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Biomedical subjects

Dominique Robertson

Publications and source records attributed to Dominique Robertson.

10 recordsLinked to original sources
PubMed2006

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing.

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections.

Arabidopsis↗
PubMed2004

Geminivirus VIGS of endogenous genes requires SGS2/SDE1 and SGS3 and defines a new branch in the genetic pathway for silencing in plants.

Virus-induced gene silencing (VIGS) is a sequence-specific RNA degradation process that can be used to downregulate plant gene expression. Both RNA and DNA viruses have been used for VIGS, but they differ in their mode of replication, gene expression, and cellular location. This study examined silencing mediated by a DNA virus, cabbage leaf curl virus (CaLCuV), in several silencing-deficient Arabidopsis mutants. A DNA VIGS vector derived from CaLCuV, which silenced chlorata42 (ChlI) needed for chlorophyll formation, was used to test endogenous gene silencing responses in suppressor of gene silencing (sgs)1, sgs2, sgs3, and Argonaute (ago)1 mutants defective in sense transgene-mediated post-transcriptional silencing (S-PTGS). SGS2/silencing defective (SDE)1, SGS3, and AGO1 are each dispensable for silencing mediated by transgenes containing inverted repeats (IR-PTGS), and SGS2/SDE1 is dispensable for RNA VIGS. We show that DNA VIGS requires both SGS2/SDE1 and SGS3, regardless of the orientation of 362 nt ChlI transcripts produced from the viral DNA promoter. Viral DNA accumulation is slightly higher, and viral symptoms increase in sgs2 and sgs3, whereas overexpression of SGS2/SDE1 mRNA results in decreased viral symptoms. Mutants affected in SGS1 and AGO1 function are only delayed in the onset of silencing, and have a small effect on chlorophyll accumulation. DNA VIGS is unaffected in defective DNA methylation (ddm)1/somniferous (som)8 and maintenance of methylation (mom)1 mutants, impaired for TGS. These results demonstrate that SGS2/SDE1 and SGS3 are needed for endogenous gene silencing from DNA viruses, and suggest that SGS2/SDE1 may reduce geminivirus symptoms by targeting viral mRNAs.

Arabidopsis↗
PubMed2004

VIGS vectors for gene silencing: many targets, many tools.

The discovery that plants recognize and degrade invading viral RNA caused a paradigm shift in our understanding of viral/host interactions. Combined with the discovery that plants cosuppress their own genes if they are transformed with homologous transgenes, new models for both plant intercellular communication and viral defense have emerged. Plant biologists adapted homology-based defense mechanisms triggered by incoming viruses to target individual genes for silencing in a process called virus-induced gene silencing (VIGS). Both VIGS- and dsRNA-containing transformation cassettes are increasingly being used for reverse genetics as part of an integrated approach to determining gene function. Virus-derived vectors silence gene expression without transformation and selection. However, because viruses also alter gene expression in their host, the process of VIGS must be understood. This review examines how DNA and RNA viruses have been modified to silence plant gene expression. I discuss advantages and disadvantages of VIGS in determining gene function and guidelines for the safe use of viral vectors.

Gene Silencing↗
PubMed2004

Geminivirus vectors for transient gene silencing in plants.

Both RNA and DNA viruses have been engineered to serve as vectors for transient silencing in intact plants. Host gene sequences carried by the virus are seen by the plant as "foreign," and homologous gene-silencing machinery acts on both the viral vector RNA and the endogenous host gene mRNA. DNA viruses, such as geminiviruses, are advantageous for silencing because only their mRNAs are silenced and their DNA genomes continue to replicate and move. The conserved genome organization of geminiviruses and the fact that they can be cloned into Escherichia coli plasmids, propagated, and then inoculated into plants for infection simplifies the procedure for silencing specific chromosomal genes in intact plants. This chapter describes the development of a silencing vector from cabbage leaf curl virus for use in Arabidopsis and procedures for silencing two genes simultaneously.

Cloning, Molecular↗
PubMed2002

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.

Agrobacterium tumefaciens↗
PubMed2002

Geminivirus-based vectors for gene silencing in Arabidopsis.

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.

Animals↗
PubMed2002

Mutations in the gravity persistence signal loci in Arabidopsis disrupt the perception and/or signal transduction of gravitropic stimuli.

Gravity plays a fundamental role in plant growth and development, yet little is understood about the early events of gravitropism. To identify genes affected in the signal perception and/or transduction phase of the gravity response, a mutant screen was devised using cold treatment to delay the gravity response of inflorescence stems of Arabidopsis. Inflorescence stems of Arabidopsis show no response to gravistimulation at 4 degrees C for up to 3 h. However, when gravistimulated at 4 degrees C and then returned to vertical at room temperature (RT), stems bend in response to the previous, horizontal gravistimulation (H. Fukaki, H. Fujisawa, M. Tasaka [1996] Plant Physiology 110: 933-943). This indicates that gravity perception, but not the gravitropic response, occurs at 4 degrees C. Recessive mutations were identified at three loci using this cold effect on gravitropism to screen for gravity persistence signal (gps) mutants. All three mutants had an altered response after gravistimulation at 4 degrees C, yet had phenotypically normal responses to stimulations at RT. gps1-1 did not bend in response to the 4 degrees C gravity stimulus upon return to RT. gps2-1 responded to the 4 degrees C stimulus but bent in the opposite direction. gps3-1 over-responded after return to RT, continuing to bend to an angle greater than wild-type plants. At 4 degrees C, starch-containing statoliths sedimented normally in both wild-type and the gps mutants, but auxin transport was abolished at 4 degrees C. These results are consistent with GPS loci affecting an aspect of the gravity signal perception/transduction pathway that occurs after statolith sedimentation, but before auxin transport.

Arabidopsis↗
PubMed2002

Host DNA replication is induced by geminivirus infection of differentiated plant cells.

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.

Aphidicolin↗
PubMed2002

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.

Base Sequence↗