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.

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.

A family portrait: structural comparison of the Whirly proteins from Arabidopsis thaliana and Solanum tuberosum.

DNA double-strand breaks are highly detrimental genomic lesions that routinely arise in genomes. To protect the integrity of their genetic information, all organisms have evolved specialized DNA-repair mechanisms. Whirly proteins modulate DNA repair in plant chloroplasts and mitochondria by binding single-stranded DNA in a non-sequence-specific manner. Although most of the results showing the involvement of the Whirly proteins in DNA repair have been obtained in Arabidopsis thaliana, only the crystal structures of the potato Whirly proteins WHY1 and WHY2 have been reported to date. The present report of the crystal structures of the three Whirly proteins from A. thaliana (WHY1, WHY2 and WHY3) reveals that these structurally similar proteins assemble into tetramers. Furthermore, structural alignment with a potato WHY2-DNA complex reveals that the residues in these proteins are properly oriented to bind single-stranded DNA in a non-sequence-specific manner.

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.

The conjugated auxin indole-3-acetic acid-aspartic acid promotes plant disease development.

Auxin is a pivotal plant hormone that regulates many aspects of plant growth and development. Auxin signaling is also known to promote plant disease caused by plant pathogens. However, the mechanism by which this hormone confers susceptibility to pathogens is not well understood. Here, we present evidence that fungal and bacterial plant pathogens hijack the host auxin metabolism in Arabidopsis thaliana, leading to the accumulation of a conjugated form of the hormone, indole-3-acetic acid (IAA)-Asp, to promote disease development. We also show that IAA-Asp increases pathogen progression in the plant by regulating the transcription of virulence genes. These data highlight a novel mechanism to promote plant susceptibility to pathogens through auxin conjugation.

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.

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.

Recombination and the maintenance of plant organelle genome stability.

Like their nuclear counterpart, the plastid and mitochondrial genomes of plants have to be faithfully replicated and repaired to ensure the normal functioning of the plant. Inability to maintain organelle genome stability results in plastid and/or mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly, deregulation of recombination in organelles results in genomic instability, often accompanied by adverse consequences for plant fitness. The recent identification of four families of proteins that prevent aberrant recombination of organelle DNA sheds much needed mechanistic light on this important process. What comes out of these investigations is a partial portrait of the recombination surveillance machinery in which plants have co-opted some proteins of prokaryotic origin but have also evolved whole new factors to keep their organelle genomes intact. These new features presumably optimized the protection of plastid and mitochondrial genomes against the particular genotoxic stresses they face.

Whirly proteins maintain plastid genome stability in Arabidopsis.

Maintenance of genome stability is essential for the accurate propagation of genetic information and cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively, little is known about how organelle genomes maintain a stable structure. Here, we report that the plastid-localized Whirly ssDNA-binding proteins are required for plastid genome stability in Arabidopsis. We show that a double KO of the genes AtWhy1 and AtWhy3 leads to the appearance of plants with variegated green/white/yellow leaves, symptomatic of nonfunctional chloroplasts. This variegation is maternally inherited, indicating defects in the plastid genome. Indeed, in all variegated lines examined, reorganized regions of plastid DNA are amplified as circular and/or head-tail concatemers. All amplified regions are delimited by short direct repeats of 10-18 bp, strongly suggesting that these regions result from illegitimate recombination between repeated sequences. This type of recombination occurs frequently in plants lacking both Whirlies, to a lesser extent in single KO plants and rarely in WT individuals. Maize mutants for the ZmWhy1 Whirly protein also show an increase in the frequency of illegitimate recombination. We propose a model where Whirlies contribute to plastid genome stability by protecting against illegitimate repeat-mediated recombination.

The transcriptional activator Pti4 is required for the recruitment of a repressosome nucleated by repressor SEBF at the potato PR-10a gene.

Transcriptional reprogramming is critical for plant disease resistance responses. In potato (Solanum tuberosum), the marker gene PATHOGENESIS-RELATED-10a (PR-10a) is transcriptionally activated by pathogens, wounding, or elicitor treatment. Activation of PR-10a requires the recruitment of the activator Why1 to its promoter. In addition, PR-10a is negatively regulated by the repressor SEBF (for Silencer Element Binding Factor). Here, we show through a yeast two-hybrid screen that SEBF interacts with Pti4, which has been shown to be a transcriptional activator. SEBF recruits Pti4 via its consensus sequence-type RNA binding domain, while Pti4 is recruited to SEBF by means of its ethylene-response factor domain. In vivo plant transcription assays confirmed that SEBF interacts with Pti4 to form a repressosome, showing that Pti4 can also play a role in transcriptional repression. Chromatin immunoprecipitation revealed that both SEBF and Pti4 are recruited to the PR-10a promoter in uninduced conditions only and that the recruitment of Pti4 is dependent on the presence of SEBF, consistent with the fact that there is no Pti4 consensus binding site in PR-10a. Unexpectedly, we also demonstrated that recruitment of SEBF was dependent on the presence of Pti4, thereby explaining why SEBF, itself a repressor, requires Pti4 for its repressing function.

Purification, crystallization and preliminary X-ray diffraction analysis of the Whirly domain of StWhy2 in complex with single-stranded DNA.

StWhy1 and StWhy2 are members of the Whirly family of single-stranded DNA (ssDNA) binding proteins. To understand the mode of binding of the Whirly proteins to single-stranded DNA, crystals of the Whirly domains of both StWhy1 and StWhy2 in complex with single-stranded DNA were obtained by the hanging-drop vapour-diffusion method. The diffraction patterns of the StWhy1-ssDNA complex crystals displayed severe anisotropy and were of low resolution, making them unsuitable for structure determination. In contrast, the crystals of the StWhy2-ssDNA complex diffracted isotropically to 2.20 A resolution. The crystallization and data collection to 2.20 A resolution of StWhy2 in the free form are also reported.

