David C. Baulcombe

RNA silencing in plants

There are at least three RNA silencing pathways for silencing specific genes in plants. In these pathways, silencing signals can be amplified and transmitted between cells, and may even be self-regulated by feedback mechanisms. Diverse biological roles of these pathways have been established, including defence against viruses, regulation of gene expression and the condensation of chromatin into heterochromatin. We are now in a good position to investigate the full extent of this functional diversity in genetic and epigenetic mechanisms of genome control.

Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.

Post‐transcriptional gene silencing (PTGS) of a green fluorescent protein (GFP) transgene is suppressed in Nicotiana benthamiana plants infected with potato virus Y (PVY) or with cucumber mosaic virus (CMV), but not in plants infected with potato virus X (PVX). By expressing PVY and CMV‐encoded proteins in a PVX vector we have shown that the viral suppressors of gene silencing are the HCPro of PVY and the 2b protein of CMV. The HCPro acts by blocking the maintenance of PTGS in tissues where silencing had already been set, whereas the 2b protein prevents initiation of gene silencing at the growing points of the plants. Combined with previous findings that viruses are both activators and targets of PTGS, these data provide compelling evidence that PTGS represents a natural mechanism for plant protection against viruses.

An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus

Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.

Two classes of short interfering RNA in RNA silencing

RNA silencing is a eukaryotic genome defence system that involves processing of double‐stranded RNA (dsRNA) into 21–26 nt, short interfering RNA (siRNA). The siRNA mediates suppression of genes corresponding to the dsRNA through targeted RNA degradation. In some plant systems there are additional silencing processes, involving systemic spread of silencing and RNA‐directed methylation/transcriptional suppression of homologous genomic DNA. We show here that siRNAs produced in plants from a green fluorescent protein (GFP) transgene are in short (21–22 nt) and long (24–26 nt) size classes, whereas those from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these classes of siRNA have different roles. The long siRNA is dispensable for sequence‐specific mRNA degradation, but correlates with systemic silencing and methylation of homologous DNA. Conversely, the short siRNA class correlates with mRNA degradation but not with systemic signalling or methylation. These findings reveal an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals.

Criteria for Annotation of Plant MicroRNAs

MicroRNAs (miRNAs) are ∼21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.

Initiation and Maintenance of Virus-Induced Gene Silencing

The phytoene desaturase (PDS) gene of Nicotiana benthamiana was silenced in plants infected with potato virus X (PVX) vectors carrying PDS inserts, and a green fluorescent protein (GFP) transgene was silenced in plants infected with PVX–GFP. This virus-induced gene silencing (VIGS) is post-transcriptional and cytoplasmic because it is targeted against exons rather than introns of PDS RNA and against viral RNAs. Although PDS and GFP RNAs are most likely targeted through the same mechanism, the VIGS phenotypes differed in two respects. PDS mRNA was targeted by VIGS in all green tissue of the PVX–PDS—infected plant, whereas PVX–PDS was not affected. In contrast, VIGS of the GFP was targeted against PVX–GFP. Initially, VIGS of the GFP was initiated in all green tissues, as occurred with PDS VIGS. However, after 30 days of infection, the GFP VIGS was no longer initiated in newly emerging leaves, although it was maintained in tissue in which it had already been initiated. Based on these analyses, we propose a model for VIGS in which the initiation of VIGS is dependent on the virus and maintenance of it is virus independent.

A Viral Movement Protein Prevents Spread of the Gene Silencing Signal in Nicotiana benthamiana

(Cell 103, 157–167; September 29, 2000) We, the editors of Cell, were contacted by the corresponding author, David Baulcombe, who informed us that this paper contains an unacknowledged image duplication. The mock control (Mock:M) lane shown in the northern blot experiment in Figure 3D is the same as the mock control lane in Figure 5D. Dr. Baulcombe informed us that these two experiments were carried out at the same time, run on a single gel, and exposed on the same autoradiograph and that they shared a negative (mock) control run in a single lane. Therefore, Figures 3D and 5D present the relevant lanes of each experiment plus the sharedmock control. Dr. Baulcombe provided us with a copy of the original autoradiograph for these experiments. We have evaluated the data and confirmed his explanation for this duplication.

Modulation of floral development by a gibberellin-regulated microRNA

Floral initiation and floral organ development are both regulated by the phytohormone gibberellin (GA). For example, in short-day photoperiods, the Arabidopsis floral transition is strongly promoted by GA-mediated activation of the floral meristem-identity gene LEAFY. In addition, anther development and pollen microsporogenesis depend on GA-mediated opposition of the function of specific members of the DELLA family of GA-response repressors. We describe the role of a microRNA (miR159) in the regulation of short-day photoperiod flowering time and of anther development. MiR159 directs the cleavage of mRNA encoding GAMYB-related proteins. These proteins are transcription factors that are thought to be involved in the GA-promoted activation of LEAFY, and in the regulation of anther development. We show that miR159 levels are regulated by GA via opposition of DELLA function, and that both the sequence of miR159 and the regulation of miR159 levels by DELLA are evolutionarily conserved. Finally, we describe the phenotypic consequences of transgenic over-expression of miR159. Increased levels of miR159 cause a reduction in LEAFY transcript levels, delay flowering in short-day photoperiods, and perturb anther development. We propose that miR159 is a phytohormonally regulated homeostatic modulator of GAMYB activity, and hence of GAMYB-dependent developmental processes.

The Rx Gene from Potato Controls Separate Virus Resistance and Cell Death Responses

Rx-mediated extreme resistance against potato virus X in potato does not involve a necrotic hypersensitive response at the site of initial infection and thereby differs from the more usual type of disease resistance in plants. However, the Rx protein is structurally similar to products of disease resistance genes conferring the hypersensitive response. We show in both Nicotiana spp and potato that Rx has the potential to initiate a cell death response but that extreme resistance is separate and epistatic to necrosis. These data indicate that cell death and pathogen arrest are separate disease resistance responses in plants.

Systemic signalling in gene silencing

Gene silencing in plants is a genetic control mechanism implicated in virus resistance,, genome maintenance and developmental control. We describe here our recent discovery that there is a systemic signal that can mediate gene silencing. From the gene-specificity of the systemic silencing, we infer that the signal molecule is likely to be a nucleic acid.