Showing papers on "Cauliflower mosaic virus published in 2014"

G-quadruplexes in viruses: function and potential therapeutic applications

Abstract: G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of ∼300,000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein-Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.

The Arabidopsis synaptotagmin SYTA regulates the cell-to-cell movement of diverse plant viruses

Abstract: Synaptotagmins are a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons, and dense core vesicle exocytosis for hormone secretion from neuroendocrine cells. Thought to be exclusive to animals, synaptotagmins have recently been characterized in Arabidopsis thaliana, in which they comprise a five gene family. Using infectivity and leaf-based functional assays, we have shown that Arabidopsis SYTA regulates endocytosis and marks an endosomal vesicle recycling pathway to regulate movement protein-mediated trafficking of the Begomovirus Cabbage leaf curl virus (CaLCuV) and the Tobamovirus Tobacco mosaic virus (TMV) through plasmodesmata (Lewis and Lazarowitz, 2010). To determine whether SYTA has a central role in regulating the cell-to-cell trafficking of a wider range of diverse plant viruses, we extended our studies here to examine the role of SYTA in the cell-to-cell movement of additional plant viruses that employ different modes of movement, namely the Potyvirus Turnip mosaic virus (TuMV), the Caulimovirus Cauliflower mosaic virus (CaMV) and the Tobamovirus Turnip vein clearing virus (TVCV), which in contrast to TMV does efficiently infect Arabidopsis. We found that both TuMV and TVCV systemic infection, and the cell-to-cell trafficking of the their movement proteins, were delayed in the Arabidopsis Col-0 syta-1 knockdown mutant. In contrast, CaMV systemic infection was not inhibited in syta-1. Our studies show that SYTA is a key regulator of plant virus intercellular movement, being necessary for the ability of diverse cell-to-cell movement proteins encoded by Begomoviruses (CaLCuV MP), Tobamoviruses (TVCV and TMV 30K protein) and Potyviruses (TuMV P3N-PIPO) to alter PD and thereby mediate virus cell-to-cell spread.

A tomato endoplasmic reticulum (ER)-type omega-3 fatty acid desaturase (LeFAD3) functions in early seedling tolerance to salinity stress

Abstract: Transgenic tomato plants overexpressing LeFAD3 sense and antisense sequences were generated. Salt stress suppressed the growth of WT and antisense plants to a higher extent than that in sense plants. In this study, we investigated the role of the LeFAD3-encoding ER-type omega-3 fatty acid desaturase in salt tolerance in tomato plants. We created transgenic tomato plants by overexpressing its sense and antisense sequences under the control of the cauliflower mosaic virus 35S promoter. Based on the results of northern and western blotting as well as quantitative reverse transcription-polymerase chain reaction, sense plants expressed more desaturase than wild-type (WT) plants, whereas antisense plants expressed less desaturase than WT. Salt stress suppressed the growth of both WT and antisense plants to a higher extent than that in sense plants, which can be attributed to the fact that sense plants performed better in maintaining the integrity of the membrane system, as revealed by electron microscopy. The concomitant increase in superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.7) may have alleviated the photoinhibition caused by the increased level of ROS in sense plants. Our results suggest that LeFAD3 overexpression can enhance the tolerance of early seedlings to salinity stress.

The Temporal Evolution and Global Spread of Cauliflower mosaic virus, a Plant Pararetrovirus

Abstract: Cauliflower mosaic virus (CaMV) is a plant pararetrovirus with a double-stranded DNA genome. It is the type member of the genus Caulimovirus in the family Caulimoviridae. CaMV is transmitted by sap inoculation and in nature by aphids in a semi-persistent manner. To investigate the patterns and timescale of CaMV migration and evolution, we sequenced and analyzed the genomes of 67 isolates of CaMV collected mostly in Greece, Iran, Turkey, and Japan together with nine published sequences. We identified the open-reading frames (ORFs) in the genomes and inferred their phylogeny. After removing recombinant sequences, we estimated the substitution rates, divergence times, and phylogeographic patterns of the virus populations. We found that recombination has been a common feature of CaMV evolution, and that ORFs I–V have a different evolutionary history from ORF VI. The ORFs have evolved at rates between 1.71 and 5.81×10−4 substitutions/site/year, similar to those of viruses with RNA or ssDNA genomes. We found four geographically confined lineages. CaMV probably spread from a single population to other parts of the world around 400–500 years ago, and is now widely distributed among Eurasian countries. Our results revealed evidence of frequent gene flow between populations in Turkey and those of its neighboring countries, with similar patterns observed for Japan and the USA. Our study represents the first report on the spatial and temporal spread of a plant pararetrovirus.

