Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

Background  

Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

Antigen Recognition By Autoreactive Cd4+ Thymocytes Drives Homeostasis Of The Thymic Medulla

The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4⁺ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4⁺ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4⁺ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.     

Phloem unloading of potato virus X movement proteins is regulated by virus and host factors

To determine the requirements for viral proteins exiting the phloem, transgenic plants expressing green fluorescent protein (GFP) fused to the Potato virus X (PVX) triple gene block (TGB)p1 and coat protein (CP) genes were prepared. The fused genes were transgenically expressed from the companion cell (CC)-specific Commelina yellow mottle virus (CoYMV) promoter. Transgenic plants were selected for evidence of GFP fluorescence in CC and sieve elements (SE) and proteins were determined to be phloem mobile based on their ability to translocate across a graft union into nontransgenic scions. Petioles and leaves were analyzed to determine the requirements for phloem unloading of the fluorescence proteins. In petioles, fluorescence spread throughout the photosynthetic vascular cells (chlorenchyma) but did not move into the cortex, indicating a specific barrier to proteins exiting the vasculature. In leaves, fluorescence was mainly restricted to the veins. However, in virus-infected plants or leaves treated with a cocktail of proteasome inhibitors, fluorescence spread into leaf mesophyll cells. These data indicate that PVX contributes factors which enable specific unloading of cognate viral proteins and that proteolysis may play a role in limiting proteins in the phloem and surrounding chlorenchyma.     

Plasmodesmata transport of GFP and GFP fusions requires little energy and transitions during leaf expansion

