Transcription is a major driving force for plastid genome instability in Arabidopsis.

Though it is an essential process, transcription can be a source of genomic instability. For instance, it may generate RNA:DNA hybrids as the nascent transcript hybridizes with the complementary DNA template. These hybrids, called R-loops, act as a major cause of replication fork stalling and DNA breaks. In this study, we show that lowering transcription and R-loop levels in plastids of Arabidopsis thaliana reduces DNA rearrangements and mitigates plastid genome instability phenotypes. This effect can be observed on a genome-wide scale, as the loss of the plastid sigma transcription factor SIG6 prevents DNA rearrangements by favoring conservative repair in the presence of ciprofloxacin-induced DNA damage or in the absence of plastid genome maintenance actors such as WHY1/WHY3, RECA1 and POLIB. Additionally, resolving R-loops by the expression of a plastid-targeted exogenous RNAse H1 produces similar results. We also show that highly-transcribed genes are more susceptible to DNA rearrangements, as increased transcription of the psbD operon by SIG5 correlates with more locus-specific rearrangements. The effect of transcription is not specific to Sigma factors, as decreased global transcription levels by mutation of heat-stress-induced factor HSP21, mutation of nuclear-encoded polymerase RPOTp, or treatment with transcription-inhibitor rifampicin all prevent the formation of plastid genome rearrangements, especially under induced DNA damage conditions.

Enabling a sustainable and prosperous future through science and innovation in the bioeconomy at Agriculture and Agri-Food Canada.

Science and innovation are important components underpinning the agricultural and agri-food system in Canada. Canada's vast geographical area presents diverse, regionally specific requirements in addition to the 21st century agricultural challenges facing the overall sector. As the broader needs of the agricultural landscape have evolved and will continue to do so in the next few decades, there is a trend in place to transition towards a sustainable bioeconomy, contributing to reducing greenhouse gas emission and our dependency on non-renewable resources. We highlight some of the key policy drivers on an overarching national scale and those specific to agricultural research and innovation that are critical to fostering a supportive environment for innovation and a sustainable bioeconomy. As well, we delineate some major challenges and opportunities facing agriculture in Canada, including climate change, sustainable agriculture, clean technologies, and agricultural productivity, and some scientific initiatives currently underway to tackle these challenges. The use of various technologies and scientific efforts, such as Next Generation Sequencing, metagenomics analysis, satellite image analysis and mapping of soil moisture, and value-added bioproduct development will accelerate scientific development and innovation and its contribution to a sustainable and prosperous bioeconomy.

Short-range inversions: rethinking organelle genome stability: template switching events during DNA replication destabilize organelle genomes.

In the organelles of plants and mammals, recent evidence suggests that genomic instability stems in large part from template switching events taking place during DNA replication. Although more than one mechanism may be responsible for this, some similarities exist between the different proposed models. These can be separated into two main categories, depending on whether they involve a single-strand-switching or a reciprocal-strand-switching event. Single-strand-switching events lead to intermediates containing Y junctions, whereas reciprocal-strand-switching creates Holliday junctions. Common features in all the described models include replication stress, fork stalling and the presence of inverted repeats, but no single element appears to be required in all cases. We review the field, and examine the ideas that several mechanisms may take place in any given genome, and that the presence of palindromes or inverted repeats in certain regions may favor specific rearrangements.

Organelle DNA rearrangement mapping reveals U-turn-like inversions as a major source of genomic instability in Arabidopsis and humans.

Failure to maintain organelle genome stability has been linked to numerous phenotypes, including variegation and cytosolic male sterility (CMS) in plants, as well as cancer and neurodegenerative diseases in mammals. Here we describe a next-generation sequencing approach that precisely maps and characterizes organelle DNA rearrangements in a single genome-wide experiment. In addition to displaying global portraits of genomic instability, it surprisingly unveiled an abundance of short-range rearrangements in Arabidopsis thaliana and human organelles. Among these, short-range U-turn-like inversions reach 25% of total rearrangements in wild-type Arabidopsis plastids and 60% in human mitochondria. Furthermore, we show that replication stress correlates with the accumulation of this type of rearrangement, suggesting that U-turn-like rearrangements could be the outcome of a replication-dependent mechanism. We also show that U-turn-like rearrangements are mostly generated using microhomologies and are repressed in plastids by Whirly proteins WHY1 and WHY3. A synergistic interaction is also observed between the genes for the plastid DNA recombinase RECA1 and those encoding plastid Whirly proteins, and the triple mutant why1why3reca1 accumulates almost 60 times the WT levels of U-turn-like rearrangements. We thus propose that the process leading to U-turn-like rearrangements may constitute a RecA-independent mechanism to restart stalled forks. Our results reveal that short-range rearrangements, and especially U-turn-like rearrangements, are a major factor of genomic instability in organelles, and this raises the question of whether they could have been underestimated in diseases associated with mitochondrial dysfunction.

