Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Authors


(fax +1 607 254 6779; e-mail mjh78@cornell.edu).

Summary

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N-terminal VAMP-associated protein (VAP)/major sperm protein (MSP) domain and a C-terminal ankyrin-repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non-plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.

Introduction

Plants have evolved various strategies to enhance their access to essential mineral nutrients, including the development of symbioses with microorganisms. One of the most widespread examples is arbuscular mycorrhizal (AM) symbiosis, formed between vascular plants and fungi of the phylum Glomeromycota. In this association, the AM fungi contribute to plant phosphate and nitrogen nutrition (Smith et al., 2003; Govindarajulu et al., 2005; Javot et al., 2007b) by delivering these mineral nutrients directly to the root cortex. In exchange, they receive carbon from their plant partners (Bago et al., 2000). This association, which arose over 400 million years ago (Remy et al., 1994; Bonfante and Genre, 2008), is estimated to occur in over 80% of the angiosperm species, and exists in terrestrial ecosystems throughout the world.

The development of AM symbiosis has been clearly described, including the existence and, more recently, the identity of some of the early signaling molecules involved. In response to strigolactones present in plant exudates, AM fungal hyphae branch, and their metabolism is activated (Akiyama et al., 2005; Besserer et al., 2006). In turn, AM fungal exudates contain an unidentified ‘myc factor’ that induces gene expression and calcium spiking in plant roots (Kosuta et al., 2003, 2008; Navazio et al., 2007). Upon contact with a root, AM fungal hyphae develop hyphopodia, and subsequently penetrate and pass through the epidermal cells guided by a preformed cellular apparatus, named the prepenetration apparatus (Genre et al., 2005). Within the cortex, hyphae grow both intra- and intercellularly, and finally enter the cortical cells, where they branch to form arbuscules, for which the symbiosis is named. Arbuscule development is accompanied by the rearrangement of the plant cortical cells, including the repositioning of organelles and the cytoskeleton, nuclear enlargement and deposition of the periarbuscular membrane (Gianinazzi-Pearson, 1996; Bonfante and Genre, 2008). The periarbuscular membrane is continuous with the plasma membrane, but is functionally distinct, and in the case of phosphate, is the site of nutrient exchange between symbionts (Javot et al., 2007a).

Plant mutants impaired at different stages of symbiotic development have been identified. Mutants blocked at the earliest phases include the tomato premycorrhizal infection 1 (pmi1) and pmi2, which suppress fungal growth and hyphopodium formation (David-Schwartz et al., 2003; Gadkar et al., 2003), and maize no perception 1 (nope1), which displays a major reduction in the formation of hyphopodia and intraradical colonization (Paszkowski et al., 2006). The most detailed genetic analyses exist for a class of mutants with a block in epidermal penetration. These mutants were identified initially in legumes, and they are also unable to form root nodule symbiosis with Rhizobia (Parniske, 2008). In Medicago truncatula and Lotus japonicus, the genes underlying eight of these mutants have been cloned, including the receptor-like kinase DMI2 (Endre et al., 2002)/SYMRK (Stracke et al., 2002), cation channels CASTOR (Imaizumi-Anraku et al., 2005) and DMI1 (Ane et al., 2004)/POLLUX (Imaizumi-Anraku et al., 2005), nucleoporins NUP85 (Saito et al., 2007) and NUP133 (Kanamori et al., 2006), the calcium- and calmodulin-dependent kinase DMI3 (Levy et al., 2004; Mitra et al., 2004)/CCaMK (Tirichine et al., 2006) and CYCLOPS (Yano et al., 2008). Mutations in these genes impair fungal passage through the epidermis and entry into the cortex, resulting in swollen hyphopodia on the root surface. Loss-of-function cyclops mutants of L. japonicus and rice (Oryza sativa) also permit a low level of cortical colonization by AM fungi, but fail to develop arbuscules (Kistner et al., 2005; Gutjahr et al., 2008; Yano et al., 2008). A Petunia (Petunia hybrida) mutant, penetration and arbuscule morphogenesis 1 (pam1), exhibits a similar phenotype, with impaired epidermal penetration, a high frequency of aborted and swollen hyphopodia and no formation of arbuscules (Reddy et al., 2007). Additional mutants impaired in cortical development include the maize (Zea mays) mutant taciturn1 (Paszkowski et al., 2006), the pea (Pisum sativum) mutant myc−2, which fails to develop mature arbuscules (Gianinazzi-Pearson et al., 1991; Gianinazzi-Pearson, 1996), and the tomato (Solanum lycopersicum)rmc mutant, in which the phenotype varies slightly with the species of AM fungus (Barker et al., 1998; Gao et al., 2001; Poulsen et al., 2005). Additionally, RNA silencing has been used to knock-down a calcium-dependent protein kinase (Ivashuta et al., 2005) and two plant subtilases (Takeda et al., 2009), both of which lead to a reduction in cortical colonization and arbuscule formation. Finally, mutating phosphate transporters MtPT4 from M. truncatula (Javot et al., 2007a) and LjPT3 from L. japonicus (Maeda et al., 2006) caused premature arbuscule degeneration and loss of functional symbiosis.

Although some details of the pathway and molecular events regulating epidermal penetration by AM fungi have been established (Parniske, 2008), relatively little is known about the genetic and cellular mechanisms that control arbuscule development within cortical cells. Transcriptome studies have identified many hundreds of M. truncatula genes induced during symbiosis (Liu et al., 2003; Guimil et al., 2005; Hohnjec et al., 2005; Gomez et al., 2009; Guether et al., 2009), some of which have specific expression patterns in the cortex coincident with fungal colonization, suggesting that the cortical cells undergo significant transcriptional reprogramming during symbiosis. The transcript profiling and spatial expression data therefore provide a useful guide for the selection of candidate genes that may play a role in arbuscule formation. Here, we report that silencing an AM symbiosis-induced gene, called Vapyrin, impairs epidermal penetration by AM fungi, and abolishes arbuscule formation. Based on expression and localization studies, and the presence of known protein domains, we hypothesize that Vapyrin plays a cellular role to enable AM fungal development within plant cells.

