Virus-induced overexpression of heterologous FLOWERING LOCUS T for efficient speed breeding in tomato.

Potato virus X (PVX) vectors expressing the Arabidopsis thaliana FLOWERING LOCUS T (FT) or tomato FT ortholog SINGLE-FLOWER TRUSS (SFT) shortened the generation time in tomato due to accelerated tomato flowering and ripening by 14-21 d, and caused a 2-3-fold increase in the number of flowers and fruits, compared with non-infected or empty vector-infected plants. The Arabidopsis FT was more effective than the tomato orthologue SFT and there was no alteration of the flower or fruit morphology. The virus was not transmitted to the next generation; therefore viral vectors with expression of a heterologous FT will be a useful approach to speed breeding in tomato and other species.

An RNA World.

My research career started with an ambition to work out how genes are regulated in plants. I tried out various experimental systems-artichoke tissue culture in Edinburgh; soybean root nodules in Montreal; soybean hypocotyls in Athens, Georgia; and cereal aleurones in Cambridge-but eventually I discovered plant viruses. Viral satellite RNAs were my first interest, but I then explored transgenic and natural disease resistance and was led by curiosity into topics beyond virology, including RNA silencing, epigenetics, and more recently, genome evolution.

Interspecific hybridization in tomato influences endogenous viral sRNAs and alters gene expression.

Background: Hybridization is associated with the activation of transposable elements and changes in the patterns of gene expression leading to phenotypic changes. However, the underlying mechanisms are not well understood.

Results: Here, we describe the changes to the gene expression in interspecific Solanum hybrids that are associated with small RNAs derived from endogenous (para)retroviruses (EPRV).

Post-transcriptional Gene Silencing Using Virus-Induced Gene Silencing to Study Plant Gametogenesis in Tomato.

Loss-of-function analyses are essential to dissect the complex nature of biological processes, including gametogenesis. Virus-induced gene silencing (VIGS) has been widely used in crop species as an amenable and rapid way to generate gene knockdowns. As a transient assay, VIGS circumvents the generation of stable transgenic lines through laborious and time-consuming tissue culture techniques.

CHROMOMETHYLTRANSFERASE3/KRYPTONITE maintains the paramutation in .

SignificanceParamutation involves the transfer of a repressive epigenetic mark between silent and active alleles. It is best known from exceptional non-Mendelian inheritance of conspicuous phenotypes in maize but also in other plants and animals. Recent genomic studies, however, indicate that paramutation may be less exceptional.

The small RNA locus map for Chlamydomonas reinhardtii.

Small (s)RNAs play crucial roles in the regulation of gene expression and genome stability across eukaryotes where they direct epigenetic modifications, post-transcriptional gene silencing, and defense against both endogenous and exogenous viruses. It is known that Chlamydomonas reinhardtii, a well-studied unicellular green algae species, possesses sRNA-based mechanisms that are distinct from those of land plants. However, definition of sRNA loci and further systematic classification is not yet available for this or any other algae.

Viral Fitness Determines the Magnitude of Transcriptomic and Epigenomic Reprograming of Defense Responses in Plants.

Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate.

Transposon age and non-CG methylation.

Silencing of transposable elements (TEs) is established by small RNA-directed DNA methylation (RdDM). Maintenance of silencing is then based on a combination of RdDM and RNA-independent mechanisms involving DNA methyltransferase MET1 and chromodomain DNA methyltransferases (CMTs). Involvement of RdDM, according to this model should decrease with TE age but here we show a different pattern in tomato and Arabidopsis.

Extensive recombination challenges the utility of Sugarcane mosaic virus phylogeny and strain typing.

Sugarcane mosaic virus (SCMV) is distributed worldwide and infects three major crops: sugarcane, maize, and sorghum. The impact of SCMV is increased by its interaction with Maize chlorotic mottle virus which causes the synergistic maize disease maize lethal necrosis. Here, we characterised maize lethal necrosis-infected maize from multiple sites in East Africa, and found that SCMV was present in all thirty samples.

Environmental and epigenetic regulation of Rider retrotransposons in tomato.

Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown.

Distinct roles of Argonaute in the green alga Chlamydomonas reveal evolutionary conserved mode of miRNA-mediated gene expression.

