Near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) were designed and interfaced with leaves of Arabidopsis thaliana plants to report hydrogen peroxide (H2O2), a key signaling molecule associated with the onset of plant stress. The sensor nIR fluorescence response (>900 nm) is quenched by H2O2 with selectivity against other stress-associated signaling molecules and within the plant physiological range (10–100 H2O2 μM). In vivo remote nIR imaging of H2O2 sensors enabled optical monitoring of plant health in response to stresses including UV-B light (−11%), high light (−6%), and a pathogen-related peptide (flg22) (−10%), but not mechanical leaf wounding (<3%). The sensor’s high biocompatibility was reflected on similar leaf cell death (<5%) and photosynthetic rates to controls without SWCNT. These optical nanosensors report early signs of stress and will improve our understanding of plant stress communication, provide novel tools for precision agriculture, and optimize the use of agrochemicals in the environment.
ORCID IDs: 0000-0003-1506-4683 (R.M.); 0000-0003-4881-6343 (V.H.M.); 0000-0002-4354-6295 (C.G.R.). TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOTS9(AIR9) are microtubule-binding proteins that localize to the division site in plants. Their function in Arabidopsis (Arabidopsis thaliana) remained unclear because neither tan1 nor air9 single mutants have a strong phenotype. We show that tan1 air9 double mutants have a synthetic phenotype consisting of short, twisted roots with disordered cortical microtubule arrays that are hypersensitive to a microtubule-depolymerizing drug. The tan1 air9 double mutants have significant defects in division plane orientation due to failures in placing the new cell wall at the correct division site. Full-length TAN1 fused to yellow fluorescent protein, TAN1-YFP, and several deletion constructs were transformed into the double mutant to assess which regions of TAN1 are required for its function in root growth, root twisting, and division plane orientation. TAN1-YFP expressed in tan1 air9 significantly rescued the double mutant phenotype in all three respects. Interestingly, TAN1 missing the first 126 amino acids, TAN1-DI-YFP, failed to rescue the double mutant phenotype, while TAN1 missing a conserved middle region, TAN1-DII-YFP, significantly rescued the mutant phenotype in terms of root growth and division plane orientation but not root twisting. We use the tan1 air9 double mutant to discover new functions for TAN1 and AIR9 during phragmoplast guidance and root morphogenesis.Plant cells are typically constrained by cell walls (Cosgrove, 2005) that are also connected via plasmodesmata (Brunkard and Zambryski, 2017), and they do not migrate relative to each other. In the absence of significant cell migration, the entire plant body must be built through elegant coordination between the division, expansion, and differentiation of cells. Therefore, division plane orientation, or the spatial control of cytokinesis, has important roles in plant development and growth (Pickett-Heaps et al., 1999).The key steps of plant division plane orientation occur during interphase, G2, and mitosis. During interphase, the cortical microtubule array typically aligns perpendicular to the cell expansion axis (Baskin, 2001), and during G2 (Gunning and Wick, 1985), this promotes the formation of a land plant-specific microtubule and microfilament array called the preprophase band (PPB;Pickett-Heaps and Northcote, 1966). Although the location of the PPB accurately predicts the future division site (Gunning et al., 1978;Van Damme et al., 2007;Rasmussen et al., 2013;Lipka et al., 2014;Martinez et al., 2017), its role in division plane establishment has recently been called into question by mutants that do not form obvious PPBs but manage to produce relatively orderly roots. These plants have mutations in the tonneau1a locus (Zhang et al., 2016) and mutations in three related tonneau1 recruiting motif (trm) loci (Schaefer et al., 2017). Whether the PPB establishes the future division site or is the signpost of an ear...
High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.
High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq'. Tiled-ClickSeq uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, obviating the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended using click-chemistry and a PCR reaction using Illumina adaptors generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.
Cell division requires spatial coordination to properly position the division plane. How division plane positioning contributes to plant growth remains unknown. Two unrelated microtubule binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOT-CULTURES9 (AIR9), are together required for normal Arabidopsis growth and division. tan1 air9 double mutants have synthetic growth and division plane orientation defects while single mutants lack obvious defects. We show that the first 132 amino acids of TAN1 (TAN11-132) rescue the tan1 air9 double mutant and localize to the division site during telophase. Loss of both rescue and division-site localization occurred when interaction between TAN1 and PHRAGMOPLAST ORIENTING KINESIN1 (POK1) was disrupted by replacing six amino acid residues with alanines in TAN11-132. However, full-length TAN1 with the same alanine substitutions significantly rescued the tan1 air9 double mutant and remained at the division site throughout mitosis, although its accumulation was reduced and phragmoplast positioning defects occurred. POK1 often fails to accumulate at the division site in tan1 air9 mutants, suggesting that both TAN1 and AIR9 stabilize POK1 there. Finally, a mitosis specific promoter driving TAN1 rescued the tan1 air9 double mutant phenotypes indicating that defects seen in the root differentiation zone reflect the loss of mitotic-specific TAN1 activity.One sentence summarySpecific amino acids within TAN1 are required for its correct localization and function partially through interaction with POK1; both TAN1 and AIR9 mediate POK1 division site localization.
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