Resetting of the 24-nt siRNA landscape in rice zygotes.

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells-the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.

The molecular pathways that trigger the initiation of embryogenesis after fertilization in flowering plants, and prevent its occurrence without fertilization, are not well understood1. Here we show in rice (Oryza sativa) that BABY BOOM1 (BBM1), a member of the AP2 family2 of transcription factors that is expressed in sperm cells, has a key role in this process. Ectopic expression of BBM1 in the egg cell is sufficient for parthenogenesis, which indicates that a single wild-type gene can bypass the fertilization checkpoint in the female gamete. Zygotic expression of BBM1 is initially specific to the male allele but is subsequently biparental, and this is consistent with its observed auto-activation. Triple knockout of the genes BBM1, BBM2 and BBM3 causes embryo arrest and abortion, which are fully rescued by male-transmitted BBM1. These findings suggest that the requirement for fertilization in embryogenesis is mediated by male-genome transmission of pluripotency factors. When genome editing to substitute mitosis for meiosis (MiMe)3,4 is combined with the expression of BBM1 in the egg cell, clonal progeny can be obtained that retain genome-wide parental heterozygosity. The synthetic asexual-propagation trait is heritable through multiple generations of clones. Hybrid crops provide increased yields that cannot be maintained by their progeny owing to genetic segregation. This work establishes the feasibility of asexual reproduction in crops, and could enable the maintenance of hybrids clonally through seed propagation5,6.

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Genome-wide redistribution of 24-nt siRNAs in rice gametes.

Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.

Somatic embryo initiation by rice BABY BOOM1 involves activation of zygote-expressed auxin biosynthesis genes.

Plant embryogenesis results from the fusion of male and female gametes but can also be induced in somatic cells. The molecular pathways for embryo initiation are poorly understood, especially in monocots. In rice, the male gamete expressed BABY BOOM1 (OsBBM1) transcription factor functions as an embryogenic trigger in the zygote and can also promote somatic embryogenesis when ectopically expressed in somatic tissues. We used gene editing, transcriptome profiling, and chromatin immunoprecipitation to determine the molecular players involved in embryo initiation downstream of OsBBM1. We identify OsYUCCA (OsYUC) auxin biosynthesis genes as direct targets of OsBBM1. Unexpectedly, these OsYUC targets in zygotes are expressed only from the maternal genome, whereas the paternal genome exclusively provides functional OsBBM1 to initiate embryogenesis. Induction of somatic embryogenesis by exogenous auxin requires OsBBM genes and downstream OsYUC targets. Ectopic OsBBM1 initiates somatic embryogenesis without exogenous auxins but requires functional OsYUC genes. Thus, an OsBBM-OsYUC module is a key player for both somatic and zygotic embryogenesis in rice. Zygotic embryo initiation involves a partnership of male and female genomes, through which paternal OsBBM1 activates maternal OsYUC genes. In somatic embryogenesis, exogenous auxin triggers OsBBM1 expression, which then activates endogenous auxin biosynthesis OsYUC genes.

Coordinated Activation of ARF1 GTPases by ARF-GEF GNOM Dimers Is Essential for Vesicle Trafficking in Arabidopsis.

Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

Acquisition of a complex root microbiome reshapes the transcriptomes of rice plants.

Soil microorganisms can colonize plant roots and assemble in communities engaged in symbiotic relationships with their host. Though the compositional dynamics of root-associated microbiomes have been extensively studied, the host transcriptional response to these communities is poorly understood. Here, we developed an experimental system by which rice plants grown under axenic conditions can acquire a defined endosphere microbiome. Using this setup, we performed a cross-sectional characterization of plant transcriptomes in the presence or absence of a complex microbial community. To account for compositional variation, plants were inoculated with soil-derived microbiomes harvested from three distinct agricultural sites. Soil microbiomes triggered a major shift in the transcriptional profiles of rice plants that included the downregulation of one-third to one-fourth of the families of leucine-rich repeat receptor-like kinases and nucleotide-binding leucine-rich repeat receptors expressed in roots. Though the expression of several genes was consistent across all soil sources, a large fraction of this response was differentially impacted by soil type. These results demonstrate the role of root microbiomes in sculpting the transcriptomes of host plants and highlight the potential involvement of the two main receptor families of the plant immune system in the recruitment and maintenance of an endosphere microbiome.

Compositional shifts in root-associated bacterial and archaeal microbiota track the plant life cycle in field-grown rice.

