Evidence for an RNAi-independent role of Arabidopsis DICER-LIKE2 in growth inhibition and basal antiviral resistance.

Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs, which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense, and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4 since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA.

Distinct ankyrin repeat subdomains control VAPYRIN locations and intracellular accommodation functions during arbuscular mycorrhizal symbiosis.

Over 70% of vascular flowering plants engage in endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. VAPYRIN (VPY) is a plant protein that is required for intracellular accommodation of AM fungi but how it functions is still unclear. VPY has a large ankyrin repeat domain with potential for interactions with multiple proteins. Here we show that overexpression of the ankyrin repeat domain results in a vpy-like phenotype, consistent with the sequestration of interacting proteins. We identify distinct ankyrin repeats that are essential for intracellular accommodation of arbuscules and reveal that VPY functions in both the cytoplasm and nucleus. VPY interacts with two kinases, including DOES NOT MAKE INFECTIONS3 (DMI3), a nuclear-localized symbiosis signaling kinase. Overexpression of VPY in a symbiosis-attenuated genetic background results in a dmi3 -like phenotype suggesting that VPY negatively influences DMI3 function. Overall, the data indicate a requirement for VPY in the nucleus and cytoplasm where it may coordinate signaling and cellular accommodation processes.

Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissues.

Arabidopsis encodes 10 ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22 nucleotide (nt) long small RNAs (sRNAs) to mediate post-transcriptional gene silencing (PTGS) or 24 nt sRNAs to promote RNA-directed DNA methylation. Using full-locus constructs, we characterized the expression, biochemical properties and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression patterns differ drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 downregulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5' adenosine bias, but unlike Arabidopsis AGO2, it binds 24 nt sRNAs most efficiently. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions in a manner reflecting their respective accumulation from their loci of origin. AGO3 localizes to the cytoplasm and co-fractionates with polysomes to possibly mediate PTGS via translation inhibition.

Author Correction: Origin and evolution of the octoploid strawberry genome.

In the version of this article originally published, author Joshua R. Puzey was incorrectly listed as having affiliation 7 (School of Plant Sciences, University of Arizona, Tucson, AZ, USA); affiliation 6 (Department of Biology, College of William and Mary, Williamsburg, VA, USA) is the correct affiliation. The error has been corrected in the HTML and PDF versions of the article.

Origin and evolution of the octoploid strawberry genome.

Cultivated strawberry emerged from the hybridization of two wild octoploid species, both descendants from the merger of four diploid progenitor species into a single nucleus more than 1 million years ago. Here we report a near-complete chromosome-scale assembly for cultivated octoploid strawberry (Fragaria × ananassa) and uncovered the origin and evolutionary processes that shaped this complex allopolyploid. We identified the extant relatives of each diploid progenitor species and provide support for the North American origin of octoploid strawberry. We examined the dynamics among the four subgenomes in octoploid strawberry and uncovered the presence of a single dominant subgenome with significantly greater gene content, gene expression abundance, and biased exchanges between homoeologous chromosomes, as compared with the other subgenomes. Pathway analysis showed that certain metabolomic and disease-resistance traits are largely controlled by the dominant subgenome. These findings and the reference genome should serve as a powerful platform for future evolutionary studies and enable molecular breeding in strawberry.

Haplotype Characterization of the sd1 Semidwarf Gene in United States Rice.