Results

Vapyrin knock-down impairs passage across the epidermis by AM fungi and abolishes arbuscule formation

To identify genes that are required for AM symbiosis, we introduced RNAi constructs targeting individual candidate genes into M. truncatula via Agrobacterium rhizogenes-mediated transformation (Boisson-Dernier et al., 2001). The resulting composite plants, which have transgenic root systems and non-transgenic shoots, were then evaluated for their ability to form an AM symbiosis (Ivashuta et al., 2005). Increased expression in mycorrhizal roots was one criterion used for the selection of candidate genes, and included in this category was a candidate gene represented by TC104091 that was induced threefold in roots colonized by AM fungi (Gomez et al., 2009). Subsequently, we named this gene Vapyrin. M. truncatula composite plants with transgenic roots expressing an RNAi construct targeting the 5′ region of TC104091, referred to as Vapyrin RNAi-1, were compared with those expressing a control RNAi construct targeting the UidA gene, referred to as GUS RNAi.

Medicago truncatula GUS RNAi roots colonized with Glomus versiforme showed a phenotype typical for this combination of plant and fungus (Harrison and Dixon, 1993). Glomus versiforme formed hyphopodia on the root surface, from which hyphae penetrated through the epidermis and grew intercellularly through the cortex, forming arbuscules in the inner cortical cells (Figure 1a,d). In contrast, in Vapyrin RNAi-1 roots a significantly higher proportion of the hyphopodia were arrested in the epidermis, indicating a partial block in root penetration (Figure 1b,f). In these arrested infections, the hyphopodia became swollen with multiple aborted projections into the root epidermis. In the infection events where hyphae successfully penetrated the epidermis, the fungus developed intercellular hyphae and grew through the cortex, but failed to form arbuscules (Figure 1c,e). In some infections, intercellular hyphal development in the cortex included many cross-connections between the individual runner hyphae, which resulted in a lattice-like appearance (Figure 1c). Short, hyphal projections, possibly attempts to penetrate cortical cells, were also apparent (Figure 1e). This aberrant mycorrhizal phenotype was observed in more than 45 transgenic plants from four independent experiments, and was also observed in Vapyrin RNAi-1 roots colonized by a different species of AM fungus, Gigaspora gigantea (Figure S1). For one of these experiments with Glomus versiforme, the cortical phenotype was quantified by scoring the presence or absence of arbuscules in infections that had reached the root cortex. The control GUS RNAi roots had arbuscules in 82% (±8% SD; n = 5) of cortical infections, whereas Vapyrin RNAi-1 roots had arbuscules in 5% (±5% SD; n = 6) (Figures 1g and S2). The A. rhizogenes transformation produces composite root systems that can include non-transgenic roots, and the few arbuscules observed in Vapyrin RNAi-1 roots probably occurred in roots not expressing the RNAi construct. Vapyrin transcript levels were assayed in RNA from these Vapyrin RNAi-1 and GUS RNAi roots by quantitative RT-PCR (qRT-PCR). Vapyrin RNAi-1 roots showed a significant (20-fold) reduction in Vapyrin transcript levels relative to the GUS RNAi control roots (Figure 2a). In addition, Vapyrin RNAi-1 roots showed a significantly reduced expression of the phosphate transporter MtPT4, which serves as an indicator of arbuscule levels (Harrison et al., 2002). Glomus versiforme α-tubulin transcripts confirmed the presence of the AM fungus in all samples (Figure 2a).

Figure 1.

 Reduced expression of Vapyrin impairs arbuscular mycorrhizal (AM) symbiosis.
Confocal microscope images of Glomus versiforme in Medicago truncatula roots expressing GUS RNAi (a, d) and Vapyrin RNAi-1 constructs (b, c, e) colonized with Glomus versiforme. Glomus versiforme stained with wheatgerm agglutinin (WGA) conjugated to Alexa Fluor 488 is shown in green. The propidium iodide (PI) overlay (a–d) stains plant cell walls red. The bright-field overlay (e) shows the outline of cortical cells. A control GUS RNAi root shows a typical mycorrhizal phenotype, with a hyphopodium penetrating the root and hyphae growing through the cortex to form arbuscules (a, d). In Vapyrin RNAi-1 roots, Glomus versiforme shows a high instance of aborted hyphopodia, and hyphae that successfully penetrate the root do not form arbuscules (b and c). Hyphal branches (arrowhead) arising from the main runner hyphae do not enter cortical cells (e). Scale bars: a–c, 50 μm; d, e, 20 μm. Key: A, arbuscule; C, cortical cell; H, hyphopodia. Roots were harvested at 4 weeks post inoculation.
(f) Total number of hyphopodia that fail to penetrate control (GUS RNAi) and Vapyrin RNAi-1 root systems.
(g) Percentage of AM infections that produce arbuscules. (f, g) Control, n = 5; Vapyrin RNAi-1, n = 6. Error bars depict the standard deviation; < 0.001 Student’s t-test.
Results shown in (f, g) are representative of four independent experiments on more than 45 Vapyrin RNAi-1 transgenic plants.

Figure 2.

 Reduction of Vapyrin transcript levels by two independent RNAi constructs: Vapyrin RNAi-1 and RNAi-2.
(a) Transcript levels in GUS RNAi control roots (black bars, n = 4) and Vapyrin RNAi-1 (white bars, n = 6), assayed by quantitative RT-PCR. RNA was extracted from samples of whole root systems used for the phenotype quantification shown in Figure 1.
(b) Transcript levels in GUS RNAi control roots (black bars, n = 2) and Vapyrin RNAi-2 (gray bars, n = 3), assayed by qRT-PCR. Single transgenic roots were excised and used for RNA extraction and phenotyping. The Vapyrin RNAi-2 roots included here showed aborted hyphopodia and infections that failed to produce arbuscules, similar to RNAi-1, whereas GUS RNAi controls showed wild-type infections.
The values represent the transcript levels of Vapyrin, MtPT4, Glomus versiforme α-tubulin and Vapyrin-like (MtVpyl) relative to the constitutively-expressed Medicago truncatula elongation factor 1-α (EΔCt), with the order of magnitude indicated above the y-axis. Vapyrin is downregulated 20-fold in RNAi-1 roots, and 60-fold in RNAi-2 roots. Error bars indicate the standard deviation; *< 0.05; ***< 0.0005.

