Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.

BACKGROUND: Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS: Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS: This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.

Viral protein suppresses oxidative burst and salicylic acid-dependent autophagy and facilitates bacterial growth on virus-infected plants.

Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection.

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.

Hitching a ride on vesicles: cauliflower mosaic virus movement protein trafficking in the endomembrane system.

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 CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.

Virus factories of cauliflower mosaic virus are virion reservoirs that engage actively in vector transmission.

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.

Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling.

Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.

Differences in the mechanism of inoculation between a semi-persistent and a non-persistent aphid-transmitted plant virus.

Inoculation of the semi-persistent cauliflower mosaic virus (CaMV, genus Caulimovirus) is associated with successive brief (5-10 s) intracellular stylet punctures (pd) when aphids probe in epidermal and mesophyll cells. In contrast to non-persistent viruses, there is no evidence for which of the pd subphases (II-1, II-2 and II-3) is involved in the inoculation of CaMV. Experiments were conducted using the electrical penetration graph (EPG) technique to investigate which particular subphases of the pd are associated with the inoculation of CaMV to turnip by its aphid vector Brevicoryne brassicae. In addition, the same aphid species/test plant combination was used to compare the role of the pd subphases in the inoculation of the non-persistent turnip mosaic virus (TuMV, genus Potyvirus). Inoculation of TuMV was found to be related to subphase II-1, confirming earlier results, but CaMV inoculation appeared to be related exclusively to subphase II-2 instead. The mechanism of CaMV inoculation and the possible nature of subphase II-2 are discussed in the scope of our findings.

Dynamics of the multiplicity of cellular infection in a plant virus.

Recombination, complementation and competition profoundly influence virus evolution and epidemiology. Since viruses are intracellular parasites, the basic parameter determining the potential for such interactions is the multiplicity of cellular infection (cellular MOI), i.e. the number of viral genome units that effectively infect a cell. The cellular MOI values that prevail in host organisms have rarely been investigated, and whether they remain constant or change widely during host invasion is totally unknown. Here, we fill this experimental gap by presenting the first detailed analysis of the dynamics of the cellular MOI during colonization of a host plant by a virus. Our results reveal ample variations between different leaf levels during the course of infection, with values starting close to 2 and increasing up to 13 before decreasing to initial levels in the latest infection stages. By revealing wide dynamic changes throughout a single infection, we here illustrate the existence of complex scenarios where the opportunity for recombination, complementation and competition among viral genomes changes greatly at different infection phases and at different locations within a multi-cellular host.

Complete genome sequence of a dahlia common mosaic virus isolate from New Zealand.

Dahlia mosaic disease of the ornamental flowering plant Dahlia is caused by two caulimoviruses, dahlia mosaic virus (DMV) and dahlia common mosaic virus (DCMV). We used a rolling-circle amplification method to amplify, clone and determine for the first time the full genome sequence of a DCMV isolate from New Zealand (DCMV-NZ). Within the 7949-bp circular double-stranded retro-transcribing DCMV-NZ DNA, we identified six putative open reading frames, typical of all genomes in the family Caulimoviridae. The availability of the complete DCMV sequence provides a reference genome against which all others can be compared.

Water deficit changes the relationships between epidemiological traits of Cauliflower mosaic virus across diverse Arabidopsis thaliana accessions.

Changes in plant abiotic environments may alter plant virus epidemiological traits, but how such changes actually affect their quantitative relationships is poorly understood. Here, we investigated the effects of water deficit on Cauliflower mosaic virus (CaMV) traits (virulence, accumulation, and vectored-transmission rate) in 24 natural Arabidopsis thaliana accessions grown under strictly controlled environmental conditions. CaMV virulence increased significantly in response to water deficit during vegetative growth in all A. thaliana accessions, while viral transmission by aphids and within-host accumulation were significantly altered in only a few. Under well-watered conditions, CaMV accumulation was correlated positively with CaMV transmission by aphids, while under water deficit, this relationship was reversed. Hence, under water deficit, high CaMV accumulation did not predispose to increased horizontal transmission. No other significant relationship between viral traits could be detected. Across accessions, significant relationships between climate at collection sites and viral traits were detected but require further investigation. Interactions between epidemiological traits and their alteration under abiotic stresses must be accounted for when modelling plant virus epidemiology under scenarios of climate change.

A role for plant microtubules in the formation of transmission-specific inclusion bodies of Cauliflower mosaic virus.

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission.

The genome of the Cauliflower mosaic virus, a plant pararetrovirus, is highly methylated in the nucleus.

