Nematode infection triggers the de novo formation of unloading phloem that allows macromolecular trafficking of green fluorescent protein into syncytia

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.     

The Arabidopsis thaliana Sucrose Transporter Gene AtSUC4 is Expressed in Meloidogyne incognita-induced Root Galls

The plant parasitic nematode Meloidogyne incognita is as an obligate parasite entirely dependent on the plants solute supply. Therefore, the nematodes induce the formation of several giant cells which are embedded into root galls. At present only little information is available about the solute transfer mechanisms of the plants to supply the induced galls and giant cells and consequently the nematodes. In the present work we could show by phloem-loading experiments that giant cells in the roots of Arabidopsis thaliana are not symplasmically connected to the phloem elements, thus differing considerably form the comparable plant–nematode interaction of Arabidopsis and Heterodera schachtii . Consequently the gene expression of the sucrose transporter AtSUC4 ( AtSUT4 ) was studied during nematode development, and its functionality was shown using RNAi gene silencing lines.     

Females and males of root-parasitic cyst nematodes induce different symplasmic connections between their syncytial feeding cells and the phloem in Arabidopsis thaliana.

Root syncytia induced by the beet cyst nematode Heterodera schachtii were thought to be symplasmically isolated. A recent study with mobile and immobile GFP constructs expressed in transgenic Arabidopsis plants under the control of pAtSUC2 showed that only mobile GFP could be detected in syncytia and suggested the existence of plasmodesmata between syncytia and the phloem. In the present study the existence of plasmodesmata between syncytia and the phloem is proven by grafting experiments. This technique rules out the possibility that GFP accumulation in syncytia is due to GFP expression in syncytia. Mobile GFP could be followed from transgenic scions carrying a pAtSUC2-gfp fusion construct via wild-type rootstocks into nematode-induced syncytia. While GFP could be detected in all syncytia associated to female nematodes, it was never observed in syncytia of male juveniles. As no GFP-mRNA could be detected in the rootstock we postulate that GFP as protein entered syncytia of females via plasmodesmata, while the protein was excluded from syncytia of male juveniles by plasmodesmata with a lower size exclusion limit.     

Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues

The Arabidopsis AtSUC3 gene encodes a sucrose (Suc) transporter that differs in size and intron number from all other Arabidopsis Suc transport proteins. Each plant species analyzed so far possesses one transporter of this special type, and several functions have been discussed for these proteins, including the catalysis of transmembrane Suc transport, and also Suc sensing and regulation of other Suc transporters. Here, we show that the AtSUC3 protein is localized in the sieve elements of the Arabidopsis phloem and is not colocalized with the companion cell-specific AtSUC2 phloem loader. Even stronger AtSUC3 expression is observed in numerous sink cells and tissues, such as guard cells, trichomes, germinating pollen, root tips, the developing seed coat, or stipules. Moreover, AtSUC3 expression is strongly induced upon wounding of Arabidopsis tissue. The physiological role of AtSUC3 in these different cells and tissues is discussed.     

AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer

The cDNA corresponding to the open reading frame T17M13.3 from Arabidopsis chromosome II was isolated and the encoded protein was characterized as a member of a subgroup of higher plant sucrose transporters. The AtSUC3 (Arabidopsis thaliana sucrose transporter 3) open reading frame encodes a protein with 594 amino acid residues, being 81 and 82 residues longer than the previously described Arabidopsis sucrose carriers AtSUC1 and AtSUC2. About 50 of these additional amino acids are part of an extended cytoplasmic loop separating the N-terminal from the C-terminal half of the protein. For functional characterization the AtSUC3 cDNA was expressed in baker's yeast. Substrate specificities, energy dependence and K(m) values of the recombinant protein were determined. Removal of the enlarged cytoplasmic loop and expression of the truncated cDNA caused no detectable change in the kinetic properties of the protein, suggesting a transport-independent function for this cytoplasmic domain. Immunolocalization with an AtSUC3-specific antiserum identified the protein in a cell layer separating the phloem from the mesophyll and in a single, subepidermal cell layer of the carpels that is important for pod dehiscence. These localizations suggest a possible role of AtSUC3 in the funnelling of sucrose from the mesophyll towards the phloem, and possibly in pod shatter.     

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.     

Plant sucrose-H+ symporters mediate the transport of vitamin H

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.     

PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major

Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters. For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias. For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions. Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family. The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose. In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago. Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues. Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed.     

Vacuoles release sucrose via tonoplast-localised SUC4-type transporters

Arabidopsis thaliana has seven genes for functionally active sucrose transporters. Together with sucrose transporters from other dicot and monocot plants, these proteins form four separate phylogenetic groups. Group-IV includes the Arabidopsis protein SUC4 (synonym SUT4) and related proteins from monocots and dicots. These Group-IV sucrose transporters were reported to be either tonoplast- or plasma membrane-localised, and in heterologous expression systems were shown to act as sucrose/H(+) symporters. Here, we present comparative analyses of the subcellular localisation of the Arabidopsis SUC4 protein and of several other Group-IV sucrose transporters, studies on tissue specificity of the Arabidopsis SUC4 promoter, phenotypic characterisations of Atsuc4.1 mutants and AtSUC4 overexpressing (AtSUC4-OX) plants, and functional comparisons of Atsuc4.1 and AtSUC4-OX vacuoles. Our data show that SUC4-type sucrose transporters from different plant families (Brassicaceae, Cucurbitaceae and Solanaceae) localise exclusively to the tonoplast, demonstrating that vacuolar sucrose transport is a common theme of all SUC4-type proteins. AtSUC4 expression is confined to the stele of Arabidopsis roots, developing anthers and meristematic tissues in all aerial parts. Analyses of the carbohydrate content of WT and mutant seedlings revealed reduced sucrose content in AtSUC4-OX seedlings. This is in line with patch-clamp analyses of AtSUC4-OX vacuoles that characterise AtSUC4 as a sucrose/H(+) symporter directly in the tonoplast membrane.     

