Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Nanobody-functionalised silica particles bind selectively to target bacteria. i) Graphical illustration of particle–bacteria binding strategy. The cysteine group on the nanobody is bound to a PEG11 linker via a maleimide bond which, in turn, is bound to streptavidin-functionalised 2.7 μm silica particles via a biotin bond. ii) Particle–bacteria binding after mixing for 30 min in a 1.5 ml Eppendorf and imaging in a 96-well plate in the phase contrast channel or iii) fluorescence channel. White arrow highlights a silica particle, blue arrow highlights a bacterium. a) Sybody-F1 (SbF1) functionalised particles bound to S. aureus GFP. b) Nanobody 01 (Nb01) functionalised particles bound to E. coli MC1061 expressing mScarlet and naturally expressing OmpA-short. c) Nanobody 41 (Nb41) functionalised particles bound to E. coli MC1061 Δ ompA expressing mNeonGreen and OmpA-long. Scale bar = 5 μm.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Bacteria, Binding Assay, Imaging, Fluorescence, Expressing

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Single species growth upon bacterial binding to sCAPA deposited particles. a) Graphical illustration of deposition and binding assay. Particles are first deposited with sCAPA, and the template is then transferred to a Petri dish and filled with BSA to cover the PDMS. Bacteria are then added to bind to the particles. Unbound bacteria are washed out with fresh PBS using a pipette. PBS is then replaced with agar media, before placing the petri dish under a microscope to image cell growth. b) Representative fluorescence images of S. aureus JE2 GFP bound to SbF1 deposited particles. i) Cells bound at t = 40 min after adding tryptone soy agar. ii) Cells growing after t = 140 min. iii) Cells growing at t = 260 min where colonies begin to merge. Scale bar = 50 μm. c) E. coli MC1061 mScarlet bound to Nb01 deposited particles. i) Cy3 image of cell binding at t = 40 min. ii) Phase contrast image of cell growth after t = 260 min. Cy3 fluorescence not visible at exponential phase due to weak fluorescence. iii) Cell growth at t = 310 min where colonies begin to merge. Scale bar = 50 μm. d) Binarised images in c used during image quantification. Binarised colony in white, red circles represent a circle with an equivalent area as overlaying colony to which a ‘colony radius’ is attributed. i) Cells bound at t = 40 min after adding tryptone soy agar, ii) colony formation after 260 min growth, iii) colonies begin to merge at 310 min. e) Quantification of the fraction of traps with observed microbial growth. Bars and errors represent mean and standard error of the mean, data points represent different fractions measured for each field of view for 3 separate templates for each bacterial strain. f) Distribution of number of bacteria bound in each trap as determined by particle localisation image analysis on ×60 magnification images of templates in PBS. Despite the particles all being the same size, the average number of bound bacteria to each particle notably varies. Mean number of bound bacteria and standard error of the mean displayed in top right. g) Distribution in time taken for individual colonies to reach a radius of 15 μm. Data points represent single growing colonies. Distributions represent all data for biological triplicates. Black lines represent mean and variance. h) Growth of indicated bacterial strains in liquid tryptone soy broth measured by optical density at 600 nm. Error bars represent standard error of the mean of 3 biological repeats and the black dotted line represents fit to a Gompertz growth law.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Bacteria, Transferring, Microscopy, Fluorescence

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Cell binding of microbial pairs with designed spatial structure. a–c) Representative images of selectively bound and growing cells across different time points. a) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in a sodium chloride lattice structure corresponding to Moran's I = −1. b) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in 8 × 8 patchy lattice structure corresponding to Moran's I = 0.75. c) S. aureus GFP and E. coli mScarlet (OmpA-long) in 1 : 24 ratio square lattice. i) Merged Cy3 and FITC image in PBS media, ii) cell growth after exchanging PBS with tryptone soy agar and growing at 37 °C. Distance between traps in all lattices is 25 μm. Time stamp in hours:mins after exchanging PBS for agar. Scale bar = 50 μm. d) Comparison of ideal vs. measured cell binding for spatial structures in a–c. Bars represent mean of 4 independent templates. e) Fraction of growing cells for sum of green and red cells. Data points represent measured growing fraction in individual microscope fields of view, for 4 independent templates. Error bars represent standard error of the mean.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Comparison, Microscopy

