Realizing the Full Potential of Advanced Microscopy Approaches for Interrogating Plant-Microbe Interactions.

Microscopy has served as a fundamental tool for insight and discovery in plant-microbe interactions for centuries. From classical light and electron microscopy to corresponding specialized methods for sample preparation and cellular contrasting agents, these approaches have become routine components in the toolkit of plant and microbiology scientists alike to visualize, probe and understand the nature of host-microbe relationships. Over the last three decades, three-dimensional perspectives led by the development of electron tomography, and especially, confocal techniques continue to provide remarkable clarity and spatial detail of tissue and cellular phenomena. Confocal and electron microscopy provide novel revelations that are now commonplace in medium and large institutions. However, many other cutting-edge technologies and sample preparation workflows are relatively unexploited yet offer tremendous potential for unprecedented advancement in our understanding of the inner workings of pathogenic, beneficial, and symbiotic plant-microbe interactions. Here, we highlight key applications, benefits, and challenges of contemporary advanced imaging platforms for plant-microbe systems with special emphasis on several recently developed approaches, such as light-sheet, single molecule, super-resolution, and adaptive optics microscopy, as well as ambient and cryo-volume electron microscopy, X-ray microscopy, and cryo-electron tomography. Furthermore, the potential for complementary sample preparation methodologies, such as optical clearing, expansion microscopy, and multiplex imaging, will be reviewed. Our ultimate goal is to stimulate awareness of these powerful cutting-edge technologies and facilitate their appropriate application and adoption to solve important and unresolved biological questions in the field. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

X-ray microscopy enables multiscale high-resolution 3D imaging of plant cells, tissues, and organs.

Capturing complete internal anatomies of plant organs and tissues within their relevant morphological context remains a key challenge in plant science. While plant growth and development are inherently multiscale, conventional light, fluorescence, and electron microscopy platforms are typically limited to imaging of plant microstructure from small flat samples that lack a direct spatial context to, and represent only a small portion of, the relevant plant macrostructures. We demonstrate technical advances with a lab-based X-ray microscope (XRM) that bridge the imaging gap by providing multiscale high-resolution three-dimensional (3D) volumes of intact plant samples from the cell to the whole plant level. Serial imaging of a single sample is shown to provide sub-micron 3D volumes co-registered with lower magnification scans for explicit contextual reference. High-quality 3D volume data from our enhanced methods facilitate sophisticated and effective computational segmentation. Advances in sample preparation make multimodal correlative imaging workflows possible, where a single resin-embedded plant sample is scanned via XRM to generate a 3D cell-level map, and then used to identify and zoom in on sub-cellular regions of interest for high-resolution scanning electron microscopy. In total, we present the methodologies for use of XRM in the multiscale and multimodal analysis of 3D plant features using numerous economically and scientifically important plant systems.

Organizing your space: The potential for integrating spatial transcriptomics and 3D imaging data in plants.

Plant cells communicate information for the regulation of development and responses to external stresses. A key form of this communication is transcriptional regulation, accomplished via complex gene networks operating both locally and systemically. To fully understand how genes are regulated across plant tissues and organs, high resolution, multi-dimensional spatial transcriptional data must be acquired and placed within a cellular and organismal context. Spatial transcriptomics (ST) typically provides a two-dimensional spatial analysis of gene expression of tissue sections that can be stacked to render three-dimensional data. For example, X-ray and light-sheet microscopy provide sub-micron scale volumetric imaging of cellular morphology of tissues, organs, or potentially entire organisms. Linking these technologies could substantially advance transcriptomics in plant biology and other fields. Here, we review advances in ST and 3D microscopy approaches and describe how these technologies could be combined to provide high resolution, spatially organized plant tissue transcript mapping.

Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector.

Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.

Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection.

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves.

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

Evaluation of multi-color genetically encoded Ca2+ indicators in filamentous fungi.

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.

Premeiotic, 24-Nucleotide Reproductive PhasiRNAs Are Abundant in Anthers of Wheat and Barley But Not Rice and Maize.