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3'UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms.

Maternal small RNAs mediate spatial-temporal regulation of gene expression, imprinting, and seed development in .

seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the -mediated pathway. To investigate their biological functions, we characterized siRNAs in the endosperm and seed coat that were separated by laser-capture microdissection (LCM) in reciprocal genetic crosses with an mutant. We also monitored the spatial-temporal activity of the -mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing.

Enhanced resistance to bacterial and oomycete pathogens by short tandem target mimic RNAs in tomato.

Nucleotide binding site leucine-rich repeat (NLR) proteins of the plant innate immune system are negatively regulated by the miR482/2118 family miRNAs that are in a distinct 22-nt class of miRNAs with a double mode of action. First, they cleave the target RNA, as with the canonical 21-nt miRNAs, and second, they trigger secondary siRNA production using the target RNA as a template. Here, we address the extent to which the miR482/2118 family affects expression of NLR mRNAs and disease resistance.

miRNA-Mediated Regulation of Synthetic Gene Circuits in the Green Alga Chlamydomonas reinhardtii.

MicroRNAs (miRNAs), small RNA molecules of 20-24 nts, have many features that make them useful tools for gene expression regulation-small size, flexible design, target predictability, and action at a late stage of the gene expression pipeline. In addition, their role in fine-tuning gene expression can be harnessed to increase robustness of synthetic gene networks. In this work, we apply a synthetic biology approach to characterize miRNA-mediated gene expression regulation in the unicellular green alga Chlamydomonas reinhardtii.

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows.

Towards annotating the plant epigenome: the Arabidopsis thaliana small RNA locus map.

Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies.

Paramutation-like features of multiple natural epialleles in tomato.

Background: Freakish and rare or the tip of the iceberg? Both phrases have been used to refer to paramutation, an epigenetic drive that contravenes Mendel's first law of segregation. Although its underlying mechanisms are beginning to unravel, its understanding relies only on a few examples that may involve transgenes or artificially generated epialleles.

Results: By using DNA methylation of introgression lines as an indication of past paramutation, we reveal that the paramutation-like properties of the H06 locus in hybrids of Solanum lycopersicum and a range of tomato relatives and cultivars depend on the timing of sRNA production and conform to an RNA-directed mechanism.

Maize chlorotic mottle virus exhibits low divergence between differentiated regional sub-populations.

Maize chlorotic mottle virus has been rapidly spreading around the globe over the past decade. The interactions of maize chlorotic mottle virus with Potyviridae viruses causes an aggressive synergistic viral condition - maize lethal necrosis, which can cause total yield loss. Maize production in sub-Saharan Africa, where it is the most important cereal, is threatened by the arrival of maize lethal necrosis.

Endogenous miRNA in the green alga Chlamydomonas regulates gene expression through CDS-targeting.

MicroRNAs (miRNAs) are 21-24-nucleotide RNAs present in many eukaryotes that regulate gene expression as part of the RNA-induced silencing complex. The sequence identity of the miRNA provides the specificity to guide the silencing effector Argonaute (AGO) protein to target mRNAs via a base-pairing process . The AGO complex promotes translation repression and/or accelerated decay of this target mRNA .

Epigenetic and Genetic Contributions to Adaptation in Chlamydomonas.

Epigenetic modifications, such as DNA methylation or histone modifications, can be transmitted between cellular or organismal generations. However, there are no experiments measuring their role in adaptation, so here we use experimental evolution to investigate how epigenetic variation can contribute to adaptation. We manipulated DNA methylation and histone acetylation in the unicellular green alga Chlamydomonas reinhardtii both genetically and chemically to change the amount of epigenetic variation generated or transmitted in adapting populations in three different environments (salt stress, phosphate starvation, and high CO2) for two hundred asexual generations.

Improved Denaturation of Small RNA Duplexes and Its Application for Northern Blotting.

Small RNAs (sRNAs) are short (18-30 nucleotide) noncoding RNA molecules, which control gene expression and pathogen response in eukaryotes. They are associated with and guide nucleases to target nucleic acids by nucleotide base pairing. We found that current techniques for small RNA detection are adversely affected by the presence of complementary RNA.