To further confirm that down-regulation of Vapyrin gene expression results in this aberrant mycorrhizal phenotype, a second Vapyrin RNAi construct targeting the 3′ region of the gene was created. Following colonization with Glomus versiforme, transgenic roots expressing Vapyrin RNAi-2 showed the same aberrant mycorrhizal phenotype observed in the initial Vapyrin RNAi-1 roots, including failed epidermal penetration attempts and cortical infections lacking arbuscules. In this experiment Vapyrin RNAi-2 and GUS RNAi transgenic roots arising from single transformation events were sampled, and in each case part of the root was stained to visualize the fungus, whereas the remainder was used for transcript analysis by qRT-PCR. Vapyrin transcript levels were reduced significantly (60-fold) in the single Vapyrin RNAi-2 roots relative to the GUS RNAi roots (Figure 2b). Vapyrin RNAi-2 single roots showed many aborted hyphopodia and no arbuscules, whereas GUS RNAi control roots showed wild-type infections with arbuscules. Consistent with this, MtPT4 transcripts were undetectable in the Vapyrin RNAi-2 roots, but were present in the GUS RNAi controls (Figure 2b). Glomus versiforme α-tubulin transcripts were detected in all samples, confirming the presence of the AM fungus (Figure 2b), but levels were lower in the Vapyrin RNAi-2 roots, where the fungus did not proliferate extensively. To test whether the Vapyrin RNAi constructs might target other genes, we monitored the expression of the gene with highest sequence similarity to Vapyrin: TC111041. This gene was named MtVpyl and is the gene most like to be cross-silenced by the RNAi constructs. MtVpyl transcript levels were slightly lower in Vapyrin RNAi-1 roots (<1.5-fold lower relative to GUS RNAi control), but did not change in the Vapyrin RNAi-2 roots relative to the GUS RNAi controls (Figure 2). Thus, the aberrant mycorrhizal phenotype observed in the Vapyrin RNAi roots cannot be attributed to MtVpyl. Overall, the data from two independent Vapryin RNAi constructs indicate that downregulation of Vapyrin expression impairs the development of AM symbiosis in M. truncatula, and in particular prevents arbuscule formation.

Vapyrin is a member of a plant-specific gene family

The expressed sequence tag (EST) contig TC104091 contained 197 nucleotides of 5′ untranslated region (UTR) sequence and a predicted open reading frame, but was not full length, and lacked the 3′ end of the gene. To obtain the missing region, 3′ RACE was performed on a cDNA library derived from M. truncatula/Glomus versiforme mycorrhizal roots. In addition, a 1761-bp DNA fragment, representing the region 5′ proximal to the predicted first ATG codon, was amplified from M. truncatula genomic DNA. The whole gene was then reconstructed and sequenced. The full-length Vapyrin gene has no introns and is predicted to encode a cytosolic protein of 541 amino acids, which contains two previously-described protein motifs (Figure 3). The N-terminal portion of the protein shares high similarity with the N-terminal end of VAMP-associated protein (VAP), which is highly conserved in plants, animals and fungi. This region is also similar to the nematode major sperm protein (MSP), an oligomeric protein that regulates the movement of nematode sperm cells (Tarr and Scott, 2005). The N-terminal domain of VAP is a protein interaction domain capable of interaction with a range of other proteins, whereas the C-terminal region of VAP includes a transmembrane domain, via which the VAPs are localized in endomembranes (Lev et al., 2008; Saravanan et al., 2009). In contrast, the C-terminal portion of Vapyrin does not contain a transmembrane domain, but instead contains eight ankyrin repeats, a widely-distributed protein domain with a helix-turn-helix structure that mediates protein–protein interactions (Rubtsov and Lopina, 2000; Li et al., 2006).

Figure 3.

 Vapyrin gene structure.
(a) The Vapyrin gene does not contain introns and is predicted to encode a protein of 541 amino acids. The N-terminal region contains a VAMP-associated protein (VAP)/major sperm protein (MSP) domain, and the C-terminal region contains eight ankyrin repeats. Domains were predicted by SMART protein motif prediction software, and the highlighted motifs are shown to scale.
(b) Amino acid sequence with the VAP domain underlined and the ankyrin repeats highlighted in gray.

BLAST searches enabled the identification of putative Vapyrin orthologs from monocot and dicot plant species, and the lycopod Selaginella moellendorffii; however, no putative ortholog was found in the non-mycorrhizal plant Arabidopsis (Figures S3 and 4). All of the putative Vapyrin genes are predicted to encode proteins with an N-terminal VAP/MSP domain and a C-terminal domain with eight or nine ankyrin repeats. The proteins share high sequence similarity, with approximately 70% identity between Vapyrin and other putative dicot orthologs, and approximately 40% with rice. Within the M. truncatula genome the gene with the highest sequence similarity to Vapyrin is TC111041, which we subsequently named MtVpyl (Vapyrin-like). MtVpyl also encodes a protein with a VAP domain and an ankyrin repeat domain. MtVpyl homologs were identified from dicots and they form a sister group to Vapyrin. Again, Arabidopsis does not contain an MtVpyl homolog, and additionally, no MtVpyl homologs were identified in monocot species. As expected, both M. truncatula and Arabidopsis have homologs of VAP and MSP, and these genes form independent sister groups to the Vapyrin group. Genes encoding proteins with the VAP and ankyrin domain combination were not present in the genomes of any non-plant species. Together, the functional data and the phylogenetic distribution of putative Vapyrin orthologs suggest that this novel combination of VAP and ankyrin domains was a plant-specific evolution that arose for AM symbiosis.

