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. 2024 Nov 2;30(11):1777–1793. doi: 10.1007/s12298-024-01523-9

Changes in soluble sugars and the expression of sugar transporter protein genes in strawberry crowns responding to Colletotrichum fructicola infection

Si-Yu Chen 1,2, Xue Li 1,3, Ke Duan 1,✉, Zi-Yi Li 1,3, Yun Bai 1,2, Xin-Yi Wang 1,3, Jing Yang 1, Xiao-Hua Zou 1, Mei-Ling Xu 4, Ying Wang 5, Qing-Hua Gao 1,✉
PMCID: PMC11646252  PMID: 39687699

Abstract

Strawberry (Fragaria × ananassa) production has been greatly hampered by anthracnose crown rot caused by Colletotrichum fructicola. Crown, the modified stem of strawberry, is a sink organ involved in sugar allocation. Some Sugar Transport Proteins (STPs) are involved in competition for sugars between pathogen and host. However, the chemical nature and involvement of strawberry STPs (FaSTPs) in crown rot development is largely elusive. To reveal how strawberry alters soluble sugars and upregulates STPs in responses to C. fructicola, high performance liquid chromatograph and FaSTP expression analysis were performed in the crowns of three strawberry varieties, following a genome-wide identification of FaSTPs. Both C. fructicola and mock treatment/control changed glucose, fructose and sucrose accumulation in strawberry crowns. With increasing infection duration, the hexose/sucrose ratio increased in all varieties; no such trend was clearly visible in mock-treated plants. A total of 56 FaSTP loci scattered across four subgenomes were identified in octoploid strawberry, and most of the protein products of these genes had a preferential location on plasma membrane. Putative fungal elicitor responsive cis-elements were identified in the promoters of more than half FaSTPs. At least eight members were upregulated in strawberry crowns during C. fructicola invasion. Of them, FaSTP8 expression was markedly enhanced in three varieties at all time points except for 3 dpi in ‘Jiuxiang’. RNAseq data retrieval further validated the expression responses of FaSTPs to Colletotrichum spp. In summary, this work identified several FaSTP candidate genes responsive to Colletotrichum fructicola invasion, demonstrated changes in soluble sugar levels in strawberry crowns as a result of infection, and laid the groundwork for future efforts to engineer strawberry resistance to Colletotrichum spp.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01523-9.

Keywords: Fragaria × ananassa, Colletotrichum fructicola, Anthracnose crown rot (ACR), Soluble sugar, Sugar transport protein (STP)

Introduction

Sugars are required by all living organisms, serving as energy storage/currency, signal molecules, carbon skeletons, transport molecules and osmolytes in cells (Chen et al. 2015). Sugars are the products of photosynthesis, which could be severely impaired upon pathogen invasion (Lopes and Berger 2001). In maize young leaves, the establishment of C4 metabolism and the sink-to-source transition were arrested upon Ustilago maydis infection, which was characterized by high hexose contents and hexose/sucrose ratio in infected tissues (Horst et al. 2008). Pathogen requires many nutrients from the host, and the most important are carbohydrates, predominantly glucose, fructose and sucrose (Talbot 2010). The elevation of extracellular soluble sugars in plants was a common response in both susceptible and resistant interactions with pathogens (Essmann et al. 2008; Siemens et al. 2011). Sugars may also contribute to plant immunity as priming signal molecules (Bolouri Moghaddam and Van den Ende 2012). Changes in soluble sugar differentially affected the outcome of plant-pathogen interactions, which was intimately correlated with the nutrition ways of pathogens. Enhanced carbohydrate accumulation inhibited the proliferation of Colletotrichum higginsianum, but promoted the colonization of biotrophic Erysiphe cruciferarum in Arabidopsis (Engelsdorf et al. 2013). Rice plants with higher levels of glucose, fructose and sucrose displayed enhanced resistance to both bacterial pathogen Xanthomonas oryzae and the hemibiotrophic fungus Magnaporthe oryzae (Sun et al. 2014).

Sugar transporters coordinately carry out carbohydrate allocation, regulating sugar partitioning between or within source/heterotrophic sink organs and in plant-pathogen interactions (Kong et al. 2019). Many pathogens could commandeer host sugar transporters to obtain carbohydrates and enhance pathogenesis (Bezrutczyk et al. 2018). By contrast, as part of non-host resistance, a nutrient deprivation strategy could effectively prevent a wide range of pathogen infections (Senthil-Kumar and Mysore 2013). Sugar transport proteins (STPs), also known as monosaccharide transporters (MSTs) or hexose transporters (HTs), transport extracellular sugars into plant cells and probably limit the sugar supply for pathogens. Expression of AtSTP4 was induced upon pathogen infection in Arabidopsis (Fotopoulos et al. 2003). High expression of AtSTP13 was closely correlated with an improved absorbance of glucose and enhanced resistance, while AtSTP13 knockout reduced glucose uptake, plant resistance and the yield traits of Arabidopsis upon infection with the necrotrophic Botrytis cinerea (Lemonnier et al. 2014). AtSTP13 mutation also weakened plant resistance to the bacterial pathogen Pst DC3000 (Yamada et al. 2016). Mutation of wheat TaSTP13 (the Lr67 gene) enhanced host resistance to multiple biotrophic pathogens including three wheat rust pathogen species and powdery mildew (Moore et al. 2015). Overexpression of TaSTP6 decreased Arabidopsis resistance to powdery mildew, while its knockout increased wheat resistance to the biotroph Puccinia striiformis (Huai et al. 2019).

Colletotrichum spp. genus was among the global top 10 genera of fungal pathogens with a wide range of hosts (Dean et al. 2012). A total of 280 Colletotrichum species grouped into 16 complexes as well as 15 additional singletons have been accepted, occurring as important plant pathogens, endophytes, saprobes and human pathogens (Liu et al. 2022). Global warming is witnessing the increasing hazard of Colletotrichum spp. on crops (Ji et al. 2023). C. fructicola and C. siamense are the top two species most frequently detected worldwide with the broadest host range (Talhinhas and Baroncelli 2021). These hemibiotrophic species live in a biotrophic way for nutrient acquisition in early infection and switch to a necrotrophic way later (Glazebrook 2005). The transition from biotrophy to necrotrophy in microbes was proposed to be mediated through quorum sensing for their population density and nutrient limitation (Fatima and Senthil-Kumar 2015). The hexose transporter ChHxt6 was found to be crucial for hexose uptake during infection at both biotrophic and necrotrophic phases for C. higginsianum (Yuan et al. 2021).

