taken by confocal micros-copy and merged. Scale bars5 10 mm.All of the assays were repeated inde-pendently at least three times withcomparable results.

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upregulation of VvHT5 expression to increase cyto-plasmic hexose accumulation in the infected cells(Hayes et al., 2010). Therefore, it is inferred that TaSTP6is 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 theapoplastic space across the plasma membrane, areencoded by 14 highly homologous genes in Arabi-dopsis (Büttner, 2010). Each AtSTP is a monosaccha-ride/H1 symporter localized to the plasma membraneand has a broad substrate spectrum, except for AtSTP9and AtSTP14 (Schneidereit et al., 2003; Büttner, 2010).The TaSTP6 protein sequence was found to share highhomology with earlier reported plasma membrane H1-monosaccharide symporters, indicating that TaSTP6potentially encodes a plasma membrane-localizedsugar transporter. To confirm this speculation, aTaSTP6-GFP fusion was expressed in N. benthamianaepidermal cells and wheat mesophyll protoplasts, andclear plasma membrane localization was shown. Theexclusive localization of the STPs is different from thatof the SWEET family members. Some SWEET familymembers have been found in different subcellularcompartments. For example, AtSWEET2, AtSWEET16,and AtSWEET17 have been reported to localize to thevacuolar membrane (Chen et al., 2015a).

Comparedwith other STPs, TaSTP6was identified asa broad-spectrum monosaccharide transporter thatcould accept Glc, Fru, and Man as a substrate. In ad-dition, TaSTP6 exhibits a preference for Glc. Its Kmvalue for Glc is comparable to the values measured formost other plant STPs characterized so far (Lecourieuxet al., 2014; Rottmann et al., 2018) with the exception ofAtSTP3, which has a lower affinity for Glc (Büttner andSauer, 2000). TaSTP6 also mediates the uptake of Xyllike some other STPs, such as AtSTP1, VvHT1, and

OsMST5 (Ngampanya et al., 2003; Büttner, 2010;Lecourieux et al., 2014). The observed inhibitory effectof CCCP indicates that TaSTP6 uses the energy of theproton gradient across the plasma membrane andfunctions as a proton symporter like all of the otherSTPs. The above-mentioned results suggest that TaSTP6is an energy-dependent hexose/H1 symporter withhigh affinity for Glc. These characterized propertiescorrelate with TaSTP6-mediated sugar transport in thewheat-Pst interaction.

Oligomerization is thought to play a role in the reg-ulation of sugar transport properties or it may con-tribute to protein stability. For example, mammaliansugar transporters, part of the same superfamily asplant sugar transporters, exhibit regulatory effectswithin a complex of dimers (Hebert and Carruthers,1992; Zottola et al., 1995; Hamill et al., 1999). Inplants, the first indication that Suc transporters exist inoligomeric complexes was derived from gel filtrationexperiments of plasma membrane fractions (Li et al.,1991). Subsequently, it was confirmed that sugartransporters function by forming homodimers (Xuanet al., 2013; Moore et al., 2015). In this study, BiFCand Y2H experiments indicated that TaSTP6 is capableof forming oligomeric structures, which might be offunctional significance for the regulation of its transportproperties. Recently, similar findings regarding oligo-merization of sugar transporters have been reported.For example, LR67res (a TaSTP13 allele) in wheat canform 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 thewheat-Pst interaction. During Pst infection of wheat,ABA levels increase and the upregulated expressionof TaSTP6, possibly through the action of an ABA-responsive transcription factor, results in enhanced

Figure 9. Possible model depicting therole of TaSTP6 in the wheat-Pst inter-action. During Pst infection of wheat,ABA levels are increased and the ex-pression of TaSTP6might be upregulatedby an ABA-responsive transcription fac-tor. Increased TaSTP6 expression resultsin enhanced import of apoplastic hex-oses into Pst-infected wheat cells. Ac-cumulation of cytoplasmic hexosepromotes infection by Pst due to a suf-ficient carbon supply for absorption byhaustoria. Expression of TaSTP6 ismaintained at a low level in the unin-oculated wheat leaves. HMC, hausto-rial mother cells; EHM, extrahaustorialmatrix; TF, transcription factor.