Plasmodesmata (Pd) are symplastic channels between neighboring plant cells and are key in plant cell-cell signaling. Viruses of proteins, nucleic acids, and a wide range of signaling macromolecules move across Pd. Protein transport Pd is regulated by development and biotic signals. Recent investigations utilizing the Arrhenius equation or Coefficient of conductivity showed that fundamental energetic measurements used to describe transport of proteins across membrane pores or the nuclear pore can also apply to protein movement across Pd. As leaves continue to expand, Pd transport of proteins declines which may result from changes in cell volume, Pd density or Pd structure.Key words: plasmodesmata, diffusion, GFP, viral transport, PVX, triple gene blockResearchers have argued for the last decade that movement of proteins and other macromolecules across Pd is regulated by development, stress and biotic signals. There are four current models describing different mechanisms of Pd transport. First is the non cell autonomous protein (NCAP) pathway that carries ribonucleoprotein complexes across Pd. NCAPs often carry RNA in a ribonucleoprotein complex to the Pd.^1–4 This mode of transport involved targeted movement, meaning that a set of proteins must dock within the Pd to gate it open to enable transfer between cells. Proteins which are normally too large to move across Pd can gate open Pd to enable its own transfer into neighboring cells. This is contrasted by nontargetted movement, which is passive movement of proteins that are sufficiently small enough to pass between cells.^5,6 The green fluorescent protein (GFP) has been described as a protein whose movement is non-targeted, meaning that it can diffuse across Pd. Reasons that we do not see continuous movement of small proteins between cells include protein compartmentalization or subcellular targeting signals. For example proteins may be synthesized and modified via the ER and Golgi networks and then transferred into vesicles and transported within cells to their destination. These proteins would not be free in the cytosol for diffusion across Pd. Alternatively, proteins which have dominant subcellular targeting signals which direct them to certain organelles such as the nucleus, peroxisome, or other destination would not be free to move across Pd.^5,6 A third model represents proteins in the ER that move laterally along the membrane or through the ER lumen into neighboring cells. This transport is quite rapid and investigations are ongoing to determine how this is regulated.^7–11 Finally, there is vesicle transport which deliver cargo to Pd.^12,13 The origin of these vesicles is still under investigation. Much more research has been accomplished toward defining non-targeted movement and the NCAP pathway while the ER and vesicle transport pathways are only recently described and very little is known about the regulatory mechanisms underlying these pathways.Pd permeability is governed in part by architecture, but also by key regulatory factors that determine Pd conductivity. Factors such as mysoin VIII, actin and calreticulin were identified in Pd which likely regulate expansion and contraction.^14–19 In addition calcium, ATP and plant hormones can downregulate Pd permeability during development and stress.^20,21 The tools for measuring Pd permeability has been to study the transport of fluorescently tagged proteins, fluorescent dextran beads, GFP or GFP fusions following microinjection or biolistic delivery to the cytoplasm of one cell.^22–26 Then video imaging or captured still images at select time intervals are used to characterize Pd transport. Until recently researchers quantified movement by the frequency they observed a certain type of movement. Therefore our ability to describe Pd permeability has been limited.Evidence that ATP impacts Pd conductivity has led investigations to explore the energy requirements for macromolecular transport across Pd. By understanding the energy requirements for transport of various proteins and nucleic acids we can better characterize passive or active transport processes. Toward this end two recent studies detailed quantitative approaches that can be employed to describe the developmental and energy requirements cell-to-cell transport of cytosolic proteins. Both papers used biolistic bombardment to deliver plasmids expressing GFP or GFP fusions to tobacco leaf epidermal cells and then captured still images of GFP fluorescence in neighboring cells. We employed the Arrhenius equation to characterize transport of GFP or GFP fused to the Potato virus X (PVX) TGBp1 movement protein. PVX TGBp1 was selected to compare with GFP alone since it is known to gate open Pd and has ATPase activity.⁴⁵ We predicted that the abilities of GFP alone and GFP-TGBp1 to move across Pd might be different and were surprised to learn that the energy for transport of both proteins was similar. This project established the principle that GFP and GFP-TGBp1 transport is temperature dependent showing a linear relationship between protein movement and the temperatures at which leaves were incubated.Green fluorescent sites on bombarded leaves were scored for the movement or no movement. Movement is defined as evidence of fluorescence in 2 or more cells at 24 h and no movement is when fluorescence is in single cells. These were then presented as a percentage of the total. So by digitizing the representation of movement we were able to represent a linear relationship between movement and temperature. Representing movement in this way also enabled us to represent movement values on a logarithm scale necessary for a classic Arrhenius plot. The activation energy (Ea) was calculated by fitting the data to the Arrhenius equation:% movement = A exp(-Ea/RT); and the Ea for GFP and GFP-TGBp1 was approximately 38 kJ/mol and 29 kJ/mol. These low activation energies are comparable to the reported 30 kJ/mol calculated for temperature dependence of protein transport through the cytosol. Evidence that GFP movement across Pd requires slightly more energy than through the cytoplasm suggests there may be some resistance within the pore. The lower energy for GFP-TGBp1 suggests that movement is facilitated, which likely reflects Pd gating by TGBp1, enabling greater transfer between cells.Liarzi and Epel define a new coefficient of conductivity of Pd.⁴² This study also concluded that cell-to-cell transport of GFP in nontransgenic or transgenic N. benthamiana plants expressing the Tobacco mosaic virus (TMV) movement protein (MP) is temperature dependent. The method was to measure the exponential decay, which is a measure of the impedance to diffusion driven cell-to-cell movement of fluorescence. The exponential decay factor? was determined by calculating the ratio of GFP fluorescence in bombarded cell 0 and neighboring cells. This was presented as a measure of fluorescence transfer from cell 0 to cell 1 to cell 2. A coefficient for conductivity C(Pd), 1/? for GFP was reflective of diffusion. Interestingly the (TMV) MP did not increase conductivity of GFP between neighboring leaf epidermal cells indicating that movement was already maximal. Considering prior reports that the TMV MP shows preferential spread into mesophyll rather than epidermal tissues during virus infection, it is possible that preferential spread into mesophyll cells would prevent experimental efforts to achieve improved conductivity of GFP between epidermal cells.