Figure 4.

 Phylogram of Vapyrin and related gene families.
The phylogram was constructed by a neighbor-joining ClustalX alignment of full-length amino acid sequences of Medicago truncatula proteins Vapyrin, MtVpyl and representative major sperm protein (MSP) and VAMP-associated protein (VAP) homologs. Included are putative Vapyrin orthologs from the plants Populus trichocarpa (Pt), Vitis vinifera (Vv), Glycine max (Gm), Solanum lycopersicum (Sl), Ricinus communis (Rc), Oryza sativa (Os), Sorghum bicolor (Sb), Zea mays (Zm) and the lycopod Selaginella moellendorffii (Sm), compared with VAP orthologs in Arabidopsis thaliana (At), Human, yeast, zebra fish, fruit fly, nematode and yeast (Scs2), and nematode MSP. Accession numbers are listed in Table S1. Distances calculated by neighbor joining and bootstraps reflect values from 100 trials. The phylogram was constructed in TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Scale bar: 0.1 substitutions per site.

Vapyrin expression is induced coincidently with the AM fungal colonization of root cells

Our previous GeneChip experiment had indicated that Vapyrin was induced in mycorrhizal roots (Gomez et al., 2009). In addition, following identification of the complete Vapyrin gene, it was apparent that the GeneChip contains a second distinct probe set (TC112496) that corresponds to the 3′ end of the Vapyrin gene. This probe set also indicated a threefold induction of Vapryin in roots colonized by Glomus intraradices (Gomez et al., 2009). To extend the GeneChip analysis, Vapyrin transcript levels were assessed by qRT-PCR in M. truncatula roots during symbiosis with two different AM fungi: Glomus versiforme and Gigaspora gigantea (Figure S4). This analysis revealed that Vapyrin transcripts are present at low levels in roots and increase fivefold during interaction with the two AM fungal species. The Medicago Gene Atlas (http://bioinfo.noble.org/gene-atlas; Benedito et al., 2008) reports that Vapyrin has a basal level of expression in roots, but no expression in aerial tissues. Interestingly, gene atlas data show that Vapyrin transcripts increase in tissue from root nodules that arise from symbiotic associations with nitrogen-fixing Rhizobia bacteria. Furthermore, some Vapyrin ESTs are derived from root nodule libraries. MtVpyl was expressed in roots, but did not show consistent differential expression during AM symbiosis, as assessed by qRT-PCR analysis (Figure S4) or in the previous GeneChip experiment (Gomez et al., 2009).

To determine the temporal and spatial expression pattern of Vapyrin, 1761 bp of genomic sequence upstream of the ATG start codon was fused to the UidA reporter and transformed into M. truncatula roots using A. rhizogenes. In plants grown without AM fungi, expression of proVapyrin:UidA was observed primarily in vascular tissue and root caps (Figure 5a–c). After colonization by Glomus versiforme, GUS staining was observed in the epidermis and outer cortical cells below penetrating hyphopodia (Figure 5d–f), and then in the cortex in cells with arbuscules, and in cells adjacent to those with arbuscules (Figure 5g–i). After fungal development in the inner cortex, GUS expression was no longer visible in the epidermis below hyphopodia, suggesting that this expression is transient and attenuated after cortical colonization. This expression pattern is consistent with the Vapyrin RNAi phenotype, in which fungal development during penetration and arbuscule formation is impaired.

Figure 5.

 Vapyrin expression in Medicago truncatula roots.
Vapyrin promoter activity revealed by histochemical staining for GUS activity in transgenic roots expressing the 1761 bp of upstream sequence fused to the UidA reporter (a–d, g). proVapyrin:UidA is expressed in the stele and root cap (a–c). Upon colonization by Glomus versiforme, GUS activity is present in epidermal and outer cortical cells beneath fungal hyphopodia, H, (d–f), and in the cortex during arbuscule formation (g–i), both in cells with arbuscules (*) and in cortical cells adjacent to those with arbuscules (arrows). Expression coincident with hyphopodia is transient, and turns off after cortical colonization (arrowhead under H in g–i). (e, h) Fungal structures are detected by wheatgerm agglutinin (WGA)-Alexa Fluor 488; (f, i) overlay of GUS and WGA. Scale bar: 100 μm.

Vapyrin protein localizes in cytoplasm and distinct, mobile complexes

To study the subcellular localization of Vapyrin during AM symbiosis, we constructed translational fusions between GFP and the 5′ and 3′ ends of the full Vapyrin coding sequence. The constructs were expressed from the native Vapyrin promoter, and were named proVapyrin:Vapyrin-GFP and proVapyrin:GFP-Vapyrin for the C- and N-terminal GFP fusions, respectively. The constructs were introduced into M. truncatula roots through A. rhizogenes transformation, inoculated with Glomus versiforme and, following the development of AM symbiosis, the roots were examined by confocal microscopy.