CORE IDEAS: Genomic data from diverse germplasm used for application in targeted breeding germplasm. Six SNPs identified that can characterize all haplotypes present at SD1 locus in diverse rice. Three alleles of the SD1 gene identified in US rice germplasm including two semidwarf alleles. Two SNPs identified and validated that differentiate the SD1 allele present in US germplasm. KASP assays designed for both SNPs for use in high-throughput breeding applications. Plant height is an important target in US rice (Oryza sativa L.) breeding programs and the large effect of the sd1 semidwarf gene makes it a suitable target for marker-assisted selection. Although the deletion underlying the semidwarf allele is known and a gel-based DNA marker is available, this marker is not ideal for applied breeding because of throughput and cost constraints. The objectives of this study were to characterize the haplotype diversity at the SD1 locus within US rice germplasm and develop a single nucleotide polymorphism (SNP) assay for breeding applications. The International Rice Research Institute (IRRI) SNP-Seek database was used to characterize the haplotype diversity present at the SD1 locus across a set of rice accessions and seven haplotypes were identified. The US rice germplasm was not well represented in the IRRI database, so a set of six SNPs was identified that could differentiate all detected haplotypes. These SNPs were designed into Kompetitive allele specific polymerase chain reaction (KASP) assays and screened across 359 elite US genotypes. Of the seven haplotypes, two were present within the US germplasm, one of which was the semidwarf deletion allele. A third haplotype was observed within the US medium-grain germplasm and demonstrated to be a semidwarf allele derived from the induced mutation in the 'Calrose76'. Two SNPs were identified that distinguish the three SD1 haplotypes present in the US germplasm. These SNPs were validated across the US germplasm and two biparental populations.

Structural Flexibility Enables Alternative Maturation, ARGONAUTE Sorting and Activities of miR168, a Global Gene Silencing Regulator in Plants.

In eukaryotes, the RNase-III Dicer often produces length/sequence microRNA (miRNA) variants, called "isomiRs", owing to intrinsic structural/sequence determinants of the miRNA precursors (pre-miRNAs). In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 (AGO1). We identified a motif conserved among plant pre-miR168 orthologs, which enables flexible internal base-pairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via "transitive RNAi", a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGO1-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of pre-miRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway.

Unlike in metazoans, plant microRNAs (miRNAs) undergo stepwise nuclear maturation before engaging cytosolic, sequence-complementary transcripts in association with the silencing effector protein ARGONAUTE1 (AGO1). Since their discovery, how and under which form plant miRNAs translocate to the cytosol has remained unclear, as has their sub-cellular AGO1 loading site(s). Here, we show that the N termini of all plant AGO1s contain a nuclear-localization (NLS) and nuclear-export signal (NES) that, in Arabidopsis thaliana (At), enables AtAGO1 nucleo-cytosolic shuttling in a Leptomycin-B-inhibited manner, diagnostic of CRM1(EXPO1)/NES-dependent nuclear export. Nuclear-only AtAGO1 contains the same 2'O-methylated miRNA cohorts as its nucleo-cytosolic counterpart, but it preferentially interacts with the miRNA loading chaperone HSP90. Furthermore, mature miRNA translocation and miRNA-mediated silencing both require AtAGO1 nucleo-cytosolic shuttling. These findings lead us to propose a substantially revised view of the plant miRNA pathway in which miRNAs are matured, methylated, loaded into AGO1 in the nucleus, and exported to the cytosol as AGO1:miRNA complexes in a CRM1(EXPO1)/NES-dependent manner.

A complex of Arabidopsis DRB proteins can impair dsRNA processing.

Small RNAs play an important role in regulating gene expression through transcriptional and post-transcriptional gene silencing. Biogenesis of small RNAs from longer double-stranded (ds) RNA requires the activity of dicer-like ribonucleases (DCLs), which in plants are aided by dsRNA binding proteins (DRBs). To gain insight into this pathway in the model plant Arabidopsis, we searched for interactors of DRB4 by immunoprecipitation followed by mass spectrometry-based fingerprinting and discovered DRB7.1. This interaction, verified by reciprocal coimmunoprecipitation and bimolecular fluorescence complementation, colocalizes with markers of cytoplasmic siRNA bodies and nuclear dicing bodies. In vitro experiments using tobacco BY-2 cell lysate (BYL) revealed that the complex of DRB7.1/DRB4 impairs cleavage of diverse dsRNA substrates into 24-nucleotide (nt) small interfering (si) RNAs, an action performed by DCL3. DRB7.1 also negates the action of DRB4 in enhancing accumulation of 21-nt siRNAs produced by DCL4. Overexpression of DRB7.1 in Arabidopsis altered accumulation of siRNAs in a manner reminiscent of drb4 mutant plants, suggesting that DRB7.1 can antagonize the function of DRB4 in siRNA accumulation in vivo as well as in vitro. Specifically, enhanced accumulation of siRNAs from an endogenous inverted repeat correlated with enhanced DNA methylation, suggesting a biological impact for DRB7.1 in regulating epigenetic marks. We further demonstrate that RNase three-like (RTL) proteins RTL1 and RTL2 cleave dsRNA when expressed in BYL, and that this activity is impaired by DRB7.1/DRB4. Investigating the DRB7.1-DRB4 interaction thus revealed that a complex of DRB proteins can antagonize, rather than promote, RNase III activity and production of siRNAs in plants.