Overexpression of mtDNA-associated AtWhy2 compromises mitochondrial function.

BACKGROUND: StWhy1, a member of the plant-specific Whirly single-stranded DNA-binding protein family, was first characterized as a transcription factor involved in the activation of the nuclear PR-10a gene following defense-related stress in potato. In Arabidopsis thaliana, Whirlies have recently been shown to be primarily localized in organelles. Two representatives of the family, AtWhy1 and AtWhy3 are imported into plastids while AtWhy2 localizes to mitochondria. Their function in organelles is currently unknown. RESULTS: To understand the role of mitochondrial Whirlies in higher plants, we produced A. thaliana lines with altered expression of the atwhy2 gene. Organellar DNA immunoprecipitation experiments demonstrated that AtWhy2 binds to mitochondrial DNA. Overexpression of atwhy2 in plants perturbs mitochondrial function by causing a diminution in transcript levels and mtDNA content which translates into a low activity level of respiratory chain complexes containing mtDNA-encoded subunits. This lowered activity of mitochondria yielded plants that were reduced in size and had distorted leaves that exhibited accelerated senescence. Overexpression of atwhy2 also led to early accumulation of senescence marker transcripts in mature leaves. Inactivation of the atwhy2 gene did not affect plant development and had no detectable effect on mitochondrial morphology, activity of respiratory chain complexes, transcription or the amount of mtDNA present. This lack of phenotype upon abrogation of atwhy2 expression suggests the presence of functional homologues of the Whirlies or the activation of compensating mechanisms in mitochondria. CONCLUSION: AtWhy2 is associated with mtDNA and its overexpression results in the production of dysfunctional mitochondria. This report constitutes the first evidence of a function for the Whirlies in organelles. We propose that they could play a role in the regulation of the gene expression machinery of organelles.

Whirly transcription factors: defense gene regulation and beyond.

Members of the Whirly family of proteins are found throughout the plant kingdom and are predicted to share the ability to bind to single-stranded DNA. Arabidopsis and potato Whirly orthologs act as transcription factors that regulate defense gene expression; the Arabidopsis Whirly protein AtWhy1 contributes to both basal and specific defense responses. Analysis of the crystal structure of potato StWhy1 has provided insight into the DNA-binding mechanism of this family of proteins, their mode of action and possible autoregulation. There is evidence to suggest that Whirly proteins might play roles in processes other than defense responses and could function in the chloroplast as well as in the nucleus.

A "Whirly" transcription factor is required for salicylic acid-dependent disease resistance in Arabidopsis.

Transcriptional reprogramming is critical for plant disease resistance responses; its global control is not well understood. Salicylic acid (SA) can induce plant defense gene expression and a long-lasting disease resistance state called systemic acquired resistance (SAR). Plant-specific "Whirly" DNA binding proteins were previously implicated in defense gene regulation. We demonstrate that the potato StWhy1 protein is a transcriptional activator of genes containing the PBF2 binding PB promoter element. DNA binding activity of AtWhy1, the Arabidopsis StWhy1 ortholog, is induced by SA and is required for both SA-dependent disease resistance and SA-induced expression of an SAR response gene. AtWhy1 is required for both full basal and specific disease resistance responses. The transcription factor-associated protein NPR1 is also required for SAR. Surprisingly, AtWhy1 activation by SA is NPR1 independent, suggesting that AtWhy1 works in conjunction with NPR1 to transduce the SA signal. Our analysis of AtWhy1 adds a critical component to the SA-dependent plant disease resistance response.

A new family of plant transcription factors displays a novel ssDNA-binding surface.

The crystal structure of p24, the single-stranded DNA (ssDNA) binding subunit of the plant defense transcription factor PBF-2, has been determined to 2.3 A resolution. p24 is representative of a novel family of ubiquitous plant-specific proteins that we refer to as the Whirly family because of their quaternary structure. PBF-2 is composed of four p24 molecules that interact through a helix-loop-helix motif. This interaction produces a central pore, with beta-strands radiating outwards, resulting in a whirligig appearance to the quaternary structure. The noncrystallographic C(4) symmetry arrangement of p24 subunits is novel for ssDNA binding proteins and may explain the binding specificity of PBF-2. This structural arrangement also supports the role of PBF-2 in binding melted promoter regions to modulate gene expression.

Crystallization and preliminary X-ray crystallographic analysis of p24, a component of the potato nuclear factor PBF-2.

The Solanum tuberosum (potato) nuclear factor PBF-2 is implicated in pathogen-induced expression of the pathogenesis-related gene PR-10a. Crystals of the DNA-binding component of PBF-2, p24, have been obtained at 277 K in 20 mM Tris-HCl pH 8.0. Recombinant protein with a His tag at its C-terminus was overexpressed in Escherichia coli in the presence and absence of selenomethionine and was purified using a combination of HiTrap affinity columns and gel-filtration chromatography. Crystals suitable for structural analysis were obtained for both native and selenomethionine-labelled proteins and yielded diffraction data at 100 K that were processed to 2.3 and 2.8 A resolution, respectively. The p24 protein crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 69.4 (69.1), b = 89.4 (90.5), c = 144.1 (144.3) A. The asymmetric unit contains four protomers, giving a crystal volume per protein mass (V(M)) of 2.23 A(3) Da(-1) and a solvent content of 45% by volume.