Silenced Suppressor Genes Research Articles

RNAi-Mediated Transgenic Tospovirus Resistance Broken by IntraspeciesSilencing Suppressor Protein Complementation

Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants additionally resistant to this strain but not to the soybean strain of this Tospovirus sp. (TYRV-s), both strains exhibiting 14.4% nucleotide sequence divergence in their N genes. Surprisingly, coinoculation of the TYRV-t-resistant transgenic lines with both TYRV-t and TYRV-s resulted in rescue of the former. Mass-spectrometric analysis of the viral ribonucleocapsids accumulating in the transgenic plants showed the presence of the N proteins of both strains excluding hetero-encapsidation as rescue mechanism and indicating suppression of TYRV-t N gene transcript breakdown by RNA interference. Prior (Potato virus X [PVX]-vector-mediated) expression of the TYRV-s silencing suppressor (NS(s)) gene also allowed TYRV-t to break the resistance. This phenomenon was also observed when the homologous (TYRV-t) NS(s) gene was provided from a PVX replicon, demonstrating that TYRV can break RNA-mediated host resistance upon a priori expression of its NS(s) protein. Remarkably, mixed inoculation of TYRV-t with other Tospovirus spp. or nonrelated viruses did not result in resistance breaking, indicating that the rescuing activity of NS(s)-though based on suppressing RNA silencing-is species-dependent.

The viral etiology of tomato yellow leaf curl disease - a review

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating plant diseases in the world. As a result of its continuing rapid spread, it now afflicts more than 30 tomato growing countries in the Mediterranean basin, southern Asia, Africa, and South, Central and North America. The disease is caused by a group of viral species of the genus <I>Begomovirus,</I> family Geminiviridae (geminiviruses), referred to as <I>Tomato yellow leaf curl virus</I> (TYLCV). These are transmitted by an insect vector, the whitefly<I> Bemisia tabaci</I>, classified in the family Aleyrodidae. The genome of TYLCV generally consists of a single circular single-stranded (ss) DNA molecule, with only one exception in which two components were identified. It encodes six open reading frames, only one of which codes for the coat protein (CP) that represents a building block of the viral particle. TYLCV, like all other members of the Geminiviridae, has geminate particles, apparently consisting of two incomplete T = 1 icosahedra joined together to produce a structure with 22 pentameric capsomers and 110 identical CP subunits. Close to 50 years of intensive research into TYLCV epidemics has been conducted to find solutions to the severe problem caused by this virus. To date, breeding for resistance appears to be the best approach to controlling this disease, although only partially resistant varieties are commercially available. Since the virus consists of a ssDNA that replicates in the host-cell nucleus, the molecular mechanisms involved in its nuclear import have been the focus of our studies in recent years and results, as well as prospects, are discussed in this review. In addition, we describe our recent finding of a suppressor of gene silencing encoded by one of the TYLCV-Isr genes. This paper provides an overview of the most outstanding achievements in TYLCV research that may lead to more effective control strategies.

A geminiviral amplicon (VA) derived from Tomato leaf curl virus (ToLCV) can replicate in a wide variety of plant species and also acts as a VIGS vector

BackgroundThe Tomato leaf curl virus (ToLCV) belongs to the genus begomoviridae of the family Geminiviridae. The 2.7 kb DNA genome of the virus encodes all the information required for viral DNA replication, transcription and transmission across the plant cells. However, all of the genome sequences are not required for viral DNA replication. We attempted to reveal the minimal essential region required for DNA replication and stable maintenance. The phenomenon of Virus Induced Gene Silencing (VIGS) has recently been observed with several geminiviruses. We investigated whether the minimal replicating region was also capable of producing siRNAs in planta and a VIGS vector could be constructed using the same minimal sequences.ResultsWe have constructed vectors containing various truncated portions of the Tomato leaf curl virus (ToLCV) genome and established that a segment spanning from common region (CR) to AC3 (ORF coding for a replication enhancer) was the minimal portion which could efficiently replicate in a variety of both monocot and dicot plants. A viral amplicon (VA) vector was constructed using this region that produced siRNAs from various sites of the vector, in a temporal manner in plants, and hence can be used as a VIGS vector. The tomato endogene PCNA was silenced using this vector. Introduction of a mutation in the ORF AC2 (a silencing suppressor) increased the silencing efficiency of the newly constructed vector several folds.ConclusionOur study reveals that the vector is capable of replicating in diverse plant species and is highly efficient in silencing endogenes like PCNA of the host plant, thus acting as a VIGS vector. We observed that the geminiviral ORF AC2 functioned as a silencing suppressor and a null mutation in this ORF increased the efficiency of silencing several fold. This is the first report of construction of improved VIGS vector by mutation of the resident silencing suppressor gene. The present study opens up the possibility of using such VIGS vectors in silencing the host genes in a broad range of plant hosts.