^27,28 In which case the absence of a trans effect of TMV MP on GFP conductivity in the epidermis may not be surprising. In fact, prior investigations of TMV MP gating activities were conducted in mesophyll cells.^29,30 The best explanation for the combined studies is that cytosolic GFP can diffuse across Pd , however viral proteins which gate Pd enable their own low energy transfer into neighboring cells without allowing other proteins to flood into neighboring cells. Therefore viral movement proteins, such as PVX TGBp1 and TMV MP, which gate Pd provide themselves with an energetic advantage for transport into neighboring cells which is essential for rapid dissemination of virus into further tissues.These studies provide an interesting contrast between PVX TGBp1 and TMV MP. Both proteins gate open Pd for virus cell-to-cell transport, but there seems to be differences in how these proteins function in epidermal cells. This is likely due to their different roles in promoting virus cell-to-cell movement. PVX TGBp1 protein is also a suppressor of RNA silencing. We recently proposed a model in which TGBp1 rapidly moves from cell-to-cell ahead of virus infection, to suppress the cell''s RNA degradation machinery, as a means to promote infection.³¹ The TMV MP on the other hand is reported to bind viral RNA for transfer into neighboring cells.^32,33 Therefore, the different observations of PVX TGBp1 and TMV MP transport between epidermal cells likely reflect their functional differences. Both proteins are required for virus cell-to-cell movement, but their exact roles in virus movement are not identical.As mentioned earlier, Pd permeability is downregulated during plant development. Research tracking GFP diffusion through Pd in embryonic cells, in young emerging leaves, and in fully expanded leaves showed that fluorescence is highly mobile between cells in young tissues but is restricted during maturation. Viral movement proteins such as Cucumber mosaic virus 3a, and PVX TGBp1 remain highly mobile in mature leaves because they gate open Pd under conditions that normally restrict movement of GFP.^34,35 Schoenknecht et al., undertook a straightforward investigation of leaf maturation describing Pd transport in relationship to leaf area expansion. The outcome of this study was evidence that GFP movement between cells declines as leaves expand.It is reasonable to consider that simultaneous changes in gene expression and physiology is reflected in a downward trend in Pd conductivity and an increased requirement for Pd gating to enable selected transport of macromolecules between cells. In Arabidopsis embryos there is an obvious transition between developmental stages which are also represented by a decline in the ability for GFP to diffuse across Pd.^36,37 A detailed analysis of Pd structure in source and sink tissues revealed that Pd are simple single channeled structures in sink tissues while source tissues contain predominantly “H” shaped branching Pd structures. The change in Pd structure has been correlated with changes in conductivity and is often correlated with changes in sink to source metabolism.^38,39 The sink-to-source transition in leaf development is typically monitored using phloem loading of carboxyfluorescein diacetate. Leaves where CF dye unloads are defined as sink leaves and leaves that were restricted in dye unloading were defined as source leaves. Then biolistic bombardment of GFP expressing plasmids to sink and source leaves revealed that GFP readily diffuses across Pd in sink leaves but is more often restricted in source leaves.^{26,34,40–42}Leaf development is typically defined as a transition from juvenile to adult which is represented by homeotic transformations as well as vegetative phase changes.^43,44 Source and sink regions of a leaf have been shown to correlate with changes in Pd structure and conductivity during leaf expansion. However, in our study we found that N. tabacum leaves identified as source during week 2 or 3 would continue to expand over an 8 week period to twice or three times the leaf area which provides a real indication that the source designation may not entirely reflect final leaf maturation or completion of leaf development.⁴⁵> For example, as cells transition from sink to source physiology it is suggested that the frequency of single channeled Pd declines while the frequency of branched Pd increases.³⁹ It is possible that even after leaves transition into photosynthetic sources that Pd architecture continues to change and there is a further decline in the proportion of single channel to branched channels. Therefore either the change in cell volume or Pd architecture or both can slow-down diffusion of GFP between cells.Researchers often point to the ER continuity between cells as a driving force for Pd formation and function. During cell division the cell wall is laid down and forms around the ER creating Pd channels.⁴⁶ However, it is also worth noting that the actin cytoskeleton is also present in Pd and is central to organ and reproductive development.^19,47 Actin and actin binding proteins are necessary for a number of plant processes determining the cell division plane, cell polarity, cell elongation, cytoplasmic streaming, transporting mRNAs and proteins, and defense.^48–51 Overexpression of ACT1 in Arabidopsis leaves can lead to changes in epidermal leaf shape and cause dwarfism in plants.⁵² Actin binding proteins are also necessary for organizing and remodeling the F-actin network which drives normal development of specific cell types and organs.⁵³ Actin filament bundling and remodeling are also seen in nonhost defense responses.⁵⁴ We do not know the effects of overexpressing certain actin homologues or actin remodeling on Pd formation or conductivity. Because the F-actin network is also central to Pd trafficking of proteins and macromolecules between cells it is worth considering F-actin as an early factor contributing to Pd formation which may be necessary to ensure cell-to-cell communication when cell polarity and elongation as well as defense machinery are being established.In summary, the novel quantitative tools developed for measuring protein movement across Pd reveal the temperature dependence of protein trafficking. Both the use of Arrhenius equation and C(Pd) provide new opportunities to measure the energy requirements for protein transport. These tools will enable researchers to quantify effects of environmental and developmental conditions on Pd conductivity, as well as comparing differences in Pd conductivity between plant species or induced by genetic mutations.     

