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In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A β-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.Potassium (K) is one of the three most important macronutrients and the most abundant cation in plants. As a major osmoticum in the vacuole, K drives the generation of turgor pressure, enabling cell expansion. In the vascular tissue, K is an important participant in the generation of root pressure (for review, see Wegner, 2014 [including his new hypothesis]). In the phloem, K is critical for the transport of photoassimilates from source to sink (Marschner, 1996; Deeken et al., 2002; Gajdanowicz et al., 2011). In addition, enhancing K absorption and decreasing sodium (Na) accumulation is a major strategy of glycophytes in salt stress tolerance (Maathuis and Amtmann, 1999; Munns and Tester, 2008; Shabala and Cuin, 2008).Plants acquire K through K-permeable proteins at the root surface. Since available K concentration in the soil may vary by 100-fold, plants have developed multiple K uptake systems for adapting to this variability (Epstein et al., 1963; Grabov, 2007; Maathuis, 2009). In a classic K uptake experiment in barley (Hordeum vulgare), root K absorption has been described as a high-affinity and low-affinity biphasic transport process (Epstein et al., 1963). It is generally assumed that the low-affinity transport system (LATS) in the roots mediates K uptake in the millimolar range and that the activity of this system is insensitive to external K concentration (Maathuis and Sanders, 1997; Chérel et al., 2014). In contrast, the high-affinity transport system (HATS) was rapidly up-regulated when the supply of exogenous K was halted (Glass, 1976; Glass and Dunlop, 1978).The membrane transporters for K flux identified in plants are generally classified into three channels and three transporter families based on phylogenetic analysis (Mäser et al., 2001; Véry and Sentenac, 2003; Lebaudy et al., 2007; Alemán et al., 2011). For K uptake, it was predicted that, under most circumstances, K transporters function as HATS, while K-permeable channels mediate LATS (Maathuis and Sanders, 1997). However, a root-expressed K channel in Arabidopsis (Arabidopsis thaliana), Arabidopsis K Transporter1 (AKT1), mediates K absorption over a wide range of external K concentrations (Sentenac et al., 1992; Lagarde et al., 1996; Hirsch et al., 1998; Spalding et al., 1999), while evidence is accumulating that many K transporters, including members of the K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) family, are low-affinity K transporters (Quintero and Blatt, 1997; Senn et al., 2001), implying that functions of plant K channels and transporters overlap at different K concentration ranges.Out of the three families of K transporters, cation proton antiporter (CPA), high affinity K/Na transporter (HKT), and KT/HAK/KUP, CPA was characterized as a K+(Na+)/H+ antiporter, HKT may cotransport Na and K or transport Na only (Rubio et al., 1995; Uozumi et al., 2000), while KT/HAK/KUP were predicted to be H+-coupled K+ symporters (Mäser et al., 2001; Lebaudy et al., 2007). KT/HAK/KUP were named by different researchers who first identified and cloned them (Quintero and Blatt, 1997; Santa-María et al., 1997). In plants, the KT/HAK/KUP family is the largest K transporter family, including 13 members in Arabidopsis and 27 members in the rice (Oryza sativa) genome (Rubio et al., 2000; Mäser et al., 2001; Bañuelos et al., 2002; Gupta et al., 2008). Sequence alignments show that genes of this family share relatively low homology to each other. The KT/HAK/KUP family was divided into four major clusters (Rubio et al., 2000; Gupta et al., 2008), and in cluster I and II, they were further separated into A and B groups. Genes of cluster I or II likely exist in all plants, cluster III is composed of genes from both Arabidopsis and rice, while cluster IV includes only four rice genes (Grabov, 2007; Gupta et al., 2008).The functions of KT/HAK/KUP were studied mostly in heterologous expression systems. Transporters of cluster I, such as AtHAK5, HvHAK1, OsHAK1, and OsHAK5, are localized in the plasma membrane (Kim et al., 1998; Bañuelos et al., 