Similar to the transcriptional reporter fusions, expression of the C-terminal GFP fusion was observed in the epidermis and outer cortex underneath the hyphopodia (Figure 6a–d). Vapyrin-GFP signal was present in cytoplasmic strands, including cytoplasmic accumulations below hyphopodia, and in some cells GFP was detected in the nucleus. Following the entry of the hypha into an epidermal cell, in addition to cytoplasmic and nuclear locations, Vapyrin-GFP accumulated in very small puncta (Figure 6c,d). After colonization of the root cortex, both Vapyrin-GFP and GFP-Vapyrin were observed in the cytoplasm of cells with arbuscules, and in adjacent cortical cells (Figure 6e–j). In cells with arbuscules the GFP signal was present in the cytoplasm, and was also concentrated in small puncta similar to those observed in the epidermal cells. These discrete, punctate accumulations occurred only in cells that contained arbuscules, and were not observed in adjacent cortical cells. Free GFP does not show any punctate accumulation in colonized cells (Figure S5), indicating that the Vapyrin GFP patterns reflect an inherent property of the Vapyrin protein. The N-terminal fusion displayed a weaker GFP signal than the C-terminal fusion, and we did not observe its expression in epidermal cells during hyphopodia formation, probably for that reason. Both N- and C-terminal Vapyrin fusions showed similar localization patterns in the cortical cells, but in some cells, the C-terminal fusion showed a GFP signal in the nucleus, whereas the N-terminal fusion was visible only in the cytoplasm, and not in the nucleus (Figure S6d,e). Interestingly, the nuclear signal was observed only in cells harboring fungal arbuscules or hyphae, but not in adjacent cells (Figures 6a,g and S6b). This could be the result of a post-translational modification, such as cleavage, that occurs specifically in cells accommodating fungal hyphae. For either construct, we were unable to detect GFP in the vascular tissue or root caps, regardless of AM colonization, suggesting the Vapyrin protein may be present at low levels, or possibly unstable in these cells.

Figure 6.

 Localization of Vapyrin tagged with GFP.
GFP signal from proVapyrin:Vapyrin-GFP (a, c, e, g) and proVapyrin:GFP-Vapyrin (i), and corresponding Nomarski (differential interference contrast, DIC) bright-field images (b, d, f, h, j).
(a, b) Vapyrin-GFP localizes in a cytoplasmic accumulation beneath a hyphopodium (arrow) and in the nuclei of two epidermal cells (N), but not in the nucleus of a nearby cell (N′).
(c, d) Vapyrin-GFP expressed in two epidermal cells penetrated by a hypha that has branched and grown into both cells. Signal accumulates in the cytoplasm and nuclei (N), and also in distinct puncta (arrowhead). (c) Projection of 22 optical sections on the z-axis taken at 0.4-μm intervals. (d) Single DIC section, zoom = 2.5.
(e, f) A cortical cell with a young arbuscule exhibits similar Vapyrin-GFP localization in the cytoplasm, nucleus (N) and puncta (arrowheads). (e) Projection of nine optical sections along the z-axis at 0.5-μm intervals. (d) Corresponding DIC of a single section, zoom = 3.5.
(g, h) Vapyrin-GFP in a cell harboring a mature arbuscule. An adjacent cell with no intracellular fungus shows GFP signal in the cytoplasm, but the GFP signal is excluded from the nucleus (N). Single optical sections, zoom = 3.2.
(i, j) The N-terminal GFP fusion to Vapyrin (GFP-Vapyrin) localizes in the cytoplasm and discrete puncta (arrowheads) in a cell with an arbuscule. (i) Projection of six optical sections along the z-axis at 0.7-μm intervals. (j) Single DIC section, zoom = 4.0. Scale bars: 20 μm.

To further evaluate the discrete GFP complexes that formed in cells with arbuscules, a series of time-lapse images were recorded using confocal microscopy. These revealed that the Vapyrin GFP puncta, seen with both N- and C-terminal fusions, can move through the cytoplasm (Figure 7; Video Clip S1), and that the style of movement resembles that of membrane-bound vesicles (daSilva et al., 2004). Vapyrin has no transmembrane domains, or predicted acylation sites, but has two protein interaction domains via which it could potentially interact with membrane-bound proteins trafficking through colonized cells.

Figure 7.

 Vapyrin-GFP moves in discrete complexes.
Three successive images from a confocal time series of transgenic roots expressing proVapyrin:Vapyrin-GFP in a cell with an arbuscule reveal Vapyrin-labeled complexes move through the cytoplasm in discrete, vesicle-like complexes (arrow). Images were taken at 4-s intervals. Zoom = 3.0. Scale bar: 10 μm.

Discussion

Vapyrin is required for AM symbiosis

There is much evidence to indicate that plant cells undergo substantial cellular alterations to accommodate their AM fungal symbionts, but the nature of the molecular components involved in the cellular reprogramming is largely unknown. Although there has been considerable progress with the identification of a signaling pathway that controls fungal entry into the root, almost nothing is known about the molecular events that enable arbuscule formation within the root cortical cells. Here, we report on the discovery and initial characterization of a gene called Vapyrin from M. truncatula that is essential for arbuscule formation, and that is also required for efficient epidermal penetration by AM fungi.

Vapyrin shows a basal level of expression in roots, and is induced transiently in the epidermis and outer cortical cells as the fungal hyphae traverse these cells. Vapyrin is also induced in cortical cells containing arbuscules, and in cells adjacent to those with arbuscules. The mycorrhizal phenotype of Vapyrin RNAi roots is consistent with the expression patterns observed, and indicates a requirement for Vapyrin function both in the epidermis and in the cortical cells. In both cases, Vapyrin is needed to enable fungal hyphae to enter the plant cell. Vapyrin RNAi roots show a high frequency of hyphopodia that attempt, but fail, to penetrate the epidermal cells, and infection arrests in the epidermis. This aspect of the phenotype is similar, although less severe, than that of the ‘common sym’ mutants (Parniske, 2008), where the fungus forms hyphopodia of which almost 100% are blocked at the epidermis. When the fungus succeeded in entering the cortex of Vapyrin RNAi roots, intercellular hyphae spread laterally through the root, but arbuscules were not formed. This phenotype is similar to that of the cyclops mutants of L. japonicus and rice (Kistner et al., 2005; Gutjahr et al., 2008; Yano et al., 2008), and the Petunia mutant pam1 (Reddy et al., 2007), where fungal penetration of the epidermis is reduced and arbuscule formation is abolished. Taken together, these mutant phenotypes support the hypothesis that a common cellular mechanism may be required to enable hyphal growth through epidermal cells and arbuscule development in cortical cells. In addition to the mutant phenotypes, the ‘pre-penetration apparatus’, an intracellular structure composed of endoplasmic reticulum (ER), cytoplasm and cytoskeletal components, that is assembled in the epidermal cells below the hyphopodia, was also observed in cortical cells prior to arbuscule development, and further supports this hypothesis (Genre et al., 2008). It could be envisaged that a single pathway evolved to enable the intracellular passage of AM fungi, with additional tissue-specific or developmental signals to specify hyphal branching and development of the periarbuscular membrane in the cortical cells. The expression patterns and RNAi phenotype of Vapyrin suggest that it is a component of the cellular machinery necessary to accommodate intracellular AM fungal growth.