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

Abstract: The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis ( Arabidopsis thaliana ) μA-adaptin subunit Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N -(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation Altogether, these results demonstrate that CaMVMP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes

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

Abstract: 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.

Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing.

Abstract: Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification.

Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots

Abstract: A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.

Activation of three pathogen-inducible promoters in transgenic citrus ( Citrus sinensis Osbeck) after Xanthomonas axonopodis pv. citri infection and wounding

Abstract: To improve the resistance of citrus to canker disease caused by Xanthomonas axonopodis pv. citri (Xac), it is important to identify gene promoters that are specifically induced by pathogen infection. Here, we evaluated the functionality of PPP1 and hsr203J (Nicotiana tabacum L.) and gst1 (potato) pathogen-inducible promoters to drive expression of the β-glucuronidase (GUS) reporter gene in transgenic citrus. The activities of these promoters in response to the Xac pathogen and wounding were determined quantitatively and qualitatively by fluorometric and histochemical GUS assays, and compared with that of the cauliflower mosaic virus (CaMV) 35S promoter. In citrus, the hsr203J promoter from tobacco was hardly activated by the Xac pathogen or wounding, whereas the PPP1 and gst1 promoters were rapidly and efficiently activated by both inducers. There was very little visible background expression from the PPP1 promoter, but a high level of background expression from the gst1 promoter. Because of its low background expression, the PPP1 promoter was more responsive to Xac and wounding than was the gst1 promoter. However, the gst1 promoter was more rapidly activated than the PPP1 promoter by Xac and wounding. The inducible activity of the two promoters was restricted to infection sites. Taken together, our results showed that the PPP1 promoter was the most efficient promoter among those evaluated in this study. Its strong responsiveness to Xac and wounding suggests that it would be a good candidate for expression of antibacterial transgenes specifically at infection sites to improve canker disease resistance in citrus.

Ectopic expression of an Arabidopsis dehydration-responsive element-binding factor DREB2C improves salt stress tolerance in crucifers.

Abstract: DREB2C acts as a transcriptional activator of the salt tolerance-related COLD - REGULATED 15A gene. DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 2C (DREB2C) regulates plant responses to heat stress. We report here that DREB2C is induced by NaCl stress in Arabidopsis, based on quantitative RT-PCR analyses of transcript levels and DREB2C promoter-controlled GUS activity assays. Constitutive overexpression of DREB2C from the cauliflower mosaic virus (CaMV) 35S promoter led to enhanced salt tolerance in transgenic Arabidopsis and canola plants that was characterized by higher chlorophyll content, lower tissue Na+ content, reduced rate of water loss, and tighter membrane integrity in plants grown in NaCl-containing medium. Basal expression of the stress-responsive genes COLD-REGULATED 15A (COR15A), RESPONSIVE TO DEHYDRATION (RD) 29A and RD29B, was higher in transgenic DREB2C-overexpressing Arabidopsis plants than in the wild-type. Promoter transactivation assays and electrophoretic mobility-shift assays showed that DREB2C interacts directly with the three DREs in the COR15A promoter, both in vivo and in vitro. Transgenic Arabidopsis constitutively overexpressing COR15A from the CaMV35S promoter exhibited greater NaCl tolerance than the untransformed wild-type. Taken together, the data suggest that DREB2C functions as transcriptional activator that promotes NaCl tolerance, in part through upregulation of the stress-responsive gene COR15A.

Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.)