Characterization of the cassava geminivirus transcription activation protein putative nuclear localization signal

The geminivirus transcription activation protein (TrAP) localizes to the nucleus and contains a putative nuclear localization signal (NLS) ( 28PRRRR 32) on the N-terminus. The role of individual residues of this putative NLS on nuclear localization and symptom induction was investigated using TrAP of East African cassava mosaic Cameroon virus (EACMCV). Subcellular localization was conducted using the green fluorescent protein (GFP). Results showed that the proline residue at position 28 (Pro-28) is essential for nuclear localization whereas individually, none of the four contiguous arginines is necessary for nuclear targeting. The role of each of the five NLS amino acid residues on TrAP-mediated disease phenotype and gene silencing suppression was investigated by expressing these mutants in Nicotiana benthamiana from the PVX vector and under the control of the Cauliflower mosaic virus 35S promoter. Results showed that all five residues of the NLS play a role on disease phenotype production in N. benthamiana plants. Furthermore, each of the NLS residues appeared to be required for suppression of VIGS but appeared not to be required for the ability of TrAP to transactivate transcription and interact with adenosine kinase (ADK).

Cucurbit yellow stunting disorder virus p25 is a suppressor of post-transcriptional gene silencing

Post-transcriptional gene silencing (PTGS) degrades RNA in a sequence-specific manner and is utilised by plants as a natural defence mechanism against virus invaders. Two members of the genus Crinivirus have been reported to encode suppressors and counter PTGS: Sweet potato chlorotic stunt virus p22 and Tomato chlorosis virus (ToCV) p22, coat protein and coat protein minor. Using an Agrobacterium-mediated transient assay on Nicotiana benthamiana wildtype and 16c plants, we screened four Cucurbit yellow stunting disorder virus (CYSDV) RNA 1-encoded proteins (papain-like protease, p25, p5.2 and p22) to determine which one possess PTGS suppressor activity. Amongst these proteins, only CYSDV p25 was able to suppress (double- and single-stranded) RNA-induced silencing of the green fluorescent protein (GFP) mRNA. Restoration of GFP expression by CYSDV p25 in both of these experiments had no apparent effect on the accumulation of the small interfering RNAs. The identification of CYSDV p25 adds to the list of suppressors encoded by crinivirus RNA 1 molecules, which are unrelated in terms of amino acid sequence homology suggesting distinct PTGS suppression mechanisms and possible roles in viral replication.

A novel shaggy-like kinase interacts with the Tomato leaf curl virus pathogenicity determinant C4 protein

Tomato leaf curl virus-Australia (ToLCV) C4 protein has been shown to be associated with virus pathogenesis. Here, we demonstrate that C4 acts as a suppressor of gene silencing. To understand the multifunctional role of C4, a novel shaggy-like kinase (SlSK) from tomato, which interacts with ToLCV C4 in a yeast two-hybrid assay, was isolated and interaction between these proteins was confirmed in vitro and in planta. Using deletion analysis of C4, a 12 amino acid region in the C-terminal part of C4 was identified which was shown to be essential for its binding to SlSK. We further demonstrate that this region is not only important for the interaction of C4 with SlSK, but is also required for C4 function to suppress gene silencing activity and to induce virus symptoms in a PVX system. The potential significance of ToLCV C4 and SlSK interaction is discussed.

Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice

Vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. Here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in Nicotiana benthamiana. The rN protein in the agrobacteria-infiltrated plant leaf accumulated up to a concentration of 79μg per g fresh leaf weight at 3 days post infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund's adjuvant. The rN protein-specific IgG in the mouse sera attained a titer about 1:1,800 following three doses of immunization, which suggested effective B-cell maturation and differentiation in mice. Antibodies of the subclasses IgG1 and IgG2a were abundantly present in the mouse sera. During vaccination of rN protein, the expression of IFN-γ and IL-10 was evidently up-regulated in splenocytes at different time points, while the expression of IL-2 and IL-4 was not. Up to now, this is the first study that plant-expressed recombinant SARS-CoV N protein can induce strong humoral and cellular responses in mice.

