Large tandem duplications affect gene expression, 3D organization, and plant-pathogen response.

Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.

The plant mobile domain proteins MAIN and MAIL1 interact with the phosphatase PP7L to regulate gene expression and silence transposable elements in Arabidopsis thaliana.

Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs.

Heat Shock Protein HSP101 Affects the Release of Ribosomal Protein mRNAs for Recovery after Heat Shock.

Heat shock (HS) is known to have a profound impact on gene expression at different levels, such as inhibition of protein synthesis, in which HS blocks translation initiation and induces the sequestration of mRNAs into stress granules (SGs) or P-bodies for storage and/or decay. SGs prevent the degradation of the stored mRNAs, which can be reengaged into translation in the recovery period. However, little is known on the mRNAs stored during the stress, how these mRNAs are released from SGs afterward, and what the functional importance is of this process. In this work, we report that Arabidopsis HEAT SHOCK PROTEIN101 (HSP101) knockout mutant (hsp101) presented a defect in translation recovery and SG dissociation after HS Using RNA sequencing and RNA immunoprecipitation approaches, we show that mRNAs encoding ribosomal proteins (RPs) were preferentially stored during HS and that these mRNAs were released and translated in an HSP101-dependent manner during recovery. By 15N incorporation and polysome profile analyses, we observed that these released mRNAs contributed to the production of new ribosomes to enhance translation. We propose that, after HS, HSP101 is required for the efficient release of RP mRNAs from SGs resulting in a rapid restoration of the translation machinery by producing new RPs.

Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana.

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5'-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5'-ribosome pausing leading to the XRN4-mediated 5'-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of 'non-aberrant' mRNAs.

Evolutionary history of double-stranded RNA binding proteins in plants: identification of new cofactors involved in easiRNA biogenesis.

In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

Parallel action of AtDRB2 and RdDM in the control of transposable element expression.

BACKGROUND: In plants and animals, a large number of double-stranded RNA binding proteins (DRBs) have been shown to act as non-catalytic cofactors of DICERs and to participate in the biogenesis of small RNAs involved in RNA silencing. We have previously shown that the loss of Arabidopsis thaliana's DRB2 protein results in a significant increase in the population of RNA polymerase IV (p4) dependent siRNAs, which are involved in the RNA-directed DNA methylation (RdDM) process. RESULTS: Surprisingly, despite this observation, we show in this work that DRB2 is part of a high molecular weight complex that does not involve RdDM actors but several chromatin regulator proteins, such as MSI4, PRMT4B and HDA19. We show that DRB2 can bind transposable element (TE) transcripts in vivo but that drb2 mutants do not have a significant variation in TE DNA methylation. CONCLUSION: We propose that DRB2 is part of a repressive epigenetic regulator complex involved in a negative feedback loop, adjusting epigenetic state to transcription level at TE loci, in parallel of the RdDM pathway. Loss of DRB2 would mainly result in an increased production of TE transcripts, readily converted in p4-siRNAs by the RdDM machinery.

The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins.

La-related proteins (LARPs) are largely uncharacterized factors, well conserved throughout evolution. Recent reports on the function of human LARP4 and LARP6 suggest that these proteins fulfill key functions in mRNA metabolism and/or translation. We report here a detailed evolutionary history of the LARP4 and 6 families in eukaryotes. Genes coding for LARP4 and 6 were duplicated in the common ancestor of the vertebrate lineage, but one LARP6 gene was subsequently lost in the common ancestor of the eutherian lineage. The LARP6 gene was also independently duplicated several times in the vascular plant lineage. We observed that vertebrate LARP4 and plant LARP6 duplication events were correlated with the acquisition of a PABP-interacting motif 2 (PAM2) and with a significant reorganization of their RNA-binding modules. Using isothermal titration calorimetry (ITC) and immunoprecipitation methods, we show that the two plant PAM2-containing LARP6s (LARP6b and c) can, indeed, interact with the major plant poly(A)-binding protein (PAB2), while the third plant LARP6 (LARP6a) is unable to do so. We also analyzed the RNA-binding properties and the subcellular localizations of the two types of plant LARP6 proteins and found that they display nonredundant characteristics. As a whole, our results support a model in which the acquisition by LARP4 and LARP6 of a PAM2 allowed their targeting to mRNA 3' UTRs and led to their neofunctionalization.

Plant mobile domain proteins ensure Microrchidia 1 expression to fulfill transposon silencing.

Silencing of transposable elements (TEs) is an essential process to maintain genomic integrity within the cell. In Arabidopsis, together with canonical epigenetic pathways such as DNA methylation and modifications of histone tails, the plant mobile domain (PMD) proteins MAINTENANCE OF MERISTEMS (MAIN) and MAIN-LIKE 1 (MAIL1) are involved in TE silencing. In addition, the MICRORCHIDIA (MORC) ATPases, including MORC1, are important cellular factors repressing TEs. Here, we describe the genetic interaction and connection between the PMD and MORC pathways by showing that MORC1 expression is impaired in main and mail1 mutants. Transcriptomic analyses of higher order mutant plants combining pmd and morc1 mutations, and pmd mutants in which MORC1 expression is restored, show that the silencing defects of a subset of TEs in pmd mutants are most likely the consequence of MORC1 down-regulation. Besides, a significant fraction of up-regulated TEs in pmd mutants are not targeted by the MORC1 pathway.

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.

Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula.

Suppressive subtractive hybridization and expressed sequence tag sequencing identified 29 plant genes which are upregulated during the appressorium stage of mycorrhiza establishment between Medicago truncatula J5 (Myc+) and Glomus mosseae. Eleven genes coding plant proteins with predicted functions in signal transduction, transcription, and translation were investigated in more detail for their relation to early events of symbiotic interactions. Expression profiling showed that the genes are activated not only from the appressorium stage up to the fully established symbiosis in the Myc+ genotype of M. truncatula, but also when the symbionts are not in direct cell contact, suggesting that diffusible fungal molecules (Myc factors) play a, role in the induction of a signal-transduction pathway. Transcript accumulation in roots of a mycorrhiza-defective Myc- dmi3 mutant of M. truncatula is not modified by appressorium formation or diffusible fungal molecules, indicating that the signal transduction pathway is required for a successful G. mosseae-M. truncatula interaction leading to symbiosis development. The symbiotic nodulating bacterium Sinorhizobium meliloti does not activate the 11 genes, which supposes early discrimination by plant roots between the microbial symbionts.

XRN4 and LARP1 are required for a heat-triggered mRNA decay pathway involved in plant acclimation and survival during thermal stress.

To survive adverse and ever-changing environmental conditions, an organism must be able to adapt. It has long been established that the cellular reaction to stress includes the upregulation of genes coding for specific stress-responsive factors. In the present study, we demonstrate that during the early steps of the heat stress response, 25% of the Arabidopsis seedling transcriptome is targeted for rapid degradation. Our findings demonstrate that this process is catalyzed from 5' to 3' by the cytoplasmic exoribonuclease XRN4, whose function is seemingly reprogrammed by the heat-sensing pathway. The bulk of mRNAs subject to heat-dependent degradation are likely to include both the ribosome-released and polysome associated polyadenylated pools. The cotranslational decay process is facilitated at least in part by LARP1, a heat-specific cofactor of XRN4 required for its targeting to polysomes. Commensurate with their respective involvement at the molecular level, LARP1 and XRN4 are necessary for the thermotolerance of plants to long exposure to moderately high temperature, with xrn4 null mutants being almost unable to survive. These findings provide mechanistic insights regarding a massive stress-induced posttranscriptional downregulation and outline a potentially crucial pathway for plant survival and acclimation to heat stress.