Sweet potato line ND98 and cv. Lizixiang were cultivated in a greenhouse under a regime of 16 h light and 8 h darkness (28°C). ND98 is resistant to F. oxysporum and Lizixiang is susceptible to this disease. ND98 was used for gene cloning in this study. One expressed sequence tag (EST) selected from the ND98 EST library constructed at our laboratory was up-regulated after the infection of F. oxysporum in ND98 and was used for gene cloning. The cloned gene was further introduced into Lizixiang for function characterization. Arabidopsis thaliana plants (Col-0) were grown in a 16-h light/8-h dark climate-controlled chamber at 22°C for analyzing the promoter of this gene.

Total RNA was extracted from the fresh leaves of 4-week-old in vitro-grown plants of ND98 using RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). The RNA samples were reverse-transcribed according to the instructions of PrimeScript^TM II 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China). Rapid Amplification of cDNA Ends (RACE) was then applied to amplify the full-length sequence of IbSWEET10 using SMARTer RACE 5′/3′ Kit (TaKaRa, Beijing, China). Gene-specific primers (GSPs) were used for 5′ RACE (5GSP1 and -2) and 3′ RACE (3GSP1 and -2). Amplification of the genomic sequence of IbSWEET10 with GS-F/R primers was performed using genomic DNA extracted from the fresh leaves of 4-week-old in vitro-grown ND98 plants, and the 5′-promoter region of IbSWEET10 was isolated using the GSPs (GW1 and -2) and Genome Walking Kit (TaKaRa, Beijing, China). All polymerase chain reaction (PCR) products were subcloned into the pMD19-T vector (TaKaRa, Beijing, China) and then transformed into competent Escherichia coli strain DH5α cells and sequenced. The primers used in this study are listed in Supplementary Table S1.

The open reading frame (ORF) of IbSWEET10 was predicted using ORF Finder¹. The molecular weight and isoelectric point (pI) of the IbSWEET10 protein were calculated at http://web.expasy.org/compute_pi/. Multiple sequence alignment of the IbSWEET10 protein with other SWEET10 proteins was performed using DNAMAN software. MEGA 6.0 software was used to construct the phylogenetic tree with the neighbour-joining (NJ) method.

Total RNA was isolated from roots, stems, leaves and petioles of 4-week-old in vitro-grown ND98 plants and storage roots, fibrous roots, stems, leaves and petioles of 3-month-old ND98 plants in the field. Total RNA was extracted as described above, and first-strand cDNA was synthesized using the PrimeScript^TM RT Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa, Beijing, China). Quantitative real-time PCR (qRT-PCR) was performed to determine the transcript levels of IbSWEET10 using SYBR Premix Ex Taq (Tli RNaseH Plus; TaKaRa, Beijing, China) and the 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers used to amplify IbSWEET10 and IbActin (internal control, AY905538) are listed in Supplementary Table S1.

To generate the P_Ibsweet10-GUS construct, the IbSWEET10 promoter fragment containing the 5′ untranslated region (5′UTR) was amplified from the ND98 genomic DNA and subcloned into the expression vector pMDC162 at PacI and AscI sites. The reconstructed plasmid was introduced into the GV3101 strain of A. tumefaciens. Arabidopsis plants were transformed using the floral dip method (Clough and Bent, 1998), and transformants were identified on 1/2 MS medium containing 60 mg L^-1 hygromycin.

The response of IbSWEET10 to Fusarium infection was investigated in ND98 plants. F. oxysporum was inoculated onto solid potato dextrose agar (PDA) medium at 28°C and cultured for 1 week in the dark. When the mycelium was fully expanded on the plate, the medium was minced, and the agar plugs were transferred to sterile distilled water and shaken for 2 h to loosen the spores from the mycelium surface. The resulting spore suspension was filtered through a nylon mesh to remove solid material; the spore concentration was determined using a haemocytometer and adjusted to ∼10⁷ spores per mL. Before being grown in 19-cm-diameter pots containing sterile sandy loam, 20-cm-cuttings of ND98 with an apical bud from the field were soaked in spore suspension for 30 min. Each pot contained three cuttings as one treatment. All leaves of each treatment were collected at 0, 3, 5, 7, and 9 days post-inoculation (dpi), and expression of IbSWEET10 was assessed by qRT-PCR.

