Hippo cooperates with p53 to maintain foregut homeostasis and suppress the malignant transformation of foregut basal progenitor cells.

Basal progenitor cells serve as a stem cell pool to maintain the homeostasis of the epithelium of the foregut, including the esophagus and the forestomach. Aberrant genetic regulation in these cells can lead to carcinogenesis, such as squamous cell carcinoma (SCC). However, the underlying molecular mechanisms regulating the function of basal progenitor cells remain largely unknown. Here, we use mouse models to reveal that Hippo signaling is required for maintaining the homeostasis of the foregut epithelium and cooperates with p53 to repress the initiation of foregut SCC. Deletion of Mst1/2 in mice leads to epithelial overgrowth in both the esophagus and forestomach. Further molecular studies find that Mst1/2-deficiency promotes epithelial growth by enhancing basal cell proliferation in a Yes-associated protein (Yap)-dependent manner. Moreover, Mst1/2 deficiency accelerates the onset of foregut SCC in a carcinogen-induced foregut SCC mouse model, depending on Yap. Significantly, a combined deletion of Mst1/2 and p53 in basal progenitor cells sufficiently drives the initiation of foregut SCC. Therefore, our studies shed light on the collaborative role of Hippo signaling and p53 in maintaining squamous epithelial homeostasis while suppressing malignant transformation of basal stem cells within the foregut.

TurboID-based proximity labelling reveals a connection between VPS34 and cellular homeostasis.

Unlike animals, plants and yeasts only have a class III phosphatidylinositol 3-kinase (PI3KC3). Its lipid product, phosphatidylinositol 3-phosphate (PtdIns-3-P, PI3P), organizes intracellular trafficking routes such as autophagosome formation, multivesicular body (MVB) formation, retro-transport from trans-Golgi network (TGN) to late Golgi, and the fusion events between autophagosomes and MVBs and the vacuole. The catalytic subunit of plant PI3KC3 is encoded by the essential gene Vacuolar Protein Sorting 34 (VPS34). Despite the importance of VPS34 in cellular homeostasis and plant development, a VPS34 interactome is lacking. Here we employed TurboID, an enzyme-catalyzed proximity labelling (PL) method, to describe a proximal interactome of Arabidopsis VPS34. TurboID catalyzed spatially restricted biotinylation and enabled VPS34-specific enrichment of 273 proteins from affinity purification coupled with mass spectrometry. The interactome confirmed known functions of VPS34 in endo-lysosomal trafficking. Intriguingly, carbohydrate metabolism was the most enriched Gene Ontology (GO) term, including glycolytic enzymes in the triose portion and enzymes functioning in chloroplast triose export and sucrose biosynthesis. The interaction between VPS34 and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1/2) was validated in planta. Also verified was the interaction between VPS34 and the plasma membrane H+-ATPase AHA2, a primary determinant of membrane potential. Our study links PI3KC3 to carbohydrate metabolism and membrane potential, two key processes that maintain cellular homeostasis.

Monitoring Autophagy in Rice With GFP-ATG8 Marker Lines.

Autophagy is a conserved intracellular trafficking pathway for bulk degradation and recycling of cellular components in eukaryotes. The hallmark of autophagy is the formation of double-membraned vesicles termed autophagosomes, which selectively or non-selectively pack up various macromolecules and organelles and deliver these cargoes into the vacuole/lysosome. Like all other membrane trafficking pathways, the observation of autophagy is largely dependent on marker lines. ATG8/LC3 is the only autophagy-related (ATG) protein that, through a covalent bond to phosphatidylethanolamine (PE), associates tightly with the isolation membrane/pre-autophagosomal structure (PAS), the growing phagophore, the mature autophagosome, and the autophagic bodies. Therefore, fluorescent protein (FP)-tagged ATG8 had been widely used for monitoring autophagosome formation and autophagic flux. In rice (Oryza sativa), FP-OsATG8 driven by Cauliflower mosaic virus (CaMV) 35S promoter had been used for imaging autophagosome and autophagic bodies. Here, we constructed three vectors carrying GFP-OsATG8a, driven by 35S, ubiquitin, and the endogenous ATG8a promoter, individually. Then, we compared them for their suitability in monitoring autophagy, by observing GFP-ATG8a puncta formation in transiently transformed rice protoplasts, and by tracking the autophagic flux with GFP-ATG8 cleavage assay in rice stable transgenic lines. GFP-Trap immunoprecipitation and mass spectrometry were also performed with the three marker lines to show that they can be used reliably for proteomic studies. We found out that the ubiquitin promoter is the best for protoplast imaging. Transgenic rice seedlings of the three marker lines showed comparable performance in autophagic flux measurement using the GFP-ATG8 cleavage assay. Surprisingly, the levels of GFP-ATG8a transcripts and protein contents were similar in all marker lines, indicating post-transcriptional regulation of the transgene expression by a yet unknown mechanism. These marker lines can serve as useful tools for autophagy studies in rice.