Strawberry (Fragaria × ananassa Duch.) is an important berry crop cultivated worldwide. Most commercial strawberries are allo-octoploid hybrid between F. virginiana and F. chiloensis (Hardigan et al. 2021). Strawberry is susceptible to many pathogens in whole life. Anthracnose caused by C. fructicola in nursery field has been one of the most destructive diseases restricting strawberry production in China, which could reduce crop yield by about 30%, or up to 90% in severe cases (Ren et al. 2008). C. fructicola has been prevailing in strawberry fields of eastern Asia–Pacific region (Ji et al. 2022). Strawberry crown, as a sink organ, the joint part between shoot and root, is indeed a condensed stem, also a part of the long-distance sugar transport system. Strawberry crowns contained both soluble and storage carbohydrates at high levels (Wang and Camp 2000; Macías-Rodríguez et al. 2002). How does Colletotrichum spp. infection affect the soluble sugar contents in strawberry crowns? It is completely obscure.

Previously, we have identified STP genes in F. vesca (Liu et al. 2020). The improved genome annotation for octoploid strawberry enable an updated gene identification (Liu et al. 2021). Concerning the importance of STP, there is an urgent need to identify this gene family in F. × ananassa. Current work aims to (i) reveal soluble sugar changes in strawberry crowns infected with C. fructicola via using high performance liquid chromatograph (HPLC); (ii) identify STP family in octoploid strawberry (FaSTPs); (iii) explore the expression responses of FaSTP members to C. fructicola infection via RT-qPCR analysis. The identification of FaSTPs responsive to C. fructicola in crowns would provide candidate genes for resistance breeding.

Materials and methods

Microbe/plant materials and growth conditions

Colletotrichum fructicola strain CGMCC3.17371 from diseased strawberry of a local field (Ren et al. 2008) was maintained in Strawberry Group of Shanghai Academy of Agricultural Sciences (SAAS). After rejuvenation on sterile strawberry seedlings from meristem tip culture, the fungus was refreshed and purified as a single spore colony on PDA medium (LOT.0091369, BD Company, USA) at 28 °C in darkness. C. fructicola mycelial plugs (about 1 cm × 1 cm squares) were cut from the advancing edge of 7-d-old plate and deposited in a conical flask containing 100 ml PDB medium (LOT.0107881, BD). Conidia were produced in a shaker (THZ-C-1 full temperature oscillator, Taicang Experimental Equipment Factory) at 28 °C and 220 rpm for 5–8 days.

Three strawberry varieties ‘Hogyoku’, ‘Sweet Charlie’ and ‘Jiuxiang’ with distinct origins and field resistance to anthracnose crown rot were used in current work. Stolons from healthy strawberry plants were rooted in 24-hole plastic tray in SAAS station at Zhuanghang Town, Fengxian District, Shanghai. Seedlings with 5 to 6 fully expanded trifoliate leaves were transplanted into pots (90/65 mm in height/upper diameter) and brought to lab. These strawberry seedlings were then cultured in an incubator (GXZ-1000, Jiangnan Instrument Factory, Ningbo, China) at 25 °C under a 14 h-light /10 h-dark rhythm. Inoculation was carried out after two weeks.

Inoculation with C. fructicola and sampling

Freshly prepared conidia solution was diluted to 2 × 105 cfu in sterile water with 0.01% Tween-20. A total of 30 plants per genotype were used in one experiment, with half for Cf-inoculation and half for mock treatment with sterile water with 0.01% Tween-20. The experiment was independently repeated twice.

Before inoculation, the lowest old leaf was removed and the plant was uniformly wounded at the basal site of crown near root via injection with a sterile needle into 2 mm in depth. Then each plant was inoculated at the wounded site with a 10-μL droplet of conidia solution or water for mock treatment. After inoculation, the plants were covered with plastic membrane to maintain 99% humidity for three days. After 3 days post inoculation (dpi), the membrane was removed. The symptom proceeding was studied at 3, 7 and 14 dpi. The root and leaves were discarded, and the joint section was transversely cut at the injection site. The segments (just 1 cm above the basal crown) from 5 plants of one genotype were sampled for further analysis. The segment below the cut surface was photographed and lesion size was calculated with ImageJ software (Rueden et al. 2017).

The symptom grading and disease index of strawberry crowns were calculated largely according to a previous report (Jin et al. 2014) slightly modified (supplementary Table S1). The crown tissues in 1 cm length were collected at 3, 7 and 14 dpi. Each sample pooled from 5 plants per genotype/treatment/timepoint was wrapped in tinfoil, quickly frozen in liquid nitrogen and stored under − 80 °C. Purification of DNA, RNA and soluble sugars were accomplished within two weeks.

DNA extraction and qPCR analysis of Colletotrichum colonization

Genomic DNA was extracted from strawberry crowns with a polysaccharide polyphenol plant Genome DNA kit (DP360, TianGen, Beijing). Nucleic acid concentration and quality was determined by spectrophotometric analysis and electrophoresis on 1% agarose gel.

The primers specific to Cutinase (gene KB020836.1) of Colletotrichum spp. and strawberry Actin gene (FvH4_1g23490) were used to detect the relative biomass during fungal colonization in strawberry (Yang et al. 2022). Real-time quantitative PCR (qPCR) and the 2−ΔCt calculation method were conducted. Three technical repeats and two independent samples were analyzed for all C. fructicola inoculation or mock-treatment/control. Primer sequence information was shown in supplementary Table S2.