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: Leaves of Arabidopsis Col-0 and the crk7 -5 mutant were infiltrated with Surfactin at concentrations from 100 µg/ml to 100 ng/µl. Heat-inactivated B . subtilis at an OD 600 of 0.02 in 10 mM MgCl 2 and 10 mM MgCl 2 were infiltrated as positive and negative controls, respectively. Pictures show three infiltrated leaves from three individual plants at 6 dpi.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) Ion leakage assay of different Arabidopsis PTI receptor mutants at 6 dpi. Leaves were infiltrated with heat-inactivated B . subtilis at an OD 600 of 0.02 in 10 mM MgCl 2 . Bars represent mean normalized conductivity ± SEM (n=12). Different letters indicate significant differences of means (One-way ANOVA with Tukey’s post hoc test, p<0.05). ( B ) Images of 5-week-old Arabidopsis Col-0, crk7 -5, and 3 individual crk7 -5 lines complemented with genomic CRK7 expressed under its native promoter ( crk7 -5/ P CRK7 :CRK7 ) at 6 dpi. ( C ) qRT-PCR to monitor PR1 expression in response to WTA treatment in Col-0, crk7 -5, and crk7 -5/ P CRK7 :CRK7 . Bars represent mean relative PR1 expression ± SEM (n>5 biological replicates, Student’s T-test between WTA and control treatment). (B and C) Leaves were infiltrated with 1 µg/ml WTA or 10 mM MgCl 2 as a control. ( D to F ) Leaves of Col-0, crk7 -5, and crk7 -5/ P CRK7 :CRK7 were pretreated for 24 h with 10 ng/ml WTA, 10 mM MgCl 2, or 100 nM flg22. Pretreated leaves were infiltrated with Pst DC3000. Colony-forming units per cm 2 leaf area (CFU/cm 2 ) were counted at 0 and 3 dpi. Bounds of boxplots represent the 25 th and 75 th percentile; the line shows the median, and the whiskers the 1.5xIQR (n=6; Welch’s T-test).

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) Infiltration of indicated mutants and corresponding wild-types (Col-0 for T-DNA lines, Col-3 gl1 for crk7 -4) with 1 µg/ml WTA in 10 mM MgCl 2 . Pictures show three infiltrated leaves of three individual plants at 6 dpi. ( B ) Identification of the crk7 -4 mutant using mapping by sequencing of F2 plants with a suppressor phenotype. Diamonds indicate the position and frequency of synonymous (light-green) and nonsynonymous (dark-green) single-nucleotide polymorphisms (SNPs). Candidate SNPs are highlighted by the red box. The red diamond indicates the crk7 -4 mutation. Grey background indicates the centromere region. ( C ) Candidate suppressor nsSNPs identified on Chromosome IV via mapping-by-sequencing. ( D ) Schematic representation of the CRK6 - CRK7 - CRK8 gene cluster on Chromosome 4 of Arabidopsis thaliana Col-0. T-DNA insertion loci of crk6 -2, crk7 -5, crk7 -6, crk7 -7, crk7 -8, and crk8 -3 are depicted, as well as the C 94 Y amino acid exchange in the crk7 -4 mutant.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) Infiltration of three independent crk7 -5 lines expressing mCitrine-tagged CRK7 under the control of the native promoter ( crk7 -5/ P CRK7 :mCitrine-CRK7 ). Col-0 and the crk7 -5 mutant were infiltrated as controls. Plants were infiltrated with 1 µg/ml WTA in 10 mM MgCl 2 . Pictures show three infiltrated leaves of individual T3 plants at 6 dpi. ( B ) Western Blot analysis with anti-GFP antibody to confirm mCitrine-CRK7 expression in the tested complementation lines. ( C ) Confocal laser scanning microscopy of the three independent crk7 -5/ P CRK7 :mCitrine-CRK7 lines. mCitrine was excited at 514 nm and detected at 525 nm to 555 nm. Chlorophyll autofluorescence was detected at 740 nm to 770 nm. Fluorescence images represent maximum projections of 12 focal planes at 1 µm distance. Scale Bar = 20 µm.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Expressing, Control, Mutagenesis, Western Blot, Confocal Laser Scanning Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) Different plant species were infiltrated with 1 µg/ml WTA in 10 mM MgCl 2 . Pictures show infiltrated leaves at 10 dpi. Black circles indicate the infiltrated area. If no circle is present, the whole leaf was infiltrated. ( B ) Taxonomic relationship of infiltrated plant species. The taxonomy tree was built using the NCBI Taxonomy Browser. BLASTp search was conducted using CRK7 and CRK43 protein sequences to identify CRK7 and CRK43 homologs in the analyzed species. Homology was accepted for hits with at least 85% identity, 95% query coverage, and a max-score per amino acid of at least 1.75.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques:

