Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.).

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

Cooperative roles of introns 1 and 2 of tobacco resistance gene N in enhanced N transcript expression and antiviral defense responses.

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.

Plant SNAREs SYP22 and SYP23 interact with Tobacco mosaic virus 126 kDa protein and SYP2s are required for normal local virus accumulation and spread.

Viral proteins often interact with multiple host proteins during virus accumulation and spread. Identities and functions of all interacting host proteins are not known. Through a yeast two-hybrid screen an Arabidopsis thaliana Qa-SNARE protein [syntaxin of plants 23 (AtSYP23)], associated with pre-vacuolar compartment and vacuolar membrane fusion activities, interacted with Tobacco mosaic virus (TMV) 126 kDa protein, associated with virus accumulation and spread. In planta, AtSYP23 and AtSYP22 each fused with mCherry, co-localized with 126 kDa protein-GFP. Additionally, A. thaliana and Nicotiana benthamiana SYP2 proteins and 126 kDa protein interacted during bimolecular fluorescence complementation analysis. Decreased TMV accumulation in Arabidopsis plants lacking SYP23 and in N. benthamiana plants subjected to virus-induced gene silencing (VIGS) of SYP2 orthologs was observed. Diminished TMV accumulation during VIGS correlated with less intercellular virus spread. The inability to eliminate virus accumulation suggests that SYP2 proteins function redundantly for TMV accumulation, as for plant development.

Combining loss of function of FOLYLPOLYGLUTAMATE SYNTHETASE1 and CAFFEOYL-COA 3-O-METHYLTRANSFERASE1 for lignin reduction and improved saccharification efficiency in Arabidopsis thaliana.

BACKGROUND: Downregulation of genes involved in lignin biosynthesis and related biochemical pathways has been used as a strategy to improve biofuel production. Plant C1 metabolism provides the methyl units used for the methylation reactions carried out by two methyltransferases in the lignin biosynthetic pathway: caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). Mutations in these genes resulted in lower lignin levels and altered lignin compositions. Reduced lignin levels can also be achieved by mutations in the C1 pathway gene, folylpolyglutamate synthetase1 (FPGS1), in both monocotyledons and dicotyledons, indicating a link between the C1 and lignin biosynthetic pathways. To test if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway, fpgs1ccoaomt1 double mutants were generated and functionally characterized. RESULTS: Double fpgs1ccoaomt1 mutants had lower thioacidolysis lignin monomer yield and acetyl bromide lignin content than the ccoaomt1 or fpgs1 mutants and the plants themselves displayed no obvious long-term negative growth phenotypes. Moreover, extracts from the double mutants had dramatically improved enzymatic polysaccharide hydrolysis efficiencies than the single mutants: 15.1% and 20.7% higher than ccoaomt1 and fpgs1, respectively. The reduced lignin and improved sugar release of fpgs1ccoaomt1 was coupled with changes in cell-wall composition, metabolite profiles, and changes in expression of genes involved in cell-wall and lignin biosynthesis. CONCLUSION: Our observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways. These improvements in sugar accessibility were achieved without introducing unwanted long-term plant growth and developmental defects.

Correction to: Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

[This corrects the article DOI: 10.1186/s13068-019-1353-7.].

Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS.

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors.

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2-year comparative analysis of field-grown lines modified for target gene or genetic element expression.

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4-KD, miRNA156-OE, MYB4-OE, COMT-KD and FPGS-KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second- versus the first-year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second-year growth of transgenics targeted for wall modification, GAUT4-KD, MYB4-OE, COMT-KD and FPGS-KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next-generation bio-feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance.

BACKGROUND: The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. RESULTS: A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. CONCLUSIONS: A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.

An efficient virus-induced gene silencing vector for maize functional genomics research.

