Plant DNA viruses and gene silencing.

Gene silencing is a multifaceted phenomenon leading to propagative down-regulation of gene expression. Gene silencing, first observed in plants containing transgenes, can operate both at the transcriptional and post-transcriptional levels. Silencing effects can be triggered by nuclear transgenes and by cytoplasmic RNA viruses, and it can be propagated between these elements and endogenous plant genes that share sequence homology. Although some aspects of gene silencing are becoming better understood, little is yet known about the relationship between nuclear and cytoplasmic events. Plant DNA viruses-- both the ssDNA geminiviruses and the reverse-transcribing pararetroviruses-- have properties with the potential to initiate gene silencing in the nucleus and in the cytoplasm. Characteristics include production of multiple copies of viral DNA genomes in the nucleus, illegitimate integration of viral DNA into host chromosomes mimicking transgene transformation, and generation of abundant viral RNAs in the cytoplasm. Evidence is emerging that geminiviruses and plant pararetroviruses can interact with the gene silencing system either from introduced DNA constructs or during viral pathogenesis. Some observations suggest there are complex relationships between DNA viral activity, transcriptional and post-transcriptional gene silencing mechanisms. DNA viruses also have properties consistent with an ability to counteract the plant silencing response. In this article, features of plant DNA viruses are discussed in relation to gene silencing phenomena, and the prospects for understanding the interaction between nuclear and cytoplasmic silencing processes.

Plants rendered herbicide-susceptible by cauliflower mosaic virus-elicited suppression of a 35S promoter-regulated transgene.

Crop plants genetically modified for herbicide tolerance were some of the first to be released into the environment. Frequently, the cauliflower mosaic virus (CaMV) 35S promoter is used to drive expression of the herbicide tolerance transgene. We analyzed the response to CaMV infection of a transgenic oilseed rape line containing the bialaphos tolerance gene (BAR) from Streptomyces hygroscopicus, regulated by the 35S promoter. Oilseed rape is susceptible to CaMV, but plants recover from infection. CaMV infection altered the expression of the herbicide tolerance gene such that plants became susceptible to the herbicide. The effect on transgene expression differed in infections with viral pathogenic variants typical of those found in natural situations worldwide. Susceptibility to the herbicide was most likely a result of transcriptional gene silencing of the transgene. Our results show that transgene phenotypes can be modified by pathogen invasion.

Pararetrovirus-crucifer interactions: attack and defence or modus vivendi?

Abstract The compatible infection of plants by viruses usually leads to the development of systemic symptoms. Symptom expression of this kind is generally understood to be a host response that indicates an inability of the host to defend itself from attack. We have been studying compatible interactions between the plant pararetrovirus cauliflower mosaic virus (CaMV) and its crucifer hosts in order to understand the relationship between viral activity, symptom expression and plant defence. A CaMV protein (P6) appears to play a major role in eliciting symptom expression. This host response leads to a regulation of the viral multiplication cycle that is associated with leaf mosaics. The host regulation of CaMV appears to operate at the transcriptional level through an effect on the 35S promoter, or at the post-transcriptional level by a process that is akin to gene silencing, and can lead to host recovery depending upon the genetic background of the host. The plant apex is a focus for antiviral defence mechanisms, presumably because viral infection of the apical meristem would rapidly compromise the ability of the plant to generate new leaves and flowers for reproduction. The balance of interactions between CaMV and crucifers can provide a sustainable source of host plants to ensure viral propagation and viral exposure allows the host to adapt and develop its repertoire of defence mechanisms.

Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene.

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.

Analysis of polypurine tract-associated DNA plus-strand priming in vivo utilizing a plant pararetroviral vector carrying redundant ectopic priming elements.