HPLC analysis of soluble sugar content in strawberry crown tissues

Soluble sugars were extracted from the same homogenized crown samples for aforementioned DNA analysis following the method reported (Liu et al. 2020) with minor modifications. In brief, 150 mg plant homogenized tissue was twice dissolved in 2 ml 80% ethanol under 37 °C for 60 min, and then centrifuged. The supernatant was collected and filled to a final 5 ml. Samples were dried via evaporation at 60 °C for 3.5 h. Furthermore, the metabolites extracted were completely dissolved in ultrapure water, then centrifuged to discard insoluble components and again filtrated through a 0.22 µm membrane. A novel HPLC column Xtimate® Sugar-H (7.8 × 300 mm, 5 μm) (Lot No. 5U02.14, Welch Materials, Inc., Shanghai, China) was used for sugar quantification, and the remaining conditions and instruments for HPLC were the same as described previously. The standard curve and correlation coefficient for sugar quantification were shown in supplementary Table S3.

The sum of sucrose, glucose and fructose contents representing the total soluble sugars was shown as the mean of six raw data from two independent experiments and three repeats in HPLC analysis. The sum of glucose and fructose contents was used as hexose contents. Hexose-to-Sucrose and Glucose-to-Total soluble sugars ratios were calculated as H/S and G/T, respectively.

Identification of FaSTP genes and phylogenetic analysis

To identify FaSTP family genes in octoploid strawberry, Fragaria × ananassa Genome CDS (v1.0.a2) at strawberry genome database (SGD, http://www.strawberryblast.ml:8080/strawberry/viroblast.php) was screened with an E-value lower than 1E-5 via TBLASTN using the protein sequences of F. vesca STPs (FvSTPs from GDR v4.0a2, Liu et al. 2020). All candidates with MFS_1 (PF07690.19) and Sugar_tr (PF00083.27) domains were further validated through PFAM analysis (http://pfam.xfam.org/).

Phylogenetic analysis was performed for a total of 92 members including 14 STP-like proteins from Arabidopsis, 22 from F. vesca, and 56 from F. × ananassa using a neighbor-joining (NJ) method in MEGA v11 (Tamura et al. 2021). The corresponding tree was constructed using 1000 re-samplings in MEGA and visualized via using the interactive Tree Of Life (iTOL) https://itol.embl.de/ (Letunic and Bork 2016).

The in-silico prediction of biochemical characteristics was performed for each strawberry STP-like member. The subcellular localization was predicted at https://wolfpsort.hgc.jp/ (Horton et al. 2007). The isoelectric point (pI) and molecular weight of the protein sequences were calculated using the online Compute pI and Mw tool at https://web.expasy.org/ (Gasteiger et al. 2003).

Domain module in deduced FaSTP proteins

The conserved protein domains of FvSTPs and FaSTPs were identified at PFAM and illustrated with IBS1.0 (Illustrator for Biological Sequences 1.0) (Liu et al. 2015). To identify and compare the conserved motifs in STP proteins, the MEME program at http://meme-suite.org/ was employed using format settings (Bailey et al. 2015) for STP proteins from F. vesca and F. × ananassa.

Chromosomal locations of FaSTP genes and the cis-elements in their promoters

The genomic locations of FaSTP genes were obtained from F. × ananassa genome databases at GDR and assigned to distinct chromosomes or subgenomes (Edger et al. 2019). The physical map of FaSTP distribution was drawn with Mapchart1.0. software (Voorrips 2002).

The promoter sequence of 2500-bp in length was extracted from GDR at the upstream of the ATG translation start codon for each STP gene in F. vesca and F. × ananassa. Cis-elements were predicted in the PlantCare database at http://bioinformatics.psb.ugent.be/webtools/plantcare/html (accessed on 9 July 2022) (Lescot et al. 2002) and illustrated via using TBtools package (Chen et al. 2020).

RNA extraction, reverse transcription and RT-qPCR

RNA was extracted from strawberry crown tissues using a Plant RNA kit (Cat#R6827, OMEGA Bio-tec, USA). cDNAs synthesis was achieved by using the HiScript III RT SuperMix kit with gDNA wiper (Vazyme, Lot#R323, China). The ChamQ™ Universal SYBR qPCR Master Mix (Vazyme, Lot#Q711, China) was used in a 12-µL cocktail following user manuals. RT-qPCR was conducted on a Light Cycler 480 (Roche, USA).

Amplification of two reference genes EF1α (FvH4_3g33150, gene28639) and GAPDH2 (FvH4_4g24420, gene07104), was used as internal control (Amil-Ruiz et al. 2013). A serial of diluted cDNAs were used for primer amplification efficiency analysis (E), and the (1 + E)−∆∆Ct method combined with the double internal reference normalization with geNorm software was used for RT-qPCR data processing (Vandesompele et al. 2002). The transcript levels of FaSTPs relative to that in mock-treated crowns at 3 dpi (set to 1) were reported as the mean of six experimental data (two independent repeats, each containing three technical replicates) ± the standard deviation (SE).

Statistical analysis

To evaluate C. fructicola colonization in strawberry crown, the effects of strawberry genotype, time point and genotype × time point on the growth of C. fructicola were detected by two-way ANOVA　(Tukey-test). One-way ANOVA was used to evaluate the dynamic difference in certain genotype/treatment.

For soluble sugars and relative expression levels, pairwise comparison between mock and Cf-inoculated was based on a Student T-test, and the significant differences were graded with asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). The Waller-Duncan method in one-way ANOVA test was used to compare the differences among distinct genotypes under mock-treatment (lowercase letters) or Cf-inoculation (uppercase letters).

Results

Distinct development of anthracnose crown rot in three strawberry varieties

Currently, three strawberry varieties were characterized in their resistance to anthracnose crown rot (ACR) caused by C. fructicola. Generally, morphological changes were largely invisible in intact plants after artificial inoculation at 25 °C for 14 days (lower part in Fig. 1A). At 3 days post C. fructicola inoculation, necrosis around the wounded site emerged, which was more frequently observed on the crown transection of cv. ‘Hogyoku’, less in ‘Jiuxiang’, and hardly in ‘Sweet Charlie’. No disease symptom was observed in mock-treated plants, regardless of genotype. Till 7 dpi, ACR symptoms appeared on the crown transections of all three genotypes. Tissue necrosis emerged on the phloem and cambium tissues of crowns in most inoculated plants of ‘Sweet Charlie’ and ‘Jiuxiang’, and the necrosis reached the inner xylem of the crowns in some individual plant of ‘Hogyoku’ (Fig. 1A). At 14 dpi, although the whole plant showed no symptom from the appearance, inner rot clearly expanded as a large black-brown necrotic region including xylem on the crown transections of three varieties.

