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. RESULTS: Antimycin A (an alternative respiratory pathway inducer and also an inducer of resistance to viruses) and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus. CONCLUSIONS: We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX.

Varied movement strategies employed by triple gene block-encoding viruses.

Several RNA virus genera belonging to the Virgaviridae and Flexiviridae families encode proteins organized in a triple gene block (TGB) that facilitate cell-to-cell and long-distance movement. The TGB proteins have been traditionally classified as hordei-like or potex-like based on phylogenetic comparisons and differences in movement mechanisms of the Hordeivirus and Potexvirus spp. However, accumulating data from other model viruses suggests that a revised framework is needed to accommodate the profound differences in protein interactions occurring during infection and ancillary capsid protein requirements for movement. The goal of this article is to highlight common features of the TGB proteins and salient differences in movement properties exhibited by individual viruses encoding these proteins. We discuss common and divergent aspects of the TGB transport machinery, describe putative nucleoprotein movement complexes, highlight recent data on TGB protein interactions and topological properties, and review membrane associations occurring during subcellular targeting and cell-to-cell movement. We conclude that the existing models cannot be used to explain all TGB viruses, and we propose provisional Potexvirus, Hordeivirus, and Pomovirus models. We also suggest areas that might profit from future research on viruses harboring this intriguing arrangement of movement proteins.

Cytoplasmic streaming enables the distribution of molecules and vesicles in large plant cells.

Recent studies of aquatic and land plants show that similar phenomena determine intracellular transport of organelles and vesicles. This suggests that aspects of cell signaling involved in development and response to external stimuli are conserved across species. The movement of molecular motors along cytoskeletal filaments directly or indirectly entrains the fluid cytosol, driving cyclosis (i.e., cytoplasmic streaming) and affecting gradients of molecular species within the cell, with potentially important metabolic implications as a driving force for cell expansion. Research has shown that myosin XI functions in organelle movement driving cytoplasmic streaming in aquatic and land plants. Despite the conserved cytoskeletal machinery propelling organelle movement among aquatic and land plants, the velocities of cyclosis in plant cells varies according to cell types, developmental stage of the cell, and plant species. Here, we synthesize recent insights into cytoplasmic streaming, molecular gradients, cytoskeletal and membrane dynamics, and expand current cellular models to identify important gaps in current research.

Analysis of potato virus X replicase and TGBp3 subcellular locations.

Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.

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.

Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya.

To test the hypothesis that many viruses remain to be discovered in plants, a procedure was developed to sequence nucleic acids cloned randomly from virus-like particle fractions of plant homogenates. As a test of the efficiency of the procedure we targeted Ambrosia psilostachya, western ragweed, plants growing at the Tallgrass Prairie Preserve of northeastern Oklahoma. Amplifiable nucleic acid was found in the fractions from six of twelve specimens and sequences were characterized from four of them. Evidence was obtained for the presence of viruses belonging to two families (Caulimoviridae, Flexiviridae). Multiple viral species were found in two of the four specimens and their level within the isolated nucleic acid population varied from less than 1-37%. None of the sequences were derived from reported sequences of known viruses. Thus, the analysis of nucleic acid from virus-like particles is a useful tool to expand our knowledge of the universe of viruses to non-cultivated species.

Mutational analysis of PVX TGBp3 links subcellular accumulation and protein turnover.

Potato virus X (PVX) TGBp3 is required for virus cell-to-cell transport, has an N-terminal transmembrane domain, and a C-terminal cytosolic domain. In the absence of virus infection TGBp3:GFP is seen in the cortical and perinuclear ER. In PVX infected cells the TGBp3:GFP fusion is also seen in the nucleoplasm indicating that events during PVX infection trigger entry into the nucleus. Mutational analysis failed to identify a nuclear targeting domain. Mutations inhibiting TGBp3 association with the ER and inhibiting virus movement did not block TGBp3:GFP in the nucleoplasm. A mutation disrupting the N-terminal transmembrane domain of TGBp3 caused the fusion to accumulate in the nucleus indicating that nuclear import is regulated by ER interactions. Tunicamycin, an ER-stress inducing chemical, caused lower levels of GFP and TGBp3:GFP to accumulate in virus infected protoplasts. MG115 and MG132 were used to demonstrate that wild-type and mutant TGBp3:GFP fusions were degraded by the 26S proteasome. These observations are consistent with an ER-associated protein degradation (ERAD) pathway suggesting that PVX TGBp3, similar to aberrant ER proteins, is translocated to the cytoplasm for degradation. Nuclear accumulation of mutant and wild-type TGBp3:GFP is independent of other PVX proteins and may be another feature of an ERAD pathway.

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.