Time- and cost-efficient identification of T-DNA insertion sites through targeted genomic sequencing.

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity.

Plastid genome instability leads to reactive oxygen species production and plastid-to-nucleus retrograde signaling in Arabidopsis.

The plastid genome is highly conserved among plant species, suggesting that alterations of its structure would have dramatic impacts on plant fitness. Nevertheless, little is known about the direct consequences of plastid genome instability. Recently, it was reported that the plastid Whirly proteins WHY1 and WHY3 and a specialized type-I polymerase, POLIB, act as safeguards against plastid genome instability in Arabidopsis (Arabidopsis thaliana). In this study, we use ciprofloxacin, an organelle double-strand break-inducing agent, and the why1why3polIb-1 variegated mutant to evaluate the impact of generalized plastid DNA instability. First, we show that in why1why3polIb-1 and ciprofloxacin-treated plants, plastid genome instability is associated with increased reactive oxygen species production. Then, using different light regimens, we show that the elevated reactive oxygen species production correlates with the appearance of a yellow-variegated phenotype in the why1why3polIb-1 population. This redox imbalance also correlates to modifications of nuclear gene expression patterns, which in turn leads to acclimation to high light. Taken together, these results indicate that plastid genome instability induces an oxidative burst that favors, through nuclear genetic reprogramming, adaptation to subsequent oxidative stresses.

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage.

All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer-tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.

Divergent roles for the two PolI-like organelle DNA polymerases of Arabidopsis.

DNA polymerases play a central role in the process of DNA replication. Yet, the proteins in charge of the replication of plant organelle DNA have not been unambiguously identified. There are however many indications that a family of proteins homologous to bacterial DNA polymerase I (PolI) is implicated in organelle DNA replication. Here, we have isolated mutant lines of the PolIA and PolIB genes of Arabidopsis (Arabidopsis thaliana) to test this hypothesis. We find that mutation of both genes is lethal, thus confirming an essential and redundant role for these two proteins. However, the mutation of a single gene is sufficient to cause a reduction in the levels of DNA in both mitochondria and plastids. We also demonstrate that polIb, but not polIa mutant lines, are hypersensitive to ciprofloxacin, a small molecule that specifically induces DNA double-strand breaks in plant organelles, suggesting a function for PolIB in DNA repair. In agreement with this result, a cross between polIb and a plastid Whirly mutant line yielded plants with high levels of DNA rearrangements and severe growth defects, indicating impairments in plastid DNA repair pathways. Taken together, this work provides further evidences for the involvement of the plant PolI-like genes in organelle DNA replication and suggests an additional role for PolIB in DNA repair.

Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co-transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing.

Two distinct transient expression approaches were compared with assess the impact of the viral suppressor p19 on a recombinant protein production performed in Nicotiana benthamiana suspension culture. A parental N. benthamiana cell line was transiently transformed with either an Agrobacterium containing a gene construct for a murine IgG1 (R514) or concurrently with two Agrobacteria containing R514 or p19. In addition, a stably transformed N. benthamiana cell line that constitutively expresses p19 was transformed with R514-containing Agrobacterium. The parental N. benthamiana cell line that had been co-cultivated with both p19 and R514 achieved the highest yield of IgG1 (1.06 mg IgG1/kg FW; 0.024% TSP) compared with that obtained without p19 (0.61 mg IgG1/kg FW; 0.014% TSP). The N. benthamiana cell line that had been stably transformed with p19 only reached 0.25 mg IgG1/kg FW (0.009% TSP) when co-cultured with R514-containing Agrobacterium. Dual agroinfiltration of N. benthamiana leaves with p19 and R514 was also performed to assess for Agrobacteria efficiencies and 147.7 mg IgG1/kg FW were obtained. Therefore, our results demonstrate that transient co-transformation of plant cell suspension culture with two transformation vectors is feasible and that the use of the viral suppressor of silencing p19 significantly raises the production of the protein of interest.

Crystal structures of DNA-Whirly complexes and their role in Arabidopsis organelle genome repair.

DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.