Abstract: A synthetic gene encoding the antimicrobial peptide magainin has been designed, cloned, and engineered for regulation by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (nos) terminator. The plant expression cassette was introduced into the vector pSB11-bar (with the glyphosate [Basta®] resistance gene, bar), and the recombinant plasmid was mobilized into Agrobacterium tumefaciens strain LBA4404 for the generation of a super-binary vector pSB111-bar-mag. Magainins, positively charged amphipathic antimicrobial peptides of 21–26 amino acid residues, are potential candidates for the development of disease resistant transgenic plants. Six-wk-old pearl millet (Pennisetum glaucum [L.] R. Br.) calli and A. tumefaciens harboring pSB111-bar-mag were cocultivated in a medium supplemented with 400 μM acetosyringone and 3.3 mM l-cysteine. Out of 3,000 infected calli subjected to selection on phosphinothricin medium, 82 calli showed sectors of healthy growth, resulting in a transformation frequency of 2.73%. Among 13 Basta-tolerant putative transformed plants, eight were fertile and their transgenic nature and expression of the transgene was characterized by Southern and Northern blot analyses, respectively. Subsequent T1 progenies co-segregated for bar and magainin genes in a 3:1 ratio. Bioassays that challenged the eight transgenic T1 plant progenies against three highly virulent strains of Sclerospora graminicola, viz., Sg 384, Sg 445, and Sg 492 failed to show resistance. The failure of synthetic magainin gene to confer resistance against downy mildew in pearl millet may be attributed to the complexity of the cell wall and cell membrane of the pathogen.

Divergence of host range and biological properties between natural isolate and full-length infectious cDNA clone of the Beet mild yellowing virus 2ITB.

Abstract: Plant infection by poleroviruses is restricted to phloem tissues, preventing any classical leaf rub inoculation with viral RNA or virions. Efficient virus inoculation to plants is achieved by viruliferous aphids that acquire the virus by feeding on infected plants. The use of promoter-driven infectious cDNA is an alternative means to infect plants and allows reverse genetic studies to be performed. Using Beet mild yellowing virus isolate 2ITB (BMYV-2ITB), we produced a full-length infectious cDNA clone of the virus (named BMYV-EK) placed under the control of the T7 RNA polymerase and the Cauliflower mosaic virus 35S promoters. Infectivity of the engineered BMYV-EK virus was assayed in different plant species and compared with that of the original virus. We showed that in vitro- or in planta-derived transcripts were infectious in protoplasts and in whole plants. Importantly, the natural aphid vector Myzus persicae efficiently transmitted the viral progeny produced in infected plants. By comparing agroinoculation and aphid infection in a host range assay, we showed that the engineered BMYV-EK virus displayed a similar host range to BMYV-2ITB, except for Nicotiana benthamiana, which proved to be resistant to systemic infection with BMYV-EK. Finally, both the BMYV-EK P0 and the full-length clone were able to strongly interfere with post-transcriptional gene silencing.

Isolation and Functional Characterization of a Novel Seed-Specific Promoter Region from Peanut

Abstract: The importance of using tissue-specific promoters in the genetic transformation of plants has been emphasized increasingly. Here, we report the isolation of a novel seed-specific promoter region from peanut and its validation in Arabidopsis and tobacco seeds. The reported promoter region referred to as groundnut seed promoter (GSP) confers seed-specific expression in heterologous systems, which include putative promoter regions of the peanut (Arachis hypogaea L.) gene 8A4R19G1. This region was isolated, sequenced, and characterized using gel shift assays. Tobacco transgenics obtained using binary vectors carrying uidA reporter gene driven by GSP and/or cauliflower mosaic virus 35S promoters were confirmed through polymerase chain reaction (PCR), RT-PCR, and computational analysis of motifs which revealed the presence of TATA, CAAT boxes, and ATG signals. This seed-specific promoter region successfully targeted the reporter uidA gene to seed tissues in both Arabidopsis and tobacco model systems, where its expression was confirmed by histochemical analysis of the transgenic seeds. This promoter region is routinely being used in the genetic engineering studies in legumes aimed at targeting novel transgenes to the seeds, especially those involved in micronutrient enhancement, fungal resistance, and molecular pharming.