Fig. 1.

Fig. 1

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Crown rot development in strawberry upon C. fructicola invasion. A Typical symptoms in crown transections and whole plants under 25 °C. B The percentage of disease grade (%) derived from lesion size on crown transversal surface. C qPCR for the colonization of C. fructicola in strawberry crowns. Different lowercase letters indicated the significant differences between mock and treatment at distinct phases in certain genotype (P < 0.05). Different uppercase letters represented the significant differences among the effects of genotype × phase (two-way ANOVA, Tukey-test)

The necrotic symptoms were graded following the criterion in supplementary Table S1. As shown in Fig. 1B, ACR symptom was developed more quickly in most individual plants of ‘Hogyoku’ at the early phase (3 dpi). Consistently, more crowns displayed a higher disease grade (grade 5, necrosis covering 11–25% of the crown transection) in ‘Hogyoku’ than in ‘Sweet Charlie’ and ‘Jiuxiang’ at 7 dpi. However, further cultivation till 14 dpi at 25 °C, more than half individual plants in ‘Jiuxiang’ developed the highest necrosis (grade 9, 50% or more of the crown transection), 20% plants of ‘Sweet Charlie’ developed the severest necrosis, while none of ‘Hogyoku’ plants reached this grade. Indeed, all C. fructicola-inoculated ‘Hogyoku’ plants at this phase displayed crow rot of grade 7 (lesion area covering 26–50% of the transection). Concerning the outcome of C. fructicola-strawberry interaction at 14 dpi, ‘Hogyoku’ was less susceptible than ‘Sweet Charlie’ and ‘Jiuxiang’, which seemed to have a capability to delay pathogen colonization since 7–14 dpi. By contrast, ‘Jiuxiang’ was the most susceptible, with pathogenesis proceeding more quickly than the rest two genotypes.

Furthermore, qPCR analysis of the relative biomass in genomic DNAs from strawberry crown tissues was conducted to reveal the proliferation of C. fructicola (Fig. 1C). The results showed that from 3 to 7 dpi, significant increase in C. fructicola biomass was observed in all three varieties, and ‘Hogyoku’ showed the highest increase in pathogen proliferation. However, from 7 to 14 dpi, significant increase of Colletotrichum biomass was only observed in ‘Jiuxiang’, although the neutrality (line) and mean values for fungal biomass were higher at 14 dpi than at 7 dpi in ‘Hogyoku’ and ‘Sweet Charlie’. In ‘Jiuxiang’, the relative biomass of C. fructicola continuously increased in the Cf-infected crowns. Plants of this cultivar exhibited the highest level of Colletotrichum colonization by 14 dpi. In sum, from the pattern of pathogen proliferation, herein strawberry varieties displayed significant differences in their defense responses to C. fructicola infection at crown tissues (P < 0.01). In addition, the background levels of latent Colletotrichum infection (endophyte) in mock-treated plants varied with strawberry variety, which was not significantly changed during 3–14 days post wounding in each variety.

Alterations of main soluble sugars in strawberry crown tissues infected with C. fructicola

As the joint part of plant shoot and root, strawberry crown accumulated a large amount of carbohydrates for long distance allocation. Intricate changes were observed in the levels of soluble sugars at this location post C. fructicola-invasion and mock-treatment (Fig. 2). Generally, the variations of fructose and glucose shared similar patterns; the levels of sucrose and the total soluble sugar fluctuated following largely consistent pattens.

Fig. 2.

Fig. 2

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Main soluble sugars in strawberry crowns revealed with HPLC. A Contents of fructose, glucose, sucrose and total soluble sugars in the crown tissues. B Composition of total soluble sugars in strawberry crowns post mock-treatment or Cf-infection. C Changes in H/S (hexose-to-sucrose ratio) and G/T (glucose-to-total soluble sugars in strawberry crowns. Six raw data were used in (A), and the average of six raw data was used in (B) and (C). Asterisks for the significant differences between mock and Cf-invasion (pairwise t-test); different lowercase and uppercase letters above the box charts indicating the differences in the dynamic and genotypic effects on sugar contents among mock or Cf-invasion, respectively (one-way ANOVA test, Waller–Duncan method)

At 3 days post mock treatment, the basal levels of crown sucrose were similar in three strawberry varieties, while the levels of crown fructose/glucose varied with genotype, and ‘Hogyoku’ harbored a significantly lower hexose than the rest two genotypes. Indeed, the soluble sugars in both mock treatment and Cf-invasion showed intricate changes depending on genotype and time duration.

At the early phase 3 dpi, the hexose level was downregulated in ‘Hogyoku’ and ‘Sweet Charlie’, while upregulated in ‘Jiuxiang’, as compared with that in mock (Fig. 2A). Simultaneously, although C. fructicola inoculation did not alter sucrose levels in ‘Hogyoku’; ‘Sweet Charlie’ and ‘Jiuxiang’ recruited more sucrose in crown tissues than that in control at 3 dpi. At 7 dpi, crown rot was gradually expanded and prominent in all three genotypes, and the levels of both hexose and sucrose were clearly upregulated in ‘Hogyoku’. On the contrary, at this phase hexose was not or only slightly altered and the sucrose decreased uniformly in ‘Sweet Charlie’ and ‘Jiuxiang’, as compared with control. By 14 dpi, crown rot continued to develop in all three genotypes with varying severity, when sucrose uniformly decreased. However, the hexose level maintained a moderate but significant upregulation in ‘Hogyoku’, just contradictory to the sharp downregulation in ‘Sweet Charlie’ and ‘Jiuxiang’ at 14 dpi.

