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.

Plant viruses cause symptoms with devastating consequences for agriculture. However, the molecular mechanisms underlying symptom development in viral infections remain largely unexplored. Here, we show that tomato yellow leaf curl virus (TYLCV) interferes with host developmental programs through a host-mimicking domain present in the viral C4 protein. This domain mediates the interaction between C4 and a family of RCC1-like domain-containing (RLD) proteins, previously shown to be required for proper plant development and environmental responses. C4 outcompetes an endogenous interactor of RLDs, hijacking RLD proteins to the plasma membrane and disrupting their function in orchestrating endomembrane trafficking and polar auxin transport. Strikingly, macroscopic symptoms do not affect viral accumulation in the plant but serve as attractants for the insect vector, presumably promoting pathogen spread in an ecological context. Our work sheds light on the molecular underpinnings and biological relevance of symptom development triggered by TYLCV in tomato. Since most plant viruses are insect-transmitted, the principles described here might have broad applicability to crop-virus interactions.

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.

ABSTRACT 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.

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. Expected final online publication date for the Annual Review of Plant Biology, Volume 74 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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). 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 a dcl2 mutant. 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.

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.

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. 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.

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.

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. VIGS involves inoculating plants during early development with genetically manipulated viral constructs carrying an endogenous gene target sequence. The viral infection triggers the host plant gene silencing machinery to process the viral genomic RNA into small RNAs (sRNAs) including the gene complementary region. The sRNAs with complementary sequences to the endogenous gene mediate posttranscriptional gene silencing of the targeted gene. Here, we provide a simple and reproducible VIGS protocol employing the tobacco rattle virus (TRV) in tomato (Solanum lycopersicum cv. M82). As it is stable at later developmental stages this approach is suitable for many traits in tomato including gametogenesis and it can be adapted to other crop species.

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.

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. Here, using data-driven machine learning approaches including Multiple Correspondence Analysis (MCA) and clustering, we have generated a comprehensively annotated and classified sRNA locus map for C. reinhardtii. This map shows some common characteristics with higher plants and animals, but it also reveals distinct features. These results are consistent with the idea that there was diversification in sRNA mechanisms after the evolutionary divergence of algae from higher plant lineages.

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 . In these species the CMTs silence long terminal repeat (LTR) transposons in the distal chromatin that are younger than those affected by RdDM. To account for these findings we propose that, after establishment of primary RdDM as in the original model, there is an RNA-independent maintenance phase involving CMTs followed by secondary RdDM. This progression of epigenetic silencing in the gene-rich distal chromatin is likely to influence the transcriptome either in cis or in trans depending on whether the mechanisms are RNA-dependent or -independent.

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. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host.

Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1-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 nrpd1 mutant. We also monitored the spatial-temporal activity of the NRPD1-mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches, we identified four distinct groups of siRNA loci dependent on or independent of the maternal NRPD1 allele in the endosperm or seed coat. A group of maternally expressed NRPD1-siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using translational promoter:AGL::GUS constructs as sensors, we demonstrate that spatial and temporal expression patterns of these genes in the endosperm are regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Moreover, altered expression of these siRNA-targeted genes affects seed size. We propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.

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. This distribution pattern indicates that SCMV is a major partner virus in the East African maize lethal necrosis outbreak. Consistent with previous studies, our SCMV isolates were highly variable with several statistically supported recombination hot- and cold-spots across the SCMV genome. The recombination events generate conflicting phylogenetic signals from different fragments of the SCMV genome, so it is not appropriate to group SCMV genomes by simple similarity.

Biotechnologists have promoted genetic manipulation in crops as being more predictable than conventional breeding, although transgene expression is well known to be unstable and to vary between lines. Normally, only a small proportion of transformants have stable and high-level expression of the