The ORF of IbSWEET10 (stop codon deleted) was amplified using primers 83S-F/R and subcloned into a modified pMDC83 vector at SpeI and AscI restriction sites. The fusion construct (IbSWEET10-GFP) and the empty vector (GFP) were separately transformed into the EHA105 strain of Agrobacterium tumefaciens, and each transformant was co-infiltrated with a plasma membrane-localized marker (CD3-1007; Nelson et al., 2007) into the leaves of Nicotiana benthamiana, as previously described by Strasser et al. (2007). Furthermore, the sequence of IbSWEET10 with GFP was amplified from IbSWEET10-GFP fusion construct using primers SG-F/R and subcloned into the pDR196 vector at SmaI and XhoI restriction sites (Rentsch et al., 1995). The constructed vector was transformed into the sucrose transport deficient Saccharomyces cerevisiae mutant SUSY7/ura3 (Riesmeier et al., 1992) for subcellular localization assay. To select positive transformants, the transformed yeast cells were grown on yeast culture medium containing 6.7 g L^-1 yeast nitrogen base, 2 g L^-1 amino acid (uracil) drop out mix and 20 g L^-1 glucose as the sole carbon source. The agroinfiltrated tobacco leaves and the yeast transformants were visualized with a laser scanning confocal microscope (Nikon Inc., Melville, NY, USA).

The ORF of IbSWEET10 was amplified using primers 196-F/R and then subcloned into the yeast expression vector pDR196 at SmaI and XhoI restriction sites (Rentsch et al., 1995). As a positive control, the AtSUT4 gene was also cloned into pDR196 (Weise et al., 2000). The constructed vectors and the empty vector were transformed into the sucrose transport deficient S. cerevisiae mutant SUSY7/ura3 (Riesmeier et al., 1992) for complementation assays. The positive transformants were selected as described above and were then cultured on medium containing 20 g L^-1 sucrose to assess the function of IbSWEET10. The average diameters of yeast colonies were measured after 1 week using the ImageJ software. In addition, the hexose transport-deficient S. cerevisiae mutant EBY.VW4000 (Wieczorke et al., 1999) and drop tests were used to examine whether IbSWEET10 transports hexose. The transformants were grown overnight to an optical density at 600 nm (OD₆₀₀) of 0.6 in liquid medium containing 20 g L^-1 maltose as the sole carbon source, and serial dilutions (OD₆₀₀ = 0.6, 0.06, 0.006, 0.0006, and 0.00006) were plated on solid media containing different carbon sources (glucose, fructose, galactose, and mannose) for 1 week at 30°C.

To explore the function of IbSWEET10, the expression cassette P_35S-IbSWEET10-T_NOS was inserted into the plant binary vector pCAMBIA3301 at SacI and XbaI sites to generate the overexpression plasmid pCIbSWEET10. To construct the RNAi plasmid pFIbSWEET10, two fragments (FF and RF) were amplified from the coding sequence of IbSWEET10 using the primers Si-UF/R and Si-DF/R and inserted into the vector pFGC5941 at XhoI/SwaI and BamHI/XbaI sites, respectively. The sequence-verified plasmids pCIbSWEET10 and pFIbSWEET10 and the empty vector were separately transfected into A. tumefaciens strain EHA105. The transformation, regeneration, and overexpression lines (OX) identification of sweet potato cv. Lizixiang plants were performed as described by Wang et al. (2016). To identify IbSWEET10 RNAi lines, genomic DNA was extracted from the leaves of putative transgenic plants, and PCR was performed using the primers int-F/R. The transformants with the empty vector pCAMBIA3301 (VCo) and ones with the empty vector pFGC5941 (VCi) were used as the controls for the OX and RNAi lines, respectively.

