Functional and Genetic Analysis Identify a Role for Arabidopsis ARGONAUTE5 in Antiviral RNA Silencing

RNA silencing functions as an antiviral defense through the action of DICER-like (DCL) and ARGONAUTE (AGO) proteins. In turn, plant viruses have evolved strategies to counteract this defense mechanism, including the expression of suppressors of RNA silencing. Potato virus X (PVX) does not systemically infect Arabidopsis thaliana Columbia-0, but is able to do so effectively in mutants lacking at least two of the four Arabidopsis DCL proteins. PVX can also infect Arabidopsis ago2 mutants, albeit less effectively than double DCL mutants, suggesting that additional AGO proteins may mediate anti-viral defenses. Here we show, using functional assays, that all Arabidopsis AGO proteins have the potential to target PVX lacking its viral suppressor of RNA silencing (VSR), P25, but that only AGO2 and AGO5 are able to target wild-type PVX. However, P25 directly affects only a small subset of AGO proteins, and we present evidence indicating that its protective effect is mediated by precluding AGO proteins from accessing viral RNA, as well as by directly inhibiting the RNA silencing machinery. In agreement with functional assays, we show that Potexvirus infection induces AGO5 expression and that both AGO2 and AGO5 are required for full restriction of PVX infection in systemic tissues of Arabidopsis.     

How Vacuolar Sorting Receptor Proteins Interact with Their Cargo Proteins: Crystal Structures of Apo and Cargo-Bound Forms of the Protease-Associated Domain from an Arabidopsis Vacuolar Sorting Receptor

In plant cells, soluble proteins are directed to vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptors (VSR). To understand how a VSR recognizes its cargo, we present the crystal structures of the protease-associated domain of VSR isoform 1 from Arabidopsis thaliana (VSR1PA) alone and complexed with a cognate peptide containing the barley (Hordeum vulgare) aleurain VSD sequence of ₁ADSNPIRPVT₁₀. The crystal structures show that VSR1PA binds the sequence, Ala-Asp-Ser, preceding the NPIR motif. A conserved cargo binding loop, with a consensus sequence of ₉₅RGxCxF₁₀₀, forms a cradle that accommodates the cargo-peptide. In particular, Arg-95 forms a hydrogen bond to the Ser-3 position of the VSD, and the essential role of Arg-95 and Ser-3 in receptor-cargo interaction was supported by a mutagenesis study. Cargo binding induces conformational changes that are propagated from the cargo binding loop to the C terminus via conserved residues in switch I-IV regions. The resulting 180° swivel motion of the C-terminal tail is stabilized by a hydrogen bond between Glu-24 and His-181. A mutagenesis study showed that these two residues are essential for cargo interaction and trafficking. Based on our structural and functional studies, we present a model of how VSRs recognize their cargos.     

Golgi-Dependent Transport of Vacuolar Sorting Receptors Is Regulated by COPII,AP1, and AP4 Protein Complexes in Tobacco

The cycling of vacuolar sorting receptors (VSRs) between early and late secretory pathway compartments is regulated by signals in the cytosolic tail, but the exact pathway is controversial. Here, we show that receptor targeting in tobacco (Nicotiana tabacum) initially involves a canonical coat protein complex II–dependent endoplasmic reticulum-to-Golgi bulk flow route and that VSR–ligand interactions in the cis-Golgi play an important role in vacuolar sorting. We also show that a conserved Glu is required but not sufficient for rate-limiting YXXɸ-mediated receptor trafficking. Protein–protein interaction studies show that the VSR tail interacts with the μ-subunits of plant or mammalian clathrin adaptor complex AP1 and plant AP4 but not that of plant and mammalian AP2. Mutants causing a detour of full-length receptors via the cell surface invariantly cause the secretion of VSR ligands. Therefore, we propose that cycling via the plasma membrane is unlikely to play a role in biosynthetic vacuolar sorting under normal physiological conditions and that the conserved Ile-Met motif is mainly used to recover mistargeted receptors. This occurs via a fundamentally different pathway from the prevacuolar compartment that does not mediate recycling. The role of clathrin and clathrin-independent pathways in vacuolar targeting is discussed.     

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.     

