Cauliflower Mosaic Virus Isolates Research Articles

Investigation of CaMV-host co-evolution through synonymous codon pattern.

Cauliflower mosaic virus (CaMV) has a double-stranded DNA genome and is globally distributed. The phylogeny tree of 121 CaMV isolates was categorized into two primary groups, with Iranian isolates showing the greatest genetic variations. Nucleotide A demonstrated the highest percentage (36.95%) in the CaMV genome and the dinucleotide odds ratio analysis revealed that TC dinucleotide (1.34 ≥ 1.23) and CG dinucleotide (0.63 ≤ 0.78) are overrepresented and underrepresented, respectively. Relative synonymous codon usage (RSCU) analysis confirmed codon usage bias in CaMV and its hosts. Brassica oleracea and Brassica rapa, among the susceptible hosts of CaMV, showed a codon adaptation index (CAI) value above 0.8. Additionally, relative codon deoptimization index (RCDI) results exhibited the highest degree of deoptimization in Raphanus sativus. These findings suggest that the genes of CaMV underwent codon adaptation with its hosts. Among the CaMV open reading frames (ORFs), genes that produce reverse transcriptase and virus coat proteins showed the highest CAI value of 0.83. These genes are crucial for the creation of new virion particles. The results confirm that CaMV co-evolved with its host to ensure the optimal expression of its genes in the hosts, allowing for easy infection and effective spread. To detect the force behind codon usage bias, an effective number of codons (ENC)-plot and neutrality plot were conducted. The results indicated that natural selection is the primary factor influencing CaMV codon usage bias.

Serological, biological and molecular characterization of viruses causing mosaic diseases on cabbage (Brassica sp L.) in Central Ethiopia.

The productivity of cabbage (Brassica oleracea var. capitata) in Ethiopia has been generally low due to several biotic and abiotic constraints among which are several viral diseases. There is a recent report indicating that this economically important vegetable is seriously affected in Ethiopia by cauliflower mosaic virus (CaMV) and turnip mosaic virus (TuMV). However, little information exists on the incidence and distribution of these viruses as the previous report is based on samples only from Addis Ababa. In this study, a total of 370 leaf samples were collected from 75 cabbage growing fields in Central Ethiopia in two rounds of survey. Two cabbage varieties locally known as "Habesha gomen" and "Tikur gomen" with virus-like symptoms were collected and tested with Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) using polyclonal antibodies specific to CaMV and TuMV. Results from serological diagnosis were confirmed with PCR and Sanger sequencing. The results indicated a high incidence and wide distribution of both viruses in Central Ethiopia with an average of 29.5% infection for CaMV and 40% for TuMV. Biological inoculation tests for CaMV or TuMV or both on healthy cabbage seedlings gave similar symptoms as those observed in the field. Symptom severity was higher with co-infection of CaMV and TuMV followed by TuMV single infection. BLAST analysis showed that TuMV and CaMV isolates from Ethiopia have nucleotide identity of 95-98% and 93-98%, respectively to previously reported isolates. Phylogenetic analysis revealed that CaMV isolates from Ethiopia are closely related to isolates from USA and Italy within Group II clade whereas TuMV isolates have close similarities with isolates from World B clade including isolates from Kenya, UK, Japan and the Netherlands. The identification of the causative agents of the mosaic disease observed on cabbage in Central Ethiopia may lay the foundation for future management studies.

Complete Genome Sequence of Isolate Bari 1, a Mild Strain of Cauliflower Mosaic Virus.

ABSTRACTWe present here the complete genome sequence of isolate Bari 1, a mild strain of cauliflower mosaic virus (CaMV). The isolate was collected from Diplotaxis tenuifolia (perennial wall-rocket) in Bari, Italy. The genome was 8,020 nucleotides long and shared ≤85.4% nucleotide identity with other CaMV isolates.

Cauliflower mosaic virus protein P6-TAV plays a major role in alteration of aphid vector feeding behaviour but not performance on infected Arabidopsis.

Emerging evidence suggests that viral infection modifies host plant traits that in turn alter behaviour and performance of vectors colonizing the plants in a way conducive for transmission of both nonpersistent and persistent viruses. Similar evidence for semipersistent viruses like cauliflower mosaic virus (CaMV) is scarce. Here we compared the effects of Arabidopsis infection with mild (CM) and severe (JI) CaMV isolates on the feeding behaviour (recorded by the electrical penetration graph technique) and fecundity of the aphid vector Myzus persicae. Compared to mock‐inoculated plants, feeding behaviour was altered similarly on CM‐ and JI‐infected plants, but only aphids on JI‐infected plants had reduced fecundity. To evaluate the role of the multifunctional CaMV protein P6‐TAV, aphid feeding behaviour and fecundity were tested on transgenic Arabidopsis plants expressing wild‐type (wt) and mutant versions of P6‐TAV. In contrast to viral infection, aphid fecundity was unchanged on all transgenic lines, suggesting that other viral factors compromise fecundity. Aphid feeding behaviour was modified on wt P6‐CM‐, but not on wt P6‐JI‐expressing plants. Analysis of plants expressing P6 mutants identified N‐terminal P6 domains contributing to modification of feeding behaviour. Taken together, we show that CaMV infection can modify both aphid fecundity and feeding behaviour and that P6 is only involved in the latter.

