Josep Casacuberta

The genome of melon (Cucumis melo L.)

We report the genome sequence of melon, an important horticultural crop worldwide. We assembled 375 Mb of the double-haploid line DHL92, representing 83.3% of the estimated melon genome. We predicted 27,427 protein-coding genes, which we analyzed by reconstructing 22,218 phylogenetic trees, allowing mapping of the orthology and paralogy relationships of sequenced plant genomes. We observed the absence of recent whole-genome duplications in the melon lineage since the ancient eudicot triplication, and our data suggest that transposon amplification may in part explain the increased size of the melon genome compared with the close relative cucumber. A low number of nucleotide-binding site–leucine-rich repeat disease resistance genes were annotated, suggesting the existence of specific defense mechanisms in this species. The DHL92 genome was compared with that of its parental lines allowing the quantification of sequence variability in the species. The use of the genome sequence in future investigations will facilitate the understanding of evolution of cucurbits and the improvement of breeding strategies.

Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae.

Tnt1 elements are a superfamily of LTR-retrotransposons distributed in the Solanaceae plant family and represent good model systems for studying regulatory and evolutionary controls established between hosts and transposable elements. Tnt1 retrotransposons tightly control their activation, by restricting expression to specific conditions. The Tnt1A element, originally discovered in tobacco, is expressed in response to stress, and its activation by microbial factors is followed by amplification, demonstrating that factors of pathogen origin can generate genetic diversity in plants. The Tnt1A promoter has the potential to be activated by various biotic and abiotic stimuli but a number of these are specifically repressed in tobacco and are revealed only when the LTR promoter is placed in a heterologous context. We propose that a tobacco- and stimulus-specific repression has been established in order to minimize activation in conditions that might generate germinal transposition. In addition to tight transcriptional controls, Tnt1A retrotransposons self-regulate their activity through gradual generation of defective copies that have reduced transcriptional activity. Tnt1 retrotransposons found in various Solanaceae species are characterized by a high level of variability in the LTR sequences involved in transcription, and have evolved by gaining new expression patterns, mostly associated with responses to diverse stress conditions. Tnt1A insertions associated with genic regions are initially favored but seem subsequently counter-selected, while insertions in repetitive DNA are maintained. On the other hand, amplification and loss of insertions may result from more brutal occurrences, as suggested by the large restructuring of Tnt1 populations observed in tobacco compared to each of its parental species. The distribution of Tnt1 elements thus appears as a dynamic flux, with amplification counterbalanced by loss of insertions. Tnt1 insertion polymorphisms are too high to reveal species relationships in the Nicotiana genus, but can be used to evaluate species relationships in the Lycopersicon and Capsicum genera. This also demonstrates that the behavior of Tnt1 retrotransposons differs between host species, most probably in correlation to differences in expression conditions and in the evolutionary and environmental history of each host.

Plant LTR-retrotransposons and MITEs: control of transposition and impact on the evolution of plant genes and genomes

Transposons are genetic elements that can move, and sometimes spread, within genomes, and that constitute an important fraction of eukaryote genomes. Two types of transposons, long terminal repeat (LTR)-retrotransposons and miniature inverted-repeat transposable elements (MITEs), are highly represented in plant genomes, and can account for as much as 50-80% of the total DNA content. In the last few years it has been shown that, in spite of their mutagenic capacity, both LTR-retrotransposons and MITEs can be found associated to genes, suggesting that their activity has influenced the evolution of plant genes. In this review we will summarise recent data on the control of the activity and the impact of both LTR-retrotransposons and MITEs on the evolution of plant genes and genomes.

A gene coding for a basic pathogenesis-related (PR-like) protein from Zea mays. Molecular cloning and induction by a fungus (Fusarium moniliforme) in germinating maize seeds

Pathogenesis-related proteins (PRs) are plant proteins produced in leaves in response to infection by pathogens including viruses, viroids, fungi and bacteria. Information on the presence and/or expression of PRs in monocotyledonous plants is scarce. Here we report the identification of cDNA and genomic clones coding for a basic form of a protein from germinating maize seeds having a high homology with the group of PR-1 from tobacco.A cDNA library enriched in aleurone-specific sequences was prepared from maize seeds two days after germination. One clone was found to contain an open reading frame encoding a protein homologous to PR proteins from tomato (p14) and tobacco (PR-1 group). Sequence analysis of the corresponding genomic clone revealed that it was encoded by a single exon. Besides, DNA blot hybridization indicates that this PR-like protein is encoded by a single-copy gene in maize. The accumulation of its mRNA increases after rehydration of desiccated seeds. Furthermore, a relationship was found between its expression and infection by a natural pathogen of maize, the fungus Fusarium moniliforme. The possible role of this protein as a response mechanism following fungal infection in cereal seeds is discussed.

Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding.

Sequence variability within the tobacco retrotransposon Tnt1 population.

Retroviruses consist of populations of different but closely related genomes referred to as quasispecies. A high mutation rate coupled with extremely rapid replication cycles allows these sequences to be highly interconnected in a rapid equilibrium. It is not known if other retroelements can show a similar population structure. We show here that when the tobacco Tnt1 retrotransposon is expressed, its RNA is not a unique sequence but a population of different but closely related sequences. Nevertheless, this highly variable population is not in a rapid equilibrium and could not be considered as a quasispecies. We have thus named the structure presented by Tnt1 RNA quasispecies‐like. We show that the expression of Tnt1 in different situations gives rise to different populations of Tnt1 RNA sequences, suggesting an adaptive capacity for this element. The analysis of the variability within the total genomic population of Tnt1 elements shows that mutations frequently occur in important regulatory elements and that defective elements are often produced. We discuss the implications that this population structure could have for Tnt1 regulation and evolution.

In vivo characterization of transcriptional regulatory sequences involved in the defence-associated expression of the tobacco retrotransposon Tnt1

The expression of the tobacco retrotransposon Tnt1 is induced by wounding, pathogen infections as well as microbial elicitors and abiotic factors known to induce the plant defence response. We report here that the LTR U3 region is sufficient to mediate transcriptional activation by biotic and abiotic elicitors in stable transgenic conditions. We have used in vivo footprinting techniques in order to analyse the cis-regulatory elements of the LTR U3 region that mediate the induction of Tnt1 expression. Our results indicate that a tandemly repeated short element, named BII box, is involved in the transcriptional activation of the tobacco retrotransposon Tnt1 in association with the plant defence signaling cascade.

Quasispecies in retrotransposons: a role for sequence variability in Tnt1 evolution

Retroviral replication is a very error-prone process. Replication of retroviruses gives rise to populations of closely related but different genomes referred to as ‘quasispecies’. This huge swarm of different sequences constitutes a reservoir of potentially useful genomes in case of an environmental change, endowing retroviruses with extreme adaptability. Retrotransposons are mobile genetic elements closely related to retroviruses, and retrotransposition is as error prone as retroviral replication. The Tnt1 retrotransposon is present in hundreds of copies in the genome of tobacco that show a high level of sequence heterogeneity. When Tnt1 is expressed, its RNA is not a single sequence but a population of sequences displaying a quasispecies-like structure. This population structure gives to Tnt1, as in the case of retroviruses, a high sequence plasticity and an adaptive capacity. We propose this adaptivity as the major reason for Tnt1 maintenance in Nicotiana genomes and we discuss in this paper the importance of sequence variability for Tnt1 evolution.

Expression of the gene encoding the PR-like protein PRms in germinating maize embryos

SummaryThe PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with E. moniliforme and studied the effect of fungal elicitors, the mycotoxin moniliformin, the hormone gibberellic acid, and specific chemical agents. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by F. monilforme, increase the steady-state level of PRms mRNA. PRms mRNA accumulation is also stimulated by the application of the hormone gibberellic acid or by treatment with silver nitrate, whereas acetylsalicylic acid has no effect. In situ RNA hybridization in isolated germinating embryo sections demonstrates that the PRms gene is expressed in the scutellum, particularly in a group of inner cells, and in the epithelium lying at the interface of the scutellum and the endosperm. The pattern of expression of the PRms gene closely resembles that found for hydrolytic enzymes, being confined to the scutellum and the aleurone layer of the germinating maize seed. Our results suggest that the PRms protein has a function during the normal process of seed germination that has become adapted to serve among the defence mechanisms induced in response to pathogens during maize seed germination.

Genome-Wide Analysis of the ‘‘Cut-and-Paste’’ Transposons of Grapevine

Background The grapevine is a widely cultivated crop and a high number of different varieties have been selected since its domestication in the Neolithic period. Although sexual crossing has been a major driver of grapevine evolution, its vegetative propagation enhanced the impact of somatic mutations and has been important for grapevine diversity. Transposable elements are known to be major contributors to genome variability and, in particular, to somatic mutations. Thus, transposable elements have probably played a major role in grapevine domestication and evolution. The recent publication of the complete grapevine genome opens the possibility for an in deep analysis of its transposon content. Principal Findings We present here a detailed analysis of the “cut-and-paste” class II transposons present in the genome of grapevine. We characterized 1160 potentially complete grapevine transposons as well as 2086 defective copies. We report on the structure of each element, their potentiality to encode a functional transposase, and the existence of matching ESTs that could suggest their transcription. Conclusions Our results show that these elements have transduplicated and amplified cellular sequences and some of them have been domesticated and probably fulfill cellular functions. In addition, we provide evidences that the mobility of these elements has contributed to the genomic variability of this species.