Virus-induced Gene Silencing (VIGS) in <em>Nicotiana benthamiana</em> and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing (VIGS) in <em>Nicotiana benthamiana</em> and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5. Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2. Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large-scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale-up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha-1-antitrypsin (rAAT) by co-infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 +/- 8.7)% and (290 +/- 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD(600) of 0.5 and a 0.25-min vacuum infiltration, and multiple 1-min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 +/- 0.09)% and (4.1 +/- 0.29)% of the total soluble protein, respectively, or (90 +/- 1.7) and (140 +/- 10) mg per kg fresh weight leaf tissue at 6 days post-induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale-up.

ARGONAUTE 1 homeostasis invokes the coordinate action of the microRNA and siRNA pathways

ARGONAUTE 1 (AGO1) slices endogenous messenger RNAs (mRNAs) during both microRNA (miRNA)- and short interfering RNA (siRNA)-guided post-transcriptional silencing. We have previously reported that AGO1 homeostasis is maintained through the repressive action of miR168 on AGO1 mRNA and the stabilizing effect of AGO1 protein on miR168, but siRNA-mediated AGO1 regulation has not been reported. Here, we show that AGO1-derived siRNAs trigger RNA DEPENDENT RNA POLYMERASE 6 (RDR6)-, SUPPRESSOR OF GENE SILENCING 3 (SGS3)- and SILENCING DEFECTIVE 5 (SDE5)-dependent AGO1 silencing, which also requires DICER-LIKE 2 (DCL2) and DCL4. By varying the efficacy of miR168-guided AGO1 mRNA cleavage, we show that siRNA-mediated AGO1 silencing depends on correct miRNA targeting, pointing to coordinated regulatory actions of the miRNA and siRNA pathways during the maintenance of AGO1 homeostasis. Finally, our results reveal that dcl2, dcl3 and dcl4 mutations similarly affect post-transcriptional gene silencing (PTGS) mediated by a sense transgene and PTGS mediated by inverted repeats, validating the branched pathway model proposed previously.

SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6-bodies

Suppressor of gene silencing 3 (SGS3) is involved in RNA-dependent RNA polymerase 6 (RDR6)-dependent small-interfering RNA (siRNA) pathways in Arabidopsis. However, the roles of SGS3 in those pathways are unclear. Here, we show that SGS3 interacts and colocalizes with RDR6 in cytoplasmic granules. Interestingly, the granules containing SGS3 and RDR6 (named SGS3/RDR6-bodies) were distinct from the processing bodies where mRNAs are decayed and/or stored. Microscopic analyses and complementation experiments using SGS3-deletion mutants suggested that proper localization of SGS3 is important for its function. These results provide novel insights into RDR6-dependent siRNA formation in plants. Structured summary MINT- 7014710: SGS3 (uniprotkb: Q9LDX1) and RDR6 (uniprotkb: Q9SG02) physically interact (MI: 0218) by bimolecular fluorescence complementation (MI: 0809) MINT- 7014697: RDR6 (uniprotkb: Q9SG02) and SGS3 (uniprotkb: Q9LDX1) colocalize (MI: 0403) by fluorescence microscopy (MI: 0416)

Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression

The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3'-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), gammab (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and gammab to the 3'-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions.

A neomorphic sgs3 allele stabilizing miRNA cleavage products reveals that SGS3 acts as a homodimer

The putative RNA-binding protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) protects RNA from degradation before transformation into dsRNA by the RNA-dependent RNA polymerase RDR6 during plant post-transcriptional gene silencing and trans-acting small interfering (siRNA) pathways. In this study, we show that SGS3 acts as a homodimer, and that the point mutation sgs3-3 impairs post-transcriptional gene silencing in a dominant-negative manner through the formation of SGS3/sgs3-3 heterodimers. Unlike complete-loss-of-function sgs3 mutants, which are impaired in the accumulation of both micro RNA-directed TAS cleavage products and mature trans-acting siRNAs, the sgs3-3 mutant overaccumulates TAS cleavage products and exhibits slightly reduced trans-acting siRNA accumulation. Together, these results suggest that sgs3-3 is a neomorphic allele that shows increased RNA protective activity, resulting in decreased RNA processing by downstream post-transcriptional gene silencing and trans-acting siRNA pathway components.

AGO1-miR173 complex initiates phased siRNA formation in plants

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation–cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c -based syn-tasiRNA formation. A single miR173 target site was sufficient to route non- TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173–AGO1 complex possesses unique functionality that many other miRNA–AGO1 complexes lack.

Plant pharming of a full‐sized, tumour‐targeting antibody using different expression strategies

The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.