An allosteric regulation mechanism of Arabidopsis Serine/Threonine kinase 1 (SIK1) through phosphorylation.

The Arabidopsis Serine/Threonine Kinase 1 (SIK1) is a Sterile 20 (STE20)/Hippo orthologue that is also categorized as a Mitogen-Activated Protein Kinase Kinase Kinase Kinase (MAP4K). Like its animal and fungi orthologues, SIK1 is required for cell cycle exit, cell expansion, polarity establishment, as well as pathogenic response. The catalytic activity of SIK1, like other MAPKs, is presumably regulated by its phosphorylation states. Since no crystal structure for SIK1 has been reported yet, we built structural models for SIK1 kinase domain in different phosphorylation states with different pocket conformation to see how this kinase may be regulated. Using computational structural biology methods, we outlined a conduction path in which a phosphorylation site on the A-loop regulates the catalytic activity of SIK1 by controlling the closing or opening of the catalytic pocket at the G-loop. Furthermore, with analyses on the dynamic motions and in vitro kinase assay, we confirmed that three key residues in this conduction path, Lys278, Glu295, and Arg370, are indeed important for the kinase activity of SIK1. Since these residues are conserved in all STE20 kinases examined, the regulatory mechanism that we discovered may be common in STE20 kinases.

Abscisic acid employs NRP-dependent PIN2 vacuolar degradation to suppress auxin-mediated primary root elongation in Arabidopsis.

How plants balance growth and stress adaptation is a long-standing topic in plant biology. Abscisic acid (ABA) induces the expression of the stress-responsive Asparagine Rich Protein (NRP), which promotes the vacuolar degradation of PP6 phosphatase FyPP3, releasing ABI5 transcription factor to initiate transcription. Whether NRP is required for growth remains unknown. We generated an nrp1 nrp2 double mutant, which had a dwarf phenotype that can be rescued by inhibiting auxin transport. Insufficient auxin in the transition zone and over-accumulation of auxin at the root tip was responsible for the short elongation zone and short-root phenotype of nrp1 nrp2. The auxin efflux carrier PIN2 over-accumulated in nrp1 nrp2 and became de-polarized at the plasma membrane, leading to slower root basipetal auxin transport. Knock-out of PIN2 suppressed the dwarf phenotype of nrp1 nrp2. Furthermore, ABA can induce NRP-dependent vacuolar degradation of PIN2 to inhibit primary root elongation. FyPP3 also is required for NRP-mediated PIN2 turnover. In summary, in growth condition, NRP promotes PIN2 vacuolar degradation to help maintain PIN2 protein concentration and polarity, facilitating the establishment of the elongation zone and primary root elongation. When stressed, ABA employs this pathway to inhibit root elongation for stress adaptation.

Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase.

BACKGROUND: The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. RESULTS: Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. CONCLUSIONS: Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

The Arabidopsis STE20/Hippo kinase SIK1 regulates polarity independently of PIN proteins.

Polarity is a feature of life. In higher plants, non-autonomous polarity is largely directed by auxin, the morphogen that drives its own polarized flow, Polar Auxin Transport (PAT), to guide patterning events such as phyllotaxis and tropism. The plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers are rate-limiting factors in PAT. In yeasts and metazoans, the STE20 kinases are key players in cell polarity. We had previously characterized SIK1 as a STE20/Hippo orthologue in Arabidopsis and confirmed its function in mitotic exit and organ growth. Here we explore the possible link between SIK1, auxin, PIN, and polarity. Abnormal phyllotaxis and gravitropism were observed in sik1. sik1 was more sensitive to exogenous auxin in primary root elongation and lateral root emergence. RNA-Seq revealed reduced expression in auxin biosynthesis genes and induced expression of auxin flux carriers in sik1. However, normal tissue- and sub-cellular localization patterns of PIN1 and PIN2 were observed in sik1. The dark-induced vacuolar degradation of PIN2 also appeared normal in sik1. An additive phenotype was observed in the sik1 pin1 double mutant, indicating that SIK1 does not directly regulate PIN1. The polarity defects of sik1 are hence unlikely mediated by PINs and await future exploration.

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy.

