The Arabidopsis Mitochondrial Nucleoid-Associated Protein WHIRLY2 Is Required for a Proper Response to Salt Stress.

In the last years, plant organelles have emerged as central coordinators of responses to internal and external stimuli, which can induce stress. Mitochondria play a fundamental role as stress sensors being part of a complex communication network between the organelles and the nucleus. Among the different environmental stresses, salt stress poses a significant challenge and requires efficient signaling and protective mechanisms. By using the why2 T-DNA insertion mutant and a novel knock-out mutant prepared by CRISPR/Cas9-mediated genome editing, this study revealed that WHIRLY2 is crucial for protecting mitochondrial DNA (mtDNA) integrity during salt stress. Loss-of-function mutants show an enhanced sensitivity to salt stress. The disruption of WHIRLY2 causes the impairment of mtDNA repair that results in the accumulation of aberrant recombination products, coinciding with severe alterations in nucleoid integrity and overall mitochondria morphology besides a compromised redox-dependent response and misregulation of antioxidant enzymes. The results of this study revealed that WHIRLY2-mediated structural features in mitochondria (nucleoid compactness and cristae) are important for an effective response to salt stress.

Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres.

Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the fluorescent coat protein-tagged for MS2 and PP7 aptamers (tdMCP-FP and tdPCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labeling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labeling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labeling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes.

Live-Cell CRISPR Imaging in Plant Cells with a Telomere-Specific Guide RNA.

Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.

Two combinatorial patterns of telomere histone marks in plants with canonical and non-canonical telomere repeats.

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non-canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate-type telomere repeat TTAGGG or Allium genus-specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non-canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR-dCas9-eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C-3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis-like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco-like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere-associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions.

Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells.

The 3D organization of chromatin plays an important role in genome stability and many other pivotal biological programs. Therefore, the establishment of imaging methods, which enable us to study the dynamics of chromatin in living cells, is necessary. Although primary live cell imaging methods were a breakthrough, there is a need to develop more specific labeling techniques. With the discovery of programmable DNA binding proteins, such zinc finger proteins (ZFP), transcription activator-like effectors (TALE), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a major leap forward was made. Here, we review the applications and potential of fluorescent repressor-operator systems, programmable DNA binding proteins with an emphasis on CRISPR-based chromatin imaging in living and fixed cells, and their potential application in plant science.

RNA-guided endonuclease - in situ labelling (RGEN-ISL): a fast CRISPR/Cas9-based method to label genomic sequences in various species.

Visualising the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN-ISL (RNA-guided endonuclease - in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans-activating crRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry. Real-time visualisation of the CRISPR/Cas9-mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.

Biotechnological advances in Lilium.

Modern powerful techniques in plant biotechnology have been developed in lilies (Lilium spp., Liliaceae) to propagate, improve and make new phenotypes. Reliable in vitro culture methods are available to multiply lilies rapidly and shorten breeding programs. Lilium is also an ideal model plant to study in vitro pollination and embryo rescue methods. Although lilies are recalcitrant to genetic manipulation, superior genotypes are developed with improved flower colour and form, disease resistance and year round forcing ability. Different DNA molecular markers have been developed for rapid indirect selection, genetic diversity evaluation, mutation detection and construction of Lilium linkage map. Some disease resistance-QTLs are already mapped on the Lilium linkage map. This review presents latest information on in vitro propagation, genetic engineering and molecular advances made in lily.

In vitro propagation of Lilium longiflorum var. ceb-dazzle through direct somatic embryogenesis.

Experiments were carried out to investigate the effects of various concentrations of Picloram (0, 1, 2, 3, 6 and 9 mg L(-1)), TDZ (0, 0.5, 1, 1.5 and 2 mg L(-1)), NAA (1.5 mg L(-1)) in combination with TDZ (0.08, 0.2 and 0.4 mg L(-1)), 2,4-D (2.5, 5 and 10 mg L(-1)) combined with BAP (0.25 mg L(-1)) and different types of explants (basal, central and distal part of the bulb scale) on direct somatic embryogenesis induction of Lilium longiflorum var. Ceb-Dazzle. The explants were surface sterilized and cultured on MS medium supplemented with 3% sucrose, 0.3% Phytagel and various concentrations of mentioned growth regulators. It was found that Picloram at a concentration of 2 mg L-' was the most effective treatment for induction of direct somatic embryogenesis and gave the highest number of embryos (18.6) on each explant. The explants from basal part of the bulb scale showed the best responses (19.9 embryos/explant). TDZ alone or combined with NAA in various concentrations was not able to induce somatic embryogenesis, but gave direct bulblet regeneration. Similar results were obtained for 2, 4-D and BAP combination treatments. Induced somatic embryos were transferred to MS medium without growth regulators for maturation and matured plantlets were successfully acclimatized and transferred to in vivo conditions.