DNA Methylation Influences the Expression of DICER-LIKE4 Isoforms, Which Encode Proteins of Alternative Localization and Function.

Plant RNA silencing operates via RNA-directed DNA-methylation (RdDM) to repress transcription or by targeting mRNAs via posttranscriptional gene silencing (PTGS). These pathways rely on distinct Dicer-like (DCL) proteins that process double-stranded RNA (dsRNA) into small-interfering RNAs (siRNAs). Here, we explored the expression and subcellular localization of Arabidopsis thaliana DCL4. DCL4 expression predominates as a transcription start site isoform encoding a cytoplasmic protein, which also represents the ancestral form in plants. A longer DCL4 transcript isoform encoding a nuclear localization signal, DCL4NLS, is present in Arabidopsis, but DNA methylation normally suppresses its expression. Hypomethylation caused by mutation, developmental reprogramming, and biotic stress correlates with enhanced DCL4NLS expression, while hypermethylation of a DCL4 transgene causes a reduction in DCL4NLS expression. DCL4NLS functions in a noncanonical siRNA pathway, producing a unique set of 21-nucleotide-long "disiRNAs," for DCL4NLS isoform-dependent siRNAs, through the nuclear RdDM dsRNA synthesis pathway. disiRNAs originate mostly from transposable elements (TEs) and TE-overlapping/proximal genes, load into the PTGS effector ARGONAUTE1 (AGO1), and display a subtle effect on transcript accumulation together with overlapping 24-nucleotide siRNAs. We propose that, via PTGS, disiRNAs could help to tighten the expression of epigenetically activated TEs and genes using the methylation-state-responsive DCL4NLS.

Genes conserved for arbuscular mycorrhizal symbiosis identified through phylogenomics.

Arbuscular mycorrhizal symbiosis (AMS), a widespread mutualistic association of land plants and fungi(1), is predicted to have arisen once, early in the evolution of land plants(2-4). Consistent with this notion, several genes required for AMS have been conserved throughout evolution(5) and their symbiotic functions preserved, at least between monocot and dicot plants(6,7). Despite its significance, knowledge of the plants' genetic programme for AMS is limited. To date, most genes required for AMS have been found through commonalities with the evolutionarily younger nitrogen-fixing Rhizobium legume symbiosis (RLS)(8) or by reverse genetic analyses of differentially expressed candidate genes(9). Large sequence-indexed insertion mutant collections and recent genome editing technologies have vastly increased the power of reverse genetics but selection of candidate genes, from the thousands of genes that change expression during AMS, remains an arbitrary process. Here, we describe a phylogenomics approach to identify genes whose evolutionary history predicts conservation for AMS and we demonstrate the accuracy of the predictions through reverse genetics analysis. Phylogenomics analysis of 50 plant genomes resulted in 138 genes from Medicago truncatula predicted to function in AMS. This includes 15 genes with known roles in AMS. Additionally, we demonstrate that mutants in six previously uncharacterized AMS-conserved genes are all impaired in AMS. Our results demonstrate that phylogenomics is an effective strategy to identify a set of evolutionarily conserved genes required for AMS.

EXO70I Is Required for Development of a Sub-domain of the Periarbuscular Membrane during Arbuscular Mycorrhizal Symbiosis.

