Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).

Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRRs), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptor-like kinases (BIRs) causes cell death, but the direct mechanisms leading to such deregulation remains unclear. Here, we found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. CSA1 mediates cell death in bak1-4 and bak1-4 bir3-2 mutants via components of effector-triggered immunity-(ETI) pathways. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, requires CSA1. Thus, we show that CSA1 guards BIR3 BAK1 homeostasis and integrates pattern- and effector-mediated cell death pathways downstream of BAK1. De-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity.

ABSTRACT Super-resolution microscopy techniques have revolutionized cell biology by providing insights into the dynamics of single molecules and nanoscale organization within living cells. However, the application of dynamic live-cell methods in plants has been limited by the lack of suitable fluorophores for simultaneous visualization of multiple proteins. To address this challenge, we implemented a two-color sptPALM approach using codon-optimized photoactivatable fluorescent proteins PA-GFP and PATagRFP. Recently, we showed their individual usability in single-color experiments in Nicotiana benthamiana and Arabidopsis thaliana cells. Here, we now demonstrate the suitability of these fluorophores and their combined use for dual-color sptPALM for the simultaneous observation of two different protein fusions in the same plant cell.

AbstractThe BRI1-associated kinase BAK1/SERK3 is a positive regulator of multiple leucine rich receptor kinase-mediated signaling pathways including pattern triggered immunity (PTI). Absence or overexpression of BAK1 leads to spontaneous cell death formation. BAK1-interacting receptors (BIR) constitutively interact with BAK1, and plants lacking or overexpressing BIR proteins phenocopy the cell death symptoms observed inbak1knock outs or overexpressors. In the interactome of BIR3, the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) was identified by mass spectrometry. CSA1 physically interacts with BIR proteins and can be detected in complexes with BAK1. Direct interaction was shown only for CSA1 with BIR proteins but not BAK1. Double mutantbak1 bir3genotypes develop strong dwarfism and cell death symptoms that are dependent on EDS1 and salicylic acid. Loss of CSA1 blocksbak1andbak1 bir3-mediated cell death formation thus demonstrating that CSA1 is causal for this type of cell death. We propose that CSA1 guards BIR proteins and initiates autoimmune cell death that is observed when BAK1 BIR complexes are impaired. Our findings reveal how cell death in the absence of BAK1 and BIR3 is executed and links BAK1, a common co-receptor of many pattern recognition receptors, to NLR proteins typically implicated in effector-triggered immunity.

Small posttranslationally modified signaling peptides are proteolytically derived from larger precursor proteins and subject to several additional steps of modification, including Pro hydroxylation, Hyp glycosylation, and/or Tyr sulfation. The processing proteases and the relevance of posttranslational modifications for peptide biogenesis and activity are largely unknown. In this study these questions were addressed for the Clavata3/Endosperm Surrounding Region (CLE) peptide CLE40, a peptide regulator of stem cell differentiation in the Arabidopsis (Arabidopsis thaliana) root meristem. We identify three subtilases (SBT1.4, SBT1.7, and SBT4.13) that cleave the CLE40 precursor redundantly at two sites. C-terminal processing releases the mature peptide from its precursor and is thus required for signal biogenesis. SBT-mediated cleavage at a second site within the mature peptide attenuates the signal. The second cleavage is prevented by Pro hydroxylation, resulting in the formation of mature and bioactive CLE40 in planta. Our data reveal a role for posttranslational modification by Pro hydroxylation in the regulation of CLE40 formation and activity.