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) Phylogenetic tree of the CRK7 ( At4g23150 ) genomic sequence of screened WTA-sensitive (black font) and insensitive (red font) Arabidopsis thaliana accessions for which a genome sequence was available from the 1001 genomes project . Neighbor joining tree was built using the Jukes-Cantor model based on a MUSCLE alignment of the full-length genomic region of CRK7, performed in Geneious©. The At4g23150 locus of the Cvi-0 accessions was set as an outgroup; branch labels indicate substitutions per site. The CRK7 gene of different accessions was separated into four groups with no CRK7 gene present (blue background), functional CRK7 (red background), potentially non-functional group I (green background), and II (yellow background). ( B ) Ion leakage assay of WTA-insensitive Arabidopsis thaliana accessions. Col-0 was included as a sensitive accession control. Accessions included in the phylogenetic tree are labeled in bold letters. Bar color represents the corresponding group as depicted in (A). Leaves were infiltrated with heat-inactivated B . subtilis at an OD 600 of 0.02 in 10 mM MgCl 2 . Bars represent mean normalized conductivity ± SEM (n=12). Different letters indicate significant differences of means (One-way ANOVA with Tukey’s post hoc test, p<0.05).

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Sequencing, Functional Assay, Control, Labeling

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) Amino acid sequence of the Col-0 CRK7 protein. Red letters indicate conserved amino acid exchanges in WTA-insensitive Arabidopsis thaliana accessions. SP: signal peptide, TM: transmembrane domain, KD: kinase domain ( B ) AlphaFold ( , ) prediction of CRK7-ectodomain homodimer. Predicted N-glycosylation sites are highlighted in purple. Red amino acids represent amino acids altered in WTA-insensitive Arabidopsis thaliana accessions. ( C ) Predicted Alignment Error (PAE) of the CRK7-ectodomain homodimer. Blue color represents high prediction confidence, red color represents low prediction confidence.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Sequencing, Glycoproteomics

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) Confocal laser scanning microscopy of mCitrine- and mScarlet-I-tagged CRK7 transiently co-expressed from the native promoter in N. benthamiana . mCitrine and mScarlet-I were excited at 514 nm or 561 nm and detected at 525 nm to 555 nm or 580 nm to 620 nm, respectively. Scale Bar = 10 µm. ( B ) Fluorescence-lifetime imaging microscopy (FLIM) of mCitrine-tagged CRK7 alone and co-expressed with mScarlet-I-tagged CRK7, infiltrated with 1 µg/ml WTA (purple) or 10 mM MgCl 2 (blue) as control 1 h before microscopy. The number of individual images (n) used for the analysis is indicated in the figure. Different letters indicate significant differences of means (One-way ANOVA with Tukey’s post hoc test, p<0.05). ( C ) Protein binding in [%] was calculated from FLIM-FRET analysis of mCitrine labeled CRK7 co-expressed with mScarlet-I labeled CRK7, treated with 1 µg/ml WTA or 10 mM MgCl 2 . (Student’s t-test, *** P<0.0001). ( D ) Anti-GFP pull-down experiment confirming the homomerization of CRK7 tagged with mCitrine and mScarlet-I. ( E ) Anti-GFP pulldown experiment demonstrating interaction between CRK7 and CRK43, which dissociates upon WTA treatment. ( F ) mCitrine-tagged CRK7 and CRK43 were transiently expressed in N. benthamiana alone, in combination, and together with 1 µg/ml WTA. Image was taken at 6 dpi ( G ). Current working model. CRK7 and CRK43 form a heteromeric complex at the plasma membrane. Upon WTA recognition, CRK43 dissociates from the complex to induce downstream signaling via the EDS1-PAD4-ADR1 signaling hub.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Confocal Laser Scanning Microscopy, Fluorescence, Imaging, Microscopy, Control, Protein Binding, Labeling, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) WTA infiltration of indicated mutants and corresponding wild types (Col-0 for T-DNA lines, Col-3 gl1 for crk7 -4), together with 4 independent complementation lines expressing mCitrine-tagged CRK43 under control of the native promoter in the crk43 -2 background. Leaves were infiltrated with 1 µg/ml WTA in 10 mM MgCl 2 . Pictures show three infiltrated leaves of three individual plants at 6 dpi. ( B ) Identification of the crk43 -2 mutant using mapping by sequencing of F2 plants with a suppressor phenotype. Diamonds indicate the position and frequency of the synonymous (light-green) and nonsynonymous (dark-green) single-nucleotide polymorphisms (SNPs). Candidate SNPs are highlighted by the red box. The red diamond indicates the crk43 -2 mutation. Grey background indicates centromere regions. ( C ) Candidate suppressor SNPs identified on Chromosome I via mapping-by-sequencing. ( D ) Schematic representation of the CRK43 gene on Chromosome I of Arabidopsis Col-0. The T-DNA insertion loci of crk43 -2 is depicted, as well as the G to A mutation (red font) in the crk43 -1 mutant. The crk43 -1 mutation converts the canonical “AG” splice acceptor at the end of intron 3 (underlined) into “AA.” cDNA analysis from Col-0 and crk43 -1 revealed that all analysed suppressor lines carry a 10 bp deletion in exon 4, consistent with the spliceosome utilizing the next downstream “AG” as an alternative acceptor (blue box). The deletion introduces a frameshift and premature stop codon between kinase subdomains V and Via , resulting in a truncated protein lacking over half of the kinase domain.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Expressing, Control, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Cysteine-rich receptor-like kinases mediate Wall Teichoic Acid perception in Arabidopsis