Caulimoviridae (Plant Pararetroviruses)

Abstract: Family Caulimoviridae comprises seven genera of plant viruses whose members replicate by reverse transcription and whose virions contain double-stranded deoxyribonucleic acid. In a recent survey of the international scientific community, Cauliflower mosaic virus (CaMV), the type species of the family, was ranked sixth in the world in a list of the most scientifically or economically important plant viruses. This notoriety is due purely to the major conceptual advances that have been made in plant virology using CaMV as a model pathogen. However, several relatively lesser known viruses in the family, such as Rice tungro bacilliform virus and Cacao swollen shoot virus, are very serious constraints to crop production in tropical regions of the world. In this article, information on the taxonomy, replication cycle, vector transmission, epidemiology and disease management of this important group of plant viruses is summarised. Key Concepts: The Caulimoviridae is the only family of plant viruses with a double-stranded deoxyribonucleic acid (dsDNA) genome, and in common with all viral retroelements, incorporates a reverse transcription step in the replication cycle. Cauliflower mosaic virus is the type species of the family, and has been a very important model plant virus for elucidating fundamental aspects of virus replication, cell-to-cell movement and aphid transmission. Members of the Caulimoviridae are most prominent in tropical regions, where they cause serious diseases such as rice tungro, cacao swollen shoot and banana streak disease. The most important component of a control programme for these viruses is to reduced inoculum levels by using clean planting material, avoiding overlapping crops, and removing diseased plants and alternative hosts of the virus. In some host species, infection can arise as a consequence of activation of viral DNA that is integrated in the nuclear genome of the plant. Keywords: plant viruses; retroelement; pararetrovirus; reverse transcription; cauliflower mosaic virus; rice tungro; cacao swollen shoot; tropical crop

Improved tolerance to boron deficiency by enhanced expression of the boron transporter BOR2

Abstract: Boron (B) cross-links the pectin polysaccharide rhamnogalacturonan II (RG-II) and thus is important for cell wall structure in plants. B deficiency is an agricultural problem that causes significant losses of crop productivity worldwide. To address this, B deficiency-tolerant plants have been generated using B transporters. With the goal of further improving plant tolerance to low-B conditions, we generated transgenic Arabidopsis thaliana (L.) Heynh. with enhanced expression of BOR2, a B transporter that promotes cross-linking of RG-II and root elongation under low B supply. We generated a DNA construct containing the cauliflower mosaic virus 35S RNA promoter, a native promoter of BOR2, a BOR2 gene and green fluorescent protein (GFP) (Pro35S-BOR2:BOR2-GFP), and obtained three independent transgenic lines with relatively high levels of BOR2-GFP expression. In the transgenic lines, BOR2-GFP was expressed mainly in the lateral root caps in the meristem zone and in the epidermal cells in the elongatio...

Two quantitative multiplex real-time PCR systems for the efficient GMO screening of food products

Abstract: According to the EU and Swiss food legislation, only deregulated traits of transgenic plants are allowed to be imported and sold to the consumer. In order to control imports of soya and maize products from retailers, efficient and reliable methods for the detection and quantification are a prerequisite. The screening for specific DNA elements characteristic of transgenic plants is crucial for further analysis and has a major impact on the efficiency of the whole analysis workflow. To allow laboratories to efficiently and reliably screen food products for transgenic plant products, two novel multiplex real-time polymerase chain reaction (PCR) systems were developed and validated. One system determines DNA contents from maize, soya, cauliflower mosaic virus (CaMV) 35S promoter (P35S), NOS terminator from Agrobacterium tumefaciens and CaMV, and the second PCR system simultaneously detects DNA sequences from figwort mosaic virus promoter (PFMV), from bar gene of Streptomyces hygroscopicus, from gene coding for phosphinothricin acetyltransferase (PAT) and from a DNA construct of enolpyruvyl shikimate phosphate synthase gene (CP4-EPSPS) and Arabidopsis thaliana (CPT2). The tests exhibit good specificity and a limit of detection of at least 0.1 % for all analytes.