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import of apoplastic hexoses into Pst-infected cells.Accumulation of cytoplasmic hexose promotes infec-tion of Pst due to sufficient carbon supply by theabsorption of haustoria, as shown in Figure 9. Never-theless, the specific transcription factor regulating theexpression of TaSTP6 and the exact functional mecha-nism of TaSTP6 during the wheat-Pst interaction stillneeds further exploration.

MATERIALS AND METHODS

Plant Materials, Pathogen Infection, andChemical Treatments

Nicotiana benthamiana and Arabidopsis (Columbia-0 background) were usedin this study. Plants were grown as described in Liu et al. (2015). Powderymildew (Golovinomyces cichoracearum) SICAU1 was maintained on Nicotianatabacum leaves at 23°C (16-h light, 8-h dark) in a growth room. Wheat (Triticumaestivum) seedlings of ‘Suwon 11’ and the Pst virulent pathotype CYR31 wasused 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 ofsterilized scissors. For the drought and salt stress treatments, the roots of wheatseedlings were immersed in 20% (w/v) PEG6000 and 200 mM of NaCl, re-spectively. For the chemical treatments, 14-d–old seedling surfaces weresprayed with 1mM of ABA or 1 mM ofMeJA in 0.05% (v/v) TWEEN 20. Controlleaveswere sprayedwith 0.05% (v/v) TWEEN 20 only. Leaveswere collected at0, 2, 6, 12, 24, and 48 hpt.

All of the sampled leaves were stored at 280°C before RNA extraction. Fordifferent treatments, three independent biological replications were performed.

Genomic DNA and Total RNA Extraction, andRT-qPCR Analysis

Genomic DNA was extracted by the cetyltrimethylammonium bromidemethod (Porebski et al., 1997). Total RNA from wheat was extracted using aQuick RNA isolation Kit (Huayueyang Biotechnology) according to the man-ufacturer’s instructions. The potential genomic DNA was digested with DNaseI. First-strand cDNA was synthesized using a RevertAid First Strand cDNASynthesis Kit (Thermo Fisher Scientific) and oligo(dT)18 primer.

Gene expression was carried out by RT-qPCR using the SYBRGreenmethodon a CFX Connect Real-Time PCR Detection System (Bio-Rad). TaEF-1a wasselected as the endogenous reference for normalization. RT-qPCR performed ina 25-mL reaction mixture containing UltraSYBR Mixture (CWBIO), 10 pmoleach of the forward and reverse gene-specific primers (Supplemental Table S2),and 2 ml of diluted cDNA (1:20). RT-qPCR analysis represented data from threebiological repeats, with each group containing three technical repeats. RT-qPCRdata were analyzed with the comparative 2-DDCt 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 wheatseedlings infected by CYR31 (Hao et al., 2016). The obtained fragment wasaligned with the T. aestivum ‘CS’ genome using data of the International WheatGenome Sequencing Consortium (https://urgi.versailles.inra.fr/blast) andportal “Ensembl Plants” (http://plants.ensembl.org/Multi/Tools/Blast), andpredicted chromosomal locations and related sequences were obtainedfrom this website. The promoter region sequences of TaSTP6 were thenisolated and putative cis-acting regulatory elements were analyzed by thedatabases “PlantCARE” (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and “PLACE” (https://www.dna.affrc.go.jp/PLACE/?action5newplace).

The amino acid sequence of TaSTP6was analyzedwith the “ProtParam” toolof “ExPASy” (http://www.expasy.org) to identify physicochemical properties.Multiple sequence alignments were carried out using the software “DNA-MAN6.0” (Lynnon Biosoft) and “ClustalW2.0” (Chenna et al., 2003), andpolygenetic relationships were inferred with the neighbor-joining method

using the software “MEGA 5.0” (Tamura et al., 2011). The transmembrane re-gion of TaSTP6 was then predicted using the “TMHMM Server v. 2.0” (http://www.cbs.dtu.dk/services/TMHMM/). The subsequent topologies of TaSTP6were visualized according to these predictions using the software “Protter”(Omasits et al., 2014).