In eukaryotic cells, polarized secretion mediated by exocytotic fusion of membrane vesicles with the plasma membrane is essential for spatially restricted expansion of the plasma membrane and for the delivery of molecules to specific locations at the membrane and/or cell surface. The EXOCYST complex is central to this process, and in yeast, regulation of the EXO70 subunit influences exocytosis and cargo specificity. In contrast to yeast and mammalian cells, plants have upwards of 23 EXO70 genes with largely unknown roles. During arbuscular mycorrhizal (AM) symbiosis, deposition of the plant periarbuscular membrane (PAM) around the fungal arbuscule creates an intracellular membrane interface between the symbionts. The PAM has two major membrane sub-domains, and symbiosis-specific transporter proteins are localized in the branch domain. Currently, the mechanisms and cellular machinery involved in biogenesis of the PAM are largely unknown. Here, we identify an EXO70I protein present exclusively in plants forming AM symbiosis. Medicago truncatula exo70i mutants are unable to support normal arbuscule development, and incorporation of two PAM-resident ABC transporters, STR and STR2, is limited. During arbuscule branching, EXO70I is located in spatially restricted zones adjacent to the PAM around the arbuscule hyphal tips where it interacts with Vapyrin, a plant-specific protein required for arbuscule development. We conclude that EXO70I provides a specific exocytotic capacity necessary for development of the main functional sub-domain of the PAM. Furthermore, in contrast to other eukaryotes, plant EXO70s have evolved distinct specificities and interaction partners to fulfill their specialized secretory requirements.

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3.

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.

DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence.

RNA silencing is a central regulator of gene expression in most eukaryotes and acts both at the transcriptional level through DNA methylation and at the post-transcriptional level through direct mRNA interference mediated by small RNAs. In plants and invertebrates, the same pathways also function directly in host defence against viruses by targeting viral RNA for degradation. Successful viruses have consequently evolved diverse mechanisms to avoid silencing, most notably through the expression of viral suppressors of RNA silencing. RNA silencing suppressors have also been recently identified in plant pathogenic bacteria and oomycetes, suggesting that disruption of host silencing is a general virulence strategy across several kingdoms of plant pathogens. There is also increasing evidence that plants have evolved specific defences against RNA-silencing suppression by pathogens, providing yet another illustration of the never-ending molecular arms race between plant pathogens and their hosts.

Polar localization of a symbiosis-specific phosphate transporter is mediated by a transient reorientation of secretion.

The arbuscular mycorrhizal (AM) symbiosis, formed by land plants and AM fungi, evolved an estimated 400 million years ago and has been maintained in angiosperms, gymnosperms, pteridophytes, and some bryophytes as a strategy for enhancing phosphate acquisition. During AM symbiosis, the AM fungus colonizes the root cortical cells where it forms branched hyphae called arbuscules that function in nutrient exchange with the plant. Each arbuscule is enveloped in a plant membrane, the periarbuscular membrane, that contains a unique set of proteins including phosphate transporters such as Medicago truncatula MtPT4 [Javot et al., (2007) Proc Natl Acad Sci USA 104:1720-1725], which are essential for symbiotic phosphate transport. The periarbuscular membrane is physically continuous with the plasma membrane of the cortical cell, but MtPT4 and other periarbuscular membrane-resident proteins are located only in the domain around the arbuscule branches. Establishing the distinct protein composition of the periarbuscular membrane is critical for AM symbiosis, but currently the mechanism by which this composition is achieved is unknown. Here we investigate the targeting of MtPT4 to the periarbuscular membrane. By expressing MtPT4 and other plasma membrane proteins from promoters active at different phases of the symbiosis, we show that polar targeting of MtPT4 is mediated by precise temporal expression coupled with a transient reorientation of secretion and alterations in the protein cargo entering the secretory system of the colonized root cell. In addition, analysis of phosphate transporter mutants implicates the trans-Golgi network in phosphate transporter secretion.

A critical role of STAYGREEN/Mendel's I locus in controlling disease symptom development during Pseudomonas syringae pv tomato infection of Arabidopsis.

