Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography.

The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.

Translatome analysis of an NB-LRR immune response identifies important contributors to plant immunity in Arabidopsis.

An important branch of plant immunity involves the recognition of pathogens by nucleotide-binding, leucine-rich repeat (NB-LRR) proteins. However, signaling events downstream of NB-LRR activation are poorly understood. We have analysed the Arabidopsis translatome using ribosome affinity purification and RNA sequencing. Our results show that the translational status of hundreds of transcripts is differentially affected upon activation of the NB-LRR protein RPM1, showing an overall pattern of a switch away from growth-related activities to defense. Among these is the central translational regulator and growth promoter, Target of Rapamycin (TOR) kinase. Suppression of TOR expression leads to increased resistance to pathogens while overexpression of TOR results in increased susceptibility, indicating an important role for translational control in the switch from growth to defense. Furthermore, we show that several additional genes whose mRNAs are translationally regulated, including BIG, CCT2, and CIPK5, are required for both NB-LRR-mediated and basal plant innate immunity, identifying novel actors in plant defense.