Two classes of premeiotic (21-nucleotides [nt]) and meiotic (24-nt) phased small interfering RNAs (phasiRNAs) and their patterns of accumulation have been described in maize (Zea mays) and rice (Oryza sativa) anthers. Their precise function remains unclear, but studies have shown that they support male fertility. The important role of phasiRNAs in anthers underpins our present study to characterize these small RNAs in wheat (Triticum aestivum) and barley (Hordeum vulgare) anthers. We staged anthers at every 0.2 mm of development for one wheat and two barley varieties. We isolated premeiotic (0.2, 0.4, and 0.6 mm), meiotic (0.8, 1.0, and 1.4 mm), and postmeiotic (1.8 mm) anthers, for which we then investigated accumulation patterns of RNAs, including reproductive phasiRNAs. We annotated a total of 12,821 and 2,897 PHAS loci in the wheat and barley genomes, respectively. By comparing the total number of PHAS loci in genomes of maize, rice, barley, and wheat, we identified an expansion of reproductive PHAS loci in the genomes of Poaceae subfamilies from Panicoideae to Oryzoideae and to Poideae. In addition to the two classes of premeiotic (21-nt) and meiotic (24-nt) phasiRNAs, previously described in maize and rice anthers, we characterized a group of 24-nt phasiRNAs that accumulate in premeiotic anthers. The absence of premeiotic 24-nt phasiRNAs in maize and rice suggests a divergence in grass species of the Poideae subfamily. Additionally, we performed a gene coexpression analysis describing the regulation of phasiRNA biogenesis in wheat and barley anthers. We highlight Argonaute 9 (AGO9) and Argonaute 6 (AGO6) as candidate binding partners of premeiotic and meiotic 24-nt phasiRNAs, respectively.

Improved Spirodela polyrhiza genome and proteomic analyses reveal a conserved chromosomal structure with high abundance of chloroplastic proteins favoring energy production.

Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. Here, we studied the genome and proteome of Spirodela polyrhiza, or greater duckweed, which has the largest body plan yet the smallest genome size in the family (1C=150 Mb). Using Oxford Nanopore sequencing combined with Hi-C scaffolding, we generated a highly contiguous, chromosome-scale assembly of S. polyrhiza line Sp7498 (Sp7498_HiC). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations relative to a recent PacBio assembly of Sp7498, highlighting the need for orthogonal long-range scaffolding techniques such as Hi-C and BioNano optical mapping. Shotgun proteomics of Sp7498 verified the expression of ~2250 proteins and revealed a high abundance of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome illustrates the utility of duckweed as a model for genomics- and proteomics-based education.

Correction: A Novel Role for the TIR Domain in Association with Pathogen-Derived Elicitors.

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

Roles of three Fusarium graminearum membrane Ca2+ channels in the formation of Ca2+ signatures, growth, development, pathogenicity and mycotoxin production.

Similar to animals and plants, external stimuli cause dynamic spatial and temporal changes of cytoplasmic Ca2+ in fungi. Such changes are referred as the Ca2+ signature and control cellular responses by modulating the activity or location of diverse Ca2+-binding proteins (CBPs) and also indirectly affecting proteins that interact with CBPs. To understand the mechanism underpinning Ca2+ signaling, therefore, characterization of how Ca2+ moves to and from the cytoplasm to create Ca2+ signatures under different conditions is fundamental. Three genes encoding plasma membrane Ca2+ channels in a Fusarium graminearum strain that expresses a fluorescent protein-based Ca2+ indicator in the cytoplasm were mutagenized to investigate their roles in the generation of Ca2+ signatures under different growth conditions and genetic backgrounds. The genes disrupted include CCH1 and MID1, which encode a high affinity Ca2+ uptake system, and FIG1, encoding a low affinity Ca2+ channel. Resulting mutants were also analyzed for growth, development, pathogenicity and mycotoxin production to determine how loss of each of the genes alters these traits. To investigate whether individual genes influence the function and expression of other genes, phenotypes and Ca2+ signatures of their double and triple mutants, as well as their expression patterns, were analyzed.

Caveolin-1 regulates P2X7 receptor signaling in osteoblasts.

The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.

Roles of three Fusarium oxysporum calcium ion (Ca(2+)) channels in generating Ca(2+) signatures and controlling growth.