Initiation of DNA plus-strand synthesis in most reverse-transcribing elements requires primer generation by reverse transcriptase-associated RNase H at one or more template polypurine tracts (PPTs). We have exploited infectious clones of the plant pararetrovirus cauliflower mosaic virus carrying redundant ectopic plus-strand priming elements to study priming in vivo. Ectopic priming generated an additional discontinuity in progeny virion DNA during infection of plants. We found that altering the length of the 13-base pair PPT by +/-25% significantly reduced priming efficiency. A short pyrimidine tract 5' to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5' end remained 3 nucleotides from the PPT 3' end in mutant primers that were longer or shorter than the wild-type primer. Use of an ectopic redundant primer to study replication-dependent priming was validated by demonstrating that it could rescue infectivity following destruction of the wild-type priming elements. We propose a model for plant pararetroviral plus-strand priming in which pyrimidines enhance PPT recognition during polymerase-dependent RNase H cleavages, and suggest that fidelity of primer maturation during polymerase-independent cleavages involves PPT length measurement and 3' end recognition by RNase H.

Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis.

Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35Stms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene. All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene. From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures.

Transgenic Arabidopsis lines expressing gene VI from cauliflower mosaic virus variants exhibit a range of symptom-like phenotypes and accumulate inclusion bodies.

Gene VI of cauliflower mosaic virus (CaMV) is an important determinant of symptom expression during infection. We have constructed a series of transgenic Arabidopsis lines that express gene VI protein (P6) from two CaMV isolates (Bari-1 and Cabb B-JI) that cause mild and severe symptoms, respectively, in Arabidopsis, and from a recombinant virus (Baji-31) with a hybrid gene VI that causes very severe symptoms. From 41 transgenic lines analyzed, 17 showed symptom-like phenotypes that ranged from mild vein chlorosis to severe chlorosis and stunting. P6 levels in transgenic lines varied from undetectable in the lowest expressors to levels greater than those in CaMV-infected plants. There was a strong correlation between phenotype severity and the level of P6, and with the gene VI origin in the order, Baji-31 > B-JI > Bari-1. This was similar to symptom severity in Arabidopsis infected with the respective CaMV variant. We also found that transgenic P6 accumulated in inclusion bodies that were similar to those found in infected plants but lacking virions. We conclude that expression of P6, in the absence of virus replication, elicits a subset of the host symptom responses normally observed during infection and that the level, sequence, and possibly the form of P6 are important in potentiating the process.

Expression of functional elements inserted into the 35S promoter region of infectious cauliflower mosaic virus replicons.

We describe experiments directed towards development of cauliflower mosaic virus (CaMV) replicons for propagation of functional elements during infection of plants. Modifications and inserts were introduced into replaceable domains associated with the 35S promoter. The 35S enhancer (-208 to -56) was found to potentiate promoter activity when in reverse orientation sufficient to establish systemic infection. However, replacement of the 35S enhancer with that from the nos promoter caused loss of infectivity. A 31 bp oligonucleotide containing a polypurine tract specifying initiation of CaMV plus strand DNA synthesis was inserted into a 35S enhancer deletion mutant and propagated in plants. Analysis of progeny DNA showed the presence of an additional discontinuity at its new location in the 35S enhancer, indicating that the artificial primer had functioned correctly in an ectopic site. An intron and flanking sequences from the RNA leader of the Arabidopsis phytoene desaturase (pds) gene, when inserted into the 35S enhancer in forward orientation was very efficiently spliced during infection. The CaMV replicon carrying the pds gene fragment produced unusual infection characteristics, with plants showing early symptoms and then recovering. We conclude that infectious CaMV replicons can be used to carry a variety of elements that target both viral and host functions.

Roles of the 35S promoter and multiple overlapping domains in the pathogenicity of the pararetrovirus cauliflower mosaic virus.