Trafficking of Vacuolar Sorting Receptors: New Data and New Problems

Vacuolar sorting receptors bind cargo ligands early in the secretory pathway and show that multivesicular body-vacuole fusion requires a Rab5/Rab7 GTPase conversion with consequences for retromer binding.To serve the purposes of controlled protein turnover, eukaryotic cells compartmentalize the required acid hydrolases in specialized digestive organelles: lysosomes in animals and vacuoles in yeasts and plants. Therefore, a reliable system must be in operation to prevent such proteolytic enzymes being released at the cell surface. Such a mechanism requires that acid hydrolases be identified and diverted away from the secretory flow to the plasma membrane (PM). This process is facilitated by receptors that recognize specific motifs in the hydrolases that are absent in secretory proteins. The most well-known example of this is the mannosyl 6-phosphate receptor (MPR), which is responsible for the sorting of lysosomal enzymes; indeed, it has become a paradigm for protein sorting in most cell biology textbooks. It entails the recognition of phosphomannan cargo ligands by MPRs in the trans-Golgi network (TGN) followed by the sequestration of the MPR-ligand complexes into specific transport vectors (clathrin-coated vesicles [CCVs]). These are then transported to an endosomal compartment (the early endosome [EE]) having a more acidic pH than the TGN, thereby causing the ligands to separate from the MPRs. The MPRs are subsequently recycled back to the TGN via retromer-coated carriers for another round of trafficking (for review, see Braulke and Bonifacino, 2009; Seaman, 2012).Many plant scientists support a scenario for the sorting of soluble vacuolar proteins and the trafficking of their receptors (vacuolar sorting receptors [VSRs]) that closely resembles that of the MPR system of mammalian cells (Hwang, 2008; De Marcos Lousa et al., 2012; Kang et al., 2012; Sauer et al., 2013; Xiang et al., 2013). This working model is based on three key observations: (1) VSRs were first identified in detergent-solubilized CCV fractions isolated from developing pea (Pisum sativum) cotyledons; (2) CCVs are regularly seen budding off the TGN in thin-sectioned plant cells; and (3) depending on the organism, VSRs and VSR-reporter constructs are found concentrated either in the TGN or in multivesicular prevacuolar compartments (PVCs) under steady-state conditions (Robinson and Pimpl, 2014a, 2014b, and refs. therein). Unfortunately, information on VSRs has not been obtained from a single experimental system. Although much work on Arabidopsis (Arabidopsis thaliana) VSR mutants has been published (for review, see De Marcos Lousa et al., 2012) and the majority of immunogold electron microscopic localization experiments have been performed in Arabidopsis, the majority of the fluorescence localizations, particularly with regard to VSR trafficking, have been carried out by transient expression in tobacco (Nicotiana tabacum; agroinfiltration for leaves and electroporation for protoplasts). Nevertheless, it should be stressed that sorting motifs for acid hydrolases and their corresponding receptors in the three major eukaryotic organismal groups differ considerably (Robinson et al., 2012). In addition, the secretory and endocytic pathways of plant cells contrast significantly with mammalian cells, the most important distinctions being (1) the lack of an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi apparatus in plants, (2) that plants have motile Golgi stacks rather than a perinuclear Golgi complex, and (3) the absence of an independent EE in plants, the function of which is assumed by the TGN (Contento and Bassham, 2012). While these differences do not automatically negate the validity of the above working model for VSR trafficking, they at least legitimize a more thorough analysis of the supporting data than has previously been the case (Robinson and Pimpl, 2014a, 2014b).The principal issues at stake are as follows. Where do VSRs bind and release their cargo ligands? What is the actual mechanism resulting in the separation of secretory from vacuolar cargo molecules? What is/are the precise role(s) of TGN-derived CCVs? And where does retromer pick up VSRs and where are they delivered to? The impact of several new publications on these points of dispute is the subject of this article.     

MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots

Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families.     

Failure of the Tomato Trans-Acting Short Interfering RNA Program to Regulate AUXIN RESPONSE FACTOR3 and ARF4 Underlies the Wiry Leaf Syndrome

Interfering with small RNA production is a common strategy of plant viruses. A unique class of small RNAs that require microRNA and short interfering (siRNA) biogenesis for their production is termed trans-acting short interfering RNAs (ta-siRNAs). Tomato (Solanum lycopersicum) wiry mutants represent a class of phenotype that mimics viral infection symptoms, including shoestring leaves that lack leaf blade expansion. Here, we show that four WIRY genes are involved in siRNA biogenesis, and in their corresponding mutants, levels of ta-siRNAs that regulate AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 are reduced, while levels of their target ARFs are elevated. Reducing activity of both ARF3 and ARF4 can rescue the wiry leaf lamina, and increased activity of either can phenocopy wiry leaves. Thus, a failure to negatively regulate these ARFs underlies tomato shoestring leaves. Overexpression of these ARFs in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) failed to produce wiry leaves, suggesting that the dramatic response in tomato is exceptional. As negative regulation of orthologs of these ARFs by ta-siRNA is common to land plants, we propose that ta-siRNA levels serve as universal sensors for interference with small RNA biogenesis, and changes in their levels direct species-specific responses.     

CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.     

Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii

Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.     

DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana Contributes to Potyvirus Movement and Transports to Plasmodesmata via the Early Secretory Pathway and the Actomyosin System

The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5′-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement.The movement of viruses in plants can be divided into three stages: intracellular, intercellular, and long-distance movement (Nelson and Citovsky, 2005; Benitez-Alfonso et al., 2010). Plasmodesmata (PD) are plasma membrane-mediated channels in cell walls that control the intercellular trafficking of micromolecules and macromolecules, including plant viruses (Boevink and Oparka, 2005; Lucas et al., 2009). Plant viruses encode movement proteins (MPs) that can regulate the size exclusion limit (SEL) of PD and mediate virus trafficking between cells (Lucas, 2006; Raffaele et al., 2009; Amari et al., 2010; Ueki et al., 2010). Based on the functions of MPs during virus movement, the viral MPs are divided into three major groups. The first group of MPs is represented by the 30-kD protein of Tobacco mosaic virus (TMV). The 30-kD proteins can interact with single-stranded RNAs and transport viral ribonucleoprotein complexes to cell walls, where they modify the SEL of PD to allow viruses to traverse the cell walls (Olesinski et al., 1996; Tzfira et al., 2000; Kawakami et al., 2004). The second group of MPs is known to form tubular structures that extend across the PD and allow virus to traverse. Viruses that encode this group of MPs include Cowpea mosaic virus, Grapevine fan leaf virus (GFLV), Cauliflower mosaic virus, and Tomato spotted wilt virus (Ritzenthaler and Hofmann, 2007; Amari et al., 2011). The third group of MPs is known as triple gene block proteins (TGBps), encoded by overlapping triple gene blocks. The three TGBps (TGBp1, TGBp2, and TGBp3) function coordinately to transport viral genomes to and through PD (Verchot-Lubicz, 2005; Jackson et al., 2009; Lim et al., 2009; Tilsner et al., 2013). Viruses that encode TGBps belong to the genera Potexvirus, Hordeivirus, and Pomovirus (Verchot-Lubicz et al., 2010). Potyviruses are different from the above viruses and lack a dedicated MP. To date, multiple potyviral proteins, including COAT PROTEIN, CYLINDRICAL INCLUSION (CI), HELPER COMPONENT PROTEINASE (HC-Pro), and VIRAL GENOME-LINKED PROTEIN, have been shown to function in the cell-to-cell movement of potyviruses (Nicolas et al., 1997; Rojas et al., 1997; Carrington et al., 1998; Wei et al., 2010).Viruses of Potyvirus (family Potyviridae), the largest genus of plant-infecting viruses, cause great economic losses to world agriculture production (Fauquet et al., 2005). The potyviral genome is a positive sense, single-stranded RNA of approximately 10 kb in length. It contains a large open reading frame (ORF) encoding a polyprotein that is later processed into 10 mature proteins by three virus-encoded proteinases (Riechmann et al., 1992; Fauquet et al., 2005). A +2 frame-shift Pretty Interesting Potyviridae (PIPO) ORF that is embedded within the P3 ORF was recently identified and proposed to produce a P3N-PIPO (for the protein encoded by 5′-terminus of P3 and frame-shift PIPO) fusion (Chung et al., 2008; Vijayapalani et al., 2012). The P3N-PIPOs of Turnip mosaic virus (TuMV) and Tobacco etch virus were previously shown to localize at PD, interact with CI in planta, and transport CI to PD in a CI:P3N-PIPO ratio-dependent manner (Wei et al., 2010). Soybean mosaic virus with a mutant PIPO domain failed to cause systemic infection in its host plant (Wen and Hajimorad, 2010). Therefore, the potyvirus P3N-PIPO has been suggested as the classical MP (Tilsner and Oparka, 2012; Vijayapalani et al., 2012).Viruses recruit host factors for their movement in plants (Chen et al., 2000; Raffaele et al., 2009; Amari et al., 2010; Ueki et al., 2010). Compared with the progresses on viral MP characterization, identifications of MP-interacting host proteins are much behind (Chen et al., 2000; Oparka, 2004; Raffaele et al., 2009; Amari et al., 2010). To date, about 20 host proteins have been identified to interact with specific viral MPs (Pallas and García, 2011). For example, the pectin methylesterase interacted with TMV MP, increased the SEL of PD, and facilitated TMV movement between cells (Chen et al., 2000); an ankyrin repeat-containing protein (ANK) interacted with TMV MP at PD, down-regulated callose formation, and aided viral movement (Ueki et al., 2010); the Arabidopsis (Arabidopsis thaliana) PLASMODESMATA-LOCALIZED PROTEIN1 (AtPDLP1) was reported to interact with GFLV MP and mediate tubule assembly during GFLV cell-to-cell movement in plants (Amari et al., 2010, 2011). TuMV P3N-PIPO was shown to interact with AtPCaP1, a plasma membrane cation-binding protein of Arabidopsis, and colocalize with this host protein at the PD. Knockout of AtPCaP1 expression resulted in a significant reduction of TuMV infection in Arabidopsis (Vijayapalani et al., 2012).Many viral MPs have been shown to traffic within plant cells via the early secretory pathway and/or along the actin filaments or microtubules. For example, the early secretory pathway and microtubules were required for GFLV MP trafficking to PD (Laporte et al., 2003). TuMV P3N-PIPO and CI were reported to utilize the early secretory pathway rather than the actomyosin motility system for their trafficking to PD (Wei et al., 2010). Several plant myosin motor proteins have been reported to participate in virus intracellular movement (Wei and Wang, 2008; Harries et al., 2010). Myosins VIII-1, VIII-2, and VIII-B were shown to transport a HEAT SHOCK PROTEIN70 homolog of Beet yellows virus to PD (Avisar et al., 2008a), but only myosin VIII-1 was needed for the nonstructural protein encoded by viral complementary strand of RNA4 (NSvc4) of Rice stripe virus traffic to PD (Yuan et al., 2011). A more recent study has indicated that both the secretory pathway and myosins XI-2 and XI-K were required for TuMV cell-to-cell movement (Agbeci et al., 2013). However, it remains largely unknown how the MP-interacting host factor(s) reach their target sites in cells.Tobacco vein banding mosaic virus (TVBMV) is a distinct potyvirus mainly infecting solanaceous crops (Tian et al., 2007; Yu et al., 2007; Zhang et al., 2011). In this article, we provide evidence showing the length requirements of the PIPO domains for its function in mediating TVBMV movement and the restoration of the movement-defective TVBMV mutants. We also show the interactions between TVBMV P3N-PIPO and CI and NbDREPP, a developmentally regulated plasma membrane protein in Nicotiana benthamiana, and the route by which NbDREPP traffics to PD. Silencing of NbDREPP expression in N. benthamiana significantly impeded the cell-to-cell movement of TVBMV.     