The contribution of three major soluble sugars to the contents of the total soluble sugar was evaluated upon C. fructicola invasion (Fig. 2B). Apparently, the changes in both hexose and sucrose jointly shaped the variation in total soluble sugar. Interestingly, the variations of total soluble sugar between Cf-invasion and mock at 3 dpi seemed to be negatively correlated with plant resistance to this pathogen at 14 dpi. Dynamically, total soluble sugar in the resistant ‘Hogyoku’ experienced a mild decrease at 3 dpi followed by an increase at 7 dpi and again decrease at 14 dpi in this study. Total soluble sugar decreased in ‘Sweet Charlie’ after 3 dpi, reaching the lowest levels at 7–14 dpi. In ‘Jiuxiang’ upon C. fructicola invasion, the changes of total soluble sugar were similar to that in ‘Sweet Charlie’ after 3 dpi, although at higher absolute levels. In addition, the hexose-to-sucrose ratio continuously went up in three genotypes upon C. fructicola-invasion, while a fluctuation in this ratio due to the decrease at 7 days post mock treatment was uniformly observed in mock (Fig. 2C).

Phylogeny and molecular features of FaSTP family members in Fragaria × ananassa

To further explore how strawberry motivate sugar transportation to limit the supply of sugar for Colletotrichum spp., we performed a genome-wide identification of FaSTP family in octoploid strawberry genome. Based on TBLASTN search, a total of 57 STP candidates were identified in the genome of octoploid strawberry. Since FvSTP7 and its homolog FxaC_15g22340 were consistently found to have only the Sugar_tr (PF00083.27) domain but without MFS_1 (PF07690.19) domain, they were not included for further analysis. An unrooted phylogenetic tree was constructed in MEGA 11.0 using the protein sequences of 22 FvSTPs, 56 FaSTPs and 14 AtSTP members (Fig. 3). This analysis supported the classification of all proteins into four groups, with seven FaSTPs of group I, six of group II, 20 of group III and 23 of group IV. FaSTP was named according to their FvSTP homolog. Each STP protein in diploid F. vesca corresponded to 1–5 homologous proteins in the octoploid F. × ananassa. It’s worth noting that since FvSTP5 and − 11 were closely clustered together, all their FaSTP homologs were named after FvSTP5. Similarly, FvSTP14 and -19 were clustered together and their octoploid strawberry homologs were named following FvSTP14. Thus, in current nomenclature system, 7, 11, 17 (discarded in FvSTPs, Liu et al. 2020) and 19 were not used for FaSTPs nomination.

Fig. 3.

Fig. 3

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The phylogenetic relationships of Sugar transport protein (STP) proteins from Arabidopsis thaliana, diploid Fragaria vesca, and octoploid F. × ananassa strawberry. The tree consisting of 14 AtSTPs, 22 FvSTPs and 56 FaSTPs was clustered into four groups (I, II, III and IV). The length of branches indicates the relative phylogenetic relationship, and the bootstrap values near branches show confidence

The genomic and biochemical features of 56 FaSTP members were summarized in Table S4. Except for FaSTP3c, FaSTP6b, FaSTP6c and FaSTP21a, the number of transmembrane helices of all FaSTP members was between 10 and 13. The isoelectric point (pI) of FaSTP members varied between 6.09 (FaSTP16e) and 9.94 (FaSTP3c). The molecular weight of FaSTP proteins varied between 24.0 (FaSTP6c) and 66.6 (FaSTP16b) kDa. PSORT analysis showed that the preferential subcellular localization of FaSTPs was the plasma membrane (53 members), and next the vacuolar membrane (41 members). In addition, 8 members were also predicted to have a second localization in the endoplasmic reticulum. FaSTP6c encoding the shortest peptide might also localize in the Golgi apparatus.

PFAM analysis provided a comparison of the protein module structures for FvSTPs and FaSTPs (Fig. S1). The module structure of certain FaSTP in the octoploid strawberry was basically the same as that of its homolog in diploid woodland strawberry. Most members have complete MFS_1 (PF07690.19) and Sugar_tr (PF00083.27) domains. Strawberry STP members generally contain one MFS_1 and one Sugar_tr domain, but FaSTP6a, 6b, 16d, 16e, 22d and 24c contain additional region of Sugar_tr domain. By contrast, the Sugar_tr domain was absent in FaSTP6c and FaSTP21a. In addition, the MFS_1 (PF07690.19) domain was obviously truncated in FaSTP3c, 6b and 6c. These variations were further observed in the motif organization revealed via using MEME online tool (supplementary Fig. S2).

Physical distribution of FaSTP loci in the genome of F. × ananassa

Generally, FaSTP genes were evenly present in four subgenomes. A, B and D subgenome each has 15 members, and C subgenome has the rest 11 FaSTPs (Fig. 4). FaSTPs on Fvb1 of each subgenome belonged to group IV. Those on Fvb2, 3, and 6 were of group III, and on Fvb7 were of group I. Fvb4 harbored members of group III and IV, and Fvb5 contained members of group I, II and III. It’s worth noting that Fvb6 (6–1 ~ 6–4), the longest chromosome of each subgenome, harbored the less FaSTPs. Two tandem duplication events involving 5–6 FaSTPs of group IV were observed. Namely, the first tandem repeat on Fvb4-4 of B-subgenome was composed of FaSTP15b, − 13, − 12, − 14b, − 18b and 8b. The second tandem repeat contained FaSTP15a, − 14c, − 20a, − 18c and -8a on Fvb4-1 of d-subgenome. Four pairs of genes in these two repeats were highly homologous and preserved orderly (FaSTP15b/c, FaSTP14b/c, FaSTP18b/c and FaSTP8b/a). This was a reminiscent of a duplication event containing 8 genes (FvSTP19, − 13, − 12, − 14, − 15, − 20, − 18 and − 8) on Chr4 in F. vesca genome (Jiu et al. 2018).

Fig. 4.

