Results

Sucrose is the most abundant sugar that accumulates in Gastrodia tubers

We first measured the concentration of various sugars at 7 developmental stages of G. elata tubers (Fig. S1). Symbiosis withArmillaria is typically established with tubers at stage 2. Tubers in stages 3-5 are actively growing and stage 6 tubers are mature and forming flowering buds. Sucrose was abundant in tubers at all stages, as high as 1% of the dry mass (Fig. 1). During the early stages of symbiosis (stages 1-2) and in the elongation stages (stages 3-4), the concentration of sucrose was two times higher than those of the hexoses, suggesting that sucrose may be the major form of transported carbon during the early stages of Gastrodia tuber growth.

Distinct apoplastic block for carbon allocation

To understand how sucrose allocation occurs within the developing tuber, we performed histological and ultrastructural observations on G. elata cortical cells in a stage 2a tuber where the basal part had been colonized by Armillaria hyphae (Fig. 2a). Histological observation revealed distinct differentiation in cortical cells (Fig. 2b). Inside the epidermis, there were three to four layers of cortical cells colonized by fungal hyphae (IC in Fig. 2b). In infected cortical cells, Armillaria rhizomorph formed a netting fungal matrix with heavily stained cell wall (Fig. 2b-d). The inner cortical cells, adjacent to infected cells, are enlarged and thus are called large cells. These large cells were filled with numerous starch grains and large vacuoles (Fig. 2b, c). The cell walls were
significantly thickened in large cells when compared to normal cortical cells. At the interface between the infected cells and adjacent large cells, papillary cell wall thickenings in contact with fungal hyphae were observed (white box and arrow in Fig. 2d, e). TEM confirmed the extensive ingrowths of cell wall and plasma membrane toward the cytoplasm of large cells (white arrow in Fig. 2e, f). These histological observations demonstrated distinct apoplastic interfaces located between three layers of cells - fungal cells, infected cells, and inner large cells – suggesting the involvement of membrane transport mechanisms for sucrose allocation in the mycoheterotrophic plant cells.

GeSUTs were highly expressed in G. elata tubers

To further examine the mechanism of sucrose allocation in G. elata, we searched for genes encoding potential sucrose transporters. An RNA-Seq dataset was generated from young mycorrhizal tubers (stage 2a in Fig. S1). All unigenes and contigs were annotated according to the sequence similarity search against the non-redundant protein sequence (nr) database in NCBI using BLASTX algorithm. Contigs 4177 and 5219 were found to encode SUT-like genes with higher FPKM (Fragments Per Kilobase Million) value of 22.77 and 7.83, respectively, suggesting that these two contigs were highly expressed in the stage 2 tubers. Full length sequences of three contigs, 4177-1, 4177-2 and 5219, were then cloned and sequenced. Phylogenetic analysis showed that contigs 4177-2 and 5219 were closely related to the SUT4 and SUT3 clades and thus namedGeSUT4 and GeSUT3 (Fig. S2), respectively (Doidy et al. 2012b). Further analysis revealed that contig 4177-1 had a 258 bp C-terminal deletion compared to contig 4177-2 and thus namedGeSUT4D (Fig. S3a). GeSUT4D may be an alternative splicing event of GeSUT4. GeSUT4 and GeSUT3 genes encoded proteins with 536 and 520 amino acids, with predicted molecular masses of 57.4 and 55.6 kDa, respectively (Fig. S3a). Both GeSUT4 and GeSUT3 proteins have 12 transmembrane domains, typical for SUTs, with both N- and C-termini on the cytoplasmic side (Fig. S3b). The GeSUT4 andGeSUT3 proteins shared only 38.7% amino acid identity. Interestingly, GeSUT4 shared high levels of amino acid identity with SUT4 clade proteins, such as rice OsSUT2 (Os12g0641400; 58.1%) and potato StSUT4 (AAG25623.2; 59.8%), while GeSUT3 shared 64% identity with OsSUT1 and was grouped into SUT2-IIB (Fig. S2). To examine developmental expression profiles of GeSUT genes, mRNA levels were first analyzed by real-time PCR (Fig. 3a). Two GeSUTgenes were constantly expressed in almost all stages, where expression of GeSUT4 was slightly higher than GeSUT3. To determine if there were possible splicing events, RNA expression was also analyzed by semi-quantitative PCR. Two GeSUT genes exhibited similar patterns where mRNA transcripts were abundant in the early stages of symbiosis (stages 1, 2a and 2b) and elongation stage (stage 4), but lower in mature G. elata tubers (stages 5 and 6) (Fig. 3a). These results suggest that functional GeSUTs are expressed in early developing tubers. To further examine sucrose metabolism in the Gastrodia- Armillariainterface, we searched our RNA-Seq dataset to identify genes involved in sucrose metabolism in the apoplasm, such as invertase (INV) (Roitsch & González 2004) and sucrose synthase (SUS) (Stein & Granot 2019). By blasting NCBI database, only one contig 5712 (Fig. S4), encoding a potential GeINV gene, was expressed in the developing tubers (Fig. 3c). In contrast, two contigs 4129 and 6657, encoding potentialGeSUS genes, were greatly expressed in all stages (Fig. 3c), indicating that GeSUS may be involved in sucrose allocation in the mycoheterotrophic cells.