β-Glucuronidase reporter gene activity in transgenic Arabidopsis was examined by histochemical staining as described by Guo et al. (2014). The incubation time for whole seedlings, roots, and flowers was 2 h; 6 h was used for leaves, stems, siliques, and seeds.

Transgenic and control sweet potato plants were cultured on solid MS medium with different sucrose concentrations (0, 1, 2, 3, 4, 5, and 6%) at 28±1°C under 16 h of cool-white fluorescent light at 54 μmol m^-2 s^-1. The standard sucrose concentration of MS medium is 3%. The growth status was observed after 4 weeks, and the fresh weight (FW) was measured immediately.

Transgenic and control sweet potato plants were transferred to the soil in a greenhouse and a field and their 20-cm-cuttings were inoculated with F. oxysporum using the same methods described above to evaluate the resistance to F. oxysporum. Growth of the transgenic and control lines was evaluated at 9 or 7 dpi, and stem samples were collected for histological examination according to the method of Zhang et al. (2014).

To determine changes in sugar composition during Fusarium infection, leaves of transgenic and control plants were harvested at 0 and 9 dpi (or 7 dpi) for quantification of sucrose, glucose, and fructose using high-performance liquid chromatography (HPLC; Goodall et al., 1995). Briefly, leaves (0.5 g) were dissolved in 10 mL 80% ethanol, vortexed thoroughly and incubated at 70°C for 30 min. The mixture was centrifuged at 12,000 g for 10 min, and the supernatant was collected into a new tube. The centrifugation step was repeated twice to remove any debris. The supernatant was dried at 70°C and redissolved into 5 mL distilled water, and 1 mL was filtered through a membrane (0.22 μm) and transferred to a glass tube for HPLC analysis.

Three biological replicates were performed and the data were presented as the mean ± SE. Data difference analysis was carried out based on Student’s t-test (two-tailed analysis) at P < 0.01 or P < 0.05.

A SWEET gene was cloned using the RACE method and we named it as IbSWEET10 based on its closest phylogenetic relationship with AtSWEET10 from A. thaliana (Eom et al., 2015). The cloned 1250-bp full-length IbSWEET10 cDNA contains a 921-bp ORF that encodes a 306-amino acid polypeptide with a molecular weight of 34.1 kDa and a pI of 9.34. A search of the IbSWEET10 protein sequence in NCBI revealed two conserved MtN3 domains. The genomic sequence of IbSWEET10 is 3269 bp long and contains six exons and five introns. The results of multiple sequence alignment show that the IbSWEET10 protein is a typical 7-TM SWEET (Figure 1A). Based on phylogenetic analysis, IbSWEET10 has the closest relationship with AtSWEET10 (Figure 1B), both belong to clade III of the SWEET family (Chen et al., 2010).

Quantitative real-time PCR analysis revealed expression level of IbSWEET10 was highest in leaves of 4-week-old in vitro-grown ND98 plants (Supplementary Figure S1A). High expression was also observed in leaves of 3-month-old ND98 plants from the field (Supplementary Figure S1B). As previously reported in sweet potato, the spatial expression pattern of a gene was correlated with its promoter activity in a heterologous system such as Arabidopsis (Noh et al., 2010, 2012; Tanabe et al., 2015). Therefore, the activity of the 1842 bp promoter including 5′UTR of IbSWEET10 was investigated in different tissues of Arabidopsis using gusA gene as a reporter gene. In 2-week-old seedlings of Arabidopsis, GUS activity driven by P_IbSWEET10 was found in all of the tissues (Figure 2A), especially in the vascular tissues of leaves and roots (Figures 2B,C). In soil-grown mature Arabidopsis plants, the leaves showed the high GUS activity, but the reduced GUS activity was detected in roots, stems, flowers, siliques, and seeds (Figures 2D–I), which are consistent with IbSWEET10 expression in sweet potato.