A tomato endoplasmic reticulum (ER)-type omega-3 fatty acid desaturase (LeFAD3) functions in early seedling tolerance to salinity stress

Abstract: Key message Transgenic tomato plants overexpressing LeFAD3 sense and antisense sequences were generated. Salt stress suppressed the growth of WT and antisense plants to a higher extent than that in sense plants. Abstract In this study, we investigated the role of the LeFAD3-encoding ER-type omega-3 fatty acid desaturase in salt tolerance in tomato plants. We created transgenic tomato plants by overexpressing its sense and antisense sequences under the control of the cauliflower mosaic virus 35S promoter. Based on the results of northern and western blotting as well as quantitative reverse transcription-poly- merase chain reaction, sense plants expressed more desat- urase than wild-type (WT) plants, whereas antisense plants expressed less desaturase than WT. Salt stress suppressed the growth of both WT and antisense plants to a higher extent than that in sense plants, which can be attributed to the fact that sense plants performed better in maintaining the integrity of the membrane system, as revealed by electron microscopy. The concomitant increase in super- oxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.7) may have alleviated the photoinhibition caused by the increased level of ROS in sense plants. Our results suggest that LeFAD3 overexpression can enhance the tolerance of early seedlings to salinity stress.

Molecular and Biological Characterization of an Isolate of Cucumber mosaic virus from Glycine soja by Generating its Infectious Full-genome cDNA Clones

Abstract: Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV) from Glycine soja (wild soybean), named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean) and Pisum sativum (pea) as well as N. benthamiana, but not the other legume species.

Plant virus-host interaction : molecular approaches and viral evolution

Abstract: 1. An overview of RNA silencing in plants against viruses: the fine tuning of defense and counter defense forces 2. Role of RNA silencing in the turnip crinkle virus-arabidopsis interaction 3. Alteration of host-encoded miRNAs in virus infected plants - experimentally verified 4. Host - virus - vector interactions with reference to banana infecting viruses 5. Geminivirus - whitefly interactions at the biological, molecular and genomic levels 6. Hosts and non-hosts in plant virology and the effect of plant viruses on host plants 7. Interference with insect transmission to control plant pathogenic viruses 8. Transmission and host interaction of geminivirus in weeds 9. Tombusvirus-induced multivesicular bodies: origin and role in virus-host interaction 10. Indian cassava mosaic virus and its vector 11. Papaya ringspot virus (PRSV) and papaya leaf curl virus (PLCV) 12. Synergism in plant virus- host interactions: a case study of CMV and PVY in tomato 13. Stability, transmission and host-interaction of papaya lethal yellowing virus in papaya 14. Establishment of endogenous pararetroviruses in the rice genome 15. Volatile organic compounds in plant virus-host interaction 16. Diversity of latent plant virus interactions and their impact on virosphere 17. Plant pathogenic viroids and the transport by insect vectors to crops in agriculture 18. Liberibacters causing greening in citrus and emerging hosts 19. Pathogen and non-pathogen derived resistance in crops against begomovirus 20. Cauliflower mosaic virus (CaMV) up-regulates translation reinitiation of its pregenomic polycistronic 35S RNA via interaction with the cell translational machinery 21. Molecular mechanism of begomovirus evolution and plant defense response 22. Impact of host on plant virus evolution 23. Chloroplast alteration induced by plant virus 24. Virus-virus interactions

Constitutive overexpression of the pollen specific gene SKS13 in leaves reduces aphid performance on Arabidopsis thaliana

Abstract: Background: Plants have developed a variety of mechanisms to counteract aphid attacks. They activate their defences by changing the expression of specific genes. Previously we identified an activation tag mutant of Arabidopsis thaliana on which Myzus persicae population development was reduced. Activation tag mutants are gain-of-function in which the expression of a gene is increased by the insertion of the Cauliflower mosaic virus 35S enhancer that acts on the natural promoter. By further characterizing this previously identified mutant we identified a gene that reduces performance of M. persicae and also provided clues about the mechanism involved. Results: We show that SKU5 SIMILAR 13 (SKS13), a gene whose expression in wild type plants is restricted to pollen and non-responsive to M. persicae attack, is overexpressed in the A. thaliana mutant showing reduced performance of M. persicae. Monitoring M. persicae feeding behaviour on SKS13 overexpressing plants indicated that M. persicae have difficulties feeding from the phloem. The constitutive expression of SKS13 results in accumulation of reactive oxygen species, which is possibly regulated through the jasmonic acid pathway. The enhanced resistance is not aphid species specific as also the population development of Brevicoryne brassicae was affected. Conclusions: We demonstrate that constitutive expression in leaves of the pollen-specific gene SKS13 can enhance plant defence, resulting in a reduction of M. persicae population development and also decreases the transmission of persistent viruses. Overexpression of SKS13 in A. thaliana also affects B. brassicae and possibly other phloem feeding insects as well. Identifying genes that can enhance plant defence against insects will be important to open up new avenues for the development of insect resistant crop plants.