Why would one process, arbuscule formation, be absolutely abolished while another, root penetration, was only impaired? For the RNAi phenotype reported here, it is possible that the incomplete knock-down of Vapyrin mRNA produced sufficient protein to enable epidermal penetration, but not arbuscule formation. This pattern of impaired epidermal penetration together with no arbuscule formation was also described for the cyclops, ccamk and pam1 mutants, and may suggest these components can be bypassed for the penetration of epidermal cells but not of inner cortical cells. Conversely, other common sym mutants, such as symrk, do allow arbuscule formation after cortical colonization (Demchenko et al., 2004; Kistner et al., 2005).

Vapyrin may function in cellular rearrangement

Whereas the common sym genes, including CYCLOPS, encode signaling proteins, we hypothesize that Vapyrin may play a role in the cellular remodeling process. In contrast to common sym mutants, in Vapyrin RNAi roots, the fungus attempts to penetrate the cells, and numerous hyphal projections are visible below the hyphopodia, but these generally fail to penetrate the cell. Similar aspects are observed in the cortex. This phenotype suggests that the signaling necessary to induce the penetration is intact, but that the cellular processes that support entry, possibly those that facilitate membrane invagination, are impaired.

The location of the Vapyrin protein is also consistent with a role in cellular remodeling. Both Vapyrin GFP fusions were localized primarily in the cytoplasm, and in cells harboring intracellular hyphae or arbuscules, the protein accumulated in distinct, mobile puncta. These puncta were not observed in non-colonized cells, which suggests that they signify a process specific to cells harboring intracellular fungal hyphae. It is possible the puncta represent vesicles or endomembrane compartments of the secretory pathway that might play a role in the deposition of membrane material. Alternatively, if Vapyrin behaves like the MSP proteins (Tarr and Scott, 2005), and has the ability to oligomerize, it is possible that the puncta represent Vapyrin oligomers. Interestingly, the C-terminal GFP fusion displayed some GFP signal in the nucleus, and although the relevance of this remains to be determined, overall, the Vapyrin location stands in sharp contrast to that of CCaMK/DMI3 and Cyclops/IPD3, which localize exclusively in the nucleus, supporting their roles in signal transduction (Smit et al., 2005; Messinese et al., 2007; Yano et al., 2008).

Finally, the Vapyrin protein is a unique combination of two domains that are well established to mediate protein–protein interactions: an N-terminal VAP/MSP homology domain and C-terminal ankyrin repeats. This composition could act as a scaffolding bridge between proteins to facilitate structural changes within the cell.

The VAP family of proteins is conserved across eukaryotes, and is characterized by an N-terminal MSP domain, a central coiled-coil domain and a C-terminal transmembrane domain. The first VAP was discovered as an interactor of VAMP (vesicle-associated membrane protein), and required the central coiled-coil domain for interaction (Lev et al., 2008). Subsequent studies have shown a multitude of interacting proteins that function in various processes from secretion to cytoskeletal to lipid signaling (Lev et al., 2008). Many of these interactions are mediated by conserved residues that bind FFAT motifs (double phenylalanine in an acidic tract) in target proteins. Hetero- and homodimerization has also been reported for VAPs, and requires the transmembrane domain (Nishimura et al., 1999). In Arabidopsis, VAP homologs have been shown to localize in the ER and vacuolar membranes, and to interact with a sterol-binding protein and a motif that mediates transport to protein storage vacuoles (Oufattole et al., 2005; Saravanan et al., 2009). Vapyrin contains the VAP/MSP region at its N terminus, but does not have the coiled-coil or transmembrane domains of the VAP family, nor the conserved residues required for binding FFAT-containing proteins.

Ankyrin repeats are one of the most widely dispersed eukaryotic protein motifs, and function exclusively in mediating protein–protein interactions (Li et al., 2006). The repeated motif consists of a well-conserved 33 amino acid sequence that forms a helix-turn-helix structure. Ankyrin repeat-containing proteins function in diverse processes within cells. Examples of plant proteins containing ankyrin repeats include ineffective greenish nodule 1 (IGN1), a protein from L. japonicus required for nodule function (Kumagai et al., 2007), and Arabidopsis accelerated cell death 6 (ACD6) (Lu et al., 2005), involved in mediating defense responses: both of the proteins also have transmembrane domains. The Arabidopsis genome is predicted to encode over 100 ankyrin repeat-containing proteins that fall into 16 classes based on additional motifs (Becerra et al., 2004). Despite the diversity of proteins containing ankyrin repeats, currently, the Vapyrin family is the only example in nature of ankyrin repeats in combination with a VAP/MSP domain.

In summary, the phylogenetic distribution of this novel protein suggests that Vapyrin is a unique evolution of the plant kingdom that arose early in the vascular plant lineage. Its absence from Arabidopsis, a non-mycorrhizal species, is consistent with its role in symbiosis, and is a pattern shared by several other genes required for AM symbiosis (Javot et al., 2007b; Parniske, 2008; Yano et al., 2008). Although the molecular function of Vapyrin remains to be determined, the domain structure of the protein along with the localization data support a structural role, mediating interactions between two or more proteins, whereas the phenotypic data indicate a role in enabling AM fungi to enter plant cells.