Elements associated with the 35S promoter involved in generating the pregenomic RNA (35S RNA) of the pararetrovirus cauliflower mosaic virus have been extensively studied in heterologous systems, but little is known about their role in viral pathogenicity. To investigate these elements, premature termination codons were progressively inserted into the 3' end of the adjacent gene VI to dissect it from colinear 35S enhancer sequences. The ability to cause a systemic infection in plants was retained with loss of up to 40 amino acids from the gene VI polypeptide, but truncations into a putative zinc finger proved lethal. In the 35S promoter, removal of the TATA box also abolished infectivity. However, upstream deletions encompassing the 35S enhancer showed that the sequence between -207 and -56 from the cap site comprised nonessential elements, although complete removal of this fragment caused loss of infectivity even when domain spacing was restored by linker insertion. Two separate enhancer domains (-207 to - 150 and -95 to -56) were identified, of which either one or the other, but not both, was required for infectivity. Some mutations affected the cellular levels of viral RNAs in unexpected ways, as with removal of the as-1 enhancer element causing an increase in 35S RNA. Others altered the relative abundance of nuclear and cytoplasmic viral DNAs. Mutations in promoter domains thought to be involved in regulating tissue-specific expression did not significantly affect virus accumulation in leaves versus roots, whereas gene VI mutants showed reduced root accumulation. We conclude that elements associated with the cauliflower mosaic virus 35S promoter contain extensive nonessential regions that can behave differently in their proper context than as isolated elements.

Retroelements: propagation and adaptation.

Retroelements are genetic entities that exist in both DNA and RNA forms generated by cyclic alternation of transcription and reverse transcription. They have in common a genetic core (the gag-pol core), encoding conserved functions of a structural protein and a replicase. These are supplemented with a variety of cis-acting nucleic acid sequences controlling transcription and reverse transcription. Most retroelements have additional genes with regulatory or adaptive roles, both within the cell and for movement between cells and organisms. These features reflect the variety of mechanisms that have developed to ensure propagation of the elements and their ability to adapt to specific niches in their hosts with which they co-evolve.

Variation in biological properties of cauliflower mosaic virus clones.

Infectious clones were prepared from virion DNA of three cauliflower mosaic virus (CaMV) isolates, 11/3, Xinjiang (XJ), and Aust, to investigate pathogenic variation in virus populations. Of 10 infectious clones obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed from those of the 11/3 wild-type. Virus from two clonal groups of 11/3 was transmissible by aphids whereas that from two others was not. Of the five infectious clones obtained from isolate XJ, two groups were identified, one of which differed symptomatically from the wild-type. Only one infectious clone was obtained from isolate Aust and this had properties similar to the wild-type. Restriction enzyme polymorphisms were found in some clonal groups and these correlated with symptoms. Other groups with different pathogenic properties could not be distinguished apart by restriction site polymorphisms. Further variation was observed in the nucleotide sequences of gene II (coding for aphid transmission factor) from these viruses as compared with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg mutation in gene II similar to that of other non-deleted non-transmissible CaMV isolates. The second had a 322 bp deletion at the site of a small direct repeat similar to that of isolate CM4-184 although occurring in a different position. The gene II deletion of isolate 11/3 produced a frame-shift that separated genes II and III by 60 bp. Most CaMV clones studied remained biologically stable producing similar symptoms during subsequent passages. However, one clone (11/3-7) produced two new biotypes during its first passage suggesting that it was relatively unstable. Our results show that wild-type populations of CaMV contain a range of infectious genome variants with contrasting biological properties and differing stability. We suggest that a variety of significant viral phenotypic changes can occur during each infection cycle resulting from relatively small genome changes.

Changes in populations of cauliflower mosaic virus DNA and RNA forms during turnip callus proliferation.