BACKGROUND: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive. RESULTS: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response. CONCLUSIONS: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.

A supramolecular hydrogel for spatial-temporal release of auxin to promote plant root growth.

Short peptide-based hydrogels have attracted extensive research interests in drug delivery because of their responsive properties. So far, most drug molecules have been conjugated with short peptides via an amide bond, restricting the release of the native drug molecules. In this study, we demonstrated the effectiveness of an auxin-based hydrogelator linked by a hydrolysable ester bond. Hydrogel I, formed by the gelator (NAA-G'FFY) linked with an ester bond, was able to release 1-naphthaleneacetic acid (NAA), whereas hydrogel II, formed by the gelator without an ester bond (NAA-GFFY), was not. By mixing NAA-G'FFY with Fmoc-GFFY to form a two-component hydrogel, the spatial and temporal release of NAA was achieved, promoting on-site auxin responses including primary root elongation and lateral root formation in the model plant Arabidopsis thaliana. The strategy of using a hydrolysable ester bond to connect drug molecules and self-assembling peptides could lead to the development of supramolecular hydrogels with more controllable drug release profiles.

Study of Autophagy in Plant Senescence.

As a major intracellular degradation pathway, autophagy contributes to nutrient recycling and is indispensable during plant senescence. Here we describe methods used for investigating the autophagic process during leaf senescence. These include transcript analysis of core machinery autophagy genes, immunoblotting of ATG8, and microscopic observation of autophagosome formation.

The Asparagine-Rich Protein NRP Facilitates the Degradation of the PP6-type Phosphatase FyPP3 to Promote ABA Response in Arabidopsis.

The phytohormone abscisic acid (ABA) plays critical roles in abiotic stress responses and plant development. In germinating seeds, the phytochrome-associated protein phosphatase, FyPP3, negatively regulates ABA signaling by dephosphorylating the transcription factor ABI5. However, whether and how FyPP3 is regulated at the posttranscriptional level remains unclear. Here, we report that an asparagine-rich protein, NRP, interacts with FyPP3 and tethers FyPP3 to SYP41/61-positive endosomes for subsequent degradation in the vacuole. Upon ABA treatment, the expression of NRP was induced and NRP-mediated FyPP3 turnover was accelerated. Consistently, ABA-induced FyPP3 turnover was abolished in an nrp null mutant. On the other hand, FyPP3 can dephosphorylate NRP in vitro, and overexpression of FyPP3 reduced the half-life of NRP in vivo. Genetic analyses showed that NRP has a positive role in ABA-mediated seed germination and gene expression, and that NRP is epistatic to FyPP3. Taken together, our results identify a new regulatory circuit in the ABA signaling network, which links the intracellular trafficking with ABA signaling.

Autophagy Is Rapidly Induced by Salt Stress and Is Required for Salt Tolerance in Arabidopsis.

Salinity stress challenges agriculture and food security globally. Upon salt stress, plant growth slows down, nutrients are recycled, osmolytes are produced, and reallocation of Na+ takes place. Since autophagy is a high-throughput degradation pathway that contributes to nutrient remobilization in plants, we explored the involvement of autophagic flux in salt stress response of Arabidopsis with various approaches. Confocal microscopy of GFP-ATG8a in transgenic Arabidopsis showed that autophagosome formation is induced shortly after salt treatment. Immunoblotting of ATG8s and the autophagy receptor NBR1 confirmed that the level of autophagy peaks within 30 min of salt stress, and then settles to a new homeostasis in Arabidopsis. Such an induction is absent in mutants defective in autophagy. Within 3 h of salt treatment, accumulation of oxidized proteins is alleviated in the wild-type; however, such a reduction is not seen in atg2 or atg7. Consistently, the Arabidopsis atg mutants are hypersensitive to both salt and osmotic stresses, and plants overexpressing ATG8 perform better than the wild-type in germination assays. Quantification of compatible osmolytes further confirmed that the autophagic flux contributes to salt stress adaptation. Imaging of intracellular Na+ revealed that autophagy is required for Na+ sequestration in the central vacuole of root cortex cells following salt treatment. These data suggest that rapid protein turnover through autophagy is a prerequisite for salt stress tolerance in Arabidopsis.

The emerging Hippo signaling pathway in plants.

How the organ size is determined is a fundamental question in developmental biology. The metazoan Hippo signaling pathway is well established to negatively regulate organ sizes. Recent studies in plants have started to shape an emerging Hippo signaling pathway. In this review, we summarize the studies in the past decade on the two known components of plant Hippo signaling pathway, the Ste20/Hippo homolog SIK1, and the MOB1/Mats homolog MOB1, with a focus on their developmental functions. Then we envision future discoveries that may shape a complete Hippo signaling pathway in plants.