2002; Gierth et al., 2005) and exhibit high-affinity K uptake in the yeast Saccharomyces cerevisiae (Santa-María et al., 1997; Fu and Luan, 1998; Rubio et al., 2000) and in Escherichia coli (Horie et al., 2011). Transporters of cluster II, like AtKUP4 (TINY ROOT HAIRS1, TRH1), HvHAK2, OsHAK2, OsHAK7, and OsHAK10, could not complement the K uptake-deficient yeast (Saccharomyces cerevisiae) but were able to mediate K fluxes in a bacterial mutant; they might be tonoplast transporters (Senn et al., 2001; Bañuelos et al., 2002; Rodríguez-Navarro and Rubio, 2006). The function of transporters in clusters III and IV is even less known (Grabov, 2007).Existing data suggest that some KT/HAK/KUP transporters also may respond to salinity stress (Maathuis, 2009). The cluster I transporters of HvHAK1 mediate Na influx (Santa-María et al., 1997), while AtHAK5 expression is inhibited by Na (Rubio et al., 2000; Nieves-Cordones et al., 2010). Expression of OsHAK5 in tobacco (Nicotiana tabacum) BY2 cells enhanced the salt tolerance of these cells by accumulating more K without affecting their Na content (Horie et al., 2011).There are only scarce reports on the physiological function of KT/HAK/KUP in planta. In Arabidopsis, mutation of AtKUP2 (SHORT HYPOCOTYL3) resulted in a short hypocotyl, small leaves, and a short flowering stem (Elumalai et al., 2002), while a loss-of-function mutation of AtKUP4 (TRH1) resulted in short root hairs and a loss of gravity response in the root (Rigas et al., 2001; Desbrosses et al., 2003; Ahn et al., 2004). AtHAK5 is the only system currently known to mediate K uptake at concentrations below 0.01 mm (Rubio et al., 2010) and provides a cesium uptake pathway (Qi et al., 2008). AtHAK5 and AtAKT1 are the two major physiologically relevant molecular entities mediating K uptake into roots in the range between 0.01 and 0.05 mm (Pyo et al., 2010; Rubio et al., 2010). AtAKT1 may contribute to K uptake within the K concentrations that belong to the high-affinity system described by Epstein et al. (1963).Among all 27 members of the KT/HAK/KUP family in rice, OsHAK1, OsHAK5, OsHAK19, and OsHAK20 were grouped in cluster IB (Gupta et al., 2008). These four rice HAK members share 50.9% to 53.4% amino acid identity with AtHAK5. OsHAK1 was expressed in the whole plant, with maximum expression in roots, and was up-regulated by K deficiency; it mediated high-affinity K uptake in yeast (Bañuelos et al., 2002). In this study, we examined the tissue-specific localization and the physiological functions of OsHAK5 in response to variation in K supply and to salt stress in rice. By comparing K uptake and translocation in OsHAK5 knockout (KO) mutants and in OsHAK5-overexpressing lines with those in their respective wild-type lines supplied with different K concentrations, we found that OsHAK5 not only mediates high-affinity K acquisition but also participates in root-to-shoot K transport as well as in K-regulated salt tolerance.  相似文献   

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Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.  相似文献   

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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.  相似文献   

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Processing (P)-bodies are cytoplasmic RNA protein aggregates responsible for the storage, degradation, and quality control of translationally repressed messenger RNAs in eukaryotic cells. In mammals, P-body-related RNA and protein exchanges are actomyosin dependent, whereas P-body movement requires intact microtubules. In contrast, in plants, P-body motility is actin based. In this study, we show the direct interaction of the P-body core component DECAPPING PROTEIN1 (DCP1) with the tails of different unconventional myosins in Arabidopsis (Arabidopsis thaliana). By performing coexpression studies with AtDCP1, dominant-negative myosin fragments, as well as functional full-length myosin XI-K, the association of P-bodies and myosins was analyzed in detail. Finally, the combination of mutant analyses and characterization of P-body movement patterns showed that myosin XI-K is essential for fast and directed P-body transport. Together, our data indicate that P-body