Cauliflower mosaic virus (CaMV) nucleic acids accumulate in the cell in different structural conformations related to their roles in gene expression, replication and virion assembly. We have characterized changes in the population CaMV DNA and RNA replication products which occur following culture of infected turnip leaves under conditions where callus proliferates. After only 5 days in culture, a significant increase in the level of genome-length and subgenomic supercoiled (SC) DNA forms was observed by two-dimensional (2D) gel electrophoresis. Open circular (OC) molecules, corresponding to these SC DNAs, with mobilities consistent with the presence of a single break in each strand, were also detected after 5 days culture. By 10 days culture, the proportion of OC molecules with only one break per double-stranded molecule had increased. After 34 days culture, SC DNA with a range of sizes predominated in the unencapsidated DNA fraction. The change in pattern of OC and SC DNA forms during callus proliferation suggests a possible precursor/product relationship involving generation of deleted molecules from gap-containing virion DNA-like molecules followed by sequential repair of the gaps to produce SC DNA. Moreover, heterogeneity in the mobility of OC DNAs in the neutral dimension of 2D electrophoresis, a feature exhibited by twisted CaMV virion DNA, changed during the time-course suggesting that untwisting occurs during gap repair. Although the relative abundance of SC DNA increased during callus proliferation, CaMV polyadenylated 35S and 19S transcripts declined together with immediate reverse transcription products. We suggest that cellular changes during callus growth lead to a decline in authentic CaMV transcripts in the cytoplasm resulting in cessation of synthesis of viral products and progeny DNA genomes. In consequence, pre-existing virion DNAs return to the nucleus, possibly as a result of a relaxation in a cytoplasmic control mechanism, where they are assembled into various forms of SC DNA. The presence of CaMV SC DNAs in replicating cells might also enhance illegitimate integration into host chromosomes, as hybridization of CaMV DNA to high M(r) DNA was observed.

Comparison of viral nucleic acid intermediates at early and late stages of cauliflower mosaic virus infection suggests a feedback regulatory mechanism.

An important phase of the multiplication cycle of the pararetrovirus cauliflower mosaic virus (CaMV) is transcription of the viral minichromosome in the nucleus. Leaves of infected turnip plants at the vein clearing stage were found to contain a relatively low level of minichromosome DNA, and abundant viral transcripts and characteristic reverse transcription products. In contrast, at the much later stage of severe leaf chlorosis, an elevated level of minichromosome DNA but less RNA, especially the 35S RNA reverse transcription template, was observed. Changes in the composition of virus nucleic acid intermediates were also seen in roots and stems early, compared with late, in infection. A possible feedback mechanism controlling the level of viral minichromosome DNA and its importance in regulation of the CaMV multiplication cycle are discussed in the light of these observations.

Susceptibility of Brassica species to cauliflower mosaic virus infection is related to a specific stage in the virus multiplication cycle.

The relative susceptibilities and symptom responses of different Brassica species to infection by cauliflower mosaic virus (CaMV) have been compared and related to molecular events of the virus multiplication cycle. Variants of B. rapa (genome descriptor aa) were highly susceptible to infection by CaMV strain Cabb B-JI and contained relatively large amounts of virus; B. oleracea (cc) variants showed low susceptibility and contained small amounts of virus. B. nigra (bb) and allotetraploid species. B. juncea (aabb), B. napus (aacc) and B. carinata (bbcc), showed moderate responses to CaMV. CaMV unencapsidated DNA forms were isolated from different Brassica plants and examined by two-dimensional gel electrophoresis and blot hybridization. Viral RNA was estimated by dot blot analysis. These analyses showed differences in accumulation of key viral replication cycle intermediates within the broad range of host plants studied. The most susceptible species contained relatively small amounts of supercoiled (SC) DNA, a component of the CaMV mini-chromosome, but abundant viral transcripts and reverse transcription replication products. Tolerant plant hosts contained high levels of SC DNA but low levels of viral transcripts and reverse transcription DNA products. Allotetraploids contained SC DNA, RNA transcripts and replication product levels which were generally intermediate between those of their respective progenitor species. Evidence is presented that accumulation of CaMV SC DNA in the less susceptible host species is probably not due to autonomous DNA replication or tissue-specific expression. We conclude that a major component of the susceptibility of Brassica plants (and probably all CaMV host species) to CaMV infection is the level of viral minichromosome expression, influenced directly by the host genotype.

Host regulation of the cauliflower mosaic virus multiplication cycle.