Subcellular targeting and interactions among the Potato virus X TGB proteins.

Potato virus X (PVX) encodes three proteins named TGBp1, TGBp2, and TGBp3 which are required for virus cell-to-cell movement. To determine whether PVX TGB proteins interact during virus cell-cell movement, GFP was fused to each TGB coding sequence within the viral genome. Confocal microscopy was used to study subcellular accumulation of each protein in virus-infected plants and protoplasts. GFP:TGBp2 and TGBp3:GFP were both seen in the ER, ER-associated granular vesicles, and perinuclear X-bodies suggesting that these proteins interact in the same subdomains of the endomembrane network. When plasmids expressing CFP:TGBp2 and TGBp3:GFP were co-delivered to tobacco leaf epidermal cells, the fluorescent signals overlapped in ER-associated granular vesicles indicating that these proteins colocalize in this subcellular compartment. GFP:TGBp1 was seen in the nucleus, cytoplasm, rod-like inclusion bodies, and in punctate sites embedded in the cell wall. The puncta were reminiscent of previous reports showing viral proteins in plasmodesmata. Experiments using CFP:TGBp1 and YFP:TGBp2 or TGBp3:GFP showed CFP:TGBp1 remained in the cytoplasm surrounding the endomembrane network. There was no evidence that the granular vesicles contained TGBp1. Yeast two hybrid experiments showed TGBp1 self associates but failed to detect interactions between TGBp1 and TGBp2 or TGBp3. These experiments indicate that the PVX TGB proteins have complex subcellular accumulation patterns and likely cooperate across subcellular compartments to promote virus infection.

Molecular biology of potexviruses: recent advances.

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5' non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3' NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr-R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

Mutations in the central domain of potato virus X TGBp2 eliminate granular vesicles and virus cell-to-cell trafficking.

Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.

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.

A new cell-to-cell transport model for Potexviruses.

In the last five years, we have gained significant insight into the role of the Potexvirus proteins in virus movement and RNA silencing. Potexviruses require three movement proteins, named triple gene block (TGB)p1, TGBp2, and TGBp3, and the viral coat protein (CP) to facilitate viral cell-to-cell and vascular transport. TGBp1 is a multifunctional protein that has RNA helicase activity, promotes translation of viral RNAs, increases plasmodesmal size exclusion limits, and suppresses RNA silencing. TGBp2 and TGBp3 are membrane-binding proteins. CP is required for genome encapsidation and forms ribonucleoprotein complexes along with TGBp1 and viral RNA. This review considers the functions of the TGB proteins, how they interact with each other and CP, and how silencing suppression might be linked to viral transport. A new model of the mechanism for Potexvirus transport is proposed.

Soilborne wheat mosaic virus (SBWMV) 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing.

Amino acid sequence analyses indicate that the Soilborne wheat mosaic virus (SBWMV) 19K protein is a cysteine-rich protein (CRP) and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX) viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP) expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.

Potato virus X TGBp1 induces plasmodesmata gating and moves between cells in several host species whereas CP moves only in N. benthamiana leaves.

Experiments were conducted to compare the plasmodesmal transport activities of Potato virus X (PVX) TGBp1 and coat protein (CP) in several plant species. Microinjection experiments indicated that TGBp1 gates plasmodesmata in Nicotiana tabacum leaves. These results support previous microinjection studies indicating that TGBp1 gates plasmodesmata in Nicotiana benthamiana and Nicotiana clevelandii leaves. To study protein movement, plasmids expressing the green fluorescent protein (GFP) gene fused to the PVX TGBp1 or CP genes were biolistically bombarded to leaves taken from four different PVX host species. GFP/TGBp1 moved between adjacent cells in N. tabacum, N. clevelandii, N. benthamiana, and Lycopersicon esculentum, whereas GFP/CP moved only in N. benthamiana leaves. Mutations m12 and m13 were introduced into the TGBp1 gene and both mutations eliminated TGBp1 ATPase active site motifs, inhibited PVX movement, reduced GFP/TGBp1 cell-to-cell movement in N. benthamiana leaves, and eliminated GFP/TGBp1 movement in N. tabacum, N. clevelandii, and L. esculentum leaves. GFP/TGBp1m13 formed aggregates in tobacco cells. The ability of GFP/CP and mutant GFP/TGBp1 fusion proteins to move in N. benthamiana and not in the other PVX host species suggests that N. benthamiana plants have a unique ability to promote protein intercellular movement.

Soilborne wheat mosaic virus movement protein and RNA and wheat spindle streak mosaic virus coat protein accumulate inside resting spores of their vector, Polymyxa graminis.