Cytosine methylation is an important defense against invasive DNAs. Here, cytosine methylation profiles of a plant pararetrovirus, Cauliflower mosaic virus (CaMV), were investigated. Nuclear CaMV DNA is highly methylated throughout the genome including at transcription regulatory regions, but the virion DNA is unmethylated. In vitro CG methylation of the viral 35S promoter reduces transcription from the downstream gene. Although nuclear CaMV DNA is highly methylated, its transcripts are accumulated in the nucleus. The data suggest that a small population of unmethylated viral genomes produced through reverse transcription are constantly delivered back to the nucleus. Small RNA profiles suggest that methylation of the CaMV DNA may be due to de novo methylation through 21-, 22-, and 24-nt small RNAs with adenines at their 5' terminus.

An update and perspectives on the use of promoters in plant genetic engineering.

Genetically engineered plants have varied applications in agriculture for enhancing the values of food and feed. Genetic engineering aims to introduce selected genetic regions with desirable traits into target plants for both spatial and temporal expressions. Promoters are the key elements responsible for regulating gene expressions by modulating the transcription factors (TFs) through recognition of RNA polymerases. Based on their recognition and expression, RNA polymerases were categorized into RNA pol II and pol III promoters. Promoter activity and specificity are the two prime parameters in regulating the transgene expression. Since the use of constitutive promoters like Cauliflower mosaic virus (CaMV) 35S may lead to adverse effects on nontarget organisms or ecosystem, inducible/tissue specific promoters and/or the RNA pol III promoters provide myriad opportunities for gene expressions with controlled regulation and with minimum adverse effects. Besides their role in transgene expression, their influence in synthetic biology and genome editing are also discussed. This review provides an update on the importance, current prospects, and insight into the advantages and disadvantages of promoters reported thus far would help to utilize them in the endeavour to develop nutritionally and agronomically improved transgenic crops for commercialization.

A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens.

Phytic acid (myo-inositol hexakisphosphate, InsP6) is an important phosphate store and signal molecule in plants. However, low-phytate plants are being developed to minimize the negative health effects of dietary InsP6 and pollution caused by undigested InsP6 in animal waste. InsP6 levels were diminished in transgenic potato plants constitutively expressing an antisense gene sequence for myo-inositol 3-phosphate synthase (IPS, catalysing the first step in InsP6 biosynthesis) or Escherichia coli polyphosphate kinase. These plants were less resistant to the avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In Arabidopsis thaliana, mutation of the gene for the enzyme catalysing the final step of InsP6 biosynthesis (InsP5 2-kinase) also diminished InsP6 levels and enhanced susceptibility to TMV and to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae. Arabidopsis thaliana has three IPS genes (AtIPS1-3). Mutant atips2 plants were depleted in InsP6 and were hypersusceptible to TMV, turnip mosaic virus, cucumber mosaic virus and cauliflower mosaic virus as well as to the fungus Botrytis cinerea and to P. syringae. Mutant atips2 and atipk1 plants were as hypersusceptible to infection as plants unable to accumulate salicylic acid (SA) but their increased susceptibility was not due to reduced levels of SA. In contrast, mutant atips1 plants, which were also depleted in InsP6, were not compromised in resistance to pathogens, suggesting that a specific pool of InsP6 regulates defence against phytopathogens.

Application of game theory to the interaction between plant viruses during mixed infections.

Natural mixed infections of plant viruses are frequent, often leading to unpredictable variations in symptoms, infectivity, accumulation and/or vector transmissibility. Cauliflower mosaic caulimovirus (CaMV) has often been found in mixed infections with turnip mosaic potyvirus (TuMV) in plants of the genus Brassica. This study addressed the effect of mixed infection on infectivity, pathogenicity and accumulation of CaMV and TuMV in Arabidopsis thaliana plants inoculated mechanically with cDNA infectious clones. In singly infected plants, TuMV accumulation was approximately 8-fold higher than that of CaMV. In co-infected plants, there was 77 % more TuMV accumulation compared with single infections, whilst the accumulation of CaMV was 56 % lower. This outcome describes a biological game in which TuMV always plays the winner strategy, leading to the competitive exclusion of CaMV. However, the infectivity of each virus was not affected by the presence of the other, and no symptom synergism was observed.

Cauliflower mosaic virus transactivator protein (TAV) can suppress nonsense-mediated decay by targeting VARICOSE, a scaffold protein of the decapping complex.

