Biology subjects

Melkonian, K.

Publications and source records attributed to Melkonian, K..

4 recordsLinked to original sources

bioRxiv2022

miniTurbo-based interactomics of two plasma membrane-localized SNARE proteins in Marchantia polymorpha

O_LIMarchantia polymorpha is a model liverwort and its overall low genetic redundancy is advantageous for dissecting complex pathways. Proximity-dependent in vivo biotin-labelling methods have emerged as powerful interactomics tools in recent years. However, interactomics studies applying proximity labelling are currently limited to angiosperm species in plants. C_LIO_LIHere, we established and evaluated a miniTurbo-based interactomics method in M. polymorpha using MpSYP12A and MpSYP13B, two plasma membrane- localized SNARE proteins, as baits. C_LIO_LIWe show that our method yields a manifold of potential interactors of MpSYP12A and MpSYP13B compared to a co-immunoprecipitation approach. Our method could capture specific candidates for each SNARE. C_LIO_LIWe conclude that a miniTurbo-based method is a feasible tool for interactomics in M. polymorpha and potentially applicable to other model bryophytes. Our interactome dataset on MpSYP12A and MpSYP13B will be a useful resource to elucidate the evolution of SNARE functions. C_LI

plant biology↗

bioRxiv2021

Differential EDS1 requirement for cell death activities of plant TIR-domain proteins

Toll/interleukin-1 Receptor (TIR) domains are integral to immune systems across all domains of life. TIRs exist as single-domain and as larger receptor or adaptor proteins. In plants, TIRs constitute N-terminal domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors. Although TIR-NLR and TIR signaling requires the Enhanced disease susceptibility 1 (EDS1) protein family, TIR domains persist in species that have incomplete or no EDS1 members. To assess whether particular TIR groups appear with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algae and land plant species, we identify four conserved TIR groups, two of which are TIR-NLRs present in eudicots and two are more widespread. Presence of one TIR-only protein group is highly correlated with EDS1 and members of this group elicit EDS1-dependent cell death. By contrast, a more widely represented TIR group of TIR-NB-WD40/TPR (TNP) proteins (formerly called XTNX) has at least one member which can induce EDS1-independent cell death. Our data provide a new phylogeny-based plant TIR classification and identify TIR groups that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity. One sentence summaryLand plants have evolved four conserved TIR groups

plant biology↗

bioRxiv2021

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying GUS genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

plant biology↗

bioRxiv2021

A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of colony counting assays. However, colony counting assays are cumbersome and time-consuming, and are unable to detect spatial patterns of bacterial colonization in plants. Here, to overcome these shortcomings, we devised a broadly applicable genetic engineering tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed plasmid vectors that have broad host ranges and enable Tn7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location ubiquitously found across bacterial phyla. These vectors allowed for generation of bioluminescent transformants of various plant pathogenic bacteria belonging to the genera Pseudomonas, Rhizobium (Agrobacterium), and Ralstonia. The bioluminescent transformant of Pseudomonas syringae pv. tomato DC3000 (Pto-lux) was as virulent in Arabidopsis thaliana as its parental strain. Direct luminescence measurements of Pto-lux-inoculated plant tissues reported bacterial titers in A. thaliana, Solanum lycopersicum, Nicotiana benthamiana, and Marchantia polymorpha as accurately as conventional colony counting assays. We further showed the utility of our vectors for converting the previously generated Pto derivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth with a dynamic range of 4 orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated spatial colonization patterns of the Pto-lux in/on inoculated plant tissues. Taken together, our vectors offer untapped opportunities for developing bioluminescence-based quantitative and spatial analysis of bacterial growth in a variety of plant-bacteria interactions. SIGNIFICANCE STATEMENTWe developed broad-host-range plasmid vectors that integrate the luciferase operon, luxCDABE, into a specific genomic location ubiquitously found across bacterial phyla. Using these vectors, we established a high-throughput method for bioluminescence-based quantitative assays of in planta bacterial growth with a dynamic range of 4 orders of magnitude and visualized spatiotemporal patterns of bacterial colonization in/on inoculated plant tissues.

plant biology↗