doi: 10.1101/2025.09.18.677107

Figure Lengend Snippet: ( A ) WTA infiltration of the pad4 -14 mutant and the corresponding wild-type Col-3 gl1 . Leaves were infiltrated with 1 µg/ml WTA in 10 mM MgCl 2 . Pictures show three infiltrated leaves of three individual plants at 6 dpi. ( B ) Identification of the pad4 -14 mutant using mapping by sequencing of F2 plants with a suppressor phenotype. Diamonds indicate the position and frequency of the synonymous (light-green) and nonsynonymous (dark-green) single-nucleotide polymorphisms (SNPs). Candidate SNPs are highlighted by the red box. The red diamond indicates the pad4 -14 mutation. Grey background indicates the centromere region. ( C ) Candidate suppressor SNPs identified on Chromosome III via mapping-by-sequencing. ( D ) Schematic representation of the PAD4 gene on Chromosome III of Arabidopsis thaliana Col-0. The amino acid exchange resulting from the pad4 -14 mutation is depicted. ( E and F ) Infiltration of eds1-2 , pad4-1 , and sag101 -3 (E) as well as adr1 triple, nrg1 triple, and adr1 nrg1 hextuple (F) mutants. Col-0 and crk7 -5 were used as positive and negative control, respectively. Leaves were infiltrated with 1 µg/ml WTA in 10 mM MgCl 2 . Pictures show three infiltrated leaves of three individual plants at 6 dpi. ( G ) Protein structure of the EDS1-(blue) PAD4 (dark-red) heterodimer bound to pRib-ADP . The EDS1 and PAD4 amino acids involved in pRib-ADP binding are depicted in yellow. Amino acid Serine 499 , which is exchanged to a Phenylalanine in the pad4 -14 mutant, is depicted in red. ( H ) Infiltration of pad4 -1/ sag101 -3 mutant plants complemented with wild-type PAD4 and a mutant allele of PAD4 with an amino acid exchange (R 314 A) in the pRib-ADP binding site, with 1 µg/ml WTA. Col-0 and the pad4 -1 mutant were used as controls.

Article Snippet: Primary antibodies used were rat monoclonal anti-GFP (Chromotek, 3H9) for the detection of mCitrine-tagged CRK7 and CRK43, rat monoclonal anti-RFP (Chromotek, 5F8) for the detection of mScarlet-tagged CRK7, and rabbit polyclonal anti-CRK7 (Agrisera) for the detection of untagged CRK7.

Techniques: Mutagenesis, Sequencing, Negative Control, Binding Assay