GeSUT4 complements sucrose uptake deficiency in yeast

To investigate the transport properties of GeSUTs, cDNAs from the GeSUT4 and
GeSUT3 genes were expressed in the yeast mutant YSL2-1, which cannot grow on medium containing monosaccharides or sucrose due to the lack of functional sugar transporters (Guo et al. 2014). Under acidic growth conditions (pH 5), expression of the Arabidopsis sucrose transporter At SUC2 enables better growth on sucrose- containing medium, especially at 5% sucrose (Fig. 4a). Expression ofGe SUT4, but not of Ge SUT3, complemented the growth deficiency of YSL2-1 on sucrose-containing media to a similar degree asAt SUC2 (Fig. 4a). In contrast, growth was not observed on medium buffered at neutral pH (pH 7), even when supplemented with 5% sucrose. Neither of the Ge SUTs could restore growth of YSL2-1 on hexose-containing media while the expression of a yeast hexose transporter HXT5 complemented the growth deficiency (Fig. S5a). Cells expressing GeSUT s showed no growth inhibition on medium containing the toxic glucose analog 2-deoxyglucose (2-DG) (Fig. S5b), indicating GeSUT s had little capacity to transport hexoses. We also examined yeast cells expressing Ge SUT4D. This protein was unable to restore growth on 5% sucrose (Fig. 4a, S5), indicating that the C-terminal was necessary for Ge SUT4’s transport activity.

GeSUT4 functions as a sucrose-specific high affinity carrier

To confirm that Ge SUT proteins were functional sucrose transporters, we performed time-dependent sucrose uptake assays using [14C]sucrose into YSL2-1 cells expressing either Ge SUT4 orGe SUT3. After 5 min more [14C]sucrose was detected in yeast cells expressing GeSUT4 compared to GeSUT3 , or the empty vector controls. The uptake of sucrose into GeSUT4 harboring cells increased linearly with time until 30 min and was much greater than observed in yeast cells expressing Ge SUT3 (Fig. 4b). Consequently,
we focused on determining the detailed transport kinetics ofGeSUT4. Uptake of [14C]sucrose was observed from as low as 0.01 mM reaching saturation close to 10 mM (Fig. 4c). The deduced KM and Vmax values of GeSUT4 catalyzed sucrose transport were 2.5 mM and 0.7 nmol 10*7 cells-1 h-1, respectively (Fig. 4c).
Competition uptake assays using [14C]sucrose and a 10-fold excess of unlabeled sugars were performed to examine transport specificity ofGe SUT4. Consistent with the growth assays (Fig. 4a, S2), only unlabeled sucrose, but none of the hexoses, was able to reduce the import of [14C]sucrose (Fig. 4d). The observation that a 10-fold excess of maltose impacts [14C]sucrose uptake (Fig. 4d) might be due to a background maltose activity of the strain (Fig. S6) (Chen et al. 2015)
As most SUT proteins function as active, proton-driven sugar carriers (Kühn & Grof 2010), we further examined the effects of various pH conditions or the protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) on sucrose transport activity. Similar to the well characterized proton-coupled sucrose symporter At SUC2 (Sauer & Stolz 1994), sucrose uptake activity of Ge SUT1 was decreased to less than 10% at neutral or alkaline pH conditions, or in the presence of CCCP (Fig. 4e). These results support the assumption that Ge SUT4 functions as a sucrose-specific H+-symporter.

