Construction and characterization of a knock-down RNA interference line of OsNRPD1 in rice (Oryza sativa ssp japonica cv Nipponbare).

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Retrotranspositional landscape of Asian rice revealed by 3000 genomes.

The recent release of genomic sequences for 3000 rice varieties provides access to the genetic diversity at species level for this crop. We take advantage of this resource to unravel some features of the retrotranspositional landscape of rice. We develop software TRACKPOSON specifically for the detection of transposable elements insertion polymorphisms (TIPs) from large datasets. We apply this tool to 32 families of retrotransposons and identify more than 50,000 TIPs in the 3000 rice genomes. Most polymorphisms are found at very low frequency, suggesting that they may have occurred recently in agro. A genome-wide association study shows that these activations in rice may be triggered by external stimuli, rather than by the alteration of genetic factors involved in transposable element silencing pathways. Finally, the TIPs dataset is used to trace the origin of rice domestication. Our results suggest that rice originated from three distinct domestication events.

Publisher Correction: Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

This article was not made open access when initially published online, which was corrected before print publication. In addition, ORCID links were missing for 12 authors and have been added to the HTML and PDF versions of the article.

Oak genome reveals facets of long lifespan.

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.

Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

The genus Oryza is a model system for the study of molecular evolution over time scales ranging from a few thousand to 15 million years. Using 13 reference genomes spanning the Oryza species tree, we show that despite few large-scale chromosomal rearrangements rapid species diversification is mirrored by lineage-specific emergence and turnover of many novel elements, including transposons, and potential new coding and noncoding genes. Our study resolves controversial areas of the Oryza phylogeny, showing a complex history of introgression among different chromosomes in the young 'AA' subclade containing the two domesticated species. This study highlights the prevalence of functionally coupled disease resistance genes and identifies many new haplotypes of potential use for future crop protection. Finally, this study marks a milestone in modern rice research with the release of a complete long-read assembly of IR 8 'Miracle Rice', which relieved famine and drove the Green Revolution in Asia 50 years ago.

Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants.

Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes.

Widespread and frequent horizontal transfers of transposable elements in plants.

Vertical, transgenerational transmission of genetic material occurs through reproduction of living organisms. In addition to vertical inheritance, horizontal gene transfer between reproductively isolated species has recently been shown to be an important, if not dominant, mechanism in the evolution of prokaryotic genomes. In contrast, only a few horizontal transfer (HT) events have been characterized so far in eukaryotes and mainly concern transposable elements (TEs). Whether these are frequent and have a significant impact on genome evolution remains largely unknown. We performed a computational search for highly conserved LTR retrotransposons among 40 sequenced eukaryotic genomes representing the major plant families. We found that 26 genomes (65%) harbor at least one case of horizontal TE transfer (HTT). These transfers concern species as distantly related as palm and grapevine, tomato and bean, or poplar and peach. In total, we identified 32 cases of HTTs, which could translate into more than 2 million among the 13,551 monocot and dicot genera. Moreover, we show that these TEs have remained functional after their transfer, occasionally causing a transpositional burst. This suggests that plants can frequently exchange genetic material through horizontal transfers and that this mechanism may be important in TE-driven genome evolution.

Small high-yielding binary Ti vectors pLSU with co-directional replicons for Agrobacterium tumefaciens-mediated transformation of higher plants.

Small high-yielding binary Ti vectors of Agrobacterium tumefaciens were constructed to increase the cloning efficiency and plasmid yield in Escherichia coli and A. tumefaciens for transformation of higher plants. We reduced the size of the binary vector backbone to 4566bp with ColE1 replicon (715bp) for E. coli and VS1 replicon (2654bp) for A. tumefaciens, a bacterial kanamycin resistance gene (999bp), and the T-DNA region (152bp). The binary Ti vectors with the truncated VS1 replicon were stably maintained with more than 98% efficiency in A. tumefaciens without antibiotic selection for 4 days of successive transfers. The transcriptional direction of VS1 replicon can be the same as that of ColE1 replicon (co-directional transcription), or opposite (head-on transcription) as in the case of widely used vectors (pPZP or pCambia). New binary vectors with co-directional transcription yielded in E. coli up to four-fold higher transformation frequency than those with the head-on transcription. In A. tumefaciens the effect of co-directional transcription is still positive in up to 1.8-fold higher transformation frequency than that of head-on transcription. Transformation frequencies of new vectors are over six-fold higher than those of pCambia vector in A. tumefaciens. DNA yields of new vectors were three to five-fold greater than pCambia in E. coli. The proper functions of the new T-DNA borders and new plant selection marker genes were confirmed after A. tumefaciens-mediated transformation of tobacco leaf discs, resulting in virtually all treated leaf discs transformed and induced calli. Genetic analysis of kanamycin resistance trait among the progeny showed that the kanamycin resistance and sensitivity traits were segregated into the 3:1 ratio, indicating that the kanamycin resistance genes were integrated stably into a locus or closely linked loci of the nuclear chromosomal DNA of the primary transgenic tobacco plants and inherited to the second generation.