Analysis of Complementarity Requirements for Plant MicroRNA Targeting Using a Nicotiana benthamiana Quantitative Transient Assay

MicroRNAs (miRNAs) guide RNA-induced silencing complexes to target RNAs based on miRNA-target complementarity. Using a dual-luciferase based sensor system in Nicotiana benthamiana, we quantitatively assessed the relationship between miRNA-target complementarity and silencing efficacy measured at both the RNA and protein levels, using several conserved miRNAs and their known target sites from Arabidopsis thaliana. We found that naturally occurring sites have variable efficacies attributable to their complementarity patterns. We also observed that sites with a few mismatches to the miRNA 3′ regions, which are common in plants, are often equally effective and sometimes more effective than perfectly matched sites. By contrast, mismatches to the miRNA 5′ regions strongly reduce or eliminate repression efficacy but are nonetheless present in several natural sites, suggesting that in some cases, suboptimal miRNA efficacies are either tolerated or perhaps selected for. Central mismatches fully abolished repression efficacy in our system, but such sites then became effective miRNA target mimics. Complementarity patterns that are functional in animals (seed sites, 3′-supplementary sites, and centered sites) did not reliably confer repression, regardless of context (3′-untranslated region or open reading frame) or measurement type (RNA or protein levels). Overall, these data provide a robust and empirical foundation for understanding, predicting, and designing functional miRNA target sites in plants.