In Arabidopsis, different SWEET proteins are localized to different cellular sites, and to date, four of the seven clade III AtSWEETs have been functionally shown to encode plasma membrane proteins (Seo et al., 2011; Chen et al., 2012; Lin et al., 2014; Chen L.Q. et al., 2015). Because IbSWEET10 is phylogenetically a clade III SWEET, we investigated whether it is also located in the plasma membrane by generating 2 × 35S_pro-IbSWEET10-GFP construct and transient expression in N. benthamiana epidermal cells. As shown in Figure 3A, the green fluorescence emitted by the IbSWEET10-GFP fusion protein is coincident with the red fluorescence of the plasma membrane-localized marker CD3-1007, indicating that IbSWEET10 was localized to the plasma membrane. The subcellular localization in yeast further revealed that IbSWEET10 is a plasma membrane protein (Figure 3B).

To investigate whether IbSWEET10 encodes a sugar transporter, the gene was expressed in the yeast mutant SUSY7/ura3, which is deficient in sucrose transport (Riesmeier et al., 1992). As shown in Figures 4A–C, SUSY7/ura3 cells expressing either AtSUT4 or IbSWEET10 grew better on sucrose media than the cells transformed with the empty pDR196 vector. The colonies transformed with IbSWEET10 were 2.09-folds of ones transformed with the empty pDR196 vector in size (Figure 4D). This was a strong indication that IbSWEET10 encodes a sucrose transporter. To examine whether IbSWEET10 transports hexose (glucose, fructose, galactose, and mannose), the yeast mutant EBY.VW4000 (Wieczorke et al., 1999), which is impaired in hexose transport, was transformed with IbSWEET10. However, no obvious signs of growth on media containing different hexoses as the main carbon source were observed (Figure 4E). These results revealed that IbSWEET10 is a sucrose transporter rather than a hexose transporter.

Transgenic Lizixiang lines were generated as described by Wang et al. (2016) (Supplementary Figure S2). At last, the 11 OX, 8 RNAi, 5 VCo and 5 VCi transgenic lines were obtained in this study. qRT-PCR analysis showed significant increases in the transcript level of IbSWEET10 in three OX lines (OX96, OX99, and OX130; Supplementary Figure S3A) and dramatic decreases in two RNAi lines (RNAi6 and RNAi7; Supplementary Figure S3B) compared with WT and VCo/VCi. Under in vitro conditions, the sucrose concentration of the growth medium has an impact on the growth of plantlets (Jo et al., 2009). Because IbSWEET10 functions as a sucrose transporter, altered IbSWEET10 expression could affect sucrose transport and thereby alter plant growth. To examine this possibility, IbSWEET10 transgenic lines were grown on media containing different sucrose concentrations. Under a normal sucrose concentration (3%), both the OX and RNAi lines showed no difference in FW from WT and VCo/VCi. However, the OX lines exhibited enhanced tolerance to low (0–2%) or high (4%) sucrose concentrations compared with WT and VCo (Figure 5A). In contrast, low sucrose concentrations resulted in decreased FW in RNAi lines, whereas high sucrose concentrations caused little change compared with WT and VCi (Figure 5B).

