David Charles Baulcombe

Transcriptome annotations provide essential information on transcript locations, sequences and structures, including transcription start, end sites and splice junctions. They underpin key biological analyses such as gene and transcript quantification, and the study of transcriptional and post-transcriptional regulation, including alternative transcription initiation, polyadenylation and splicing. Accurate characterisation of transcript isoforms is critical for understanding how gene expression relates to functional protein products. However, for many species—including Solanaceae crops such as potato and tomato—current annotations suffer from limited isoform coverage, with tens or hundreds of thousands of splice junctions and transcript isoforms missing. This undermines the completeness and accuracy of transcript-level analyses. Here, by generating Iso-seq and RNA-seq on a range of tissues and samples, we have produced transcriptome annotations for both potato and tomato with improved coverage, diversity, accurate splice junctions, and transcript start and end sites. We have also made these high-quality resources accessible through genome browsers. These enhanced annotations will enable more accurate transcriptome analyses, supporting higher-resolution and novel biological discoveries.

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.

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. On the way, I have learned about approaches to research, finding tractable systems, and taking academic research into the real world. I have always tried to consider the broader significance of our work, and my current projects address the definition of epigenetics, the arms race concept of disease resistance, and Darwin's abominable mystery.

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

Adaptive antiviral immunity in plants is an RNA-based mechanism in which small RNAs derived from both strands of the viral RNA are guides for an Argonaute (AGO) nuclease. The primed AGO specifically targets and silences the viral RNA. In plants this system has diversified to involve mobile small interfering RNAs (siRNAs), an amplification system involving secondary siRNAs and targeting mechanisms involving DNA methylation. Most, if not all, plant viruses encode multifunctional proteins that are suppressors of RNA silencing that may also influence the innate immune system and fine-tune the virus-host interaction. Animal viruses similarly trigger RNA silencing, although it may be masked in differentiated cells by the interferon system and by the action of the virus-encoded suppressor proteins. There is huge potential for RNA silencing to combat viral disease in crops, farm animals, and people, although there are complications associated with the various strategies for siRNA delivery including transgenesis. Alternative approaches could include using breeding or small molecule treatment to enhance the inherent antiviral capacity of infected cells.

RNA silencing is a well-established antiviral immunity system in plants, in which small RNAs guide Argonaute proteins to targets in viral RNA or DNA, resulting in virus repression. Virus-encoded suppressors of silencing counteract this defence system. In this Review, we discuss recent findings about antiviral RNA silencing, including the movement of RNA through plasmodesmata and the differentiation between plant self and viral RNAs. We also discuss the emerging role of RNA silencing in plant immunity against non-viral pathogens. This immunity is mediated by transkingdom movement of RNA into and out of the infected plant cells in vesicles or as extracellular nucleoproteins and, like antiviral immunity, is influenced by the silencing suppressors encoded in the pathogens’ genomes. Another effect of RNA silencing on general immunity involves host-encoded small RNAs, including microRNAs, that regulate NOD-like receptors and defence signalling pathways in the innate immunity system of plants. These RNA silencing pathways form a network of processes with both positive and negative effects on the immune systems of plants.

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 interspecificSolanumhybrids that are associated with small RNAs derived from endogenous (para)retroviruses (EPRV). There were prominent changes to sRNA profiles in these hybrids involving 22-nt species produced in the DCL2 biogenesis pathway, and the hybridization-induced changes to the gene expression were similar to those in adcl2mutant.Conclusions: These findings indicate that hybridization leads to activation of EPRV, perturbation of small RNA profiles, and, consequently, changes in the gene expression. Such hybridization-induced variation in the gene expression could increase the natural phenotypic variation in natural evolution or in breeding for agriculture.

Significance: Paramutation 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. It may be a consequence of wide-cross hybridization and may contribute to quantitative trait variation or unstable phenotypes in crops. Using the sulfurea ( sulf ) locus in tomato, we demonstrate that a self-reinforcing feedback loop involving DNA- and histone-methyl transferases CHROMOMETHYLTRANSFERASE3 (CMT3) and KRYPTONITE (KYP) is required for paramutation of sulf and that there is a change in chromatin organization. These findings advance the understanding of non-Mendelian inheritance in plants.

Hybridization and environmental stress trigger genome shock that perturbs patterns of gene expression leading to phenotypic changes. In extreme examples it is associated to transposon mobilization and genome rearrangement. Here we discover a novel alternative mechanism in interspecific Solanum hybrids in which changes to gene expression were associated with DCL2-mediated small (s)RNAs derived from endogenous (para)retroviruses (EPRVs). Correspondingly, the altered patterns of gene expression overlapped with the effects of dcl2 mutation and the changes to sRNA profiles involved 22nt species produced in the DCL2 biogenesis pathway. These findings implicate hybridization-induced genome shock leading to EPRV activation and sRNA silencing as causing changes in gene expression. Such hybridization-induced variation in gene expression could increase the range of traits available for selection in natural evolution or in breeding for agriculture.

Paramutation involves the transfer of a repressive epigenetic mark from a silent allele to an active homologue and, consequently, non-Mendelian inheritance. In tomato the sulfurea ( sulf ) paramutation is associated with a high level of CHG hypermethylation in a region overlapping the transcription start site of the SlTAB2 gene that affects chlorophyll synthesis. The CCG sub-context hypermethylation is under-represented at this region relative to CTG or CAG implicating the CHROMOMETHYLTRANSFERASE3 (CMT3) in paramutation at this locus. Consistent with this interpretation, loss of CMT 3 function leads to loss of the sulf chlorosis, the associated CHG hypermethylation and paramutation. Loss of KRYPTONITE (KYP) histone methyl transferase function has a similar effect linked to reduced H3K9me2 at the promoter region of SlTAB2 and a shift in higher order chromatin structure at this locus. Mutation of the largest subunit of RNA polymerase V ( PolV ) in contrast does not affect sulf paramutation. These findings indicate the involvement of a CMT3/KYP dependent feedback rather than the PolV-dependent pathway leading to RNA directed DNA methylation (RdDM) in the maintenance of paramutation.