Fig. 4

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Chromosomal distribution of FaSTPs in the genome of octoploid strawberry F. × ananassa. The localization information was obtained from GDR, and visualized in MapChart software. The length of each chromosome was indicated below in megabases (Mb). Arrows and lines indicated the transcription orientation and the transcription starting site for each locus

Cis-elements in the promoter regions of STP genes from F. vesca and F. × ananassa

Studying the cis-elements in gene promoter region will help to understand the regulation of gene expression. These elements identified in the 2500 bp upstream sequence of STP genes were classified into four types, including hormone response, plant growth, stimulus, and others (Table S5). Notably, TC-rich repeats (dark red circles) were involved in defense and stress responses (Germain et al. 2012). W box (TTGACC) (dark green circles) is a fungal elicitor-induced element that is also the binding site of WRKY responsible for plant defense response (Chen and Chen 2002). TCA element (yellow triangles) is a salicylic acid response element (Goldsbrough et al. 1993). The as-1 (dark blue triangle) sequence involved in the auxin and salicylic acid responses is the same as the MeJA response element, which consists of two CGTCA or TGACG motifs that constitute the TGA binding site, involved in the response of plants to plant growth hormone (SA, MeJA) and other stresses including hydrogen peroxide (dehydration, salt stress), etc. (Ulmasov et al. 1994; Krawczyk et al. 2002).

Promoter of strawberry Group I STPs uniformly possessed at least one W box the putative fungal elicitor-induced element (dark green circle), which was enriched in the promoters of FaSTP1a/b (Fig. 5). The TC-rich repeats involved in defense response (dark red circle) was observed in the FvSTP1 and FaSTP1a, − 1b, and − 1c promoters, and enriched in FaSTP1c promoter. This element was not present in the promoters of rest of the members in Group I. Interestingly, all members of Group I except for FvSTP2 and FaSTP2 contain TCA element (yellow triangle), which was enriched in FaSTP1a/b and FaSTP3a/b.

Fig. 5.

Fig. 5

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Cis-elements predicted in the 2500 bp promoter regions of STP genes in Fragaria vesca and F. × ananassa. The promoter sequences of 78 STP genes (56 from F. × ananassa and 22 from F. vesca) were analyzed at online PlantCare tool. Geometric shapes of different colors and shapes represent elements involved in different processes

In Group II, the TC-rich repeats and W box elements were simultaneously present in FvSTP21, FaSTP21a/b and FaSTP23c, while FvSTP23 and FaSTP23a/b/d had no such elements. Great variations could be observed in the types and locations of cis-elements in diploid woodland strawberry and octoploid cultivated strawberry.

Group III contained more members than Group I and II. The W-box (putative fungus-induced element) in FvSTP4 promoter was lost in FaSTP4a/b promoters. FaSTP4c/d promoters not only maintained W-box but had enriched TC-rich repeats for defense responses. In addition, two copies of TCA-element for SA response were similarly present in the promoters of FvSTP4 and FaSTP4a/b/c. The scarcity of cis-elements in FaSTP6b/c promoters was largely caused by the incomplete upstream sequence, although it’s also possible that these two loci were pseudogenes. FvSTP9 promoter contained four copies of TC-rich repeats, which were lost in most octoploid homologs, and only one copy was maintained in FaSTP9b promoter. As compared with that in FvSTP9 promoter, the TCA-element was enriched in FaSTP9a/b promoters, and the TC-rich repeats were enriched in FaSTP9a/b/c promoters. By contrast, the locations of cis-elements in FvSTP24 promoter were largely maintained in FaSTP24a/b/c promoters.

The W-box for putative fungus-induced element was observed in half members of Group IV. One or two copies of W-box were identified in the promoters of FaSTP14a/b/c and FaSTP22a/b/c/d, two to three copies were found for FaSTP8a/b and FaSTP20a/b. Notably, FvSTP18 and FaSTP18a/b/c promoters were rich in TC-rich repeats for defense response. This cis-element was also enriched in the promoters of FvSTP22 and FaSTP22a. Generally, more variations than conservation were observed in the composition and locations of cis-elements in promoters of STP genes from F. × ananassa and F. vesca.

Comparative transcriptional responses of FaSTPs to C. fructicola in strawberry crowns

To identify defense-related members in responses to crown rot caused by C. fructicola, RT-qPCR analysis was performed for FaSTP genes with cDNAs from the crown tissues of three varieties displaying varying soluble sugar changes and pathogenesis processes. Those genes displaying significant changes in transcripts levels during ACR development as compared with mock treatment were identified. A set of eight genes including FaSTP1-4, 8, 21–23 were detected in three varieties (Fig. 6).

Fig. 6.

Fig. 6

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RT-qPCR analysis of strawberry FaSTP genes upon C. fructicola invasion in the crown tissues of cultivars ‘Hogyoku’, ‘Sweet Charlie’ and ‘Jiuxiang’. The relative transcript levels of STP genes were normalized with two reference genes EF1a and GAPDH2 (Amil-Ruiz et al. 2013) and reported as the mean of six replicates ± SE from two independent experiments. Pairwise T-test analysis was conducted for mock and C.f-invasion and indicated as asterisk (*P < 0.05; **P < 0.01; ***P < 0.001) for certain gene/genotype/phase. Distinct lowercase and uppercase letters indicated significant differences (P < 0.05) among relative mRNA levels during the dynamic process for mock and C.f-invasion, respectively

FaSTP1 expression was induced at 7 dpi but inhibited at 14 dpi in ‘Sweet Charlie’. FaSTP3 was upregulated in cv. ‘Jiuxiang’ at 3 dpi and 7 dpi, and in ‘Hogyoku’ at 14 dpi. FaSTP4 was induced in ‘Sweet Charlie’ at 3 dpi and in ‘Jiuxiang’ at 7 dpi, and it was upregulated similarly in ‘Sweet Charlie’ and ‘Jiuxiang’ at 14 dpi. Three additional genes were induced during C. fructicola invasion in all three cultivars. FaSTP2 and FaSTP8 were highly induced in the crowns of three varieties at 14 dpi, but FaSTP8 was also induced in ‘Sweet Charlie’ at 7 dpi. FaSTP21 was induced upon pathogen infection in three varieties with varying dynamic patterns: in ‘Hogyoku’ being upregulated at 14 dpi, in ‘Sweet Charlie’ upregulated at 3 dpi and 7 dpi, while in ‘Jiuxiang’ induced at three phases. The rest two genes showed contrasting expression patterns in different strawberry cultivars. FaSTP22 was induced at 7 dpi and 14 dpi in ‘Hogyoku’ while suppressed in ‘Sweet Charlie’ at 3 dpi and 7 dpi. FaSTP22 was also upregulated in ‘Jiuxiang’ at 7 dpi. FaSTP23 was suppressed at 7 dpi in ‘Hogyoku’. On the contrary, this gene was uniformly upregulated in ‘Sweet Charlie’ and ‘Jiuxiang’ at 3 dpi and 14 dpi.