The asparagine-rich protein NRP interacts with the Verticillium effector PevD1 and regulates the subcellular localization of cryptochrome 2.

The soil-borne fungal pathogen Verticillium dahliae infects a wide range of dicotyledonous plants including cotton, tobacco, and Arabidopsis. Among the effector proteins secreted by V. dahliae, the 16 kDa PevD1 induces a hypersensitive response in tobacco. Here we report the high-resolution structure of PevD1 with folds resembling a C2 domain-like structure with a calcium ion bound to the C-terminal acidic pocket. A yeast two-hybrid screen, designed to probe for molecular functions of PevD1, identified Arabidopsis asparagine-rich protein (NRP) as the interacting partner of PevD1. Extending the pathway of V. dahliae effects, which include induction of early flowering in cotton and Arabidopsis, NRP was found to interact with cryptochrome 2 (CRY2), leading to increased cytoplasmic accumulation of CRY2 in a blue light-independent manner. Further physiological and genetic evidence suggests that PevD1 indirectly activates CRY2 by antagonizing NRP functions. The promotion of CRY2-mediated flowering by a fungal effector outlines a novel pathway by which an external stimulus is recognized and transferred in changing a developmental program.

Jasmonate inhibits COP1 activity to suppress hypocotyl elongation and promote cotyledon opening in etiolated Arabidopsis seedlings.

A germinating seedling undergoes skotomorphogenesis to emerge from the soil and reach for light. During this phase, the cotyledons are closed, and the hypocotyl elongates. Upon exposure to light, the seedling rapidly switches to photomorphogenesis by opening its cotyledons and suppressing hypocotyl elongation. The E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is critical for maintaining skotomorphogenesis. Here, we report that jasmonate (JA) suppresses hypocotyl elongation and stimulates cotyledon opening in etiolated seedlings, partially phenocopying cop1 mutants in the dark. We also find that JA stabilizes several COP1-targeted transcription factors in a COP1-dependent manner. RNA-seq analysis further defines a JA-light co-modulated and cop1-dependent transcriptome, which is enriched for auxin-responsive genes and genes participating in cell wall modification. JA suppresses COP1 activity through at least two distinct mechanisms: decreasing COP1 protein accumulation in the nucleus; and reducing the physical interaction between COP1 and its activator, SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA1). Our work reveals that JA suppresses COP1 activity to stabilize COP1 targets, thereby inhibiting hypocotyl elongation and stimulating cotyledon unfolding in etiolated Arabidopsis seedlings.

Arabidopsis COP1-interacting protein 1 is a positive regulator of ABA response.

COP1-interacting protein 1 (CIP1, At5g41790) was the first reported interacting protein for CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) of Arabidopsis; however its physiological function has remained unknown for two decades. Here we show that CIP1 is a positive regulator of abscisic acid (ABA) response. CIP1 is mainly expressed in the photosynthetic cells and the vascular tissue, and its promoter activity can be induced by osmotic stress and ABA. The CIP1 protein is localized to the plasma membrane. A T-DNA insertion mutant cip1-1 was then characterized. The mutant is sensitive to osmotic stress and has ABA insensitive phenotypes. RNA sequencing showed that cip1-1 has lower levels of gene expression in abiotic stress response compared with the wild-type. Meanwhile, transcript levels of ABA biosynthesis genes are higher in cip1-1 than in the wild-type. These results suggested that CIP1 is positively involved in ABA response.

The Hippo/STE20 homolog SIK1 interacts with MOB1 to regulate cell proliferation and cell expansion in Arabidopsis.

Multicellular organisms co-ordinate cell proliferation and cell expansion to maintain organ growth. In animals, the Hippo tumor suppressor pathway is a master regulator of organ size. Central to this pathway is a kinase cascade composed of Hippo and Warts, and their activating partners Salvador and Mob1/Mats. In plants, the Mob1/Mats homolog MOB1A has been characterized as a regulator of cell proliferation and sporogenesis. Nonetheless, no Hippo homologs have been identified. Here we show that the Arabidopsis serine/threonine kinase 1 (SIK1) is a Hippo homolog, and that it interacts with MOB1A to control organ size. SIK1 complements the function of yeast Ste20 in bud site selection and mitotic exit. The sik1 null mutant is dwarf with reduced cell numbers, endoreduplication, and cell expansion. A yeast two-hybrid screen identified Mob1/Mats homologs MOB1A and MOB1B as SIK1-interacting partners. The interaction between SIK1 and MOB1 was found to be mediated by an N-terminal domain of SIK1 and was further confirmed by bimolecular fluorescence complementation. Interestingly, sik1 mob1a is arrested at the seedling stage, and overexpression of neither SIK1 in mob1a nor MOB1A in sik1 can rescue the dwarf phenotypes, suggesting that SIK1 and MOB1 may be components of a larger protein complex. Our results pave the way for constructing a complete Hippo pathway that controls organ growth in higher plants.

