Effect of tris(2-carboxyethyl)phosphine and tertiary butyl alcohol on the performance of convection polymerase chain reaction.

Rapid and on-site DNA-based molecular detection has become increasingly important for sensitive, specific, and timely detection and treatment of various diseases. To prepare and store biomolecule-containing reagents stably, reducing agents are used during protein preparation, and freeze-drying technology has been applied to the protein reagents. Some of the additives used during these processes may affect subsequent processes such as polymerase chain reaction (PCR). In this study, we evaluated the impact of TCEP, a reducing agent, and TBA, a freeze-drying medium, on the performance of convection PCR (cPCR) using a battery-operable PCR device. Singleplex cPCR detection of a 249 bp amplicon from human genomic DNA suggested that approximately 82% of performance was achieved in the presence of 0.1 mM TCEP and 1% TBA. The limit of detection and the minimum number of cycles at which amplicons began to appear was a little lower (~ 82% efficiency) or higher (20 vs 15 cycles), respectively, in the chemical-treated group than in the control group. With larger amplicons of 500 bp, the chemical-treated group revealed approximately 78% of performance and amplicons started to appear at 20 cycles of cPCR in both groups. Similar results were obtained with multiplex cPCR amplification.

Development of fast and sensitive protocols for the detection of viral pathogens using a small portable convection PCR platform.

One of the most crucial steps for preventing viral pandemics is the early detection of the causative virus on site. Various molecular and immunological approaches have been developed for virus detection. In this study, we investigated the utility of the recently introduced convection polymerase chain reaction (cPCR) platform for the rapid and sensitive detection of various animal viruses in the field, including the foot-and-mouth disease virus (FMDV) and avian influenza viruses (AIVs). Primer sets were designed to simultaneously detect two highly conserved regions of the FMDV, including the 5' untranslated region (5'-UTR) and 3D gene, and to specifically amplify the NP and hemagglutinin (HA) genes of H5 and H9 subtypes of AIVs. The portable cPCR system was able to amplify from as low as 1 to 10 copies of viral cDNAs in the singleplex mode and 10 to 100 copies of viral cDNAs in the duplex mode within 21 min. Thus, our data suggest that the cPCR protocols developed in this study are highly sensitive and enable quick detection of animal viruses in biological samples.

Adaptor protein complex 2-mediated endocytosis is crucial for male reproductive organ development in Arabidopsis.

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2-dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.

The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis.

Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, brassinosteroid insensitive1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.

Arabidopsis µ-adaptin subunit AP1M of adaptor protein complex 1 mediates late secretory and vacuolar traffic and is required for growth.

Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, µ-adaptin, that selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their µ subunits in plants. Because of uncertain homology, the µ-adaptins of Arabidopsis have been designated muA through muD [Happel et al. (2004) Plant J 37(5):678-693]. Furthermore, only muD has been assigned to a specific AP complex, AP-3, involved in Golgi-vacuolar trafficking [Niihama et al. (2009) Plant Cell Physiol 50(12):2057-2068, Zwiewka et al. (2011) Cell Res 21(12):1711-1722, and Wolfenstetter et al. (2012) Plant Cell 24(1):215-232]. In contrast, the µ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 µ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans-Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans-Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.

Trafficking of vacuolar proteins: the crucial role of Arabidopsis vacuolar protein sorting 29 in recycling vacuolar sorting receptor.

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.

Recruitment of Arf1-GDP to Golgi by Glo3p-type ArfGAPs is crucial for golgi maintenance and plant growth.