To verify whether IbSWEET10 contributes to pathogen resistance, we first examined the changes in IbSWEET10 expression in ND98 plants infected by F. oxysporum. qRT-PCR analysis indicated that IbSWEET10 expression was significantly induced by the pathogen. The highest expression level was detected at 7 dpi: 3.46-fold of that at 0 dpi (Supplementary Figure S4). We further examined the severity of fusarium wilt in OX or RNAi lines at 9 or 7 dpi, respectively. At 9 dpi, almost half of the WT and VCo leaves were withered and yellow, whereas only a few OX leaves showed this symptom. The length of stem browning of the OX lines was also shorter than that of WT and VCo. Some new roots were formed on the OX stems, but no root formation was observed in WT and VCo (Figure 6A). Furthermore, RNAi lines exhibited more severe symptoms compared with WT and VCi, most of the leaves became wilted and yellow, the stems turned brown to black, and no root formation was found on the stems (Figure 6B). Histological observation of stems showed that the cells in the pith of WT and VCo were deformed and became loosely arranged, whereas the stem structure of OX lines remained intact, with compact cells in the pith and cortex (Figure 7A). Cross-sectional observation of the stems of RNAi lines exhibited a destroyed pith structure and distortion of cortex cells, which were not serious in WT and VCi (Figure 7B). These results indicate that IbSWEET10 plays a positive role in enhancing sweet potato resistance to F. oxysporum.

The carbohydrate status of plants affects the development/resistance of disease and vice versa (Berger et al., 2007; Gamm et al., 2011; Li et al., 2014). Because we identified the sucrose transporter activity of IbSWEET10, the altered resistance of transgenic plants to F. oxysporum was most likely caused by an uneven distribution of sucrose and other soluble sugars. Therefore, the sucrose, glucose and fructose contents in the leaves of transgenic and control plants were examined. For OX lines, a significant decrease in each of these three sugars was observed at both 0 and 9 dpi compared with WT and VCo (Figures 8A,B). However, RNAi lines contained higher levels of each sugar at 0 and 7 dpi compared with WT and VCi (Figures 8C,D). We also noticed that compared with 0 dpi, the sucrose content in each plant line was increased at 9 or 7 dpi.

Sugars Will Eventually be Exported Transporters have been demonstrated to have sugar transport capacity, mainly transporting sucrose and glucose in many plants (Chen et al., 2010; Chen L.Q. et al., 2015). In the present study, assays using sucrose transport-deficient yeast SUSY7/ura3 cells revealed that IbSWEET10 is a sucrose transporter (Figure 4). Based on expression in hexose transport-deficient yeast EBY.VW4000 cells, IbSWEET10 did not transport glucose, fructose, galactose or mannose (Figure 4D). This result is consistent with other reported SWEETs that belong to clade III (Chen L.Q. et al., 2015).

It is reported that the physiological role of sucrose transporters can be predicted through analysis of their tissue-specific expression and subcellular localization. The most possible route of sucrose transport into the SE/CC might begin directly from its production site in mesophyll cells through the apoplasm and into phloem parenchyma cells (Chen et al., 2012). Analysis of the GUS expression patterns of P_IbSWEET10-GUS-transgenic Arabidopsis revealed that IbSWEET10 is more abundantly expressed in leaves of mature plants, especially in vascular tissues (Figure 2F). This finding indicates that IbSWEET10 functions to export sucrose from source leaves and participates in phloem loading. A two-step model of sucrose apoplasmic phloem loading shows that sucrose is exported by SWEETs residing in the plasma membrane of phloem parenchyma cells and feeds the sucrose transporter SUT in the SE/CC (Chen et al., 2012). Our study reveals that IbSWEET10 is located in the plasma membrane (Figure 3). Thus, we suggest that IbSWEET10 participates in the first step of sucrose apoplasmic phloem loading across the plasma membrane.