The DNA genome of cauliflower mosaic virus (CaMV) replicates in the cytoplasm of infected plant cells by reverse transcription of an RNA template. Viral RNA is generated in the nucleus by transcription of an episomal minichromosome containing supercoiled DNA. We have assessed the relative activities of the nuclear and cytoplasmic phases of the CaMV multiplication cycle by monitoring unencapsidated viral DNA forms and polyadenylylated RNAs in different organs of one host plant and in different host species. Systemically infected leaves of a highly susceptible host, turnip (Brassica rapa), contained abundant 35S RNA and 19S RNA transcripts and unencapsidated reverse transcription products but relatively little supercoiled DNA. In contrast, supercoiled DNA accumulated in roots and other tissues of turnip plants but without significant amounts of steady-state viral RNA. Infected but asymptomatic leaves of a less susceptible CaMV host, kohlrabi (Brassica oleracea), contained supercoiled DNA almost exclusively but negligible viral RNA and DNA products of reverse transcription. An allotetraploid species, rape (Brassica napus), exhibited infection characteristics and minichromosome expression levels intermediate between the other two species from which it was derived. We conclude that expression of the CaMV minichromosome is a key phase of the virus multiplication cycle, which is regulated differentially in organs of a highly susceptible host species. Furthermore, this regulation exhibits genetic variation among different Brassica species and controls host susceptibility to CaMV infection.

Segregation of cauliflower mosaic virus symptom genetic determinants.

We have created a series of hybrid cauliflower mosaic virus (CaMV) genomes between a severe virus strain (Cabb BJI) and a mild strain (Bari 1) to map the virus genetic loci responsible for specific systemic symptom characters produced in infected turnip plants. Recombinants were generated in vivo by recombinational rescue and in vitro by restriction enzyme fragment exchange. On infection, hybrids induced either parental (wild-type) symptoms or segregated parental characters. Some of the engineered hybrid genomes produced novel symptomatic effects not observed in either of the parental strains whilst others reverted to express parental symptom characters following passaging. Determinants defining differences between the two CaMV strains in respect of four specific symptom characters were delimited to separate genome regions. A locus involved in determining the rate of spread of systemic vein clearing symptoms mapped to a region containing part of gene VII and gene I (nts 109-780). This phenomenon is consistent with the putative involvement of the CaMV gene I product in mediating virus movement within infected plants. Determinants influencing the degree of leaf chlorosis were located in a separate genome domain encompassing part of gene VI together with the large intergenic region and part of gene VII (nts 6103-90). Determinants controlling timing of initial systemic symptom appearance were mapped to a region between nts 2150 and 4438 containing part of gene III, gene IV, and part of gene V. Plant stunting was influenced by loci in at least two separate regions, one containing parts of gene I and II, and a second within the reverse transcriptase gene (V). We conclude that symptoms produced by CaMV infection can be subdivided into individual characters, the genetic determinants of which segregate to different virus genetic loci and are not restricted to a single gene product.

Structure and replication of caulimovirus genomes.

In this paper the current state of knowledge of the replication of cauliflower mosaic virus (CaMV) is reviewed and the DNA intermediates and enzymes involved in replication are discussed. Based on this information a model for the replication complex is developed. In this model it is suggested that replication complexes resemble virus particles and that, in their assembly, there are close interactions between the inclusion body protein, the virus coat protein, the replicase enzyme, the tRNA primer and the 35S RNA template. The similarities between CaMV replication complexes and those of retroviruses are discussed, and we extend this discussion to a comparison between CaMV and reverse transcribing elements.

Hairpin DNAs of cauliflower mosaic virus generated by reverse transcription in vivo.

Cauliflower mosaic virus (CaMV) is a DNA plant virus which replicates by reverse transcription. During our examination of CaMV replication intermediates by 2-D gel electrophoresis, we have discovered a population of bizarre linear double-stranded hairpin DNAs. The largest hairpin is the size of the CaMV genome; hairpin loop ends of smaller molecules map to several sites around the genome but the open ends are all located close to the origin of reverse transcription at the primer binding site. We believe that the hairpin DNAs are generated in vivo by reverse transcription of CaMV RNA followed by self-primed second strand synthesis. The accumulation of hairpin DNAs in vivo might represent a side reaction of the CaMV reverse transcriptase although an essential role for them in the virus replication cycle cannot be discounted. The structure of the hairpin DNAs provides further evidence for the location of the start site and of the polarity of reverse transcription in CaMV.