Plasmid Construction

To further verify that TaSTP6 responds toABA treatments, a 2,000-bp regionupstream of TaSTP6-2D was amplified by PCR using genomic DNA as a tem-plate. The promoter fragments were cloned into the XbaI/BamHI restrictionsites in plasmid pCB308 (Xiang et al., 1999), yielding construct pCB308-TaSTP6np.

To analyze the subcellular localization of TaSTP6 in plants, we generatedTaSTP6-GFP fusion constructs by using the SalI/BamHI restriction sites ofvector pTF486 (Yu et al., 2008), under the control of the Cauliflower mosaic virus35S promoter.

To overexpress TaSTP6 in plants, the full-length TaSTP6ORFwas amplifiedand 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 inyeast, the ORF of TaSTP6 was amplified with primers for insertion into theXhoI/BamHI sites in vector pDR195. Similarly, the GFP-fusion TaSTP6 and freeGFP 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 andpDR195-GFP, respectively, for expression in yeast.

To investigate oligomerization of TaSTP6, the TaSTP6 ORF was cloned intothe SfiI restriction site of vectors pBT3-SUC-Cub and pPR3N-NubG (Stagljaret al., 1998) to construct pBT3-SUC -Cub-TaSTP6 and pPR3N-NubG-TaSTP6recombinant plasmids. In addition, the coding regions of TaSTP6 were subcl-oned into vector pSPYNE(R)173 and pSPYCE(M; Waadt et al., 2008) with re-striction sites BamHI and XhoI to generate the pSPYNE(R)173-TaSTP6 andpSPYCE(M)-TaSTP6 vectors, respectively.

The plasmids used for silencing TaSTP6 were constructed as described inHolzberg 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 aBLASTn (National Institutes of Health) search of the National Center for Bio-technology (http://www.ncbi.nlm.nih.gov/) show the lowest sequence simi-larity with other wheat genes and the highest polymorphism within the STPfamily. Three copies shared 98.98% and 99.75% nucleotide identity, respec-tively, with each other for the two VIGS fragments. Consequently, two cDNAfragments were amplified by PCR to construct the recombination plasmidsTaSTP6-as1 and TaSTP6-as2, respectively, in an antisense orientation.

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

Arabidopsis Transformation and Inoculation

Theplasmid pCB308-TaSTP6np and pK7FWG2-TaSTP6were sequenced andtransferred into Agrobacterium tumefaciens strain GV3101 and subsequentlytransformed into Arabidopsis using the floral dip method (Clough and Bent,1998). Transgenic Arabidopsis were identified on half-strength Murashige andSkoog medium containing 10 mg/L of BASTA (Bayer) or 50 mg/mL of kana-mycin. Selection-marker–resistant seedlings were checked and transferred intosingle pots filled with soil. Then, the seedlings were transferred to a growthchamber and they were allowed to grow for the next generation of seeds.Methods of inoculationwith powderymildew and conidiophore counting werethe same as those described by Xiao et al. (2005).

GUS Activity Assay

For the measurement of GUS activity, 6-week–old transgenic lines weresprayed with 1 mM of ABA and 0.05% (v/v) TWEEN 20. Leaves were sampledjust before ABA spraying to measure basal GUS activity, and at 2 6, 12, 24, and48 hpt to determine ABA-induced GUS activity. For the parallel control, leaveswere sprayed with 0.05% (v/v) TWEEN 20 only. Total proteins extraction andquantitative GUS assays were performed as described previously by Jeffersonet al. (1987). Protein concentrations were determined as described by Bradford(1976). The standard curves were prepared with 4-methylumbelliferone.