Bacterial communities associated with roots impact the health and nutrition of the host plant. The dynamics of these microbial assemblies over the plant life cycle are, however, not well understood. Here, we use dense temporal sampling of 1,510 samples from root spatial compartments to characterize the bacterial and archaeal components of the root-associated microbiota of field grown rice (Oryza sativa) over the course of 3 consecutive growing seasons, as well as 2 sites in diverse geographic regions. The root microbiota was found to be highly dynamic during the vegetative phase of plant growth and then stabilized compositionally for the remainder of the life cycle. Bacterial and archaeal taxa conserved between field sites were defined as predictive features of rice plant age by modeling using a random forest approach. The age-prediction models revealed that drought-stressed plants have developmentally immature microbiota compared to unstressed plants. Further, by using genotypes with varying developmental rates, we show that shifts in the microbiome are correlated with rates of developmental transitions rather than age alone, such that different microbiota compositions reflect juvenile and adult life stages. These results suggest a model for successional dynamics of the root-associated microbiota over the plant life cycle.

Reproductive Long Intergenic Noncoding RNAs Exhibit Male Gamete Specificity and Polycomb Repressive Complex 2-Mediated Repression.

Long noncoding RNAs (lncRNAs) have been characterized extensively in animals and are involved in several processes, including homeobox gene expression and X-chromosome inactivation. In comparison, there has been much less detailed characterization of plant lncRNAs, and the number of distinct lncRNAs encoded in plant genomes and their regulation by developmental and epigenetic mechanisms remain largely unknown. Here, we analyzed transcriptome data from Asian rice (Oryza sativa) and identified 6,309 long intergenic noncoding RNAs (lincRNAs), focusing on their expression in reproductive tissues and organs. Most O. sativa lincRNAs were expressed in a highly tissue-specific manner, with an unexpectedly high fraction specifically expressed in male gametes. Mutation of a component of the Polycomb Repressive Complex2 (PRC2) resulted in derepression of another large class of lincRNAs, whose expression is correlated with H3K27 trimethylation in developing panicles. Overlap with the sperm cell-specific lincRNAs suggests that epigenetic repression of lincRNAs in the panicles was partially relieved in the male germline. Expression of a subset of lincRNAs also showed modulation by drought in reproductive tissues. Comparison with other cereal genomes showed that the lincRNAs generally have low levels of conservation at both the sequence and structural levels. Use of a novelty detection support vector machine model enabled the detection of nucleotide sequence and structural homology in ∼10% and ∼4% of the lincRNAs in genomes of purple false brome (Brachypodium distachyon) and maize (Zea mays), respectively. This is the first study to report on a large number of lncRNAs that are targets of repression by PRC2 rather than mediating regulation via PRC2. That the vast majority of the lincRNAs reported here do not overlap with those of other rice studies indicates that these are a significant addition to the known lincRNAs in rice.

[Screening and identification of CKI1 upstream transcription regulators in Arabidopsis].

Arabidopsis CKI1 (cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique. Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.

ARF2-ARF4 and ARF5 are Essential for Female and Male Gametophyte Development in Arabidopsis.

The plant hormone auxin plays critical roles in plant growth and development. Auxin response factors (ARFs) are a class of transcription factors which regulate auxin-mediated gene expression. While the functions of ARFs in sporophytic development have been well characterized, their functions specific to gametophytic development have not been studied extensively. In this study, Arabidopsis ARF genes were selectively down-regulated in gametophytes by misexpression of targeted microRNAs (amiRARF234, amiRARFMP and MIR167a) to silence AtARF2-AtAEF4, AtARF5, AtARF6 and AtARF8. Embryo sacs in amiRARF234- and amiRARFMP-expressing plants exhibited identity defects in cells at the micropylar pole, such as formation of two cells with egg cell-like morphology, concomitant with loss of synergid marker expression and seed abortion. The pollen grains of the transgenic plants were morphologically aberrant and unviable, and the inclusions and nuclei were lost in the abnormal pollen grains. However, plants misexpressing MIR167a showed no obvious abnormal phenotypes in the embryo sacs and pollen grains. Overall, these results provide evidence that AtARF2-AtARF4 and AtARF5 play significant roles in regulating both female and male gametophyte development in Arabidopsis.

The gymnosperm ortholog of the angiosperm central cell-specification gene CKI1 provides an essential clue to endosperm origin.

A defining feature of angiosperms is double fertilization involving the female gametophyte central cell and formation of a nutrient-storing tissue called endosperm. The route for the evolutionary origin of endosperm from a gymnosperm ancestor, particularly the molecular steps involved, has remained elusive. Recently, the histidine kinase gene Cytokinin-Independent 1 (CKI1), an activator of cytokinin signaling, was described as a key to specification of the endosperm precursor central cell in Arabidopsis. Here, we have investigated the function and expression of a putative ortholog of CKI1 in the gymnosperm Ginkgo biloba. We demonstrate that Ginkgo CKI1 can partially rescue an Arabidopsis cki1 mutant and promote weak activation of the cytokinin signaling pathway in the Arabidopsis embryo sac, but does not confer central cell specification. Ginkgo CKI1 is expressed in both male and female gametophytes of Ginkgo. In the latter, it is expressed in the ventral canal cell, which is sister to the egg cell in the archegonium. As in Arabidopsis, Ginkgo CKI1 is not expressed in the egg cell. The similarities in expression patterns of CKI1 in Ginkgo and Arabidopsis female gametophytes suggest that extant gymnosperms possess an essential component of the molecular machinery required for angiosperm endosperm development, and provide new insights into endosperm origin from a gymnospermous ancestor.