movement in plants is governed by myosin XI members through direct binding to AtDCP1 rather than through an adapter protein, as known for membrane-coated organelles. Interspecies and intraspecies interaction approaches with mammalian and yeast protein homologs suggest that this mechanism is evolutionarily conserved among eukaryotes.Newly synthesized mRNAs are processed and transported from the nucleus into the cytoplasm. Their subsequent fate depends on a regulatory balance between cytoplasmic translation and mRNA degradation. Both processes are modulated by mRNA-binding proteins, which influence the translational efficiency, stability, and localization of mRNA molecules. Over the last decade, it became apparent that messenger ribonucleoproteins that are translationally repressed and associated with the cytoplasmic 5′ to 3′ decapping machinery tend to assemble into a newly discovered class of highly dynamic mRNA granules, termed processing (P)-bodies (Sheth and Parker, 2003; Anderson and Kedersha, 2006; Parker and Sheth, 2007; Franks and Lykke-Andersen, 2008). P-bodies were initially identified as discrete cytoplasmic punctae to which the mouse 5′ to 3′ exonuclease localizes (Bashkirov et al., 1997). Since then, they have been established as major cytoplasmic sites for the storage of translationally repressed mRNAs, 5′ to 3′ mRNA degradation, quality control, and microRNA-dependent gene silencing (Dunckley and Parker, 1999; Sheth and Parker, 2003; Cougot et al., 2004; Sen and Blau, 2005).Although their complete composition is still not known, a set of proteins related to these different functions has been identified and shown to colocalize with each other in these cytoplasmic foci (Ingelfinger et al., 2002; van Dijk et al., 2002; Sheth and Parker, 2003; Eulalio et al., 2007; Parker and Sheth, 2007). P-bodies are commonly described as highly mobile and dynamic structures (Yang et al., 2004; Aizer et al., 2008; Loschi et al., 2009). In Drosophila melanogaster and mammals, they show a microtubule-dependent pattern of motility (Aizer et al., 2008; Loschi et al., 2009), which is inhibited by drugs interfering with the microtubule cytoskeleton (Aizer et al., 2008). Actin, by contrast, is important for the delivery of RNA and P-body components to P-bodies and for their assembly. This is suggested by the colocalization of mammalian myosin Va with the P-body-associated protein eIF4E (for Eukaryotic Translation Initiation Factor4E) and further by the reduction of P-body number and transport of eIF4E to P-bodies when expressing dominant-negative myosin Va fragments (Lindsay and McCaffrey, 2011). An association of myosins with P-bodies was also reported in yeast. In yeast (Saccharomyces cerevisiae), Myo2p was successfully used to immunoprecipitate complex ribonucleoprotein particles, including hundreds of mRNAs and some P-body components, such as DECAPPING PROTEIN1 (Dcp1p), SM-LIKE PROTEIN1, DNA TOPOISOMERASE 2-ASSOCIATED PROTEIN1, DExD/H-BOX HELICASE1, and EXORIBONUCLEASE1 (Chang et al., 2008).In plants, P-body movement appears to be based on the actin cytoskeleton (Hamada et al., 2012), although myosins have not been implicated in P-body movement. In Arabidopsis (Arabidopsis thaliana), the myosin family contains 17 proteins (Reddy and Day, 2001; Peremyslov et al., 2011), which are grouped into two classes, XI and VIII. Members of the class VIII myosins are associated with endosomal structures, the endoplasmic reticulum, and plasmodesmata (Avisar et al., 2008a; Golomb et al., 2008; Sattarzadeh et al., 2008). The 13 class XI myosins show a domain structure that is similar to those of fungi and animal myosin V proteins. They are composed of an N-terminal motor domain, for both actin association as well as ATP hydrolysis (Kinkema et al., 1994; Tominaga et al., 2003), an internal neck domain, comprising a stretch of six calmodulin-binding IQ motifs (Kinkema and Schiefelbein, 1994), and the C-terminal tail region, which contains a coiled-coil domain of variable extent and a bipartite globular cargo-binding domain (Pashkova et al., 2006; Li and Nebenführ, 2007). The Arabidopsis XI myosins have been implicated in the movement of various membranous organelles and vesicles (for