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Fluorescence strength was measured on an Infinite 200 PRO Multimode PlateReader (Tecan Life Sciences) with the excitation at 455 nm and emission at 365nm. 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 infectedby CYR31.Wheat leaveswere harvested at 8 and 10 hpi. The levels of ABAweredetermined by the company Shanghai Applied Protein Technology. One-hundred milligrams of infected leaves was ground in a precooled mortar andextracted at 4°C for 12 h in 1mL 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 thensamples were homogenized and vortexed. After centrifugation at 14,000g for10 min at 4°C, supernatants were transferred to fresh 2-mL Eppendorf tubes.The extractions were combined and dried under N2 gas, then resuspended in100 mL of 50% (v/v) ACN and subsequently filtered through a 0.22-mm Milli-pore filter. The supernatants were transferred to glass bottles and then analyzedby liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Subsequently, 5 mL of supernatant was injected into an Acquity I-Class LCUltra-Performance Liquid Chromatograph (Waters) and a 5500 QTrap Qua-druple Tandem Mass Spectrometer (AB Sciex). HPLC analysis was performedusing an Acquity UPLC BEHC18 (Waters) column (2.1 mm3 100mm; 1.7mm).The mobile phase A solvents consisted of ultrapure water/0.05% (v/v) FA, andthemobile phase B solvents consisted of 0.05% (v/v) FA in ACN.MS conditionswere as follows: the pressure of the air curtain gas, ion Source gas1, and ionSource gas1 were 30, 45, and 45 psi, respectively; the ion spray voltage was4,500 V; and the source temperature was 500°C. Three biological replicationswere performed.

BSMV-Mediated TaSTP6 Gene Silencing

Infectious BSMV RNAs were prepared as described by Wang et al. (2012).Four BSMV viruses (BSMV:g, BSMV:TaPDS, BSMV:TaSTP6-as1, andBSMV:TaSTP6-as2) were inoculated individually into the second fully ex-panded leaves of wheat seedlings as described previously by Scofield et al.(2005). MOCK-treated seedlings were inoculated with 1 3 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 agrowth chamber at 25°C. When the virus phenotype was observed and pho-tographed (12 d after BSMV treatment), the fourth leaves were further treatedwith CYR31. The plants were then maintained at 16°C and the fourth leaveswere sampled at 0, 24, 48, 72, and 120 hpi for histological observation and RNAisolation. We used RT-qPCR to confirm the silencing efficiency of TaSTP6 foreach assay. At 14 dpi, when extensive fungal growth was visible in leaves, theinfection phenotypes of Pst were examined and photographed. Three inde-pendent sets of inoculations were performed, which consisted of 50 seedlingsinoculated for each BSMV virus.

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

Histological Observations of Fungal Growth

To identify the function of TaSTP6 in the wheat-Pst infections, the fungaldevelopment was observed by microscopy. The leaf fractions were fixed andcleared as described inWang et al. (2007). Wheat germ agglutinin conjugated toAlexa Fluor 488 (Invitrogen) was used to stain the Pst infection structures asdescribed in Ayliffe et al. (2011). The hyphal branches, haustorial mother cells,haustoria hyphal, length, and infection areawere observed andmeasured usingthe software “cellSens Entry” (Olympus). Stained tissue was examined underblue-light excitation (excitation wavelength 450–480 nm, emission wavelength515 nm) with a BX51 Microscope (Olympus). The final data of each index werethe mean of at least 50 infection sites for each of the five randomly selected leafsegments per treatment.

Extraction and Determination ofWater-Soluble Carbohydrates

The leaves of 4-week–old transgenic plants were weighed to obtain 2 g offresh 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 leaveswere infiltrated with GV3101 carrying pK7FWG2-TaSTP6. For plasma mem-brane staining by FM4-64 (Thermo Fisher Scientific; Fischer-Parton et al., 2000),leaves were immersed in a phosphate-buffered saline of 5 mM of FM4-64. Wheatleaf protoplast isolation and the transient expression of pTF486-TaSTP6 con-structs were performed as described by Ahmed et al. (2017). Fluorescence sig-nals were monitored 2 d later or 18-h post transformations using an FV1000Confocal Laser Microscope (Olympus). All of the assays were performed induplicate and repeated at least three times.