Structure, variation, and assembly of the root-associated microbiomes of rice.

Plants depend upon beneficial interactions between roots and microbes for nutrient availability, growth promotion, and disease suppression. High-throughput sequencing approaches have provided recent insights into root microbiomes, but our current understanding is still limited relative to animal microbiomes. Here we present a detailed characterization of the root-associated microbiomes of the crop plant rice by deep sequencing, using plants grown under controlled conditions as well as field cultivation at multiple sites. The spatial resolution of the study distinguished three root-associated compartments, the endosphere (root interior), rhizoplane (root surface), and rhizosphere (soil close to the root surface), each of which was found to harbor a distinct microbiome. Under controlled greenhouse conditions, microbiome composition varied with soil source and genotype. In field conditions, geographical location and cultivation practice, namely organic vs. conventional, were factors contributing to microbiome variation. Rice cultivation is a major source of global methane emissions, and methanogenic archaea could be detected in all spatial compartments of field-grown rice. The depth and scale of this study were used to build coabundance networks that revealed potential microbial consortia, some of which were involved in methane cycling. Dynamic changes observed during microbiome acquisition, as well as steady-state compositions of spatial compartments, support a multistep model for root microbiome assembly from soil wherein the rhizoplane plays a selective gating role. Similarities in the distribution of phyla in the root microbiomes of rice and other plants suggest that conclusions derived from this study might be generally applicable to land plants.

AHP2, AHP3, and AHP5 act downstream of CKI1 in Arabidopsis female gametophyte development.

Histidine phosphotransfer proteins (HPs) are key elements of the two-component signaling system, which act as a shuttle to transfer phosphorylation signals from histidine kinases (HKs) to response regulators (RRs). CYTOKININ INDEPENDENT 1 (CKI1), a key regulator of central cell specification in the Arabidopsis female gametophyte, activates the cytokinin signaling pathway through the Arabidopsis histidine phosphotransfer proteins (AHPs). There are five HP genes in Arabidopsis, AHP1-AHP5, but it remains unknown which AHP genes act downstream of CKI1 in Arabidopsis female gametophyte development. Promoter activity analysis of AHP1-AHP5 in embryo sacs revealed AHP1, AHP2, AHP3, and AHP5 expression in the central cell. Phenotypic studies of various combinations of ahp mutants showed that triple mutations in AHP2, AHP3, and AHP5 resulted in defective embryo sac development. Using cell-specific single and double markers in the female gametophyte, the ahp2-2 ahp3 ahp5-2/+ triple mutant ovules showed loss of central cell and antipodal cell fates and gain of egg cell or synergid cell attributes, resembling the cki1 mutant phenotypes. These data suggest that AHP2, AHP3, and AHP5 are the major factors acting downstream of CKI1 in the two-component cytokinin signaling pathway to promote Arabidopsis female gametophyte development.

oiwa, a female gametophytic mutant impaired in a mitochondrial manganese-superoxide dismutase, reveals crucial roles for reactive oxygen species during embryo sac development and fertilization in Arabidopsis.

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice.

Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes.

Molecular characterization of the glauce mutant: a central cell-specific function is required for double fertilization in Arabidopsis.

Double fertilization of the egg cell and the central cell by two sperm cells, resulting in the formation of the embryo and the endosperm, respectively, is a defining characteristic of flowering plants. The Arabidopsis thaliana female gametophytic mutant glauce (glc) can exhibit embryo development without any endosperm. Here, we show that in glc mutant embryo sacs one sperm cell successfully fuses with the egg cell but the second sperm cell fails to fuse with the central cell, resulting in single fertilization. Complementation studies using genes from the glc deletion interval identified an unusual genomic locus having homology to BAHD (for BEAT, AHCT, HCBT, and DAT) acyl-transferases with dual transcription units and alternative splicing that could rescue the sterility defect of glc. Expression of these transcripts appears restricted to the central cell, and expression within the central cell is sufficient to restore fertility. We conclude that the central cell actively promotes its own fertilization by the sperm cell through a signaling mechanism involving products of At1g65450. Successful fertilization of the egg cell is not blocked in the glc mutant, suggesting that evolution of double fertilization in flowering plants involved acquisition of specific functions by the central cell to enable its role as a second female gamete.

The Armadillo repeat gene ZAK IXIK promotes Arabidopsis early embryo and endosperm development through a distinctive gametophytic maternal effect.

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin-dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of miniseed3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways.

Nonredundant regulation of rice arbuscular mycorrhizal symbiosis by two members of the phosphate transporter1 gene family.

Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.