Experimental procedures

Plant growth and transformation

Medicago truncatula cv. Jemalong, line A17, was used for all of the experiments described. Transgenic plants were produced by A. rhizogenes-mediated root transformation (Boisson-Dernier et al., 2001). Briefly, seeds were surface sterilized, germinated, and root tips were excised and inoculated with A. rhizogenes strain ARqua1 harboring the appropriate vectors. Inoculated seedlings were grown on modified Fahraeus media with 25 mg L−1 kanamycin to select transformed roots (Liu et al., 2003). After 3 weeks, seedlings were transferred to sterile turface, with between four and six plants per 11-inch diameter pot, and were grown for 10 days and then inoculated with surface-sterilized Glomus versiforme spores or mock-inoculated with the final spore rinse water, as described previously (Liu et al., 2004). Plants were grown in growth rooms under a 16-h light (25°C)/8-h dark (22°C) regime, and were fertilized once a week with a modified half-strength Hoagland’s solution with full-strength nitrogen and 20 μm potassium phosphate (Arnon and Hoagland, 1940). Plants were analyzed at 21–35 days post-inoculation (21–35 dpi) with Glomus versiforme.

Cloning and vector construction

The Vapyrin promoter was amplified from an M. truncatula genomic library (Harrison et al., 2002) using the iProof enzyme (Bio-Rad, http://www.bio-rad.com), with a Vapyrin primer, R1, and a standard T3 primer present in the vector. After amplification, the reaction was diluted and used as a template for a second and third PCR reaction using nested primers R2 and R3, resulting in a band of 1761 bp, which was cloned and sequenced. To obtain the full-length Vapyrin open reading frame (ORF), first-strand cDNA was synthesized from RNA from M. truncatula/Glomus versiforme roots by the SMART cDNA synthesis kit (Clontech, http://www.clontech.com). The cDNA was then used as a template for nested PCR with Vapyrin primers F1, F2, F3 (Table S2) and the provided 3′ primer, and was then sequenced.

The Vapyrin RNAi-1 and -2 plasmids were created by Gateway cloning (Invitrogen, http://www.invitrogen.com) in a modified pHellsgate 8 (Helliwell et al., 2002), that includes a Ubiquitin promoter:dsRed reporter cassette (J. Liu and M.J. Harrison, unpublished data). The Vapyrin RNAi-1 construct was amplified from the EST clone CF069730 by a gene-specific reverse primer and vector-specific forward primer, producing an RNAi hairpin corresponding to a 383 nucleotide region, −150 to +233 nucleotides relative to the ATG. The Vapyrin RNAi-2 construct was amplified from genomic DNA with specific forward and reverse primers, creating an RNAi hairpin corresponding to a 262 nucleotide region 1375–1748 nucleotides downstream of the ATG. This includes 238 nucleotides at the 3′ end of the coding sequence and 24 nucleotides of 3′ UTR. This region is downstream of the ankyrin repeats to avoid the possible silencing of other genes containing ankyrin repeats. A control RNAi vector was created, similarly targeting a 400-bp fragment of the GUS (UidA) gene. Primer sequences are shown in Table S2.

pVapyrin:UidA was created in pCAMBIA2301. A fragment corresponding to 1761 bp upstream of the Vapyrin ATG start codon was amplified from M. truncatula genomic DNA with primers that add 5′SacI and 3′PstI restriction sites, and was ligated into SacI- and PstI-digested pCAMBIA2301. The UidA coding sequence was amplified from pCAMBIA2301 with primers that add a 5′PstI restriction site directly upstream of the ATG start codon, and include an endogenous 3′BstEII site. pCAMBIA2301 containing the Vapyrin promoter was digested with PstI and BstEII, and the UidA gene was inserted between these sites.

pVapyrin:GFP-Vapyrin was assembled beginning with the pCAMBIA2301 vector containing the Vapyrin promoter digested with PstI and BstEII. The Vapyrin open reading frame from ATG to TAG was amplified by PCR using primers that introduce a 5′PstI site directly upstream of the ATG, and a 3′BstEII site directly downstream of the TAG. This fragment was ligated into the pCAMBIA2301 vector containing the Vapyrin promoter. The green fluorescent protein S65T variant (Chiu et al., 1996) was amplified using primers that introduce 5′ and 3′PstI restriction sites, and was ligated into pCAMBIA2301 between the Vapyrin promoter and the ORF after digestion with the PstI restriction enzyme, resulting in an in-frame fusion of GFP to the 5′ end of the Vapyrin ORF.

pVapyrin:Vapyrin-GFP was created using a pCAMBIA2301 vector with GFP S65T and the NOS terminator cloned into the multiple cloning site between the BamHI and EcoRI restriction sites. The Vapyrin promoter and ORF minus the stop codon were amplified with primers that added a 5′SalI site and a 3′BamHI site (Table S2). This fragment was inserted between the SalI and BamHI sites of modified pCAMBIA2301 containing GFP. This created an in-frame fusion of GFP to the 3′ end of the Vapyrin ORF.

All vectors were sequenced to confirm the correct insertions and absence of mutations.

Protein predictions, alignments and phylogram construction

Protein domains were predicted by SMART through the ELM server (http://elm.eu.org; Puntervoll et al., 2003). The phylogram was constructed by a neighbor-joining ClustalX alignment of full-length amino acid sequences of genes with highest sequence similarity to M. truncatula Vapyrin, and included representative members of VAP and MSP gene families from plants, animals and fungi. Accession numbers are listed in Table S1. Distances were calculated by neighbor joining, and bootstraps reflect values from 100 trials. The phylogram was constructed in TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Expression analysis

Induction of Vapyrin on an Affymetrix GeneChip array was reported in Gomez et al. (2009) as probe sets Mtr.39050.1.S1, corresponding to the 5′ end of Vapyrin (TC104091) and Mtr.42828.1.S1, representing the 3′ end of Vapyrin (TC112496). MtVpyl is represented by Mtr.11779.1.S1 (TC111041), and is not significantly differentially regulated on this array. These probe sets were queried in the Medicago Gene Atlas (http://bioinfo.noble.org/gene-atlas). Gene expression in AM roots was analyzed using RNA extracted from M. truncatula/Glomus versiforme mycorrhizal roots and corresponding mock-inoculated controls, and M. truncatula/Gigaspora gigantea mycorrhizal roots and corresponding mock-inoculated controls. In both cases, roots were harvested at 28 dpi and colonization levels were 76 and 44% root length colonized (RLC), respectively. RNA was extracted from these samples, and also from Vapyrin RNAi and GUS RNAi roots, using the Trizol method (Invitrogen), and 1 μg was used as the template for cDNA synthesis with SuperScript III Reverse Transcriptase (Invitrogen), according to the protocol outlined in Gomez et al. (2009).

