GeSUT4 is a sucrose-specific active importer in G. elata

We also identified genes encoding sucrose transporters, Ge SUT4 andGe SUT3, that are expressed in mycoheterotrophic Gastradiacells (Fig. 3a, b). The high expression of GeSUT genes in young and elongating tubers (stages 2A-2B and 4) coincides with high levels of sucrose, but not of glucose, in developing tubers (Fig. 1). As been observed in most plant-fungal interfaces (Doidy et al. 2012a; Garcia et al. 2016; Hennion et al. 2019), distinct expression of GeSUT genes occurred in the absence of any symplastic connections in infected cortical cells and large cells (Fig. 2 and Fig. 5c). The sucrose- specific transport activity ofGe SUT4 was demonstrated by complementation and uptake kinetics in the yeast mutant (Fig. 4). The sensitivity of Ge SUT4-mediated
sucrose import to alkaline pH and the protonophore CCCP confirmed that this transporter is a proton-dependent transporter (Fig. 4e), as shown for most characterized SUT4 type carriers (Kühn & Grof 2010; Reinders, Sivitz & Ward 2012; Schulz et al. 2011; Weise et al.2000). Considering the high amino acid identity (up to 58 %) to theOs SUT2 symporter (Eom et al. 2011), it is possible thatGe SUT4 functions as a proton-symporter. The presence of an active uptake mechanism like Ge SUT4 can also be supported by expression of various P-type ATPases in the G. elata genome (Yuan et al. 2018). The absence of Ge SUT3 transport activity in yeast may be due to inappropriate expression in a heterologous system andGe SUT3 may still be involved in sucrose homeostasis in vivo .
GeSUT4 exhibits a high substrate affinity (KM ~2.5 mM) for sucrose (Fig. 4c), similar to its close homolog in rice, OsSUT2 (KM ~1.8mM) (Eom et al. 2011). The feature nicely fits with the low sucrose contents measured in the plant-microbe interfaces (Naher, Radziah, Halimi, Shamsuddin & Razi 2008). Sucrose transporters characterized in some pathogenic fungal cells also exhibit high affinity to sucrose (Sutton, Henry & Hall 1999; Wahl, Wippel, Goos, Kamper & Sauer 2010). The transport properties of GeSUT4 would enable strong competition for carbon nutrients from the symbiotic fungal cells and could provide a strategy to avoid rapid metabolism of sucrose into hexoses during carbon allocation.