Receptor Specificity of Influenza A H3N2 Viruses Isolated in Mammalian Cells and Embryonated Chicken Eggs

Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of α2-6 and α2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to α2-6- and α2-3-type receptors but retained substantial binding to specific O- and N-linked α2-3 glycans, including α2-3GalNAc and fucosylated α2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.Influenza A viruses are generally isolated and propagated in embryonated chicken eggs or in cultures of cells of mammalian origin. Human influenza viruses were previously noted to acquire mutations in the hemagglutinin (HA) gene upon isolation and culture in the allantoic sac of embryonated chicken eggs (herein simply referred to as “eggs”) compared to the sequences of those isolated in mammalian cell substrates (herein referred to as “cells”) (29, 30, 44, 53, 58). These mutations resulted in amino acid substitutions that were found to mediate receptor specificity changes and improved viral replication efficiency in eggs (37). In general, cell-grown viruses are assumed to be more similar than their egg-grown counterparts to the viruses present in respiratory secretions (30, 56). Since their emergence in 1968, influenza A (H3N2) viruses have evolved and adapted to the human host while losing their ability to be efficiently isolated and replicate in eggs, particularly after 1992 (37, 42, 48). The rate of isolation of H3N2 clinical specimens after inoculation into eggs can be up to ∼30 times lower than that in mammalian cell cultures, highlighting the strong selective pressure for the emergence of sequence variants (77).Virtually all influenza vaccines for human use were licensed decades ago by national regulatory authorities, which used a product manufactured from influenza viruses isolated and propagated exclusively in eggs; therefore, cell culture isolates have been unacceptable for this purpose (41, 71). The antigen composition of influenza vaccines requires frequent updates (every 2 years, on average) to closely match their antigenic properties to the most prevalent circulating antigenic drift variant viruses (51). The limited availability of H3N2 viruses isolated in eggs has on one or more occasions delayed vaccine composition updates and may have reduced the efficacy of vaccination against new antigenically drifted viruses (3, 34, 37).Entry of influenza viruses into host cells is mediated by HA, which binds to sialic acid containing glycoconjugates on the surface of epithelial cells in the upper respiratory tract (2, 13). The nature of the linkage between sialic acid and the vicinal sugar (usually galactose) varies in different host species and tissues and may therefore determine whether an influenza virus binds to and infects avian or human cells (40, 46, 59, 62, 72-75). Human influenza viruses preferentially bind to α2-6-linked sialic acids, and avian viruses predominantly bind to α2-3-linked sialic acids (59). Previous studies with chicken embryo chorioallantoic membranes revealed differential lectin binding, suggesting that α2-3-linked but not α2-6-linked sialosides are present on the epithelial cells (28). Human H3N2 viruses isolated in cell culture were reported to bind with a high affinity to α2-6-linked sialosides, while viruses isolated in eggs often had increased specificity for α2-3-linked sialosides (19, 20, 28). The functional classification of avian and mammalian influenza virus receptors is further complicated since in vitro and tissue-binding assays have led to new working hypotheses involving glycan chain length, topology, and the composition of the inner fragments of the carbohydrate chain as additional receptor specificity determinants (9, 17, 65, 66, 82). However, the significance of these in vitro properties remains unknown, since the structures of the natural sialosides on host cells that are used for infectious virus entry are undefined.The techniques most widely used to study the interactions of the influenza virus with host cell receptors employ animal cells in various assay formats (36, 57, 59, 64, 69). To overcome the problems of cell-based techniques, new assays that rely on labeled sialyl-glycoproteins or polymeric sialoglycans have been developed (18). However, these assays are limited by having only a few glycans available in polymeric form and offer low throughput. In contrast, glycan microarrays can assess virus binding to multiple well-defined glycans simultaneously. Previous work with influenza live or β-propiolactone (BPL)-inactivated virions as well as recombinantly produced HAs revealed a good correlation with receptor specificity compared to that achieved by other methods of analysis (4, 11, 57, 58, 65-68).Here we have compared paired isolates derived in eggs or cell cultures from the single clinical specimen to better understand their receptor binding specificity and its implications for vaccine production. We examined the differences in the sequences of the HAs between egg- and cell-grown isolates and analyzed their receptor binding profiles using glycan microarrays. Sequence analysis of the HA and glycan binding results revealed two distinct groups of viruses, with many egg isolates showing unexpectedly reduced levels of binding to α2-3 and α2-6 sialosides compared to the levels for the viruses isolated in mammalian cells. Furthermore, these studies highlighted that specific glycans may be important for H3N2 virus growth in eggs.     

A negative electrostatic determinant mediates the association between the Escherichia coli trp repressor and its operator DNA.

The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan-analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove. However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove. This negative potential is significantly neutralized in the protein conformation that is bound to DNA. Positive potential is also associated with the tryptophan binding-site surface, a consequence of the tryptophan- or tryptophan analogue-induced allosteric change. This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein-DNA complex. The effects of charge-change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein-DNA binding constants. A consistent model is formed that defines a role for long-range electrostatics early in the protein-DNA association process and complements previous structural, molecular association, and mutagenesis studies.