ADP-ribosylation factor1 (Arf1), a member of the small GTP-binding proteins, plays a pivotal role in protein trafficking to multiple organelles. In its GDP-bound form, Arf1 is recruited from the cytosol to organelle membranes, where it functions in vesicle-mediated protein trafficking. However, the mechanism of Arf1-GDP recruitment remains unknown. Here, we provide evidence that two Glo3p-type Arf GTPase-activating proteins (ArfGAPs), ArfGAP domain8 (AGD8) and AGD9, are involved in the recruitment of Arf1-GDP to the Golgi apparatus in Arabidopsis (Arabidopsis thaliana). RNA interference plants expressing low levels of AGD8 and AGD9 exhibited abnormal Golgi morphology, inhibition of protein trafficking, and arrest of plant growth and development. In RNA interference plants, Arf1 was poorly recruited to the Golgi apparatus. Conversely, high levels of AGD8 and AGD9 induced Arf1 accumulation at the Golgi and suppressed Golgi disruption and inhibition of vacuolar trafficking that was caused by overexpression of AGD7. Based on these results, we propose that the Glo3p-type ArfGAPs AGD8 and AGD9 recruit Arf1-GDP from the cytosol to the Golgi for Arf1-mediated protein trafficking, which is essential for plant development and growth.

Functional identification of sorting receptors involved in trafficking of soluble lytic vacuolar proteins in vegetative cells of Arabidopsis.

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis ßFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

An A/ENTH domain-containing protein functions as an adaptor for clathrin-coated vesicles on the growing cell plate in Arabidopsis root cells.

Cytokinesis is the process of partitioning the cytoplasm of a dividing cell, thereby completing mitosis. Cytokinesis in the plant cell is achieved by the formation of a new cell wall between daughter nuclei using components carried in Golgi-derived vesicles that accumulate at the midplane of the phragmoplast and fuse to form the cell plate. Proteins that play major roles in the development of the cell plate in plant cells are not well defined. Here, we report that an AP180 amino-terminal homology/epsin amino-terminal homology domain-containing protein from Arabidopsis (Arabidopsis thaliana) is involved in clathrin-coated vesicle formation from the cell plate. Arabidopsis Epsin-like Clathrin Adaptor1 (AtECA1; At2g01600) and its homologous proteins AtECA2 and AtECA4 localize to the growing cell plate in cells undergoing cytokinesis and also to the plasma membrane and endosomes in nondividing cells. AtECA1 (At2g01600) does not localize to nascent cell plates but localizes at higher levels to expanding cell plates even after the cell plate fuses with the parental plasma membrane. The temporal and spatial localization patterns of AtECA1 overlap most closely with those of the clathrin light chain. In vitro protein interaction assays revealed that AtECA1 binds to the clathrin H chain via its carboxyl-terminal domain. These results suggest that these AP180 amino-terminal homology/epsin amino-terminal homology domain-containing proteins, AtECA1, AtECA2, and AtECA4, may function as adaptors of clathrin-coated vesicles budding from the cell plate.

Development and validation of a fast quantitative real-time PCR assay for the detection of African swine fever virus.

African swine fever virus (ASFV) is a double-stranded DNA virus that causes African swine fever (ASF), a lethal hemorrhagic fever that is highly contagious among domestic pigs and wild boars. Due to the high mortality rates and highly contagious nature of the ASF, it is important to develop a fast detection method for ASFV with high sensitivity and specificity to take an immediate action to stop wide spread of the virulent disease. Therefore, a fast and quantitative molecular detection method of ASFV is presented in this study. A total of 24 genotypes of ASFV have been identified based on nucleic acid sequences of the major capsid protein p72. The primers and probe of the present assay was designed to detect all of the p72-based genotypes of ASFV. The turnaround time for PCR detection was within 50 min which is at least about two-times faster compared to other PCR assays. Limit of detection (LoD) was 6.91 genomic copies/reaction for the most virulent genotype II. LoD values for other genotypes were within 10-20 copies/reaction. Cross-reactivity of the assay was validated using a panel of pathogens related to swine disease, and no cross-reactivity was observed. Positive and negative clinical samples (50 samples each) obtained from sick and healthy animals, were used to validate the assay. The results showed that 100% agreement for both positive and negative samples. In summary, the assay described in this study offers the advantage of rapid detection of all genotypes of ASFV with high sensitivity and specificity. The assay is a valuable tool both in clinical and laboratory uses for sensitive and fast detection of ASFV.

