Biology subjects

Sugano, S. S.

Publications and source records attributed to Sugano, S. S..

6 recordsLinked to original sources

bioRxiv2024

AistSeq: An In-House Tn5-Based Plasmid Sequencing Platform Using A Compact Benchtop Sequencer

Sequence verification of plasmids is a fundamental process in synthetic biology. For plasmid sequence verification using next-generation sequencing (NGS) library preparation, Tn5 transposase is widely used. Streamlined sequencing workflow for laboratory-scale applications is important; however, recombinant Tn5 production in-house can be laborious. In this study, we demonstrated that the addition of a large soluble tag was not essential for purification and that the fusion of a His10 tag and protein A was sufficient to purify sufficient amounts of active Tn5 transposase. In addition, we evaluated exonuclease-based genomic DNA digestion for plasmid sequencing from an E. coli lysate and the data analysis pipeline of sequences derived from the Illumina iSeq100 platform for de novo assembly, reference mapping, and annotation. This study proposes a simple workflow of an in-house Tn5-based plasmid Sequencing platform using a compact benchtop sequencer (AistSeq). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/618112v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@f1546corg.highwire.dtl.DTLVardef@1dd947aorg.highwire.dtl.DTLVardef@3efb78org.highwire.dtl.DTLVardef@efbde1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Genetic and phosphoproteomic basis of LysM-mediated immune signaling in Marchantia polymorpha highlights conserved elements and new aspect of pattern-triggered immunity in land plants

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in plant immunity. The model liverwort Marchantia polymorpha is emerging as a popular model for investigating the evolution of plant-microbe interactions. M. polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha have not yet been described. We show that, in M. polymorpha, which has four LysM receptor homologs, lysin motif (LysM) receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR are required for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for a negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and demonstrates the utility of M. polymorpha as a plant model for discovering novel or fundamental molecular mechanisms underlying PRR-triggered immune signaling in plants.

plant biology↗

bioRxiv2022

Comprehensive Analysis of Peptide-Coding Genes Identifies an LRR-only Microprotein That Regulates Reproductive Organs in Marchantia polymorpha

1In the past two decades, many plant peptides have been found to play crucial roles in various biological events by mediating cell-to-cell communications. However, a large number of small open reading frames (sORFs) or short genes capable of encoding peptides remain uncharacterized. In this study, we examined several candidate genes for peptides conserved between two model plants: Arabidopsis thaliana and Marchantia polymorpha. We examined the expression pattern in M. polymorpha and subcellular localization using a transient assay with Nicotiana benthamiana. We found that one candidate, MpSGF10B, was expressed in meristems, gemma cups, and male reproductive organs called antheridiophores. MpSGF10B has an N-terminal signal peptide followed by two leucine-rich repeat (LRR) domains and was secreted to the extracellular region in N. benthamiana and M. polymorpha. Compared with the wild type, two independent knockout mutants had an increased number of antheridiophores, which emerged from meristems. It was revealed in gene ontology enrichment analysis that MpSGF10B was significantly co-expressed with genes related to cell cycle and development. These results suggest that MpSGF10B may regulate the reproductive development in M. polymorpha. Our research should shed light on the unknown role of LRR-only proteins in land plants.

plant biology↗

bioRxiv2020

Stomatal density affects gas diffusion and CO2 assimilation dynamics in Arabidopsis under fluctuating light

Stomatal density (SD) is closely associated with photosynthetic and growth characteristics in plants. In the field, light intensity can fluctuate drastically throughout a day. The objective of the present study is to examine how the change in SD affects stomatal conductance (gs) and CO2 assimilation rate (A) dynamics, biomass production, and water use under fluctuating light. Here, we compared the photosynthetic and growth characteristics under constant and fluctuating light among four lines of Arabidopsis thaliana (L.): a wild-type (WT), a STOMAGEN/EPFL9-overexpressing line, STOMAGEN/EPFL9-silencing line, and an EPIDERMAL PATTERNING FACTOR 1 knockout line (epf1). Lower SD resulted in faster response of A owing to the faster response of gs to fluctuating light and higher water use efficiency without decreasing A. Higher SD resulted in a faster response of A because of the higher initial gs. epf1, with a moderate increase in SD, showed the larger carbon gain, attributable to the high capacity and fast response of A, yielding higher biomass production than WT under fluctuating light. The present study suggests that higher SD can be beneficial to improve biomass production in the plant under field conditions.

plant biology↗

bioRxiv2019

Two Y chromosome-encoded genes determine sex in kiwifruit

Dioecy, the presence of male and female individuals, has evolved independently in multiple flowering plant lineages. Although theoretical models for the evolution of dioecy, such as the \"two-mutation\" model, are well established, little is known about the specific genes determining sex and their evolutionary history. Kiwifruit, a major tree crop consumed worldwide, is a dioecious species. In kiwifruit, we had previously identified a Y-encoded sex-determinant candidate gene acting as the suppressor of feminization (SuF), named Shy Girl (SyGI). Here, we identified a second Y-encoded sex-determinant that we named Friendly boy (FrBy), which exhibits strong expression in tapetal cells. Gene-editing and complementation analyses in Arabidopsis thaliana and Nicotiana tabacum indicated that FrBy acts for the maintenance of male (M) functions, independently of SyGI, and that these functions are conserved across angiosperm species. We further characterized the genomic architecture of the small (< 1 Mb) male specific region of the Y-chromosome (MSY), which harbors only two genes significantly expressed in developing gynoecia and androecia, respectively: SyGI and FrBy. Resequencing of the genome of a natural hermaphrodite kiwifruit revealed that this individual is genetically male but carries deletion(s) of parts of the Y-chromosome, including SyGI. Additionally, expression of FrBy in female kiwifruit resulted in hermaphrodite plants. These results clearly indicate that Y-encoded SyGI and FrBy act independently as the SuF and M factors in kiwifruit, respectively, and provide insight into the evolutionary path leading to a two-factor sex determination system but also a new breeding approach for dioecious species.

plant biology↗

bioRxiv2018

Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.

plant biology↗