Quantitative RT-PCR was performed using an ABI PRISM 7900 HT sequence detection system in optical 384-well plates with SYBR Green PCR Master Mix (Applied Biosystems, http://www.appliedbiosystems.com). Three technical replicates were performed for each primer/cDNA combination in a 10-μl reaction including 40 ng cDNA and 200 nmoles of each primer. The PCR cycle conditions were: 50°C for 2 min, 95°C for 10 min, 43 cycles of 95°C for 30 s and 60°C for 30 s, except for Glomus versiforme α-tubulin, which annealed at 57°C. Following amplification, a dissociation cycle of 95°C for 15 s, 60°C for 15 s and 95°C for 15 s was performed, and primers used in this study were confirmed to amplify one specific PCR product. The cycle threshold (Ct) values were calculated with sds 2.3 (Applied Biosystems) using manual baseline and threshold values. Primer efficiency was calculated independently for each plate by LinRegPCR (Ramakers et al., 2003), and was then used in calculating the relative transcript abundance (EΔCt) after normalizing to M. truncatula elongation factor 1α. The relative transcript levels in Glomus versiforme or Gigaspora gigantea mycorrhizal roots versus non-colonized roots was determined by the EΔΔCt method. Primers are listed in Table S2.

Analysis of RNAi plants

Vapyrin RNAi and control plants were harvested 21–28 dpi with Glomus versiforme. For Vapyrin RNAi-1 and its GUS RNAi control, a representative sample of root tissue was excised from each whole root system and frozen in liquid nitrogen for RNA extraction. The remaining root samples were stained with wheatgerm agglutinin (WGA) conjugated to Alexa Fluor 488 (Molecular Probes, http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes.html) to visualize fungal structures (Javot et al., 2007a). For Vapyrin RNAi-2 and corresponding GUS RNAi controls, individual transgenic roots were excised, and part of the root was sampled for RNA extraction, whereas the remainder was stained as described above and the mycorrhizal phenotype was evaluated. Some samples were counter-stained with propidium iodide (PI; 10 μg ml−1) to visualize plant cell walls (De Smet et al., 2008). Roots were examined microscopically, and Vapyrin RNAi-1 and GUS RNAi control phenotypes were quantified by counting. For each individual root system, the number of hyphopodia that did not penetrate into the inner cortex, infections that penetrated the cortex and spread laterally but did not form arbuscule structures, and infections that penetrated and did form arbuscules were counted.

Conclusions were drawn from four independent experiments, which all showed similar phenotypes, and included more than 45 individual Vapyrin RNAi-1 plants. Individual root sections from Vapyrin RNAi-2 and GUS RNAi root systems used for qRT-PCR showed similar phenotypes as those shown for Vapyrin RNAi-1 and GUS RNAi controls, respectively, and the phenotype was observed in more than 10 individual Vapyrin RNAi-2 transgenic root systems. Samples that contained no fungus or no fungal α-tubulin expression were not included in the qRT-PCR analysis. Representative infections from control and Vapyrin RNAi roots were imaged on a Leica TCS-SP5 confocal microscope (Leica, http://www.leica.com) with a 63× or 20× water immersion objective, with numerical apertures of 1.2 and 0.7, respectively. WGA was excited with a blue argon ion laser (488 nm), and emitted fluorescence was collected from 510 to 540 nm; PI was excited with a diode-pumped solid state (DPSS) laser at 561 nm, and fluorescence was collected from 640 to 710 nm.

Analysis of GUS reporter fusions

The expression of pVapyrin:UidA was evaluated in transgenic plants grown alone or grown with Glomus versiforme for 21–28 days. Histochemical staining for GUS activity was performed as previously described (Liu et al., 2008).

Analysis of GFP fusion proteins

Roots expressing Vapyrin GFP fusions were identified using a dissecting microscope (Olympus SZX-12 stereo microscope; Olympus, http://www.olympus.com). To study protein localization during hyphopodia formation, roots expressing epidermal GFP were excised and imaged on a Leica TCS-SP5 confocal microscope. To analyze GFP expression in the inner cortex, roots were bisected longitudinally through the stele, and were imaged as described previously (Pumplin and Harrison, 2009). Only undisrupted cells in layers below the cut surface were imaged. GFP was excited with the blue argon ion laser (488 nm), and emitted fluorescence was collected from 505 to 545 nm; differential interference contrast (DIC) images were collected together with fluorescence images using the transmitted light detector. GmMan1-mCherry was excited with a DPSS laser at 561 nm, and emitted fluorescence was collected from 590 to 640 nm. Controls were performed to check that there was no crossover between channels. Images were processed using Leica las-af software (versions 1.6.3 and 1.7.0) and Adobe Photoshop cs2 7 (Adobe, http://www.adobe.com).

Vapyrin and Vapyrin-like (MtVpyl) gene sequences were deposited in GenbankTM under accession numbers GQ423209 (Vapyrin) and GQ423210 (Vapyrin-like).

Acknowledgements

The authors thank Aynur Cakmak for technical assistance, and Karen Gomez, Jinyuan Liu and Laura Blaylock for RNA samples. Financial support for this project was provided by the US National Science Foundation, grants DBI-0421676 and DBI-0618969.

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