Characterization of Subcellular Dynamics of Sterol Methyltransferases Clarifies Defective Cell Division in smt2 smt3, a C-24 Ethyl Sterol-Deficient Mutant of Arabidopsis.

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes.

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A. thaliana and the single LRL/DROP protein MpLRL in M. polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A. thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M. polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.

The SYP123-VAMP727 SNARE complex delivers secondary cell wall components for root hair shank hardening in Arabidopsis.

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.

Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha.

Gametogenesis is an essential event for sexual reproduction in various organisms. Bryophytes employ motile sperm (spermatozoids) as male gametes, which locomote to the egg cells to accomplish fertilization. The spermatozoids of bryophytes harbor distinctive morphological characteristics, including a cell body with a helical shape and two flagella. During spermiogenesis, the shape and cellular contents of the spermatids are dynamically reorganized. However, the reorganization patterns of each organelle remain obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to changes in cellular and nuclear shapes and flagellar development. We then examined the remodeling of microtubules and the reorganization of endomembrane organelles. The results indicated that the state of glutamylation of tubulin changes during formation of the flagella and spline. We also found that the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner, which involves the functions of endosomal sorting complexes required for transport (ESCRT) machineries in endocytic and vacuolar transport. These findings are expected to provide useful indices to classify developmental and subcellular processes of spermiogenesis in bryophytes.

Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis.

Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.

Membrane trafficking functions of the ANTH/ENTH/VHS domain-containing proteins in plants.

Subcellular localization of proteins acting on the endomembrane system is primarily regulated via membrane trafficking. To obtain and maintain the correct protein composition of the plasma membrane and membrane-bound organelles, the loading of selected cargos into transport vesicles is critically regulated at donor compartments by adaptor proteins binding to the donor membrane, the cargo molecules and the coat-protein complexes, including the clathrin coat. The ANTH/ENTH/VHS domain-containing protein superfamily generally comprises a structurally related ENTH, ANTH, or VHS domain in the N-terminal region and a variable C-terminal region, which is thought to act as an adaptor during transport vesicle formation. This protein family is involved in various plant processes, including pollen tube growth, abiotic stress response and development. In this review, we provide an overview of the recent findings on ANTH/ENTH/VHS domain-containing proteins in plants.

Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.

The liverwort oil body is formed by redirection of the secretory pathway.

Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We find that two paralogous syntaxin-1 homologs in the liverwort Marchantia polymorpha are distinctly targeted to forming cell plates and the oil body, suggesting that these structures share some developmental similarity. Oil body formation is regulated by an ERF/AP2-type transcription factor and loss of the oil body increases M. polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical redirection of the secretory pathway depending on cellular phase transition.

Longin R-SNARE is retrieved from the plasma membrane by ANTH domain-containing proteins in Arabidopsis.

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.

Deep Imaging Analysis in VISUAL Reveals the Role of YABBY Genes in Vascular Stem Cell Fate Determination.

Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial-abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.

Enrichment of Phosphatidylinositol 4,5-Bisphosphate in the Extra-Invasive Hyphal Membrane Promotes Colletotrichum Infection of Arabidopsis thaliana.

Pathogenic fungi from the genus Colletotrichum form invasive hyphae; the hyphae are surrounded by an extra-invasive hyphal membrane (EIHM), which is continuous with the plant plasma membrane. Although the EIHM plays a crucial role as the interface between plant and fungal cells, its precise function during Colletotrichum infection remains elusive. Here, we show that enrichment of phosphoinositides (PIs) has a crucial role in Colletotrichum infection. We observed the localization of PIs in Arabidopsis thaliana cells infected by A. thaliana-adapted Colletotrichum higginsianum (Ch), and found that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was extremely enriched in the EIHM during Ch infection. We also found that phosphatidylinositol 4-phosphate-5 kinase (PIP5K), which catalyzes production of PI(4,5)P2, also accumulated at the EIHM. The overexpression of PIP5K3 in A. thaliana increased hyphal invasion by Ch. An exocytic factor, EXO84b, was targeted to the EIHM during Ch infection, although endocytic factors such as CLATHRIN LIGHT CHAIN 2 and FLOTILLIN 1 did not. Intriguingly, the interfacial membranes between A. thaliana and powdery mildew- or downy mildew-causing pathogens did not accumulate PI(4,5)P2. These results suggest that Ch could modify the PI(4,5)P2 levels in the EIHM to increase the exocytic membrane/protein supply of the EIHM for successful infection. Our results also suggest that PI(4,5)P2 biosynthesis is a promising target for improved defense against Colletotrichum infection.

