Radim Cegan

High-frequency, precise modification of the tomato genome

BackgroundThe use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.ResultsHere, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences.ConclusionsHigh-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.

Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR–Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR–Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.

A multi-purpose toolkit to enable advanced genome engineering in plants

An integrated reagent toolkit and streamlined protocols work across diverse plant species to enable sophisticated genome edits. We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

Structure and evolution of Apetala3, a sex-linked gene in Silene latifolia

BackgroundThe evolution of sex chromosomes is often accompanied by gene or chromosome rearrangements. Recently, the gene AP3 was characterized in the dioecious plant species Silene latifolia. It was suggested that this gene had been transferred from an autosome to the Y chromosome.ResultsIn the present study we provide evidence for the existence of an X linked copy of the AP3 gene. We further show that the Y copy is probably located in a chromosomal region where recombination restriction occurred during the first steps of sex chromosome evolution. A comparison of X and Y copies did not reveal any clear signs of degenerative processes in exon regions. Instead, both X and Y copies show evidence for relaxed selection compared to the autosomal orthologues in S. vulgaris and S. conica. We further found that promoter sequences differ significantly. Comparison of the genic region of AP3 between the X and Y alleles and the corresponding autosomal copies in the gynodioecious species S. vulgaris revealed a massive accumulation of retrotransposons within one intron of the Y copy of AP3. Analysis of the genomic distribution of these repetitive elements does not indicate that these elements played an important role in the size increase characteristic of the Y chromosome. However, in silico expression analysis shows biased expression of individual domains of the identified retroelements in male plants.ConclusionsWe characterized the structure and evolution of AP3, a sex linked gene with copies on the X and Y chromosomes in the dioecious plant S. latifolia. These copies showed complementary expression patterns and relaxed evolution at protein level compared to autosomal orthologues, which suggests subfunctionalization. One intron of the Y-linked allele was invaded by retrotransposons that display sex-specific expression patterns that are similar to the expression pattern of the corresponding allele, which suggests that these transposable elements may have influenced evolution of expression patterns of the Y copy. These data could help researchers decipher the role of transposable elements in degenerative processes during sex chromosome evolution.

Comparative analysis of a plant pseudoautosomal region (PAR) in Silene latifolia with the corresponding S. vulgaris autosome

BackgroundThe sex chromosomes of Silene latifolia are heteromorphic as in mammals, with females being homogametic (XX) and males heterogametic (XY). While recombination occurs along the entire X chromosome in females, recombination between the X and Y chromosomes in males is restricted to the pseudoautosomal region (PAR). In the few mammals so far studied, PARs are often characterized by elevated recombination and mutation rates and high GC content compared with the rest of the genome. However, PARs have not been studied in plants until now. In this paper we report the construction of a BAC library for S. latifolia and the first analysis of a > 100 kb fragment of a S. latifolia PAR that we compare to the homologous autosomal region in the closely related gynodioecious species S. vulgaris.ResultsSix new sex-linked genes were identified in the S. latifolia PAR, together with numerous transposable elements. The same genes were found on the S. vulgaris autosomal segment, with no enlargement of the predicted coding sequences in S. latifolia. Intergenic regions were on average 1.6 times longer in S. latifolia than in S. vulgaris, mainly as a consequence of the insertion of transposable elements. The GC content did not differ significantly between the PAR region in S. latifolia and the corresponding autosomal region in S. vulgaris.ConclusionsOur results demonstrate the usefulness of the BAC library developed here for the analysis of plant sex chromosomes and indicate that the PAR in the evolutionarily young S. latifolia sex chromosomes has diverged from the corresponding autosomal region in the gynodioecious S. vulgaris mainly with respect to the insertion of transposable elements. Gene order between the PAR and autosomal region investigated is conserved, and the PAR does not have the high GC content observed in evolutionarily much older mammalian sex chromosomes.

Impact of repetitive DNA on sex chromosome evolution in plants

Structurally and functionally diverged sex chromosomes have evolved in many animals as well as in some plants. Sex chromosomes represent a specific genomic region(s) with locally suppressed recombination. As a consequence, repetitive sequences involving transposable elements, tandem repeats (satellites and microsatellites), and organellar DNA accumulate on the Y (W) chromosomes. In this paper, we review the main types of repetitive elements, their gathering on the Y chromosome, and discuss new findings showing that not only accumulation of various repeats in non-recombining regions but also opposite processes form Y chromosome. The aim of this review is also to discuss the mechanisms of repetitive DNA spread involving (retro) transposition, DNA polymerase slippage or unequal crossing-over, as well as modes of repeat removal by ectopic recombination. The intensity of these processes differs in non-recombining region(s) of sex chromosomes when compared to the recombining parts of genome. We also speculate about the relationship between heterochromatinization and the formation of heteromorphic sex chromosomes.

