Dominique Robertson

Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation.

Geminiviruses have small, single-stranded DNA genomes that replicate through double-stranded intermediates in the nuclei of infected plant cells. Viral double-stranded DNA also assembles into minichromosomes and is transcribed in infected cells. Geminiviruses encode only a few proteins for their replication and transcription and rely on host enzymes for these processes. However, most plant cells, which have exited the cell cycle and undergone differentiation, do not contain the replicative enzymes necessary for viral DNA synthesis. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells, most likely by modifying cell cycle and transcriptional controls. In animals, several DNA viruses depend on host replication and transcription machinery and can alter their hosts to create an environment that facilitates efficient viral replication. Analysis of these viruses and their proteins has contributed significantly to our understanding of DNA replication, transcription, and cell cycle regulation in mammalian cells. Geminiviruses have the same potential for plant systems. Plants offer many advantages for these types of studies, including ease of transformation, well-defined cell populations and developmental programs, and greater tolerance of cell cycle perturbation and polyploidy. Our knowledge of the molecular and cellular events that mediate geminivirus infection has increased significantly during recent years. The goal of this review is to summarize recent research addressing geminivirus DNA replication and its integration with transcriptional and cell cycle regulatory processes.

Geminiviruses: masters at redirecting and reprogramming plant processes

The family Geminiviridae is one of the largest and most important families of plant viruses. The small, single-stranded DNA genomes of geminiviruses encode 5–7 proteins that redirect host machineries and processes to establish a productive infection. These interactions reprogramme plant cell cycle and transcriptional controls, inhibit cell death pathways, interfere with cell signalling and protein turnover, and suppress defence pathways. This Review describes our current knowledge of how geminiviruses interact with their plant hosts and the functional consequences of these interactions.

A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants.

Geminiviruses replicate in nuclei of mature plant cells after inducing the accumulation of host DNA replication machinery. Earlier studies showed that the viral replication factor, AL1, is sufficient for host induction and interacts with the cell cycle regulator, retinoblastoma (pRb). Unlike other DNA virus proteins, AL1 does not contain the pRb binding consensus, LXCXE, and interacts with plant pRb homo logues (pRBR) through a novel amino acid sequence. We mapped the pRBR binding domain of AL1 between amino acids 101 and 180 and identified two mutants that are differentially impacted for AL1–pRBR interactions. Plants infected with the E‐N140 mutant, which is wild‐type for pRBR binding, developed wild‐type symptoms and accumulated viral DNA and AL1 protein in epidermal, mesophyll and vascular cells of mature leaves. Plants inoculated with the KEE146 mutant, which retains 16% pRBR binding activity, only developed chlorosis along the veins, and viral DNA, AL1 protein and the host DNA synthesis factor, proliferating cell nuclear antigen, were localized to vascular tissue. These results established the importance of AL1–pRBR interactions during geminivirus infection of plants.

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.

Reprogramming plant gene expression: a prerequisite to geminivirus DNA replication.

SUMMARY Geminiviruses constitute a large family of plant-infecting viruses with small, single-stranded DNA genomes that replicate through double-stranded intermediates. Because of their limited coding capacity, geminiviruses supply only the factors required to initiate their replication and use plant nuclear DNA polymerases to amplify their genomes. Many geminiviruses replicate in differentiated cells that no longer contain detectable levels of host DNA polymerases and associated factors. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells by reprogramming host gene expression. The mammalian DNA tumour viruses activate host genes required for DNA replication by binding to the retinoblastoma protein, a negative regulator of cell cycle progression, and relieving repression through the E2F family of transcription factors. In this review, we discuss recent experiments showing that geminiviruses also modulate components of the retinoblastoma/E2F transcription regulatory network to induce quiescent plant cells to re-enter the cell cycle and regain the capacity to support high levels of DNA replication. Regulation of the cell division cycle and its integration with developmental pathways is complex, with many factors, including hormones, sucrose and environmental signals, controlling re-entry into the plant cell cycle. Geminivirus interactions with these regulatory networks are likely to determine if and where they can replicate their genomes in different plant tissues and hosts.

Proliferating Cell Nuclear Antigen Transcription Is Repressed through an E2F Consensus Element and Activated by Geminivirus Infection in Mature Leaves

The geminivirus tomato golden mosaic virus (TGMV) amplifies its DNA genome in differentiated plant cells that lack detectable levels of DNA replication enzymes. Earlier studies showed that TGMV induces the accumulation of proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerase δ, in mature cells of Nicotiana benthamiana. We sought to determine if PCNA protein accumulation reflects transcriptional activation of the host gene. RNA gel blot analysis detected an ∼1200-nucleotide PCNA transcript in young leaves. The same RNA was found in mature leaves of infected but not healthy plants. Reporter gene analysis showed that a 633-bp promoter fragment of the N. benthamiana PCNA gene supports high levels of expression in cultured cells and in young but not mature leaves of healthy transgenic plants. In contrast, PCNA promoter activity was detected in both young and mature leaves of TGMV-infected plants. Developmental studies established a strong relationship between symptom severity, viral DNA accumulation, PCNA promoter activity, and endogenous PCNA mRNA levels. Mutation of an E2F consensus element in the PCNA promoter had no effect on its activity in young leaves but increased transcription in healthy mature leaves. Unlike the wild-type PCNA promoter, TGMV infection had no detectable effect on the activity of the mutant E2F promoter. Together, these results demonstrate that geminivirus infection induces the accumulation of a host replication factor by activating transcription of its gene in mature tissues, most likely by overcoming E2F-mediated repression.

Geminivirus-Mediated Gene Silencing from Cotton Leaf Crumple Virus Is Enhanced by Low Temperature in Cotton

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar ‘Deltapine 5415’ bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30°C/26°C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22°C/18°C. However, endogenous gene silencing decreased at 30°C/26°C. There was an approximately 7 d delay in the onset of gene silencing at 22°C/18°C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

Two E2F Elements Regulate the Proliferating Cell Nuclear Antigen Promoter Differently during Leaf Development

E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.

Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20–50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9–35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment.

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for β-glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.