Data for the optimization of conditions for meat species identification using ultra-fast multiplex direct-convection PCR.

This article contains data related to the research article entitled "Ultra-fast DNA-based multiplex convection PCR method for meat species identification with possible on-site applications" (Song et al., 2017 [1]). Direct PCR that does not require prior DNA extraction is critical for ultra-fast molecular detection of meat species. We successfully acquired DNA by swab sampling in Taq DNA polymerase buffer. To reduce DNA sample preparation time, proteinase K incubation (0.2 µg/mL) and heat inactivation times were decreased to 10 min and 1 min, respectively. The analysis of swabbed DNA samples from mixed meat could differentiate meat species within the mixed sample. The swabbed DNA samples could be diluted 100 times without losing detection sensitivity.

The A/ENTH Domain-Containing Protein AtECA4 Is an Adaptor Protein Involved in Cargo Recycling from the trans-Golgi Network/Early Endosome to the Plasma Membrane.

Endocytosis and subsequent trafficking pathways are crucial for regulating the activity of plasma membrane-localized proteins. Depending on cellular and physiological conditions, the internalized cargoes are sorted at (and transported from) the trans-Golgi network/early endosome (TGN/EE) to the vacuole for degradation or recycled back to the plasma membrane. How this occurs at the molecular level remains largely elusive. Here, we provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane in Arabidopsis thaliana. AtECA4:sGFP primarily localized to the TGN/EE and plasma membrane (at low levels). Upon NaCl or mannitol treatment, AtECA4:sGFP accumulated at the TGN/EE at an early time point but was released from the TGN/EE to the cytosol at later time points. The ateca4 mutant showed higher resistance to osmotic stress and more sensitive to exogenous abscisic acid (ABA) than the wild type, as well as increased expression of ABA-inducible genes RD29A and RD29B. Consistently, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the prevacuolar compartment in ateca4, indicating a defect in recycling to the plasma membrane. However, the role of AtECA4 in cargo recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a crucial role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane.

Ultra-fast DNA-based multiplex convection PCR method for meat species identification with possible on-site applications.

The aim of this study was to develop an ultra-fast molecular detection method for meat identification using convection Palm polymerase chain reaction (PCR). The mitochondrial cytochrome b (Cyt b) gene was used as a target gene. Amplicon size was designed to be different for beef, lamb, and pork. When these primer sets were used, each species-specific set specifically detected the target meat species in singleplex and multiplex modes in a 24min PCR run. The detection limit was 1pg of DNA for each meat species. The convection PCR method could detect as low as 1% of meat adulteration. The stability of the assay was confirmed using thermal processed meats. We also showed that direct PCR can be successfully performed with mixed meats and food samples. These results suggest that the developed assay may be useful in the authentication of meats and meat products in laboratory and rapid on-site applications.

An ankyrin repeat domain of AKR2 drives chloroplast targeting through coincident binding of two chloroplast lipids.

In organellogenesis of the chloroplast from endosymbiotic cyanobacteria, the establishment of protein-targeting mechanisms to the chloroplast should have been pivotal. However, it is still mysterious how these mechanisms were established and how they work in plant cells. Here we show that AKR2A, the cytosolic targeting factor for chloroplast outer membrane (COM) proteins, evolved from the ankyrin repeat domain (ARD) of the host cell by stepwise extensions of its N-terminal domain and that two lipids, monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG), of the endosymbiont were selected to function as the AKR2A receptor. Structural analysis, molecular modeling, and mutational analysis of the ARD identified two adjacent sites for coincidental and synergistic binding of MGDG and PG. Based on these findings, we propose that the targeting mechanism of COM proteins was established using components from both the endosymbiont and host cell through a modification of the protein-protein-interacting ARD into a lipid binding domain.