GeSUT4 functions on the plasma membrane and tonoplast

The subcellular localization of Ge SUT4-GFP was examined in Arabidopsis mesophyll protoplasts. When co-transformed with a plasma membrane marker At PIP2-RFP (Nelson et al. 2007), green fluorescence derived from Ge SUT4-GFP appeared in a plasma-membrane ring on the outer side of chloroplasts (shown in red auto- fluorescence, indicated “c” in Fig. 5a). The co-localization ofGe SUT4-GFP signals on the plasma membrane was further confirmed by overlapping with red fluorescence
derived from At PIP2-RFP (Fig. 5a). In some cases, we also observed expression of Ge SUT4-GFP fusions on the inner side of chloroplasts (arrowhead in Fig. 5a). To examine if co-expression of two fusion proteins may result in mis-targeting of Ge SUT4-GFP, cells expressing only Ge SUT4-GFP were stained with the plasma membrane stain, FM4-64. Consistently, co-localization of Ge SUT4-GFP signals on the plasma membrane (asterisks) and inner membrane (arrowhead), likely the tonoplast, was observed (Fig. S7a). To confirm the tonoplast membrane localization, protoplasts were co-transformed with Ge SUT4-GFP and a tonoplast marker, At γTIP-RFP (Jauhet al. 1999). Yellow signals derived from the overlapping expression of both fusion proteins were clearly observed (arrowhead in Fig. 5b). In a lysed protoplast where the tonoplast membrane can separate from the plasma membrane, yellow fluorescence derived from co-localization of Ge SUT4-GFP and At γTIP-RFP fusions was observed (arrowhead in Fig. S7b). These observations demonstrated dual targeting of Ge SUT4 to both plasma membrane and the tonoplast.

GeSUT4 expressed in symbiotic cells and large cells.

To determine the cell-specific localization of GeSUT4 RNA expression, we performed in situ hybridization using a gene-specific probe. A low level of GeSUT4 RNA was detected in most cells, but extremely strong signals (brown color) were detected in cells surrounded by netting hyphae in Armillaria inoculated cells (Fig. 5C). Distinct signals were also detected in nuclei of large cells adjacent to infected cells.

Ectopic expression of GeSUT4 promotes leaf growth

Currently it is not possible to produce transgenic Gastrodiaorchid and this limits the experimental approaches available to understand the function of GeSUT4 . An alternative is to examine the effect of ectopic expression of GeSUT4 in transgenic Arabidopsis, as has been done with orchid genes (He et al. 2019; Hsu, Huang, Chou &
Yang 2003) and other sugar transporters from non-model plants(Maet al. 2016; Wang et al. 2018). Arabidopsis plants expressing high levels of GeSUT4 RNA were generated (Fig. 6a). IfGe SUT4 is an active, sucrose-specific importer at the plasma membrane (Fig. 4, 5), accumulation of sucrose in the cytosol due toGe SUT4 activity may result in sucrose toxicity inhibiting root growth (Lu et al. 2014). As expected, while no consistent growth differences were observed under conditions without sucrose, root growth of GeSUT4 overexpressor lines (OE-12, 13, 19) was significantly reduced compared to Arabidopsis transformed with the empty vector (vector, Fig. 6b). In contrast, when transgenic seedlings were transferred to soil for long-term growth, total leaf area of overexpressor plants was 140% to 190% higher than corresponding controls (Fig. 6c, S8). However, no consistent differences in sugar contents in leaves and roots were measured (data not shown).

GeSUT4 activity limits root colonization with Bacillus subtilis

To examine whether Ge SUT4 uptake activity could play a role in plant-microbe interactions, we examined the ability of B. subtilis , a plant growth-promoting rhizobacterium, to colonize roots of transgenic Arabidopsis seedlings expressing GeSUT4(Allard-Massicotte et al. 2016). In 9-d-old seedlings, overexpression of GeSUT4 (OE-12, 13) significantly decreased colonization efficiency in roots compared to control plants (Col, vector) (Fig. 6d), even when root lengths of all seedlings were similar (Fig. S9).