Improved detection and quantification of cauliflower mosaic virus in food crops: assessing false positives in GMO screening based on the 35S promoter

The 35S promoter (P35S) of the cauliflower mosaic virus (CaMV) is frequently used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in food and feed samples. However, PCR reactions targeting P35S do not distinguish between GMOs and naturally occurring CaMV introduced by infection or contamination, resulting in false positives. Initial attempts at specific CaMV detection were based on a limited number of CAMV sequences. To account for the broad variability in the viral genome, we designed a new real-time PCR assay based on the genomic sequences of 96 CaMV isolates collected worldwide. This system amplified a 112 bp fragment of ORFV in the double-stranded genome, with complete sequence homology to the primer and probe targets of the tested genomes. Tests on a CaMV isolate collection and GMOs revealed improved specificity compared to previously published methods as well as high sensitivity, with limits of detection (LOD) and quantification (LOQ) of three copies of CaMV.

Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV.

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11–13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.

First fully sequenced genome of an Australian isolate of Cauliflower mosaic virus

We report the first fully sequenced genome of an Australian isolate of Cauliflower mosaic virus. The circular genome is 8022 base pairs in length. A phylogenetic analysis suggests recent common ancestry of this virus isolate with those from Japan and the USA, and origins of the clade in western Europe.

Detection of herbaceous-plant pararetrovirus in lichen herbarium samples.

Cauliflower mosaic virus (CaMV) - a plant pararetrovirus that naturally causes diseases in Brassicaceae and Solanaceae plant hosts worldwide - has been detected by PCR for the first time in herbarium samples of Usnea sp. lichens. The virus's presence in these lichens did not result in any micro- or macromorphological changes, and the herbarium records were classified as representative for the distinct species. Sequence analyses classified all the detected viruses into one lineage of CaMV isolates. We have shown here that herbarium samples could be a good source for virus study, especially where a longer time span is involved.

Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern.

The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.

A Distinct Subpopulation of Cauliflower mosaic virus (CaMV) in South Iran

Cauliflower mosaic virus (CaMV) with a high incidence and widespread distribution on Brassica crops in Iran reduces the yield and quality of these crops. The complete sequences of three open reading frames (ORFs) 2, 4 and 6 coding for aphid transmission (AT), coat protein (CP) and inclusion body protein/translation transactivator (TAV) genes, respectively, were determined for two Iranian CaMV isolates from Kerman (south Iran). They induced latent or mild mottle (L/MMo) infection in Brassica oleracea var. capitata so are considered as the (L/MMo) biotype. Clear recombination breakpoints were detected between ORF2 and ORF6 in two Kerman isolates using concatenate fragments. Phylogenetic analysis revealed three Iranian CaMV subpopulations in which the two Kerman isolates in the new subgroup C were added to the two previously reported Iranian subpopulations A (central and west Iran) and B (north-east Iran). Also three regions of pairwise identity were detected which representing: 97.1–100, 93.8–97.1 and 90.6–93.8% for subgroups A, C and B, respectively. Our analysis showed the high variability of Iranian CaMV population and provided valuable new information for understanding the diversity and evolution of caulimoviruses. Furthermore, star phylogeny was found in the subgroup C with overall lack of nt diversity and high haplotype diversity as evidence of a recent population expansion after a genetic bottleneck although this may have been modified subsequently by clinal genetic drift. The appearance of new genetic types demonstrates a high potential of risks and should be considered in the planning of efficient control programmes.

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

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.

Unconventional P-35S sequence identified in genetically modified maize

The Cauliflower Mosaic Virus 35S promoter sequence, CaMV P-35S, is one of several commonly used genetic targets to detect genetically modified maize and is found in most GMOs. In this research we report the finding of an alternative P-35S sequence and its incidence in GM maize marketed in Jordan. The primer pair normally used to amplify a 123 bp DNA fragment of the CaMV P-35S promoter in GMOs also amplified a previously undetected alternative sequence of CaMV P-35S in GM maize samples which we term V3. The amplified V3 sequence comprises 386 base pairs and was not found in the standard wild-type maize, MON810 and MON 863 GM maize. The identified GM maize samples carrying the V3 sequence were found free of CaMV when compared with CaMV infected brown mustard sample. The data of sequence alignment analysis of the V3 genetic element showed 90% similarity with the matching P-35S sequence of the cauliflower mosaic virus isolate CabbB-JI and 99% similarity with matching P-35S sequences found in several binary plant vectors, of which the binary vector locus JQ693018 is one example. The current study showed an increase of 44% in the incidence of the identified 386 bp sequence in GM maize sold in Jordan’s markets during the period 2009 and 2012.