Maize is a major crop whose rich genetic diversity provides an advanced resource for genetic research. However, a tool for rapid transient gene function analysis in maize that may be utilized in most maize cultivars has been lacking, resulting in reliance on time-consuming stable transformation and mutation studies to obtain answers. We developed an efficient virus-induced gene silencing (VIGS) vector for maize based on a naturally maize-infecting cucumber mosaic virus (CMV) strain, ZMBJ-CMV. An infectious clone of ZMBJ-CMV was constructed, and a vascular puncture inoculation method utilizing Agrobacterium was optimized to improve its utility for CMV infection of maize. ZMBJ-CMV was then modified to function as a VIGS vector. The ZMBJ-CMV vector induced mild to moderate symptoms in many maize lines, making it useful for gene function studies in critically important maize cultivars, such as the sequenced reference inbred line B73. Using this CMV VIGS system, expression of two endogenous genes, ZmPDS and ZmIspH, was found to be decreased by 75% and 78%, respectively, compared with non-silenced tissue. Inserts with lengths of 100-300 bp produced the most complete transcriptional and visual silencing phenotypes. Moreover, genes related to autophagy, ZmATG3 and ZmATG8a, were also silenced, and it was found that they function in leaf starch degradation. These results indicate that our ZMBJ-CMV VIGS vector provides a tool for rapid and efficient gene function studies in maize.

Salicylic acid treatment and expression of an RNA-dependent RNA polymerase 1 transgene inhibit lethal symptoms and meristem invasion during tobacco mosaic virus infection in Nicotiana benthamiana.

BACKGROUND: Host RNA-dependent RNA polymerases (RDRs) 1 and 6 contribute to antiviral RNA silencing in plants. RDR6 is constitutively expressed and was previously shown to limit invasion of Nicotiana benthamiana meristem tissue by potato virus X and thereby inhibit disease development. RDR1 is inducible by salicylic acid (SA) and several other phytohormones. But although it contributes to basal resistance to tobacco mosaic virus (TMV) it is dispensable for SA-induced resistance in inoculated leaves. The laboratory accession of N. benthamiana is a natural rdr1 mutant and highly susceptible to TMV. However, TMV-induced symptoms are ameliorated in transgenic plants expressing Medicago truncatula RDR1. RESULTS: In MtRDR1-transgenic N. benthamiana plants the spread of TMV expressing the green fluorescent protein (TMV.GFP) into upper, non-inoculated, leaves was not inhibited. However, in these plants exclusion of TMV.GFP from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by SA. TMV normally kills N. benthamiana plants but although MtRDR1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. Recovery from disease was markedly enhanced by SA treatment in MtRDR1-transgenic plants whereas in control plants SA delayed but did not prevent systemic necrosis and death. Following SA treatment of MtRDR1-transgenic plants, extractable RDR enzyme activity was increased and Western blot analysis of RDR extracts revealed a band cross-reacting with an antibody raised against MtRDR1. Expression of MtRDR1 in the transgenic N. benthamiana plants was driven by a constitutive 35S promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to SA. This suggests that the effects of SA on MtRDR1 are exerted at a post-transcriptional level. CONCLUSIONS: MtRDR1 inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants. Thus, RDR1 may act in a similar fashion to RDR6. MtRDR1 and SA acted additively to further promote recovery from disease symptoms in MtRDR1-transgenic plants. Thus it is possible that SA promotes MtRDR1 activity and/or stability through post-transcriptional effects.

A model for intracellular movement of Cauliflower mosaic virus: the concept of the mobile virion factory.

The genomes of many plant viruses have a coding capacity limited to <10 proteins, yet it is becoming increasingly clear that individual plant virus proteins may interact with several targets in the host for establishment of infection. As new functions are uncovered for individual viral proteins, virologists have realized that the apparent simplicity of the virus genome is an illusion that belies the true impact that plant viruses have on host physiology. In this review, we discuss our evolving understanding of the function of the P6 protein of Cauliflower mosaic virus (CaMV), a process that was initiated nearly 35 years ago when the CaMV P6 protein was first described as the 'major inclusion body protein' (IB) present in infected plants. P6 is now referred to in most articles as the transactivator (TAV)/viroplasmin protein, because the first viral function to be characterized for the Caulimovirus P6 protein beyond its role as an inclusion body protein (the viroplasmin) was its role in translational transactivation (the TAV function). This review will discuss the currently accepted functions for P6 and then present the evidence for an entirely new function for P6 in intracellular movement.

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae.

As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pitting-associated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step towards the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acids in the methyl-transferase domain responsible for the formation of these punctate structures. The punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane is likely through an amphipathic α helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.

Association of the P6 protein of Cauliflower mosaic virus with plasmodesmata and plasmodesmal proteins.

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.