Genomic diversity in two related plant species with and without sex chromosomes--Silene latifolia and S. vulgaris.

Background Genome size evolution is a complex process influenced by polyploidization, satellite DNA accumulation, and expansion of retroelements. How this process could be affected by different reproductive strategies is still poorly understood. Methodology/Principal Findings We analyzed differences in the number and distribution of major repetitive DNA elements in two closely related species, Silene latifolia and S. vulgaris. Both species are diploid and possess the same chromosome number (2n = 24), but differ in their genome size and mode of reproduction. The dioecious S. latifolia (1C = 2.70 pg DNA) possesses sex chromosomes and its genome is 2.5× larger than that of the gynodioecious S. vulgaris (1C = 1.13 pg DNA), which does not possess sex chromosomes. We discovered that the genome of S. latifolia is larger mainly due to the expansion of Ogre retrotransposons. Surprisingly, the centromeric STAR-C and TR1 tandem repeats were found to be more abundant in S. vulgaris, the species with the smaller genome. We further examined the distribution of major repetitive sequences in related species in the Caryophyllaceae family. The results of FISH (fluorescence in situ hybridization) on mitotic chromosomes with the Retand element indicate that large rearrangements occurred during the evolution of the Caryophyllaceae family. Conclusions/Significance Our data demonstrate that the evolution of genome size in the genus Silene is accompanied by the expansion of different repetitive elements with specific patterns in the dioecious species possessing the sex chromosomes.

Identification of a Novel Retrotransposon with Sex Chromosome-Specific Distribution in Silene latifolia

Silene latifolia is a dioecious plant species with chromosomal sex determination. Although the evolution of sex chromosomes in S. latifolia has been the subject of numerous studies, a global view of X chromosome structure in this species is still missing. Here, we combine X chromosome microdissection and BAC library screening to isolate new X chromosome-linked sequences. Out of 8 identified BAC clones, only BAC 86M14 showed an X-preferential signal after FISH experiments. Further analysis revealed the existence of the Athila retroelement which is enriched in the X chromosome and nearly absent in the Y chromosome. Based on previous data, the Athila retroelement belongs to the CL3 group of most repetitive sequences in the S. latifolia genome. Structural, transcriptomics and phylogenetic analyses revealed that Athila CL3 represents an old clade in the Athila lineage. We propose a mechanism responsible for Athila CL3 distribution in the S. latifolia genome.

Impact of Repetitive Elements on the Y Chromosome Formation in Plants

In contrast to animals, separate sexes and sex chromosomes in plants are very rare. Although the evolution of sex chromosomes has been the subject of numerous studies, the impact of repetitive sequences on sex chromosome architecture is not fully understood. New genomic approaches shed light on the role of satellites and transposable elements in the process of Y chromosome evolution. We discuss the impact of repetitive sequences on the structure and dynamics of sex chromosomes with specific focus on Rumex acetosa and Silene latifolia. Recent papers showed that both the expansion and shrinkage of the Y chromosome is influenced by sex-specific regulation of repetitive DNA spread. We present a view that the dynamics of Y chromosome formation is an interplay of genetic and epigenetic processes.

Expression response of duplicated metallothionein 3 gene to copper stress in Silene vulgaris ecotypes

Metallothioneins (MTs) were identified as important players in metal metabolism. MT3 gene presents a key metallothionein controlling copper homeostasis in plants. We have selected one cupricolous and one non-cupricolous ecotype to isolate and analyse the MT3 gene in Silene vulgaris. For expression data comparison, we have also included other metal-tolerant ecotypes. Based on a S. vulgaris BAC library screening, we have identified and sequenced a genomic clone containing MT3 gene (SvMT3). We found that SvMT3 gene has been locally duplicated in a tandem arrangement. Expression analysis and complementation studies using yeast mutants showed that both copies of the SvMT3 gene were functional. Moreover, we examined the expression of MT3 gene(s) in selected ecotypes under different copper treatments to show the tissue-specific expression response to copper stress. We demonstrated that higher copper concentrations specifically affected MT3 expression among ecotypes. Our analysis shows that MT3a has similar expression pattern in cupricolous ecotypes while MT3b has common expression features shared by all metallophyte S. vulgaris ecotypes. Our data indicate that down-regulation of MT3b root expression in higher copper concentrations is associated with copper stress. We propose that there might be a specific regulation of SvMT3s transcription depending on the type of heavy metal tolerance.