Screening for Gene Function Using the FOX ( F ull-Length cDNA O vere X pressor Gene) Hunting System

Abstract: Mutant resources are indispensable for the characterization of the functions of genes. There are two types of mutants, loss-of-function and gain-of-function mutants. Recently, we have developed a novel system in plants that uses a gain-of-function approach and is named as the FOX (full-length cDNA overexpressor gene) hunting system. In this system, Arabidopsis full-length cDNAs (fl-cDNAs) are randomly over-expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis plants. These transgenic plants, or Arabidopsis FOX lines, possess ectopically expressed fl-cDNAs in their genome. Chemical genomics is a newly emerging field that connects chemical biology with genomes. Since each FOX line expresses an excess amount of the protein from the transgene it can be resistant or hypersensitive to bioactive chemicals when the protein is the target for the chemical. In this protocol, we describe the procedure for identification of the fl-cDNAs responsible for the target of the chemical or for the signal transduction pathway involving the chemical.

Potato transformation with the HvNHX3 gene and the improvement of transformant salt tolerance

Abstract: Transformation by genes of various ion transporters is a tool to study plant salt tolerance mechanisms. In this work, the gene of a vacuolar NHX antiporter HvNHX3 was introduced in two potato Solanum tuberosum L. cultivars, Yubiley Zhukova and Skoroplodny-7. The binary vector pCambia-HvNHX3 was made and used for transformation. This vector carries HvNHX3 and NPTII for kanamycin selection each driven by 35S promoter from cauliflower mosaic virus. The presence of the target gene HvNHX3 and its mRNA were confirmed in 35 out of 48 kanamycin-resistant transformants. The growth parameters of 13 transgenic lines were measured in control and NaCl-containing media. The transformation of a relatively salt-tolerant cv. Yubiley Zhukova did not significantly change its growth characteristics and salt tolerance. In control conditions, most transgenic lines of cv. Skoroplodny-7 had the larger biomass and height. Unlike untransformed plants, they rooted and grew on 100 mM NaCl, indicating their greater salt tolerance.

Addressing the issue of horizontal gene transfer from a diet containing genetically modified components into rat tissues

Abstract: Genetically modified (GM) food crops are considered to have the potential of providing food security especially in developing countries. Scientists have raised concern over the hazards associated with the consumption of genetically modified organisms (GMOs). One of these hazards, which have great controversy reports, is the possible horizontal gene transfer from GM-food or feed to human or animal tissues. Many researches were conducted to investigate the presence of some transgenic sequences in animal tissues fed on GM- crops. Many of the inserted genes in the GM-crops are under the control of the promoter of the Cauliflower mosaic virus (CaMVP35S) and produce insecticidal proteins. Health hazards are suggested to accompany the ingestion of this promoter. CaMVP35S can function in a wide range of organisms (plants and animals). It has also been demonstrated that the CaMV-P35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. The present work was conducted to evaluate the possibility of horizontal gene transfer from a diet containing DNA segments from Cauliflower mosaic virus -35S promoter (CaMVP-35S) to the cells of different organs of rats fed for three months on diets containing genetically modified components. Analysis of the results revealed that: 1) ingested fragments from the CaMV-35S promoter incorporated into blood, liver, and brain tissues of experimental rats, 2) The total mean of transfer of GM target sequences increased significantly by increasing the feeding durations, and 3) The affinity of different transgenic fragments from the ingested GM-diet, to be incorporated into the different tissues of rats varied from one target sequence to the other. Key words: Genetic modification, transgenic sequences, GM-crops, gene flow, CaMV P-35S.

Study on distribution and detection of cauliflower mosaic virus (CaMV) in Dezful region of Iran

Abstract: Cauliflower mosaic virus (CaMV) belong to Caulimovirus is one of the most important causal agent of viral diseases. During autumn and late winter of 2012, 180 canola (Brassica napus) leaf samples were randomly collected from fields around Dezful region in Iran. DAS-ELISA test proved CaMV presence serologically in some samples. RT-PCR reaction using specific primers confirmed ELISA tests. Seven samples out of total samples (3.9%) showed CaMV infection using serological and RT-PCR assays, hence incidence of CaMV in canola fields in Dezful area was confirmed.

