The leaf vasculature plays a key role in solute translocation. Veins consist of at least seven distinct cell types, with specific roles in transport, metabolism, and signaling. Little is known about leaf vascular cells, in particular the phloem parenchyma (PP). PP effluxes sucrose into the apoplasm as a basis for phloem loading, yet PP has been characterized only microscopically. Here, we enriched vascular cells from Arabidopsis leaves to generate a single-cell transcriptome atlas of leaf vasculature. We identified at least 19 cell clusters, encompassing epidermis, guard cells, hydathodes, mesophyll, and all vascular cell types, and used metabolic pathway analysis to define their roles. Clusters comprising PP cells were enriched for transporters, including SWEET11 and SWEET12 sucrose and UmamiT amino acid efflux carriers. We provide evidence that PP development occurs independently from ALTERED PHLOEM DEVELOPMENT, a transcription factor required for phloem differentiation. PP cells have a unique pattern of amino acid metabolism activity distinct from companion cells (CCs), explaining differential distribution/metabolism of amino acids in veins. The kinship relation of the vascular clusters is strikingly similar to the vein morphology, except for a clear separation of CC from the other vascular cells including PP. In summary, our single-cell RNA-sequencing analysis provides a wide range of information into the leaf vasculature and the role and relationship of the leaf cell types.

Targeted deletions of exons in FLM, generated through CRISPR/Cas9 technology, clarify how specific splice variants contribute to the regulation of temperature-dependent flowering time in Arabidopsis thaliana.

Recent findings suggest that alternative splicing has a critical role in controlling the responses of plants to temperature variations. However, alternative splicing factors in plants are largely uncharacterized. Here we establish the putative splice regulator, PORCUPINE (PCP), as temperature-specific regulator of development in Arabidopsis thaliana. Our findings point to the misregulation of WUSCHEL and CLAVATA3 as the possible cause for the meristem defects affecting the pcp-1 loss-of-function mutants at low temperatures.

A central goal in microbiome research is to learn what distinguishes a healthy from a dysbiotic microbial community. Shifts in diversity and taxonomic composition are important indicators of dysbiosis, but a full understanding also requires knowledge of absolute microbial biomass. Simultaneous information on both microbiome composition and the quantity of its components can provide insight into microbiome function and disease state. Here we use shotgun metagenomics to simultaneously assess microbiome composition and microbial load in the phyllosphere of wild populations of the plant Arabidopsis thaliana. We find that wild plants vary substantially in the load of colonizing microbes, and that high loads are typically associated with the proliferation of single taxa, with only a few putatively pathogenic taxa achieving high abundances in the field. Our results suggest (i) that the inside of a plant leaf is on average sparsely colonized with an estimated two bacterial genomes per plant genome and an order of magnitude fewer eukaryotic microbial genomes, and (ii) that higher levels of microbial biomass often indicate successful colonization by pathogens. Lastly, our results show that load is a significant explanatory variable for loss of estimated Shannon diversity in phyllosphere microbiomes, implying that reduced diversity may be a significant predictor of microbial dysbiosis in a plant leaf.

BackgroundOur knowledge of natural genetic variation is increasing at an extremely rapid pace, affording an opportunity to come to a much richer understanding of how effects of specific genes are dependent on the genetic background. To achieve a systematic understanding of such GxG interactions, it is desirable to develop genome editing tools that can be rapidly deployed across many different genetic varieties.ResultsWe present an efficient CRISPR/Cas9 toolbox of super module (SM) vectors. These vectors are based on a previously described fluorescence protein marker expressed in seeds allowing identification of transgene-free mutants. We have used this vector series to delete genomic regions ranging from 1.7 to 13 kb in different natural accessions of the wild plant Arabidopsis thaliana. Based on results from 53 pairs of sgRNAs targeting individual nucleotide binding site leucine-rich repeat (NLR) genes, we provide a comprehensive overview of obtaining heritable deletions.ConclusionsThe SM series of CRISPR/Cas9 vectors enables the rapid generation of transgene-free, genome edited plants for a diversity of functional studies.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0330-7) contains supplementary material, which is available to authorized users.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the first exon of the HTT gene. Affected individuals inherit more than 40 repeats and the CAG repeat is genetically unstable in both the germline and soma. Molecular diagnosis and genotyping of the CAG repeat is traditionally performed by estimation of PCR fragment size. However, this approach is complicated by the presence of an adjacent polymorphic CCG repeat and provides no information on the presence of variant repeats, flanking sequence variants or on the degree of somatic mosaicism. To overcome these limitations, we have developed an ampliconsequencing protocol that allows the sequencing of hundreds of samples in a single MiSeq run. The composition of the HTT exon one trinucleotide repeat locus can be determined from the MiSeq sequencing reads generated. With sufficient sequencing depth, such MiSeq data can also be used to quantify the degree of somatic mosaicism of the HTT CAG repeat in the tissue analysed.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the first exon of the HTT gene. Affected individuals inherit more than 40 repeats and the CAG repeat is genetically unstable in both the germline and soma. Molecular diagnosis and genotyping of the CAG repeat is traditionally performed by estimation of PCR fragment size. However, this approach is complicated by the presence of an adjacent polymorphic CCG repeat and provides no information on the presence of variant repeats, flanking sequence variants or on the degree of somatic mosaicism. To overcome these limitations, we have developed an ampliconsequencing protocol that allows the sequencing of hundreds of samples in a single MiSeq run. The composition of the HTT exon one trinucleotide repeat locus can be determined from the MiSeq sequencing reads generated. With sufficient sequencing depth, such MiSeq data can also be used to quantify the degree of somatic mosaicism of the HTT CAG repeat in the tissue analysed.

A phospholipase D is a negative regulator of plant immunity and forms complexes with regulatory receptor-like kinases.