A phylogeographical study of the cauliflower mosaic virus population in mid-Eurasia Iran using complete genome analysis

The full-length sequences of 34 Iranian cauliflower mosaic virus (CaMV) isolates were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic variability and patterns of genetic exchange in CaMV isolates from Iran. Based on the severity of symptoms and their ability to infect Brassica oleracea var. capitata, Iranian CaMV isolates were grouped into two distinct biotypes: latent/mild mottle (LI/MMo) and severe (S) infection. Recombination breakpoints were detected between the large intergenic region (LIR) and open reading frame (ORF) V (event 2); between ORF VII and ORF II (event 3), between ORF I and ORF III (event 4), and within ORF VI (event 1). Phylogenetic analysis indicated that Iranian CaMV isolates clustered into two subgroups belonging to group I (GI) that were distinct from North American and European isolates from group II (GII). Northeast Iranian isolates (subgroup B) and CaMV isolates from subgroup A closely corresponded to the S and LI/MMo biological groups, respectively. Genome-wide pairwise identity analysis of the CaMV isolates revealed three regions of pairwise identity representation: 92-94 % for GII and 94-96 % and 98-100 % for subgroups A and B. The within-population diversity was lower than the between-population diversity, suggesting the contribution of a founder effect on diversification of CaMV isolates. Amino acid sequences were conserved, with ω values ranging from 0.074 to 0.717 in different proteins. Thirteen amino acids in the deduced proteins of ORFs I, II, III, VI and VII were under positive selection (ω > 1), whereas purifying selection applied to the proteins encoded by ORFs IV and V. This study suggests that variation in the CaMV population can be explained by host-range differentiation and selection pressure. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of CaMV strains, providing valuable new information for understanding the diversity and evolution of caulimoviruses.

Biological and molecular variation of Iranian Cauliflower mosaic virus (CaMV) isolates

Seventeen provinces of Iran were surveyed during 2003-2012 to find Brassicaceae hosts of Cauliflower mosaic virus (CaMV). A total 397 samples were collected from plants with virus-like symptoms. Among those tested by ELISA, 255 samples (67.2%) were found to be infected with CaMV. Mechanical transmission tests showed that the Iranian isolates have similar biological properties on a number of Brassica and Raphanus plant species and cultivars tested. However, the isolates varied in the severity of symptoms they induced and in the capacity to infect B. oleracea var. capitata, on the basis of which they were grouped into two distinct biotypes L/MMo (latent/mild mottle) and severe (S) infection. The molecular diversity of natural population of CaMV were investigated based on the complete sequences of OFR 6 of 36 Iranian isolates collected from different geographically distant regions in Iran alongside the sequences of 14 previously reported isolates. Phylogenetic analyses indicated that the Iranian CaMV isolates belong to two groups (GI and GII). Most of the Iranian isolates fell into GI with other exotic isolates; however, the isolates from North-East Iran with Xinjiang from China fell into GII. The phylogenetic group GII (the North-East Iranian isolates) closely corresponded to the S biological group however other Iranian isolates corresponded to the L/MMo biological group. The within-population diversity was lower than the between population diversity suggesting the contribution of a founder effect on diversification of CaMV isolates. The Iranian isolates were differentiated from other exotic CaMV isolates and clustered into two RFLP groups using Hpy99I which closely corresponded to the biological and phylogenetic groups. This study showed the evolutionary process in CaMV isolates is shaped by a combination of host range differentiation and nucleotide substitution using the approach of population genetics.

Reduction of leaf area and symptom severity as proxies of disease-induced plant mortality: The example of the Cauliflower mosaic virus infecting two Brassicaceae hosts

Disease induced effects on host survival are important to understand the evolution of parasitic virulence and host resistance/tolerance. Unfortunately, experiments evaluating such effects are in most cases logistically demanding justifying the measurement of survival proxies. For plant hosts commonly used proxies are leaf area and the nature and severity of visual qualitative disease symptoms. In this study we tested whether these traits are indeed correlated to the host mortality rate induced by viral infection. We infected Brassica rapa and Arabidopsis thaliana plants with different natural isolates of Cauliflower mosaic virus (CaMV) and estimated over time the development of symptoms and the relative reduction of leaf area compared to healthy plants and followed plant mortality. We observed that the mortality of infected plants was correlated with the relative reduction of leaf area of both B. rapa and A. thaliana. Measures of mortality were also correlated with the severity of visual qualitative symptoms but the magnitude of the correlations and the time frame at which they were significant depended on the host plant: stronger and earlier correlations were observed on A. thaliana.