During pathogenesis, viruses hijack the host cellular machinery to access molecules and sub-cellular structures needed for infection. We have evidence that the multifunctional viral translation transactivator/viroplasmin (TAV) protein from Cauliflower mosaic virus (CaMV) can function as a suppressor of nonsense-mediated mRNA decay (NMD). TAV interacts specifically with a scaffold protein of the decapping complex VARICOSE (VCS) in the yeast two-hybrid system, and co-localizes with components of the decapping complex in planta. Notably, plants transgenic for TAV accumulate endogenous NMD-elicited mRNAs, while decay of AU-rich instability element (ARE)-signal containing mRNAs are not affected. Using an agroinfiltration-based transient assay we confirmed that TAV specifically stabilizes mRNA containing a premature termination codon (PTC) in a VCS-dependent manner. We have identified a TAV motif consisting of 12 of the 520 amino acids in the full-length sequence that is critical for both VCS binding and the NMD suppression effect. Our data suggest that TAV can intercept NMD by targeting the decapping machinery through the scaffold protein VARICOSE, indicating that 5'-3' mRNA decapping is a late step in NMD-related mRNA degradation in plants.

Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus.

The split GFP technique is based on the auto-assembly of GFP when two polypeptides-GFP1-10 (residues 1-214; the detector) and GFP11 (residues 215-230; the tag)-both non-fluorescing on their own, associate spontaneously to form a fluorescent molecule. We evaluated this technique for its efficacy in contributing to the characterization of Cauliflower mosaic virus (CaMV) infection. A recombinant CaMV with GFP11 fused to the viral protein P6 (a key player in CaMV infection and major constituent of viral factory inclusions that arise during infection) was constructed and used to inoculate transgenic Arabidopsis thaliana expressing GFP1-10. The mutant virus (CaMV11P6) was infectious, aphid-transmissible and the insertion was stable over many passages. Symptoms on infected plants were delayed and milder. Viral protein accumulation, especially of recombinant 11P6, was greatly decreased, impeding its detection early in infection. Nonetheless, spread of infection from the inoculated leaf to other leaves was followed by whole plant imaging. Infected cells displayed in real time confocal laser scanning microscopy fluorescence in wild type-looking virus factories. Thus, it allowed for the first time to track a CaMV protein in vivo in the context of an authentic infection. 11P6 was immunoprecipitated with anti-GFP nanobodies, presenting a new application for the split GFP system in protein-protein interaction assays and proteomics. Taken together, split GFP can be an attractive alternative to using the entire GFP for protein tagging.

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5) to 4 x 10(-5). This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

Membrane and protein dynamics in virus-infected plant cells.

In terms of functional genomics research, Nicotiana benthamiana, more so than other model plants, is highly amenable to high-throughput methods, especially those employing virus-induced gene silencing and agroinfiltration. Furthermore, through recent and ongoing sequencing projects, there are now upward of 18,000 unique N. benthamiana ESTs to support functional genomics research. Despite these advances, the cell biology of N. benthamiana itself, and in the context of virus infection, lags behind that of other model systems. Therefore, to meet the challenges of diverse cell biology studies that will be derived from ongoing functional genomics projects, a series of methods relevant to the characterization of membrane and protein dynamics in virus-infected cells are provided here. The data presented here were derived from our studies with plant rhabdoviruses. However, the employed techniques should be broadly applicable within the field of plant virology. We report here on the use of a novel series of binary vectors for the transient or stable expression of autofluorescent protein fusions in plants. Use of these vectors in conjunction with advanced microscopy techniques such as fluorescent recovery after photobleaching and total internal fluorescence microscopy, has revealed novel insight into the membrane and protein dynamics of virus-infected cells.

The P1 gene of Cauliflower mosaic virus is responsible for breaking resistance in Arabidopsis thaliana ecotype Enkheim (En-2).

Arabidopsis thaliana ecotype En-2 is resistant to several strains of Cauliflower mosaic virus (CaMV), including strain W260, but is susceptible to strain NY8153. Resistance in En-2 is conditioned by a single, semi-dominant gene called CAR1. We constructed several recombinant infectious clones between W260 and NY8153 and evaluated their capability to infect En-2. This analysis showed that the capacity of NY8153 to break resistance in En-2 was conditioned by mutations within the CaMV gene 1, a gene that encodes a protein dedicated to cell-to-cell movement (P1), and conversely, that P1 of W260 is responsible for eliciting the plant defense response. A previous study had shown that P6 of W260 was responsible for overcoming resistance in Arabidopsis ecotype Tsu-0 and that P6 of CaMV strain CM1841 was responsible for triggering resistance. The present study now shows that a second gene of CaMV is targeted by Arabidopsis for plant immunity.