To study virus-vector interactions between Soilborne wheat mosaic virus (SBWMV) or Wheat spindle streak mosaic virus (WSSMV) and Polymyxa graminis Ledingham, P. graminis was propagated in plants grown hydroponically. P. graminis accumulated to high levels in several barley cultivars tested. Multiple developmental stages of P. graminis could be identified in infected barley roots. Accumulation of SBWMV and WSSMV inside P. graminis sporosori in the roots of soil-grown winter wheat and hydroponically grown barley was compared to determine if data obtained from plants naturally infected plants and plants infected by manual inoculation were similar. WSSMV coat protein (CP), SBWMV RNAs, SBWMV movement protein but not SBWMV CP were detected in both soil-grown winter wheat and hydroponically grown barley roots. These data are the first direct evidence that SBWMV and WSSMV are internalized by P. graminis.

Evidence that the 37 kDa protein of Soil-borne wheat mosaic virus is a virus movement protein.

Experiments were conducted to determine if the 37 kDa protein (37K) of Soil-borne wheat mosaic virus (SBWMV) is a virus movement protein. First, evidence was obtained that indicated that 37K has the ability to move from cell to cell, similar to other virus movement proteins (MPs). Plasmids containing the GFP gene fused to the SBWMV 37K, the coat protein (CP) or the CP readthrough domain (RT) ORFs were delivered by biolistic bombardment to wheat and tobacco leaves. In wheat leaves, cell-to-cell movement of GFP-37K was observed, while GFP, GFP-CP and GFP-RT accumulated primarily in single cells. All fusion proteins accumulated in single cells in tobacco leaves. Thus, cell-to-cell movement is a specific property of 37K that occurs in SBWMV host plants. Subcellular accumulation of 37K was studied using SBWMV-infected and 37K-expressing transgenic wheat. In infected and transgenic wheat leaves, 37K accumulated in the cell wall, similar to other virus MPs, and in aggregates in the cytoplasm. Phylogenetic studies were conducted to compare the furovirus 37K proteins with members of the 30K superfamily of virus MPs. Amino acid sequences of the furovirus 37K proteins were aligned with the MPs from 43 representative viruses. The furovirus 37K proteins were found to reside in a clade that also contained the dianthovirus MPs. Combined, these data suggest that SBWMV 37K is probably a virus MP.

The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement.

Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement. Plasmids expressing GFP fused to TGBp2 were bombarded to leaf epidermal cells and GFP:TGBp2 moved cell to cell in Nicotiana benthamiana leaves but not in Nicotiana tabacum leaves. GFP:TGBp2 movement was observed in TGBp1-transgenic N. tabacum, indicating that TGBp2 requires TGBp1 to promote its movement in N. tabacum. In this study, GFP:TGBp2 was detected in a polygonal pattern that resembles the endoplasmic reticulum (ER) network. Amino acid sequence analysis revealed TGBp2 has two putative transmembrane domains. Two mutations separately introduced into the coding sequences encompassing the putative transmembrane domains within the GFP:TGBp2 plasmids and PVX genome, disrupted membrane binding of GFP:TGBp2, inhibited GFP:TGBp2 movement in N. benthamiana and TGBp1-expressing N. tabacum, and inhibited PVX movement. A third mutation, lying outside the transmembrane domains, had no effect on GFP:TGBp2 ER association or movement in N. benthamiana but inhibited GFP:TGBp2 movement in TGBp1-expressing N. tabacum and PVX movement in either Nicotiana species. Thus, ER association of TGBp2 may be required but not be sufficient for virus movement. TGBp2 likely provides an activity for PVX movement beyond ER association.

The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement.

Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.

Cell-to-cell movement of the PVX 12K, 8K, or coat proteins may depend on the host, leaf developmental stage, and the PVX 25K protein.

The requirements for intercellular movement of Potato virus X (PVX) 12K, 8K, and coat proteins (CP) differed in two Nicotiana spp. Plasmids containing the green fluorescent protein (GFP) gene fused to PVX 12K, 8K, or CP genes were bombarded to tobacco leaves. Protein movement was observed in N. benthamiana but not N. tabacum leaves. GFP:12K and GFP:8K moved cell-to-cell in 25K-expressing transgenic N. tabacum source but not sink leaves. In N. tabacum, GFP:12K and GFP:8K intercellular movement depends on the 25K and leaf developmental stage. Leaves were bombarded using two biolistic delivery methods and the results were similar indicating that movement of GFP:12K or GFP:8K is independent of the delivery system. Mutations in 12K, 8K, and CP genes within the PVX genome inhibited viral intercellular movement in both Nicotiana spp. Thus plasmodesmata gating is not an essential function of these proteins for virus cell-to-cell movement. These proteins likely provide additional activities for virus cell-to-cell movement.