review, see Sparkes, 2010, 2011). Colocalization studies and the analysis of movement in myosin mutants revealed that plant XI myosins are involved in the rapid trafficking of Golgi stacks, mitochondria, and peroxisomes (Avisar et al., 2008b, 2009; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008), the long-distance transport of endomembrane vesicles (Peremyslov et al., 2012), plant-specific transport vesicles (Peremyslov et al., 2013), motility of plastids (Natesan et al., 2009; Sattarzadeh et al., 2009), as well as endoplasmic reticulum flow (Peremyslov et al., 2010; Ueda et al., 2010) and remodeling (Sparkes et al., 2009).In the light of recent data available for the mammalian and yeast systems, myosins appear to be good candidates to also mediate the movement of membraneless RNA granules, such as P-bodies. The molecular basis linking P-bodies to the actin cytoskeleton is not known in any system. In this study, we demonstrate that the P-body core component DECAPPING PROTEIN1 (DCP1) physically interacts with myosins in mammals, yeast, and Arabidopsis and also in interspecies experiments. In support of a direct interaction between myosins and P-bodies, Arabidopsis DCP1 (AtDCP1) colocalizes with the full-size myosin XI-K. Overexpression of dominant-negative myosin tail domains inhibited P-body movement efficiently. The analysis of the respective mutants revealed that in two xi-k alleles, P-body movement was almost completely abolished. This defective motility was efficiently restored in triple myosin knockout plants expressing full-size myosin XI-K. Taken together, these data demonstrate that P-body movement is governed by unconventional myosins and that their anchoring to myosins occurs by direct binding to a core component rather than by connecting to specific adapter proteins, as known for eukaryotic organelle transport.  相似文献   

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Heavy metal-transporting P-type ATPase (HMA) has been implicated in the transport of heavy metals in plants. Here, we report the function and role of an uncharacterized member of HMA, OsHMA5 in rice (Oryza sativa). Knockout of OsHMA5 resulted in a decreased copper (Cu) concentration in the shoots but an increased Cu concentration in the roots at the vegetative stage. At the reproductive stage, the concentration of Cu in the brown rice was significantly lower in the mutants than in the wild-type rice; however, there was no difference in the concentrations of iron, manganese, and zinc between two independent mutants and the wild type. The Cu concentration of xylem sap was lower in the mutants than in the wild-type rice. OsHMA5 was mainly expressed in the roots at the vegetative stage but also in nodes, peduncle, rachis, and husk at the reproductive stage. The expression was up-regulated by excess Cu but not by the deficiency of Cu and other metals, including zinc, iron, and manganese, at the vegetative stage. Analysis of the transgenic rice carrying the OsHMA5 promoter fused with green fluorescent protein revealed that it was localized at the root pericycle cells and xylem region of diffuse vascular bundles in node I, vascular tissues of peduncle, rachis, and husk. Furthermore, immunostaining with an antibody against OsHMA5 revealed that it was localized to the plasma membrane. Expression of OsHMA5 in a Cu transport-defective mutant yeast (Saccharomyces cerevisiae) strain restored the growth. Taken together, OsHMA5 is involved in loading Cu to the xylem of the roots and other organs.Plants require nutrient elements to maintain normal growth and development. A number of different transporters, such as Cation Diffusion Facilitator, Natural resistance-associated macrophage protein, ATP-Binding Cassette, Zinc- and Iron-regulated-like Protein, and P-type ATPase, have been reported to be involved in the uptake, translocation, distribution, and homeostasis of nutrients (Hall and Williams, 2003; Krämer et al., 2007; Palmer and Guerinot, 2009). Among them, heavy metal-transporting P-type ATPase (HMA), the P1B subfamily of the P-type ATPase superfamily, has been implicated in heavy metal transport (Williams and Mills, 2005; Grotz and Guerinot, 2006; Argüello et al., 2007; Burkhead et al., 