ANTH domain-containing proteins are required for the pollen tube plasma membrane integrity via recycling ANXUR kinases.

During plant reproduction, sperm cells are delivered to ovules through growing pollen tubes. This process involves tip-localized receptor kinases regulating integrity and/or guidance of pollen tubes, whose localizations must be strictly regulated. However, the molecular basis for tip-localization of these molecules remains largely elusive. Here we show that a pair of AP180 N-terminal homology domain-containing proteins, PICALM5a and PICALM5b, is responsible for the tip-localization of ANXUR receptor kinases acting in an autocrine signaling pathway required for pollen tube integrity in Arabidopsis thaliana. The picalm5a picalm5b double mutant exhibits reduced fertility, and the double mutant pollen is defective in pollen tube integrity with premature bursts. The tip localization of ANXUR proteins is severely impaired in picalm5a picalm5b pollen tubes, whereas another receptor kinase PRK6 acting in pollen tube guidance is not affected. Based on these results, we propose that PICALM5 proteins serve as specific loading adaptors to recycle ANXUR proteins.

Transcriptional switch for programmed cell death in pith parenchyma of sorghum stems.

Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.

Integration of two RAB5 groups during endosomal transport in plants.

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells.

RAB GTPases in the Basal Land Plant Marchantia polymorpha.

The RAB GTPase is an evolutionarily conserved machinery component of membrane trafficking, which is the fundamental system for cell viability and higher order biological functions. The composition of RAB GTPases in each organism is closely related to the complexity and organization of the membrane trafficking pathway, which has been developed uniquely to realize the organism-specific membrane trafficking system. Comparative genomics has suggested that terrestrialization and/or multicellularization were associated with the expansion of membrane trafficking pathways in green plants, which has yet to be validated in basal land plant lineages. To obtain insight into the diversification of membrane trafficking systems in green plants, we analyzed RAB GTPases encoded in the genome of the liverwort Marchantia polymorpha in a comprehensive manner. We isolated all genes for RAB GTPases in Marchantia and analyzed their expression patterns and subcellular localizations in thallus cells. While a majority of MpRAB GTPases exhibited a ubiquitous expression pattern, specific exceptions were also observed; MpRAB2b, which contains a sequence similar to an intraflagellar transport protein at the C-terminal region; and MpRAB23, which has been secondarily lost in angiosperms, were specifically expressed in the male reproductive organ. MpRAB21, which is another RAB GTPase whose homolog is absent in Arabidopsis, exhibited endosomal localization with RAB5 members in Marchantia. These results suggest that Marchantia possesses unique membrane trafficking pathways involving a unique repertoire of RAB GTPases.

Distinct sets of tethering complexes, SNARE complexes, and Rab GTPases mediate membrane fusion at the vacuole in Arabidopsis.

Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily.

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2 Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

Constitutive activation of plant-specific RAB5 GTPase confers increased resistance against adapted powdery mildew fungus.

The obligate biotrophic fungal pathogens that cause powdery mildew disease establish infection in living host cells by modifying host cellular functions, including membrane trafficking. Previously, we reported that two Arabidopsis thaliana RAB5 GTPases, plant-specific ARA6/RABF1 and canonical ARA7/RABF2b, accumulate at the extrahaustorial membrane (EHM), which surrounds the specialized infection hypha called the haustorium. In this study, we examined the role of ARA6 and ARA7, which regulate distinctive endosomal trafficking pathways, in plant-powdery mildew fungus interactions. Although ARA6- and ARA7-related mutants did not exhibit altered susceptibility to the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii, overexpression of constitutively active ARA6, but not constitutively active ARA7, repressed proliferation of G. orontii. The repression of fungal proliferation was associated with accelerated formation of the callosic encasement around the haustorium. Furthermore, microscopic observation revealed an accumulation of the constitutively active form of ARA6, but not active ARA7, at the EHM. These results indicate that plant-specific ARA6 has a specific role in plant-powdery mildew fungus interaction, and manipulation of ARA6 activity could be a novel tool to overcome this plant disease.

Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2.

BACKGROUND: Rab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown. METHODS: A complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy. RESULTS: PfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b. CONCLUSION: N-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.

Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.