Functional Characterization of TaSTP6 by HeterologousExpression in Yeast

For the complementation andsubcellular localizationassays inSaccharomycescerevisiae, pDR195, pDR195-TaSTP6, pDR195-GFP, and pDR195-TaSTP6-GFPwere transformed into a hexose transporter-deficient yeast strain EBY.VW4000(Wieczorke et al., 1999) using the lithium acetate method (Soni et al., 1993). Thetransformants were selected on SD medium supplemented with maltose as thesole carbon source without uracil at 30°C for 4 d, and transformants wereconfirmed via PCR. In addition, to determine the expression of TaSTP6 in S.cerevisiae, the yeast cells harboring pDR195-TaSTP6-GFP were observed byconfocal microscopy. The transformants expressing the free GFP construct wereused for the control. The positive transformants were grown on SD mediumsupplemented with 2% (w/v) maltose for 1 d. Dilutions of 103, 104, 105, and 106

cells mL21 in water were quantified with a hemocytometer, and 6 mL of cellswas dropped on solid SD medium containing Glc, Fru, or Man as the uniquecarbon source. Three biological replicates were performed.

For the Glc uptake assays, the transformants preparation was performed asdescribed 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 per-formed as described by Sauer and Stadler (1993). Whatman syringe filter andWhatman glass microfiber filters, Grade GF/B (Sigma-Aldrich), were used. Allof 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 investigatepolymerization of TaSTP6. To ensure correct expression and functionality ofthis system, the bait construct pBT3-SUC-Cub-TaSTP6was cotransformed withthe prey control vector, which express the wild-type N-terminal half of ubiq-uitin (NubI ; positive) portion or the NubG (negative) portion bearing an Ile toGly mutation. Self-interaction of TaSTP6 was determined by cotransformationof pBT3-SUC-Cub-TaSTP6 and pPR3N-NubG-TaSTP6. The transformedS. cerevisiae NMY51 were cultured on solid SD-Leu-Trp (SD-LW) medium at30°C for 3 d.

A single colony was cultured in liquid SD-LW medium for 36 h, and thenserial 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 betaA4 precursor protein-binding family B member 1 (pNubG-Fe65) were com-bined as the positive control. Interactions were tested by analysis of reporters(i.e. His auxotrophy and b-galactosidase activity). All of the assays were re-peated at least three times.

BiFC Assay

nYFP and cYFP sequences were fused to the N-terminal and C-terminalsequences 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 electro-poration and coinfiltrated into N. benthamiana leaves (Xuan et al., 2013). YFPfluorescence was detected by confocal microscopy, with an excitation laser at488 nm. All of the assays were repeated independently at least three times withcomparable results.

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Statistical Analysis

Data analysis was performed using the software “SPSS v17.0” (IBM). Sta-tistical analyses of independent experiments were reported as the mean 6 SE

(SE). Significance was determined by conducting Student’s t tests or one-wayANOVA 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 theNational 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-1a (Q03033), AtUBC21 (AT5G25760).

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

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Phylogenetic analysis of TaSTP6 and the STPsfrom Arabidopsis and rice.

Supplemental Figure S2. Multialignment of the encoding sequences of thethree 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 selectedhomologous proteins from other plants.

Supplemental Figure S6. Expression profiles of TaSTP6 in response toabiotic stress.

Supplemental Figure S7. Analysis of the promoter region of TaSTP6 andPCR identification of transgenic Arabidopsis plants.

Supplemental Figure S8. Standard curves generated for the absolute quan-tification of genomic DNA of Pst and wheat.

Supplemental Figure S9. Quantitative assessment of histological obser-vations of fungal development in the control (BSMV:g) and TaSTP6-silenced (BSMV:TaSTP6-as1 and BSMV: TaSTP6-as2) wheat plantschallenged with Pst CYR31.

Supplemental Figure S10. RT-PCR analysis of TaSTP6 expression inTaSTP6-OE and wild-type leaves.

Supplemental Table S1. Motifs detected in the TaSTP6 promoter sequencebased 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 se-quences used in this study.

ACKNOWLEDGMENTS

We thank Professor Eckhard Boles for providing the EBY.VW4000 mutant,Professor Wenming Wang for offering tobacco powdery mildew, and North-west A&F University, State Key Laboratory of Crop Stress Biology for AridAreas’ shared instrument platform.

Received May 28, 2019; accepted September 10, 2019; published September 20,2019.

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