Duckweeds are the smallest angiosperms, possessing a simple body architecture and highest rates of biomass accumulation. They can grow near-exponentially via clonal propagation. Understanding their reproductive biology, growth, and development is essential to unlock their potential for phytoremediation, carbon capture, and nutrition. However, there is a lack of non-laborious and convenient methods for spatially and temporally imaging an array of duckweed plants and growth conditions in the same experiment. We developed an automated microscopy approach to record time-lapse images of duckweed plants growing in 12-well cell culture plates. As a proof-of-concept experiment, we grew duckweed on semi-solid media with and without sucrose and monitored its effect on their growth over 3 days. Using the PlantCV toolkit, we quantified the thallus area of individual plantlets over time, and showed that L. minor grown on sucrose had an average growth rate four times higher than without sucrose. This method will serve as a blueprint to perform automated high-throughput growth assays for studying the development patterns of duckweeds from different species, genotypes, and conditions.

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues (Hordeum vulgare), nematode (Caenorhabditis elegans) and yeast (Saccharomyces cerevisiae). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.

Following the recognition of pathogen-encoded effectors, plant TIR-NB-LRR immune receptors induce defense signaling by a largely unknown mechanism. We identify a novel and conserved role for the SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-domain transcription factor SPL6 in enabling the activation of the defense transcriptome following its association with a nuclear-localized immune receptor. During an active immune response, the Nicotiana TIR-NB-LRR N immune receptor associates with NbSPL6 within distinct nuclear compartments. NbSPL6 is essential for the N-mediated resistance to Tobacco mosaic virus. Similarly, the presumed Arabidopsis ortholog AtSPL6 is required for the resistance mediated by the TIR-NB-LRR RPS4 against Pseudomonas syringae carrying the avrRps4 effector. Transcriptome analysis indicates that AtSPL6 positively regulates a subset of defense genes. A pathogen-activated nuclear-localized TIR-NB-LRR like N can therefore regulate defense genes through SPL6 in a mechanism analogous to the induction of MHC genes by mammalian immune receptors like CIITA and NLRC5.

Plant innate immunity is mediated by Resistance (R) proteins, which bear a striking resemblance to animal molecules of similar function. Tobacco N is a TIR-NB-LRR R gene that confers resistance to Tobacco mosaic virus, specifically the p50 helicase domain. An intriguing question is how plant R proteins recognize the presence of pathogen-derived Avirulence (Avr) elicitor proteins. We have used biochemical cell fraction and immunoprecipitation in addition to confocal fluorescence microscopy of living tissue to examine the association between N and p50. Surprisingly, both N and p50 are cytoplasmic and nuclear proteins, and N's nuclear localization is required for its function. We also demonstrate an in planta association between N and p50. Further, we show that N's TIR domain is critical for this association, and indeed, it alone can associate with p50. Our results differ from current models for plant innate immunity that propose detection is mediated solely through the LRR domains of these molecules. The data we present support an intricate process of pathogen elicitor recognition by R proteins involving multiple subcellular compartments and the formation of multiple protein complexes. © 2007 Burch-Smith et al.

[This corrects the article DOI: 10.1371/journal.pbio.0050068.].

Inter-organellar communication is vital for successful innate immune responses that confer defense against pathogens. However, little is known about how chloroplasts, which are a major production site of pro-defense molecules, communicate and coordinate with other organelles during defense. Here we show that chloroplasts send out dynamic tubular extensions called stromules during innate immunity or exogenous application of the pro-defense signals, hydrogen peroxide (H2O2) and salicylic acid. Interestingly, numerous stromules surround nuclei during defense response, and these connections correlate with an accumulation of chloroplast-localized NRIP1 defense protein and H2O2 in the nucleus. Furthermore, silencing and knockout of chloroplast unusual positioning 1 (CHUP1) that encodes a chloroplast outer envelope protein constitutively induces stromules in the absence of pathogen infection and enhances programmed cell death. These results support a model in which stromules aid in the amplification and/or transport of pro-defense signals into the nucleus and other subcellular compartments during immunity.

Genetically encoded Ca²⁺ indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca²⁺ signaling in fungi, ranging from documenting Ca²⁺ signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca²⁺ signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca²⁺ indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 and F. verticillioides elongation factor-1α gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca²⁺ indicator. Wild-type and three Ca²⁺ signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.

C₄ plants frequently experience high light and high temperature conditions in the field, which reduce growth and yield. However, the mechanisms underlying these stress responses in C₄ plants have been under-explored, especially the coordination between mesophyll (M) and bundle sheath (BS) cells. We investigated how the C₄ model plant Setaria viridis responded to a four-hour high light or high temperature treatment at photosynthetic, transcriptomic, and ultrastructural levels. Although we observed a comparable reduction of photosynthetic efficiency in high light or high temperature treated leaves, detailed analysis of multi-level responses revealed important differences in key pathways and M/BS specificity responding to high light and high temperature. We provide a systematic analysis of high light and high temperature responses in S. viridis, reveal different acclimation strategies to these two stresses in C₄ plants, discover unique light/temperature responses in C₄ plants in comparison to C₃ plants, and identify potential targets to improve abiotic stress tolerance in C₄ crops.