Production of disease symptoms represents the final phase of infectious diseases and is a main cause of crop loss and/or marketability. However, little is known about the molecular basis of disease symptom development. In this study, a genetic screening was conducted to identify Arabidopsis (Arabidopsis thaliana) mutants that are impaired specifically in the development of disease symptoms (leaf chlorosis and/or necrosis) after infection with the bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000. An ethyl methanesulfonate-induced Arabidopsis mutant (no chlorosis1 [noc1]) was identified. In wild-type plants, the abundance of chlorophylls decreased markedly after Pst DC3000 infection, whereas the total amount of chlorophylls remained relatively unchanged in the noc1 mutant. Interestingly, noc1 mutant plants also exhibited reduced disease symptoms in response to the fungal pathogen Alternaria brassicicola. Genetic and molecular analyses showed that the nuclear gene STAYGREEN (SGR; or Mendel's I locus) is mutated (resulting in the aspartic acid to tyrosine substitution at amino acid position 88) in noc1 plants. Transforming wild-type SGR cDNA into the noc1 mutant rescued the chlorosis phenotype in response to Pst DC3000 infection. The SGR transcript was highly induced by Pst DC3000, A. brassicicola, or coronatine (COR), a bacterial phytotoxin that promotes chlorosis. The induction of SGR expression by COR is dependent on COI1, a principal component of the jasmonate receptor complex. These results suggest that pathogen/COR-induced expression of SGR is a critical step underlying the development of plant disease chlorosis.

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

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.

Live-cell imaging reveals periarbuscular membrane domains and organelle location in Medicago truncatula roots during arbuscular mycorrhizal symbiosis.

In the arbuscular mycorrhizal symbiosis, the fungal symbiont colonizes root cortical cells, where it establishes differentiated hyphae called arbuscules. As each arbuscule develops, the cortical cell undergoes a transient reorganization and envelops the arbuscule in a novel symbiosis-specific membrane, called the periarbuscular membrane. The periarbuscular membrane, which is continuous with the plant plasma membrane of the cortical cell, is a key interface in the symbiosis; however, relatively little is known of its composition or the mechanisms of its development. Here, we used fluorescent protein fusions to obtain both spatial and temporal information about the protein composition of the periarbuscular membrane. The data indicate that the periarbuscular membrane is composed of at least two distinct domains, an "arbuscule branch domain" that contains the symbiosis-specific phosphate transporter, MtPT4, and an "arbuscule trunk domain" that contains MtBcp1. This suggests a developmental transition from plasma membrane to periarbuscular membrane, with biogenesis of a novel membrane domain associated with the repeated dichotomous branching of the hyphae. Additionally, we took advantage of available organelle-specific fluorescent marker proteins to further evaluate cells during arbuscule development and degeneration. The three-dimensional data provide new insights into relocation of Golgi and peroxisomes and also illustrate that cells with arbuscules can retain a large continuous vacuolar system throughout development.

Closely related members of the Medicago truncatula PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities.

Phosphorus is one of the essential mineral nutrients required by all living cells. Plants assimilate phosphate (Pi) from the soil, and their root systems encounter tremendous variation in Pi concentration, both temporally and spatially. Genome sequence data indicate that plant genomes contain large numbers of genes predicted to encode Pi transporters, the functions of which are largely unexplored. Here we present a comparative analysis of four very closely related Pi transporters of the PHT1 family of Medicago truncatula. Based on their sequence similarity and locations in the genome, these four genes probably arose via recent gene duplication events, and they form a small subfamily within the PHT1 family. The four genes are expressed in roots with partially overlapping but distinct spatial expression patterns, responses to Pi and expression during arbuscular mycorrhizal symbiosis. The proteins are located in the plasma membrane. Three members of the subfamily, MtPT1, MtPT2, and MtPT3, show low affinities for Pi. MtPT5 shares 84% amino acid identity with MtPT1, MtPT2, and MtPT3 but shows a high affinity for Pi with an apparent Km in yeast of 13 microm. Sequence comparisons and protein modeling suggest that amino acid residues that differ substantially between MtPT5 and the other three transporters are clustered in two regions of the protein. The data provide the first clues as to amino acid residues that impact transport activity of plant Pi transporter proteins.