AN EXPERIMENTAL TEST OF THE TRANSMISSION-VIRULENCE TRADE-OFF HYPOTHESIS IN A PLANT VIRUS

The transmission-virulence trade-off hypothesis is one of the few adaptive explanations of virulence evolution, and assumes that there is an overall positive correlation between parasite transmission and virulence. The shape of the transmission-virulence relationship predicts whether virulence should evolve toward either a maximum or to an intermediate optimum. A positive correlation between each of these traits and within-host growth is often suggested to underlie the relationship between virulence and transmission. There are few experimental tests of this hypothesis; this study reports on the first empirical test on a plant pathogen. We infected Brassica rapa plants with nine natural isolates of Cauliflower mosaic virus and then estimated three traits: transmission, virulence, and within-host viral accumulation. As predicted by the trade-off hypothesis, we observed a positive correlation between transmission and virulence, suggestive of the existence of an intermediate optimum. We discovered the unexpected existence of two groups of within-host accumulation, differing by at least an order of magnitude. When accumulation groups were not accounted for, within-host accumulation was correlated neither to virulence nor transmission, although our results suggest that within each group these correlations exist.

Cauliflower mosaic virus major inclusion body protein interacts with the aphid transmission factor, the virion-associated protein, and gene VII product

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral infection. In order to perform its various tasks, P6 interacts with both viral and host factors, as well as forming electron-dense cytoplasmic inclusion bodies. Here we investigate the interactions of P6 with three CaMV proteins: P2 (aphid transmission factor), P3 (virion-associated protein), and P7 (protein of unknown function). Based on yeast two-hybrid and maltose-binding protein pull-down experiments, P6 interacted with all three of these CaMV proteins. P2 helps to stabilize P6 inclusion bodies. Although the P2s from two CaMV isolates (W260 and CM1841) differ in the ability to stabilize inclusion bodies, both interacted similarly with P6. This suggests that inclusion body stability may not be dependent on the efficiency of P2–P6 interaction. However, neither P2 nor P3 interacted with P7 in yeast two-hybrid assays.

Cauliflower mosaic virus gene VI product N-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein

Cauliflower mosaic virus (CaMV) gene VI encodes a multifunctional protein (P6) involved in the translation of viral RNA, the formation of inclusion bodies, and the determination of host range. Arabidopsis thaliana ecotype Tsu-0 prevents the systemic spread of most CaMV isolates, including CM1841. However, CaMV isolate W260 overcomes this resistance. In this paper, the N-terminal 110 amino acids of P6 (termed D1) were identified as the resistance-breaking region. D1 also bound full-length P6. Furthermore, binding of W260 D1 to P6 induced higher β-galactosidase activity and better leucine-independent growth in the yeast two-hybrid system than its CM1841 counterpart. Thus, W260 may evade Tsu-0 resistance by mediating P6 self-association in a manner different from that of CM1841. Because Tsu-0 resistance prevents virus movement, interaction of P6 with P1 (CaMV movement protein) was investigated. Both yeast two-hybrid analyses and maltose-binding protein pull-down experiments show that P6 interacts with P1. Although neither half of P1 interacts with P6, the N-terminus of P6 binds P1. Interestingly, D1 by itself does not interact with P1, indicating that different portions of the P6 N-terminus are involved in different activities. The P1–P6 interactions suggest a role for P6 in virus transport, possibly by regulating P1 tubule formation or the assembly of movement complexes.

Carbohydrate partitioning and sugar signalling in Cauliflower mosaic virus-infected turnip and Arabidopsis

We analyzed the correlation between the development of symptoms, virus titre and carbohydrate levels in turnip and Arabidopsis during infection with four isolates of Cauliflower mosaic virus (CaMV). Infection did not significantly affect sugar levels in source leaves except at the very late stages (28 d.p.i.), but induced a three-fold but short lived increase in sink leaves. Titres of severe isolates were greater than mild isolates, but we observed no obvious correlation between sugar levels and symptom appearance. In wild-type Arabidopsis, infection did not stimulate increased sugar levels, but did so in a mutant, cnr160, which shows altered growth-response to high carbon and low nitrogen. These results do not support a direct role for sugar-mediated control of symptom development during virus infection.

Real-Time Polymerase Chain Reaction Detection of Cauliflower mosaic virus to Complement the 35S Screening Assay for Genetically Modified Organisms

Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.