The P0 gene of Sugarcane yellow leaf virus encodes an RNA silencing suppressor with unique activities

The Sugarcane yellow leaf virus (SCYLV) P0, a member of the highly heterologous proteins of poleroviruses, is a suppressor of posttranscriptional gene silencing (PTGS) and has additional activities not seen in other P0 proteins. The P0 protein in previously tested poleroviruses ( Beet western yellows virus and Cucurbit aphid-borne yellows virus), suppresses local, but not systemic, PTGS induced by both sense GFP and inverted repeat GF using its F-box-like domain to mediate destabilization of the Argonaute1 protein. We now report that the SCYLV P0 protein not only suppressed local PTGS induced by sense GFP and inverted repeat GF in Nicotiana benthamiana, but also triggered a dosage dependent cell death phenotype in infiltrated leaves and suppressed systemic sense GFP-PTGS. Deletion of the first 15 N-terminal amino acid residues of SCYLV P0 abolished suppression of both local and systemic PTGS and the induction of cell death. In contrast, only systemic PTGS and cell death were lost when the 15 C-terminal amino acid residues were deleted. We conclude that the 15 C-terminal amino acid residue region of SCYLV P0 is necessary for suppressing systemic PTGS and inducing cell death, but is not required for suppression of local PTGS.

Abstract A27: Contribution of DNA methylation in development of lung cancer in former smokers

Abstract A27 Background Lung cancer is the most common cause of cancer death worldwide with more than 1.2 million people dying of the disease each year. Half of newly diagnosed lung cancer patients are former smokers. Understanding why former smokers develop lung cancer is clearly important to the development of early detection, prevention and treatment strategies for these people. The effects of cigarette smoke on the epigenome are widespread as both global DNA methylation and local DNA methylation have been identified, associated with genomic instability and tumour suppressor gene (TSG) silencing, respectively. In a cancer-specific context, upregulation of oncogenes and silencing of tumour suppressor genes both occur as a result of tobacco smoke exposure. Therefore, molecular studies examining tumors at genomic and epigenomic levels will likely identify causal genetic events involved in cancer development in former smokers. Objective The objective of this study is to determine the contribution of DNA methylation as a mechanism to lung cancer development in former smokers. Hypothesis As smoking induces methylation changes in bronchioepithelial cells, we hypothesize that this constitutes the first hit to TSG inactivation. In order to inactivate both alleles, these methylation changes would be maintained in tumors where a second hit would be found at the same gene loci. Materials and Methods Epithelial cells from former smokers (those with >10 years of smoking cessation) were collected from peripheral airways during routine bronchoscopy. Half of the cells were fixed in Cytolyt and the other half in RNAlater for DNA and RNA extraction, respectively. Copy number profiling was performed by array comparative genomic hybridization (aCGH) using whole-genome tiling path SMRT v2 BAC array. Methylation analysis was performed by coupling affinity based enrichment of methylated sequences with hybridization to the same aCGH platform described above. Expression status of genes was determined by gene expression microarray analysis using Agilent 44K expression arrays. RESULTS: Preliminary MeDIP aCGH of bronchial brush cells from eight former smokers who had previous surgical removal of Stage I NSCLC, and eight former smokers without NSCLC, of similar age (68±7 versus 62±6), revealed distinct differences in frequency of DNA methylation between cancer and non-cancer groups. For example, at 11p13 the cancer group shows hypermethylation at the WT1 locus, a known TSG. Analysis in 62 NSCLC tumors for gene dosage, showed a high frequency of loss at the WT1 locus. To examine downstream effects of these events in tumours, expression of WT1 was assessed and found to be significantly underexpressed in the majority of NSCLC tumours compared to a normal lung reference. Collection of more samples and further integrative analysis is currently underway. Conclusion Differences in methylation between these two groups may explain why some former smokers develop cancer while others remain cancer free despite similar lifestyle changes. As methylation is a reversible DNA modification, this knowledge would prompt the development and application of DNA demethylation chemopreventative agents and unique therapeutic strategies. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A27.

Replication and encapsidation of recombinant Turnip yellow mosaic virus RNA

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding beta-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing beta-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

The Effect of Cucumber mosaic virus 2b Protein to Transient Expression and Transgene Silencing Mediated by Agro-infiltration

The transient and rapid expression system of a foreign protein in planta is a very useful technique in biotechnology application. We have investigated optimum condition of Agrobacterium-infiltration technique in which expression level of foreign proteins were maximized without detrimental effects on plants using GFP and Cucumber mosaic virus 2b protein, which is known as an enhancer of gene expression and a suppressor of post-transcriptional gene silencing(PTGS). The optimum expression level of both RNA and protein of GFP with minimum leaf impairment was obtained at