RLK7, a leucine-rich repeat receptor-like kinase, is required for proper germination speed and tolerance to oxidative stress in Arabidopsis thaliana.

The leucine-rich repeat class of receptor-like kinase (LRR-RLKs) encoding genes represents the largest family of putative receptor genes in the Arabidopsis thaliana genome. However, very little is known about the range of biological process that they control. We present in this paper the functional characterization of RLK7 that has all the structural features of a receptor-like kinase of the plant-specific LRR type. To this end, we identified and characterized three independent T-DNA insertion mutants, constructed lines carrying truncated versions of this putative receptor, one lacking the cytoplasmic kinase domain (RLK7Δkin) and the other one lacking 14 LRR repeats (RLK7ΔLRR) and generated RLK7 overexpressing lines. We thus provide evidences that RLK7 is involved in the control of germination speed and the tolerance to oxidant stress. First, consistent with the expression kinetics of the RLK7 gene in the seeds, we found that all three mutants showed a delay in germination, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines displayed a phenotype of more precocious germination. Second, a non-hypothesis driven proteomic approach revealed that in the seedlings of the three T-DNA insertion lines, four enzymes directly or indirectly involved in reactive oxygen species detoxification, were significantly less abundant. Consistent with this finding, the three mutants were less tolerant than the wild type to a hydrogen peroxide treatment, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines presented the opposite phenotype.

Identification of an active LTR retrotransposon in rice.

Transposable elements are ubiquitous components of plant genomes. When active, these mobile elements can induce changes in the genome at both the structural and functional levels. Availability of the complete genome sequence for several model plant species provides the opportunity to study TEs in plants at an unprecedented scale. In the case of rice, annotation of the genomic sequence of the variety Nipponbare has revealed that TE-related sequences form more than 25% of its genome. However, most of the elements found are inactive, either because of structural alterations or because they are the target of various silencing pathways. In this paper, we propose a new post-genomic strategy aimed at identifying active TEs. Our approach relies on transcript profiling of TE-related sequences using a tiling microarray. We applied it to a particular class of TEs, the LTR retrotransposons. A transcript profiling assay of rice calli led to identification of a new transpositionally active family, named Lullaby. We provide a complete structural description of this element. We also show that it has recently been active in planta in rice, and discuss its phylogenetic relationships with Tos17, the only other active LTR retrotransposon described so far in the species.

AtERF38 (At2g35700), an AP2/ERF family transcription factor gene from Arabidopsis thaliana, is expressed in specific cell types of roots, stems and seeds that undergo suberization.

An inverse genetic approach was used to gain insight into the role of AP2/ERF-type transcription factors genes during plant development in Arabidopsis thaliana. Here we show that the expression pattern of AtERF38, which is, among the organs tested, more intensively expressed in mature siliques and floral stems, is closely associated with tissues that undergo secondary cell wall modifications. Firstly, public microarray data sets analysis indicates that AtERF38 is coregulated with several genes involved in secondary wall thickening. Secondly, this was experimentally confirmed in different types of cells expressing a Pro(AtERF38)::GUS fusion: histochemical analysis revealed strong and specific GUS activity in outer integument cells of mature seeds, endodermal cells of the roots in the primary developmental stage and some sclerified cells of mature inflorescence stems. All of these cells are known or shown here to be characterized by a reinforced wall. The latter, which have not been well characterized to date in Arabidopsis and may be suberized, could benefit of the use of AtERF38 as a specific marker. We were not able to detect any phenotype in an insertion line in which ectopic expression of AtERF38 is caused by the insertion of a T-DNA in its promoter. Nevertheless, AtERF28 may be considered as a candidate regulator of secondary wall metabolism in particular cell types that are not reinforced by the typical deposition of lignin and cellulose, but that have at least in common accumulation of suberin-like lipid polyesters in their walls.

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana.

We analyzed the Arabidopsis thaliana genome sequence to detect Late Embryogenesis Abundant (LEA) protein genes, using as reference sequences proteins related to LEAs previously described in cotton or which present similar characteristics. We selected 50 genes representing nine groups. Most of the encoded predicted proteins are small and contain repeated domains that are often specific to a unique LEA group. Comparison of these domains indicates that proteins with classical group 5 motifs are related to group 3 proteins and also gives information on the possible history of these repetitions. Chromosomal gene locations reveal that several LEA genes result from whole genome duplications (WGD) and that 14 are organized in direct tandem repeats. Expression of 45 of these genes was tested in different plant organs, as well as in response to ABA and in mutants (such as abi3, abi5, lec2 and fus3) altered in their response to ABA or in seed maturation. The results demonstrate that several so-called LEA genes are expressed in vegetative tissues in the absence of any abiotic stress, that LEA genes from the same group do not present identical expression profile and, finally, that regulation of LEA genes with apparently similar expression patterns does not systematically involve the same regulatory pathway.