Plants growing under tissue culture conditions are semiautotrophic, and supplementation of sucrose in the medium meets the energy demand for growth and physiological functions (Hazarika, 2003). The atsweet11;12 mutants show reduced root length when germinated on sugar-free media (Chen et al., 2012) and atsuc2 mutant seedlings are smaller than wild-type under the same conditions (Gottwald et al., 2000). In this study, a significant increase in fresh weigh of the OX plants was observed compared with the controls under low sucrose concentration (0–2%), suggesting that overexpression of IbSWEET10 increases the efflux of sucrose from leaves and promotes the plant growth (Figure 5A). Under high sucrose concentration (4%), the OX plants also exhibited the increased FW compared with the controls, thinking that its overexpression might facilitate sucrose transport into the OX plant roots from leaves and medium, which induces osmotic stress balance in the rhizosphere (Jo et al., 2009; Chen L.Q. et al., 2015). However, when expression of IbSWEET10 was suppressed, low sucrose concentrations in the medium caused the decreased FW in RNAi plants. No significant difference in FW was observed between RNAi plants and controls at high sucrose concentrations (Figure 5B). One possible explanation for this result is that reduced expression of IbSWEET10 might lead to impaired export of sucrose from leaves and thus negatively affect the plant growth (Burkle et al., 1998). When the sucrose supply was sufficient, the RNAi plants were able to acquire sucrose from the medium for growth.

Access to nutrients from hosts for reproduction is the primary objective of infection (Chen et al., 2010), and sugars are among the most important nutrients providing energy for pathogen growth. For many diseases caused by soil pathogens, the nutrients provided by the host are considered to be a main factor in deciding the success or failure of symptomatic development (Hancock and Huisman, 1981). The observed high incidence of Fusarium with the shrunken-2 mutation was proposed to result from the elevated sugar content in kernels (Headrick et al., 1990). Additionally, AtSWEET2 activity reduced sugar availability in the rhizosphere, thus contributing to Pythium resistance (Chen H.Y. et al., 2015). Thus, lack of carbohydrates will limit the growth of a pathogenic fungus and reduce the ability of the pathogen to infect plants.

Alteration of the expression levels of sucrose transporter genes might cause changes in the sugar content in plants. Leaves of atsweet11:12 double mutants accumulate high levels of sugars, indicating an impaired ability to export sugar from the leaves (Chen et al., 2012). In addition, disruption of AtSUC4 or AtSUC2 caused sucrose accumulation in source leaves possibly by interfering with phloem loading (Gong et al., 2015), and the sucrose concentration in the vacuole of PatSUT4-RNAi lines was higher than in WT under well-watered conditions (Frost et al., 2012). In our study, overexpression of IbSWEET10 might enhance sugar export from leaves, which can explain the lower sugar contents in the leaves of OX plants. The opposite was observed for RNAi plants (Figure 8A). Long-distance transport of sucrose between leaves and roots occurs in the phloem sieve elements of the stem vascular system (Williams et al., 2000), and blocked phloem loading causes more accumulation of sugars in leaves (Chen et al., 2012). After F. oxysporum infection, the stems of OX plants displayed only slight damage and phloem loading block, which facilitated the transport of sugars from the leaves compared with WT, VCo, VCi and RNAi plants which showed serious damage and phloem loading block (Figure 7). As a result, the sucrose content was significantly decreased in the OX leaves than in others after F. oxysporum infection (Figures 8B,D). These results indicated that IbSWEET10 plays an important role in sugar transport in sweet potato plants.

The soil-borne F. oxysporum proliferates in the vascular of sweet potato stems and then spreads to the leaves, which causes wilting of stems and leaves. Therefore, the lower level of sugars in the OX stems and leaves might limit the proliferation of the pathogen. Meanwhile, closely arranged cell structure of OX stems might prevent the invasion and spread of F. oxysporum. As a result, the OX plants exhibited the enhanced resistance to F. oxysporum (Figure 6).

In addition, the overexpression of OsSWEET11, -13, and -14 enhanced the pathogenicity of X. oryzae in rice (Yang et al., 2006; Antony et al., 2010; Liu et al., 2011). The AtSWEET2-overexpressing Arabidopsis plants showed the increased resistance to P. irregulare (Chen H.Y. et al., 2015). Our results revealed that overexpression of IbSWEET10 enhanced the resistance of sweet potato to F. oxysporum. Therefore, it is thought that different members of the SWEET gene family differentially function in the responses to plant pathogens.