2009). There are eight and nine members of P1B-ATPase in Arabidopsis thaliana and rice (Oryza sativa), respectively (Williams and Mills, 2005). They are divided into two groups: zinc (Zn)/cadmium (Cd)/cobalt/lead (Pb) and copper (Cu)/silver transporters (Williams and Mills, 2005). AtHMA1 to AtHMA4 in A. thaliana and OsHMA1 to OsHMA3 in rice belong to the former group, while AtHMA5 to AtHMA8 and OsHMA4 to OsHMA9 belong to the latter group, although AtHMA1 has also been shown to transport Zn, Cu, and calcium (Axelsen and Palmgren, 2001; Williams and Mills, 2005; Seigneurin-Berny et al., 2006; Moreno et al., 2008; Kim et al., 2009).All members of HMAs in A. thaliana have been functionally characterized. AtHMA1 is involved in delivering Cu to the stroma, exporting Zn2+ from the chloroplast, or as a Ca2+/heavy metal transporter to the intracellular organelle (Seigneurin-Berny et al., 2006; Moreno et al., 2008; Kim et al., 2009). AtHMA2 and AtHMA4 localized at the pericycle are partially redundant and responsible for the release of Zn into the xylem (xylem loading) as well as Cd (Hussain et al., 2004; Verret et al., 2004; Wong and Cobbett, 2009; Wong et al., 2009), while AtHMA3 localized at the tonoplast plays a role in the detoxification of Zn/Cd/cobalt/Pb by mediating them into the vacuole (Morel et al., 2009; Chao et al., 2012). On the other hand, AtHMA5 is involved in the Cu translocation from roots to shoots or Cu detoxification of roots (Andrés-Colás et al., 2006; Kobayashi et al., 2008). AtHMA6 (PAA1, for P-type ATPase of Arabidopsis1) localized at the chloroplast periphery has been proposed to transport Cu over the chloroplast envelope, whereas AtHMA8 (PAA2) localized at the thylakoid membranes most likely transports Cu into the thylakoid lumen to supply plastocyanin (Shikanai et al., 2003; Abdel-Ghany et al., 2005). Finally, AtHMA7 (RESPONSIVE-TO-ANTAGONIST1) is responsible for delivering Cu to ethylene receptors and Cu homeostasis in the seedlings (Hirayama et al., 1999; Woeste and Kieber, 2000; Binder et al., 2010).By contrast, only three out of nine P-type ATPase members have been functionally characterized in rice. OsHMA2 was recently reported to be involved in the root-shoot translocation of Zn and Cd (Satoh-Nagasawa et al., 2012; Takahashi et al., 2012; Yamaji et al., 2013). Furthermore, OsHMA2 at the node is required for preferential distribution of Zn to young leaves and panicles (Yamaji et al., 2013). OsHMA3 is localized to the tonoplast of the root cells and responsible for the sequestration of Cd into the vacuoles (Ueno et al., 2010; Miyadate et al., 2011). On the other hand, OsHMA9 was mainly expressed in vascular tissues, including the xylem and phloem (Lee et al., 2007). The knockout lines accumulated more Zn, Cu, Pb, and Cd, suggesting its role in the efflux of these metals from the cells (Lee et al., 2007).Some members of P-type ATPase have also been identified in other plant species, including barley (Hordeum vulgare), wheat (Triticum aestivum), Thlaspi caerulescens (Noccaea caerulescens), and Arabidopsis halleri. HvHMA1 from barley might be involved in mobilizing Zn and Cu during the stage of grain filling (Mikkelsen et al., 2012). HvHMA2 from barley and TaHMA2 from wheat showed similar functions as OsHMA2 in rice (Mills et al., 2012; Tan et al., 2013). AhHMA3 in A. halleri, a Zn hyperaccumulator, is probably involved in high Zn accumulation (Becher et al., 2004; Chiang et al., 2006). Furthermore, AhHMA4 for Zn translocation showed a higher expression level (Chiang et al., 2006; Hanikenne et al., 2008). On the other hand, TcHMA3 from ecotype Ganges of T. caerulescens, a Cd hyperaccumulator, plays an important role in the detoxification of Cd by sequestering Cd into the vacuole of the leaves (Ueno et al., 2011). High expression of TcHMA4 (NcHMA4) was also reported in T. caerulescens (Bernard et al., 2004; Papoyan and Kochian, 2004; Craciun et al., 2012).In this study, we investigated the function and role of an uncharacterized member of P-type ATPase in rice, OsHMA5. We found that OsHMA5 is involved in the xylem loading of Cu at both the vegetative and reproductive growth stages.  相似文献   

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