Substrate specificity of the Arabidopsis thaliana sucrose transporter AtSUC2

The Arabidopsis sucrose transporter AtSUC2 is expressed in the companion cells of the phloem (specialized vascular tissue) and is essential for the long distance transport of carbohydrates within the plant. A variety of glucosides are known to inhibit sucrose uptake into yeast expressing AtSUC2; however, it remains unknown whether glucosides other than sucrose could serve as transported substrates. By expression of AtSUC2 in Xenopus oocytes and two-electrode voltage clamping, we have tested the ability of AtSUC2 to transport a range of physiological and synthetic glucosides. Sucrose induced inward currents with a K0.5 of 1.44 mM at pH 5 and a membrane potential of -137 mV. Of the 24 additional sugars tested, 8 glucosides induced large inward currents allowing kinetic analysis. These glucosides were maltose, arbutin (hydroquinone-beta-D-glucoside), salicin (2-(hydroxymethyl)phenyl-beta-D-glucoside), alpha-phenylglucoside, beta-phenylglucoside, alpha-paranitrophenylglucoside, beta-paranitrophenylglucoside, and paranitrophenyl-beta-thioglucoside. In addition, turanose and alpha-methylglucoside induced small but significant inward currents indicating that they were transported by At-SUC2. The results indicate that AtSUC2 is not highly selective for alpha-over beta-glucosides and may function in transporting glucosides besides sucrose into the phloem, and the results provide insight into the structural requirements for transport by AtSUC2.     

The Arabidopsis thaliana AtSUC2 Gene is Specifically Expressed in Companion Cells

Polyclonal antisera against a fusion protein of β-galactosidase and the 20 C-terminal amino acids of the Arabidopsis thaliana sucrose carrier AtSUC2 were used to determine the cellular localization of the AtSUC2 protein. Using fluorescence-labelling on sections from different organs of Arabidopsis the AtSUC2 protein was immunolocalized exclusively in companion cells. The presented data indicate that phloem loading in Arabidopsis may be catalyzed by the AtSUC2 sucrose carrier which transports sucrose into the companion cells. No evidence for a participation of the second Arabidopsis sucrose transporter AtSUC1 has been obtained.     

AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes

Three members of the Arabidopsis sucrose transporter gene family, AtSUC6-AtSUC8 (At5g43610; At1g66570; At2g14670), share a high degree of sequence homology in their coding regions and even in their introns and in their 5'- and 3'-flanking regions. A fourth sucrose transporter gene, AtSUC9 (At5g06170), which is on the same branch of the AtSUC-phylogenetic tree, shows only slightly less sequence homology. Here we present data demonstrating that two genes from this subgroup, AtSUC6 and AtSUC7, encode aberrant proteins and seem to represent sucrose transporter pseudogenes, whereas AtSUC8 and AtSUC9 encode functional sucrose transporters. These results are based on analyses of splice patterns and polymorphic sites between these genes in different Arabidopsis ecotypes, as well as on functional analyses by cDNA expression in baker's yeast. For one of these genes, AtSUC7 (At1g66570), different, ecotype-specific splice patterns were observed in Wassilewskija (Ws), C24, Columbia wild type (Col-0) and Landsberg erecta (Ler). No incorrect splicing and no sequence polymorphism were detected in the cDNAs of AtSUC8 and AtSUC9, which encode functional sucrose transporters and are expressed in floral tissue. Finally, promoter-reporter gene plants and T-DNA insertion lines were analyzed for AtSUC8 and AtSUC9.     

Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.     

Syncytia formed by adult female Heterodera schachtii in Arabidopsis thaliana roots have a distinct cell wall molecular architecture

? Plant-parasitic cyst nematodes form a feeding site, termed a syncytium, through which the nematode obtains nutrients from the host plant to support nematode development. The structural features of cell walls of syncytial cells have yet to be elucidated. ? Monoclonal antibodies to defined glycans and a cellulose-binding module were used to determine the cell wall architectures of syncytial and surrounding cells in the roots of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii. ? Fluorescence imaging revealed that the cell walls of syncytia contain cellulose and the hemicelluloses xyloglucan and heteromannan. Heavily methyl-esterified pectic homogalacturonan and arabinan are abundant in syncytial cell walls; galactan could not be detected. This is suggestive of highly flexible syncytial cell walls. ? This work provides important information on the structural architecture of the cell walls of this novel cell type and reveals factors that enable the feeding site to perform its functional requirements to support nematode development.