Maize Elongin C interacts with the viral genome-linked protein, VPg, of Sugarcane mosaic virus and facilitates virus infection.

The viral genome-linked protein, VPg, of potyviruses is involved in viral genome replication and translation. To determine host proteins that interact with Sugarcane mosaic virus (SCMV) VPg, a yeast two-hybrid screen was used and a maize (Zea mays) Elongin C (ZmElc) protein was identified. ZmELC transcript was observed in all maize organs, but most highly in leaves and pistil extracts, and ZmElc was present in the cytoplasm and nucleus of maize cells in the presence or absence of SCMV. ZmELC expression was increased in maize tissue at 4 and 6 d post SCMV inoculation. When ZmELC was transiently overexpressed in maize protoplasts the accumulation of SCMV RNA was approximately doubled compared with the amount of virus in control protoplasts. Silencing ZmELC expression using a Brome mosaic virus-based gene silencing vector (virus-induced gene silencing) did not influence maize plant growth and development, but did decrease RNA accumulation of two isolates of SCMV and host transcript encoding ZmeIF4E during SCMV infection. Interestingly, Maize chlorotic mottle virus, from outside the Potyviridae, was increased in accumulation after silencing ZmELC expression. Our results describe both the location of ZmElc expression in maize and a new activity associated with an Elc: support of potyvirus accumulation.

Contribution of host intracellular transport machineries to intercellular movement of turnip mosaic virus.

The contribution of different host cell transport systems in the intercellular movement of turnip mosaic virus (TuMV) was investigated. To discriminate between primary infections and secondary infections associated with the virus intercellular movement, a gene cassette expressing GFP-HDEL was inserted adjacent to a TuMV infectious cassette expressing 6K2:mCherry, both within the T-DNA borders of the binary vector pCambia. In this system, both gene cassettes were delivered to the same cell by a single binary vector and primary infection foci emitted green and red fluorescence while secondarily infected cells emitted only red fluorescence. Intercellular movement was measured at 72 hours post infiltration and was estimated to proceed at an average rate of one cell being infected every three hours over an observation period of 17 hours. To determine if the secretory pathway were important for TuMV intercellular movement, chemical and protein inhibitors that blocked both early and late secretory pathways were used. Treatment with Brefeldin A or Concanamycin A or expression of ARF1 or RAB-E1d dominant negative mutants, all of which inhibit pre- or post-Golgi transport, reduced intercellular movement by the virus. These treatments, however, did not inhibit virus replication in primary infected cells. Pharmacological interference assays using Tyrphostin A23 or Wortmannin showed that endocytosis was not important for TuMV intercellular movement. Lack of co-localization by endocytosed FM4-64 and Ara7 (AtRabF2b) with TuMV-induced 6K2-tagged vesicles further supported this conclusion. Microfilament depolymerizing drugs and silencing expression of myosin XI-2 gene, but not myosin VIII genes, also inhibited TuMV intercellular movement. Expression of dominant negative myosin mutants confirmed the role played by myosin XI-2 as well as by myosin XI-K in TuMV intercellular movement. Using this dual gene cassette expression system and transport inhibitors, components of the secretory and actomyosin machinery were shown to be important for TuMV intercellular spread.

The P6 protein of Cauliflower mosaic virus interacts with CHUP1, a plant protein which moves chloroplasts on actin microfilaments.

The gene VI product, protein 6 (P6), of Cauliflower mosaic virus (CaMV) assembles into large, amorphous inclusion bodies (IBs) that are considered sites for viral protein synthesis and viral genome replication and encapsidation. P6 IBs align with microfilaments and require them for intracellular trafficking, a result implying that P6 IBs function to move virus complexes or virions within the cell to support virus physiology. Through a yeast two-hybrid screen we determined that CHUP1, a plant protein allowing chloroplast transport through an interaction with chloroplast and microfilament, interacts with P6. The interaction between CHUP1 and P6 was confirmed through colocalization in vivo and co-immunoprecipitation assays. A truncated CHUP1 fused with enhanced cyan fluorescent protein, unable to transport chloroplasts, inhibited intracellular movement of P6-Venus inclusions. Silencing of CHUP1 in N. edwardsonii impaired the ability of CaMV to infect plants. The findings suggest that CHUP1 supports CaMV infection through an interaction with P6.