Nematode Infection Triggers the de Novo Formation of Unloading Phloem That Allows Macromolecular Trafficking of Green Fluorescent Protein into Syncytia

Abstract

Syncytial feeding complexes induced by the cyst nematode Heterodera schachtii represent strong metabolic sinks for photoassimilates. These newly formed structures were described to be symplastically isolated from the surrounding root tissue and their mechanism of carbohydrate import has repeatedly been under investigation. Here, we present analyses of the symplastic connectivity between the root phloem and these syncytia in nematode-infected Arabidopsis (Arabidopsis thaliana) plants expressing the gene of the green fluorescent protein (GFP) or of different GFP fusions under the control of the companion cell (CC)-specific AtSUC2 promoter. In the same plants, phloem differentiation during syncytium formation was monitored using cell-specific antibodies for CCs or sieve elements (SEs). Our results demonstrate that free, CC-derived GFP moved freely from the phloem into the syncytial domain. No or only marginal cell-to-cell passage of GFP was observed into other root cells adjacent to these syncytia. In contrast, membrane-anchored GFP variants as well as soluble GFP fusions with increased molecular masses were restricted to the SE-CC complex. The presented data also show that nematode infection triggers the de novo formation of phloem containing an approximately 3-fold excess of SEs over CCs. This newly formed phloem exhibits typical properties of unloading phloem previously described in other sink tissues. Our results reveal the existence of a symplastic pathway between phloem CCs and nematode-induced syncytia. The plasmodesmata responsible for this symplastic connectivity allow the cell-to-cell movement of macromolecules up to 30 kD and are likely to represent the major or exclusive path for the supply of assimilates from the phloem into the syncytial complex.

Starch Serves as Carbohydrate Storage in Nematode-Induced Syncytia

“…In fact, a massive transfer of sugars from the host plant's phloem into syncytia occurs, which leads to the necessary high levels of sugars (Hofmann et al, 2007). Sugar import has been shown to be performed via transporters at the beginning of syncytium formation and via plasmodesmata at later stages (starting around 10 dai; Juergensen et al, 2003;Hoth et al, 2005;Grundler, 2006, 2007a;Hofmann et al, 2007). It could be anticipated that continuous sugar demand induces an equivalent transport, thus establishing physiological equilibrium.…”

Differential vascularization of nematode-induced feeding sites

“…The formation of unloading phloem around syncytia during the infestation with the CN H. schachtii has been described (11). In contrast to the situation in RKs, the phloem surrounding syncytia consists of CCs and SEs.…”

“…It was postulated that sucrose transporters are responsible for this accumulation (9,10). However, despite of the presence of transcripts for these transporters in syncytia, the proteins were not detected (11). On the contrary, it could be demonstrated that soluble green fluorescent protein (sGFP) synthesized in companion cells (CCs) of P AtSUC2 :sGFP plants (12) entered the syncytia induced by females of H. schachtii in roots of these plants.…”

“…This suggested that, despite the predicted low number of non-functional PD, symplastic transport is fundamental for movement of nutrients into syncytia. Furthermore, the induction of CCs and sieve elements (SEs) around syncytia suggested effective nutrient supply by this symplastic pathway (11).…”

“…In case of RKNs, we compared the distribution of sGFP to the localization of membrane-anchored GFP (tmGFP9). These ORFs were expressed under the control of the CC-specific AtSUC2 promoter (11,12,16). tmGFP9 fluorescence, an indicator of CC identity and thus for the sites of sGFP synthesis, was found exclusively in the vasculature outside the root-knot (RK), but absent from cells surrounding the GCs.…”