In sum, transcription of all eight genes was reprogrammed in strawberry crowns during C. fructicola invasion. Only three of eight were occasionally downregulated in certain cultivar at specific phase: in ‘Sweet Charlie’ FaSTP1 (14 dpi) and FaSTP22 (3 and 7 dpi) were suppressed; in ‘Hogyoku’ FaSTP23 (7 dpi) was downregulated. Indeed, all eight members were upregulated in certain phase in some varieties. There were more FaSTP members motivated at 14 dpi than other phases, when distinct sets of 4–5 members were upregulated in three varieties.

Transcriptome profiles of FaSTP genes in distinct strawberries infected with Colletotrichum spp.

To improve the understanding of FaSTPs expression, we revisited available RNA sequencing (RNA-seq) data related to strawberry defenses against Colletotrichum spp. invasion. First of all, the RNA-seq data from the previous research in our laboratory (Zhang et al. 2018) were retrieved. The results showed that in the mature leaves of strawberry ‘Jiuxiang’, a set of 9 STP genes were detected, and the relative levels of STP transcripts were ranked as follows: FvSTP4 > FvSTP8 > FvSTP2 > FvSTP22 > FvSTP23 > FvSTP3 > FvSTP1 > FvSTP15 > FvSTP21 (Fig. 7A). C. fructicola infection significantly increased the expression of nearly all except for two members at 72 hpi (3 dpi) and 96 hpi (4 dpi). Only FvSTP15 expression was inhibited upon C. fructicola infection at 72 hpi and 96 hpi. FvSTP22 was the unique member not significantly altered during pathogen infection (Fig. 7A).

Fig. 7.

Fig. 7

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RNA-seq data indicated the variations in the expression responses of STP family genes in octoploid strawberry upon Colletotrichum spp. infection. The heat map was produced with Log2-transformed RPKM (mapping readings per kilobytes) values for each transcript. A In leaves from cv. ‘Jiuxiang’ infected with C. fructicola (Zhang et al. 2018). B In leaves from cv. ‘Yanli’ and ‘Benihoppe’ with C. gloeosporioides (Wang et al. 2017). C In crowns from cv. ‘Elyana’ and ‘Festival’ infected with C. gloeosporioides (Chandra et al. 2021)

An additional work reported the comparative transcriptome in the leaves of the resistant ‘Yanli’ and the susceptible ‘Benihoppe’ at 72 h post infection with C. gloeosporioides (Wang et al. 2017). A total of seven STP genes were screened. The transcript levels of FaSTP15, − 16 and − 24 were very low and not significantly altered. In mock at 0 h, the relative transcript levels of the rest four members were ranked as the following: FaSTP22 > FaSTP10 > FaSTP9 > FaSTP8. As compared with mock treatment at 72 h, these four genes were uniformly suppressed in the resistant ‘Yanli’ while upregulated in the susceptible ‘Benihoppe’ upon C. gloeosporioides infection (Fig. 7B).

Expression of strawberry STP genes was also examined in the crowns of the resistant ‘Elyana’ and susceptible ‘Festival’ during infection with C. gloeosporioides (Chandra et al. 2021). Only three loci of FaSTP genes were identified (Fig. 7C). FaSTP3b was weakly expressed in ‘Elyana’ while not detected in ‘Festival’. FaSTP8b and FaSTP8a were consistently upregulated in two cultivars at 72 h post inoculation, but there were a 3–4.5-fold increase in the resistant ‘Elyana’ while more than tenfold increase in the susceptible ‘Festival’. These RNAseq data expanded our knowledge for the involvement of FaSTP genes in strawberry defense response to Colletotrichum spp.

Discussion

This work revealed the genotype-specific shift of soluble sugar contents upon C. fructicola infection in strawberry crowns. Based on genome-wide characterization of the STP family in octoploid strawberry, a set of FaSTP genes responsive to C. fructicola were identified in strawberry crowns.

Previous report suggested that the soluble sugars in strawberry crowns varied with temperature, with higher amounts of fructose at higher day/night temperatures, while more sucrose at cooler temperatures (Wang and Camp 2000). Sugar content and composition were also related with crown sites. A total of soluble carbohydrates over 100 mg/g dry weight was detected in the crown of ‘Camarosa’, with glucose most abundant in the upper and basal sections, and sucrose richest in middle section (Macías-Rodríguez et al. 2002). Currently, both C. fructicola inoculation and mock treatment after wounding altered the accumulation of soluble carbohydrates in strawberry crowns. Neither C. fructicola growth nor strawberry defense might be limited by soluble sugars. As compared with mock treatment, hexose/sucrose ratio kept on increasing after C. fructicola challenge, which was consistent with previous reports in tomato infected with Botrytis cinerea (Berger et al. 2004) and strawberry fruits challenged by C. nymphaeae (Weber et al. 2015). Strawberry ripened fruits accumulated higher total soluble sugars with decrease in sucrose and increase in hexoses, similarly observed in both susceptible and tolerant cultivars upon infection C. nymphaeae (Mikulic-Petkovsek et al. 2013). In most cases, whether the kind of sugar changes was beneficial for the host or pathogen is elusive and yet to be further investigated (Kanwar and Jha 2019).

Previously, a negative correlation between disease severity caused by C. hingginsianum and total leaf sugars was reported (Engelsdorf et al. 2013). In present study, the soluble sugar contents in crowns challenged by C. fructicola at 3 dpi was positively correlated with the disease severity at 14 dpi. In chickpea root, a rapid reduction in the levels of sucrose and fructose was observed after 2 days post infection with Fusarium oxysporum, which was more prominent in the susceptible genotype (Kumar et al. 2016). In this work, at 3 dpi only glucose and fructose contents decreased in cv. ‘Hogyoku’ and ‘Sweet Charlie’. Indeed, the level of sucrose was even elevated in the crowns of ‘Sweet Charlie’ and ‘Jiuxiang’ at 3 dpi in comparison with that in mock. In addition, the proportion of fructose in total soluble sugars was proposed as a major marker of tomato stem defense, negatively correlated with the severity of symptoms caused by B. cinerea (Lecompte et al. 2017). Current work did not allow similar conclusions, hinting the complexity between soluble sugar homeostasis in crown and the resistance of strawberry to C. fructicola. It remains puzzling how soluble sugar changes favor strawberry or Colletotrichum spp. Undoubtedly, the competition for sugars between plant phloem cells and fungal hyphae may manifest through very subtle changes in the distribution of sugars, rather than their total content in a whole organ such as the crown. It is worth exploring this hypothesis further in near future.