Functions of autophagy in plant carbon and nitrogen metabolism.

Carbon and nitrogen are essential components for plant growth. Although models of plant carbon and nitrogen metabolisms have long been established, certain gaps remain unfilled, such as how plants are able to maintain a flexible nocturnal starch turnover capacity over various light cycles, or how nitrogen remobilization is achieved during the reproductive growth stage. Recent advances in plant autophagy have shed light on such questions. Not only does autophagy contribute to starch degradation at night, but it participates in the degradation of chloroplast proteins and even chloroplasts after prolonged carbon starvation, thus help maintain the free amino acid pool and provide substrate for respiration. The induction of autophagy under these conditions may involve transcriptional regulation. Large-scale transcriptome analyses revealed that ATG8e belongs to a core carbon signaling response shared by Arabidopsis accessions, and the transcription of Arabidopsis ATG7 is tightly co-regulated with genes functioning in chlorophyll degradation and leaf senescence. In the reproductive phase, autophagy is essential for bulk degradation of leaf proteins, thus contributes to nitrogen use efficiency (NUE) both under normal and low-nitrogen conditions.

Nitric oxide induces cotyledon senescence involving co-operation of the NES1/MAD1 and EIN2-associated ORE1 signalling pathways in Arabidopsis.

After germination, cotyledons undertake the major role in supplying nutrients to the pre-photoautorophy angiosperm seedlings until they senesce. Like other senescence processes, cotyledon senescence is a programmed degenerative process. Nitric oxide can induce premature cotyledon senescence in Arabidopsis thaliana, yet the underlying mechanism remains elusive. A screen for genetic mutants identified the nes1 mutant, in which cotyledon senescence was accelerated by nitric oxide. Map-based cloning revealed that NES1 is allelic to a previously reported mitotic checkpoint family gene, MAD1. The nes1/mad1 mutants were restored to the wild type, in response to nitric oxide, by transforming them with pNES1::NES1. Ectopic expression of NES1 in the wild type delayed nitric oxide-mediated cotyledon senescence, confirming the repressive role of NES1. Moreover, two positive regulators of leaf senescence, the ethylene signalling component EIN2 and the transcription factor ORE1/AtNAC2/ANAC092, were found to function during nitric oxide-induced senescence in cotyledons. The block of ORE1 function delayed senescence and ectopic expression induced the process, revealing the positive role of ORE1. EIN2 was required to induce ORE1. Furthermore, the genetic interaction analysis between NES1 and ORE1 showed that the ore1 loss-of-function mutants were epistatic to nes1, suggesting the dominant role of ORE1 and the antagonistic role of NES1 during nitric oxide-induced cotyledon senescence in Arabidopsis.

ROOT HAIR DEFECTIVE3 family of dynamin-like GTPases mediates homotypic endoplasmic reticulum fusion and is essential for Arabidopsis development.

In all eukaryotic cells, the endoplasmic reticulum (ER) forms a tubular network whose generation requires the fusion of ER membranes. In Arabidopsis (Arabidopsis thaliana), the membrane-bound GTPase ROOT HAIR DEFECTIVE3 (RHD3) is a potential candidate to mediate ER fusion. In addition, Arabidopsis has two tissue-specific isoforms of RHD3, namely RHD3-like (RL) proteins, and their function is not clear. Here, we show that a null allele of RHD3, rhd3-8, causes growth defects and shortened root hairs. A point mutant, rhd3-1, exhibits a more severe growth phenotype than the null mutant, likely because it exerts a dominant-negative effect on the RL proteins. Genetic analysis reveals that the double deletion of RHD3 and RL1 is lethal and that the rhd3 rl2 plants produce no viable pollen, suggesting that the RL proteins are redundant to RHD3. RHD3 family proteins can replace Sey1p, the homolog of RHD3 in yeast (Saccharomyces cerevisiae), in the maintenance of ER morphology, and they are able to fuse membranes both in vivo and in vitro. Our results suggest that RHD3 proteins mediate ER fusion and are essential for plant development and that the formation of the tubular ER network is of general physiological significance.