Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus.

Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.

Single-cell transcriptomics unveils xylem cell development and evolution.

BACKGROUND: Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types. RESULTS: Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers. CONCLUSIONS: This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history.

A novel synthetic-genetic-array-based yeast one-hybrid system for high discovery rate and short processing time.

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.

NINJA-associated ERF19 negatively regulates Arabidopsis pattern-triggered immunity.

Recognition of microbe-associated molecular patterns (MAMPs) derived from invading pathogens by plant pattern recognition receptors (PRRs) initiates a subset of defense responses known as pattern-triggered immunity (PTI). Transcription factors (TFs) orchestrate the onset of PTI through complex signaling networks. Here, we characterized the function of ERF19, a member of the Arabidopsis thaliana ethylene response factor (ERF) family. ERF19 was found to act as a negative regulator of PTI against Botrytis cinerea and Pseudomonas syringae. Notably, overexpression of ERF19 increased plant susceptibility to these pathogens and repressed MAMP-induced PTI outputs. In contrast, expression of the chimeric dominant repressor ERF19-SRDX boosted PTI activation, conferred increased resistance to the fungus B. cinerea, and enhanced elf18-triggered immunity against bacteria. Consistent with a negative role for ERF19 in PTI, MAMP-mediated growth inhibition was weakened or augmented in lines overexpressing ERF19 or expressing ERF19-SRDX, respectively. Using biochemical and genetic approaches, we show that the transcriptional co-repressor Novel INteractor of JAZ (NINJA) associates with and represses the function of ERF19. Our work reveals ERF19 as a novel player in the mitigation of PTI, and highlights a potential role for NINJA in fine-tuning ERF19-mediated regulation of Arabidopsis innate immunity.