Currently, a continuous elevation of hexose/sucrose ratio was observed in the crowns of three strawberry cultivars upon C. fructicola infection. This might be caused by sucrose hydrolysis and consistent with previous reports in the infected tissues of other plants (Herbers et al. 2000; Fotopoulos et al. 2003; Hayes et al. 2010; Tauzin and Giardina 2014). Both plant and pathogen originated invertases as well as sucrose synthases might be responsible for this alteration (Cabello et al. 2014). This change in monosaccharide proportion might promote fungal colonization and influence strawberry defense responses. C. fructicola caused changes in soluble sugar accumulation was significantly affected by strawberry genotype. The hexose/sucrose ratios at 14 dpi in strawberry crowns were largely meeting with their relative susceptibility.

Transporters in the fight for sugars between pathogen and host plant at least include three types. In addition to pathogen sugar transporters, there in host exist some sugar transporters manipulated by pathogen to plunder carbohydrates and some others for defending plunderage. In Arabidopsis, all characterized STPs function as plasma membrane-localized H + /hexose symporters, and several STP members has been reported to respond to pathogen infection. AtSTP4 was rapidly induced upon powdery mildew fungi Erysiphe cichoracearum infection in mature leaves, while it was mainly expressed in sink tissues such as the roots under normal conditions (Fotopoulos et al. 2003). AtSTP1 and AtSTP4 jointly introduced mesophyll derived glucose into leaf guard cells serving as an important mediator linking stomatal movement and photosynthesis (Flütsch et al. 2020). AtSTP13 and its homolog in wheat were confirmed to contribute to plant resistance to necrotrophic pathogens while enhance susceptibility to biotrophic pathogens (Lemonnier et al. 2014; Moore et al. 2015; Yamada et al. 2016; Huai et al. 2019). Interestingly, highly homologous to AtSTP13 and harboring at least two copies of putative cis-elements responsive to fungal elicitor in its promoter, FaSTP8 mRNA was significantly upregulated upon C. fructicola infection in the crowns of three strawberry cultivars in similar patterns, except for a delay of such upregulation in the susceptible ‘Jiuxiang’. Similarly, FaSTP21 contained three copies of putative cis-elements responsive to fungal elicitor and one copy responsive to defense and stress in its promoter, whose expression was enhanced in three strawberry cultivars in most stages, with the exception of an early down-regulation at 3 dpi in ‘Hogyoku’.

Based on characterizing the dynamic chemical variations and screening FaSTP responsive genes in strawberry crowns following C. fructicola infection, our work opened a new chapter on sugar changes and FaSTP motivation in Colletotrichum spp.-strawberry interaction. And we offer two tantalizing hypotheses. Could FaSTP8 uptake hexose from the apoplasm into the cytoplasm and sequestering sugars away from the hemibiotrophic Colletotrichum spp.? Does FaSTP21 also function to disable Colletotrichum fungal ability to access host carbohydrates? It’s highly expected to determine the physiological role of these C. fructicola-responsive genes in near future. To reveal their subcellular localization, monosaccharide transport activity and substrate specificity (Wieczorke et al. 1999; Liu et al. 2018) would provide valuable information. Functional study via Crispr/Cas9 genome editing in strawberry (Zhou et al. 2018) would greatly improve our understanding of FaSTP candidates in mediating strawberry resistance to ACR. Furthermore, the fine-tuning transcriptional control of these STP candidates could be studied via novel economic DNA–protein interaction technique such as CUT&Tag (Kaya-Okur et al. 2020). Current work and future efforts would finally shed lights on sugar conundrum in strawberry-Colletotrichum spp. interactions and facilitate breeding via engineering strawberry resistance to anthracnose crown rot.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 262 KB) (262.4KB, docx)
Supplementary file2 (DOCX 705 KB) (705KB, docx)
Supplementary file3 (XLSX 17 KB) (17.4KB, xlsx)
Supplementary file4 (DOCX 39 KB) (38.8KB, docx)

Acknowledgements

The authors are grateful to Mr. Zhicheng Gao for improving Fig. 6 and Fig. 7 during revision. Thanks are due to anonymous reviewers for valuable comments which improved this manuscript.

Author contributions

S-Y. C.: investigation, data curation, visualization, writing; X.L.: data curation, visualization, qPCR; K.D.: conceptualization, funding acquisition, writing; Z-Y. L.: RNA extraction; Y.B.: software, sequence retrieval; X–Y. W.: pathogen inoculation; J. Y.: quantifying microbes in planta; X–H Z. qPCR normalization; M-L X.: sampling; Y W.: materials; Q-H.G.: conceptualization, funding acquisition, editing.

Funding

This research was funded by Shanghai Agriculture Applied Technology Development Program, China to Qing-Hua Gao (2019-02-08-00-08-F01108) and Ke Duan (Grant No. 2016-06-01-04) as well as by funds from Shanghai Academy of Agricultural Sciences (Grants No. JCYJ242201, JD2422).

Data availability

The datasets generated during the current study are available from the corresponding author kduan936@126.com on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Submitted to: “Physiology and Molecular Biology of Plants”

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Ke Duan, Email: kduan936@126.com.

Qing-Hua Gao, Email: qhgao20338@sina.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 262 KB) (262.4KB, docx)
Supplementary file2 (DOCX 705 KB) (705KB, docx)
Supplementary file3 (XLSX 17 KB) (17.4KB, xlsx)
Supplementary file4 (DOCX 39 KB) (38.8KB, docx)

Data Availability Statement

The datasets generated during the current study are available from the corresponding author kduan936@126.com on reasonable request.

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