Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas.In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5 untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-␣/␤) activity in VEE pathogenesis. In IFN-␣/␤R ؊/؊ mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000-and V3043-infected IFN-␣/␤R ؊/؊ mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-␣/␤ pathway. In cells derived from IFN-␣/␤R ؊/؊ mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-␣/␤ plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-␣/␤ between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-␣/␤ in two separate in vitro assays, suggesting that the increased sensitivity to IFN-␣/␤ plays a major role in the in vivo attenuation of V3043.Venezuelan equine encephalitis virus (VEE) is a member of the Alphavirus genus in the Togaviridae family. The genome of this enveloped virus is a single-stranded, messenger-sense RNA molecule of approximately 11.5 kb (24), capped at the 5Ј end and polyadenylated at the 3Ј end. The genomic RNA encodes four nonstructural proteins (nsP1 through -4) and three structural proteins (capsid and two envelope glycoproteins, E1 and E2). The 5Ј untranslated region (UTR) in VEE is 45 nucleotides (nt) long, and although its sequence is not conserved among alphaviruses, the sequence predicts a stemloop structure that is conserved across the Alphavirus genus (55). It has been proposed that the complementary sequence at the 3Ј end of the minus strand also folds into a conserved secondary structure that may play a role as a promoter for the initiation of genome RNA synthesis from the minus-strand template (10,38,55). The nonstructural proteins are translated direct...

(Y.Z.).In plant cells, secretory and endocytic routes intersect at the trans-Golgi network (TGN)/early endosome (EE), where cargos are further sorted correctly and in a timely manner. Cargo sorting is essential for plant survival and therefore necessitates complex molecular machinery. Adaptor proteins (APs) play key roles in this process by recruiting coat proteins and selecting cargos for different vesicle carriers. The m1 subunit of AP-1 in Arabidopsis (Arabidopsis thaliana) was recently identified at the TGN/EE and shown to be essential for cytokinesis. However, little was known about other cellular activities affected by mutations in AP-1 or the developmental consequences of such mutations. We report here that HAPLESS13 (HAP13), the Arabidopsis m1 adaptin, is essential for protein sorting at the TGN/EE. Functional loss of HAP13 displayed pleiotropic developmental defects, some of which were suggestive of disrupted auxin signaling. Consistent with this, the asymmetric localization of PIN-FORMED2 (PIN2), an auxin transporter, was compromised in the mutant. In addition, cell morphogenesis was disrupted. We further demonstrate that HAP13 is critical for brefeldin A-sensitive but wortmannin-insensitive post-Golgi trafficking. Our results show that HAP13 is a key link in the sophisticated trafficking network in plant cells.

These authors contributed equally to this work. SUMMARYSuccessful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)-anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C-terminal hydrophobic residues. GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell-cell communications in plants.

Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis.

ORCID IDs: 0000-0001-6345-6855 (J.-G.W.); 0000-0002-3501-5857 (Y.Z.).Plant vacuoles are versatile organelles critical for plant growth and responses to environment. Vacuolar proteins are transported from the endoplasmic reticulum via multiple routes in plants. Two classic routes bear great similarity to other phyla with major regulators known, such as COPII and Rab5 GTPases. By contrast, vacuolar trafficking mediated by adaptor protein-3 (AP-3) or that independent of the Golgi has few recognized cargos and none of the regulators. In search of novel regulators for vacuolar trafficking routes and by using a fluorescence-based forward genetic screen, we demonstrated that the multispan transmembrane protein, Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is an AP-3-mediated vacuolar cargo. We show that the tonoplast targeting of PAT10 is mediated by the AP-3 complex but independent of the Rab5-mediated post-Golgi trafficking route. We also report that AP-3-mediated vacuolar trafficking involves a subpopulation of COPII and requires the vacuolar tethering complex HOPS. In addition, we have identified two novel mutant alleles of AP-3d, whose point mutations interfered with the formation of the AP-3 complex as well as its membrane targeting. The results presented here shed new light on the vacuolar trafficking route mediated by AP-3 in plant cells.

Double fertilization in angiosperms requires the delivery of immotile sperm through pollen tubes, which enter embryo sacs to initiate synergid degeneration and to discharge. This fascinating process, called pollen tube reception, involves extensive communications between pollen tubes and synergids, within which few intracellular regulators involved have been revealed. Here, we report that vacuolar acidification in synergids mediated by AP1G and V-ATPases might be critical for pollen tube reception. Functional loss of AP1G or VHA-A, encoding the γ subunit of adaptor protein 1 or the shared component of two endomembrane V-ATPases, respectively, impaired synergid-controlled pollen tube reception and caused partial female sterility. AP1G works in parallel to the plasma membrane-associated receptor FERONIA in synergids, suggesting that synergid-mediated pollen tube reception requires proper sorting of vacuolar cargos by AP1G. Although AP1G did not mediate the targeting of V-ATPases, AP1G loss of function or the expression of AP1G-RNAi compromised vacuolar acidification mediated by V-ATPases, implying their genetic interaction. We propose that vacuolar acidification might represent a distinct cell-death mechanism specifically adopted by the plant phylum, which is critical for synergid degeneration during pollen tube reception. . The female gametophyte (FG), i.e., the embryo sac, is formed inside the ovules and contains an egg cell, a central cell, two synergid cells, and three antipodal cells (3). Upon landing on a receptive pistil, a pollen grain forms a long cylindrical extension, called a pollen tube, which extends inside female sporophytic tissues. To deliver immotile sperm, pollen tubes perceive attractive signals sent out by the FG, change their growth axis, and finally enter the embryo sacs through the micropyle (1-4). A process called "pollen tube reception" instructs the cessation and discharge of the penetrating pollen tube, leading to sperm release and fertilization (1-4).Studies have uncovered key female factors controlling pollen tube reception, including FERONIA (FER) (5-7), LORELEI (LRE) (8, 9), and early nodulin-like proteins (ENODLs) (10) and NORTIA (NTA) (11). These synergid receptors likely operate in the same genetic pathway (10-12) whose functional loss caused the failure of pollen tube discharge and led to reduced female fertility (5-9, 11). Male ligands are yet to be identified. However, recent studies showed that the production of reactive oxygen species (ROS) and Ca 2+ spiking are downstream events of FER-controlled pollen tube reception (13-16).Synergid degeneration, a form of programmed cell death (PCD), is a key step during pollen tube reception. Between the two synergid cells, a receptive synergid cell is the one that succeeds in interacting with the pollen tube and induces it to burst and undergoes cell death first. The other synergid cell that continues to persist and then undergoes cell death orchestrated by the fertilized egg cell and the central cell is the persistent synergid (3,11,1...

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