Transgene instability due to promoter hypermethylation and deletion in transgenic Nicotiana benthamiana

Abstract: Epigenetics is the study of heritable alternations in gene expression resulting in phenotypic changes without modification of the nucleotide sequence, which could be called a successive evolution. In our study, a plant transformation strategy was employed by using Nicotiana benthamiana (Domin.) to study the heritable stability of the introduced transgenes through the T8 to T9 generations and represented transgene silencing phenomenon at the T9 generation. Through the introduction of the green fluorescent protein (GFP) reporter gene, characteristics of the gene-silencing phenomenon were analyzed. Individual plants were classified as follows according to their phenotype: GFP expression line, GFP mosaic expression line, and silenced line. This phenomenon resulted from by hypermethylation and nucleotide deletion in the cauliflower mosaic virus (CaMV) 35S promoter region. In particular, there was obvious differences in the levels of the expression of the introduced GFP gene and DNA methylation-related endogenous genes including domains rearranged methylase 2 (DRM2), SU(VAR)3-9 homolog 9 (SUVH9), and histone deacetylase 1 (HDA1). Our study demonstrates that the stability of transgenes was controlled by expression levels of genes involved in endogenous DNA methylations and hypermethylation on the introduced promoter, which triggered promoter region deletions or modifications.

Development and Validation of a P-35S, T-nos, T-35S and P-FMV Tetraplex Real-time PCR Screening Method to Detect Regulatory Genes of Genetically Modified Organisms in Food.

Abstract: In routine analysis screening methods based on real-time PCR (polymerase chain reaction) are most commonly used for the detection of genetically modified (GM) plant material in food and feed. Screening tests are based on sequences frequently used for GM development, allowing the detection of a large number of GMOs (genetically modified organisms). Here, we describe the development and validation of a tetraplex real-time PCR screening assay comprising detection systems for the regulatory genes Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens nos terminator, Cauliflower Mosaic Virus 35S terminator and Figwort Mosaic Virus 34S promoter. Three of the four primer and probe combinations have already been published elsewhere, whereas primers and probe for the 35S terminator have been developed in-house. Adjustment of primer and probe concentrations revealed a high PCR sensitivity with insignificant physical cross-talk between the four detection channels. The sensitivity of each PCR-system is sufficient to detect a GMO concentration as low as 0.05% of the containing respective element. The specificity of the described tetraplex is high when tested on DNA from GM maize, soy, rapeseed and tomato. We also demonstrate the robustness of the system by inter-laboratory tests. In conclusion, this method provides a sensitive and reliable screening procedure for the detection of the most frequently used regulatory elements present in GM crops either authorised or unauthorised for food.

Sri Lankan cassava mosaic virus replication associated protein (Rep) triggers transposition of IS426 in Agrobacterium

Abstract: We report a high rate of IS426 transposition in Agrobacterium tumefaciens in the presence of the Sri Lankan cassava mosaic virus (SLCMV) replication associated protein gene (Rep). Upon conjugal transfer of the binary plasmid pCam-SLCMV-Rep with the SLCMV Rep gene in the sense orientation under the transcriptional control of the Cauliflower mosaic virus (CaMV) 35S promoter into the A. tumefaciens vir helper strain EHA105, the binary plasmid size increased in all 15 transconjugants studied. Southern blot analysis of the transconjugants with the binary plasmid probe revealed that the 35S promoter and its proximal sequences in the T-DNA were rearranged. The rearranged sequences harboured the 1.3-kb IS426 element of A. tumefaciens. Conjugal mobilisation of the binary plasmid pCam-SLCMV-asRep, with the SLCMV Rep gene in antisense orientation, did not cause DNA rearrangement in EHA105. A mutated SLCMV Rep, in which a frame shift mutation caused retention of only 27 of the 351 amino acids, did not cause IS426 transposition in A. tumefaciens. These findings show that the multifunctional begomoviral Rep protein of SLCMV triggers transposition of IS426 in Agrobacterium.