Spatial and temporal changes of cytoplasmic calcium ions ([Ca(2+)]c), caused by external stimuli, are known as the Ca(2+) signature and presumably control cellular and developmental responses. Multiple types of ion channels, pumps, and transporters on plasma and organellar membranes modulate influx and efflux of Ca(2+) to and from the extracellular environment and internal Ca(2+) stores to form Ca(2+) signatures. Expression of a fluorescent protein-based Ca(2+) probe, Cameleon YC3.60, in Fusarium oxysporum enabled us to study how disruption of three Ca(2+) channel genes, including FoCCH1, FoMID1 and FoYVC1, affects Ca(2+) signature formation at polarized hyphal tips and whether specific changes in the Ca(2+) signature caused by these mutations are related to growth-related phenotypes. Resulting mutants displayed altered amplitude, interval, and duration of Ca(2+) pulses under various external Ca(2+) concentrations as well as changes in sporulation and growth. Loss of FoMID1 and FoCCH1, genes encoding putative plasma membrane channel proteins, had a major impact on Ca(2+) signatures and growth, while disruption of FoYVC1, which encodes a vacuolar channel, only subtly affected both traits. Results from our study provide new insights into the underpinning of Ca(2+) signaling in fungi and its role in controlling growth and also raise several new questions.

Novel positive regulatory role for the SPL6 transcription factor in the N TIR-NB-LRR receptor-mediated plant innate immunity.

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.

P2Y2 receptors regulate osteoblast mechanosensitivity during fluid flow.

Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Furthermore, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that small interfering RNA (siRNA) suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 min after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the Rho kinase (ROCK)1/LIM kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the intracellular Ca(2+) concentration ([Ca(2+)]i) response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.

Transient Influx of nickel in root mitochondria modulates organic acid and reactive oxygen species production in nickel hyperaccumulator Alyssum murale.

Mitochondria are important targets of metal toxicity and are also vital for maintaining metal homeostasis. Here, we examined the potential role of mitochondria in homeostasis of nickel in the roots of nickel hyperaccumulator plant Alyssum murale. We evaluated the biochemical basis of nickel tolerance by comparing the role of mitochondria in closely related nickel hyperaccumulator A. murale and non-accumulator Alyssum montanum. Evidence is presented for the rapid and transient influx of nickel in root mitochondria of nickel hyperaccumulator A. murale. In an early response to nickel treatment, substantial nickel influx was observed in mitochondria prior to sequestration in vacuoles in the roots of hyperaccumulator A. murale compared with non-accumulator A. montanum. In addition, the mitochondrial Krebs cycle was modulated to increase synthesis of malic acid and citric acid involvement in nickel hyperaccumulation. Furthermore, malic acid, which is reported to form a complex with nickel in hyperaccumulators, was also found to reduce the reactive oxygen species generation induced by nickel. We propose that the interaction of nickel with mitochondria is imperative in the early steps of nickel uptake in nickel hyperaccumulator plants. Initial uptake of nickel in roots results in biochemical responses in the root mitochondria indicating its vital role in homeostasis of nickel ions in hyperaccumulation.

Uptake, efflux, and mass transfer coefficient of fluorescent PAMAM dendrimers into pancreatic cancer cells.

Targeted delivery of imaging agents to cells can be optimized with the understanding of uptake and efflux rates. Cellular uptake of macromolecules is studied frequently with fluorescent probes. We hypothesized that the internalization and efflux of fluorescently labeled macromolecules into and out of mammalian cells could be quantified by confocal microscopy to determine the rate of uptake and efflux, from which the mass transfer coefficient is calculated. The cellular influx and efflux of a third generation poly(amido amine) (PAMAM) dendrimer labeled with an Alexa Fluor 555 dye was measured in Capan-1 pancreatic cancer cells using confocal fluorescence microscopy. The Capan-1 cells were also labeled with 5-chloromethylfluorescein diacetate (CMFDA) green cell tracker dye to delineate cellular boundaries. A dilution curve of the fluorescently labeled PAMAM dendrimer enabled quantification of the concentration of dendrimer in the cell. A simple mass transfer model described the uptake and efflux behavior of the PAMAM dendrimer. The effective mass transfer coefficient was found to be 0.054±0.043µm/min, which corresponds to a rate constant of 0.035±0.023min(-1) for uptake of the PAMAM dendrimer into the Capan-1 cells. The effective mass transfer coefficient was shown to predict the efflux behavior of the PAMAM dendrimer from the cell if the fraction of labeled dendrimer undergoing non-specific binding is accounted for. This work introduces a novel method to quantify the mass transfer behavior of fluorescently labeled macromolecules into mammalian cells.

Function of endoplasmic reticulum calcium ATPase in innate immunity-mediated programmed cell death.

Programmed cell death (PCD) initiated at the pathogen-infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca(2+)-ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N- and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptor-mediated PCD. Our results indicate that ER-Ca(2+)-ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response.