ABSTRACT
A path to sustainably reduce world hunger, food insecurity, and malnutrition is to close the crop yield gap, particularly, losses due to pathogens. Breeding resistant crops is key to achieving this goal, an effort requiring collaboration among stakeholders, scientists, breeders, farmers and policymakers. During a disease outbreak, epidemiologists survey the occurrence of a disease after which pathologists investigate mechanisms to stop an infection. Policymakers then implement strategies with farmers and breeders to overcome the outbreak. Information flow from the field to the lab and back to the field involves several processing hubs that require different information inputs. Failure to communicate the necessary information results in the transfer of meaningless data. Here, we discuss gaps in information acquisition and transfer between the field and laboratory. Using rice bacterial blight disease as an example, we discuss pathogen biology and disease resistance to point out the importance of reporting pathogen strains that caused an outbreak to optimize the deployment of resistant crop varieties. We examine differences between infection in the field and assays performed in the laboratory to draw awareness of possible misinformation concerning plant resistance or susceptibility. We discuss key data considered useful for reporting disease outbreaks, sampling bias, and suggestions for improving data quality. We also touch on the knowledge gap in the state-of-the-art literature regarding disease dispersal and transmission. We use a recent case study to exemplify the gaps mentioned. We conclude by highlighting potential actions that may contribute to food security and to closing of the yield gap.
ABSTRACT
The phytohormone gibberellic acid (GA) is critical for environmentally sensitive plant development including germination, skotomorphogenesis, and flowering. The Förster resonance energy transfer biosensor GIBBERELLIN PERCEPTION SENSOR1, which permits single-cell GA measurements in vivo, has been used to observe a GA gradient correlated with cell length in dark-grown, but not light-grown, hypocotyls. We sought to understand how light signaling integrates into cellular GA regulation. Here, we show how the E3 ligase CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) play central roles in directing cellular GA distribution in skoto- and photomorphogenic hypocotyls, respectively. We demonstrate that the expression pattern of the GA biosynthetic enzyme gene GA20ox1 is the key determinant of the GA gradient in dark-grown hypocotyls and is a target of COP1 signaling. We engineered a second generation GPS2 biosensor with improved orthogonality and reversibility. GPS2 revealed a previously undetectable cellular pattern of GA depletion during the transition to growth in the light. This GA depletion partly explains the resetting of hypocotyl growth dynamics during photomorphogenesis. Achieving cell-level resolution has revealed how GA distributions link environmental conditions with morphology and morphological plasticity. The GPS2 biosensor is an ideal tool for GA studies in many conditions, organs, and plant species.
ABSTRACT
Plant roots are functionally heterogeneous in cellular architecture, transcriptome profile, metabolic state, and microbial immunity. We hypothesized that axial differentiation may also impact spatial colonization by root microbiota along the root axis. We developed two growth systems, ArtSoil and CD-Rhizotron, to grow and then dissect Arabidopsis thaliana roots into three segments. We demonstrate that distinct endospheric and rhizosphere bacterial communities colonize the segments, supporting the hypothesis of microbiota differentiation along the axis. Root metabolite profiling of each segment reveals differential metabolite enrichment and specificity. Bioinformatic analyses and GUS histochemistry indicate microbe-induced accumulation of SWEET2, 4, and 12 sugar uniporters. Profiling of root segments from sweet mutants shows altered spatial metabolic profiles and reorganization of endospheric root microbiota. This work reveals the interdependency between root metabolites and microbial colonization and the contribution of SWEETs to spatial diversity and stability of microbial ecosystem.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Microbiota , Arabidopsis/microbiology , Bacteria/metabolism , Rhizosphere , Sugars/metabolism , Plant Roots/microbiology , Monosaccharide Transport Proteins/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolismABSTRACT
Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.
Subject(s)
Arabidopsis , Cell Nucleus , Optical Imaging , Arabidopsis/metabolism , Cell Nucleus/metabolism , Optical Imaging/methods , Phytochrome/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Plant Roots/metabolism , Plant Roots/cytology , FluorescenceABSTRACT
Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.
Subject(s)
Arabidopsis , Oryza , Plant Viruses , Oryza/genetics , Nuclear Pore Complex Proteins/genetics , Arabidopsis/genetics , Gene EditingABSTRACT
Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo. To develop intensiometric sensors with the capacity for ratiometric quantification, dual-FP Matryoshka sensors were generated by using a single cassette with a large Stokes shift (LSS) reference FP nested within the reporter FP (cpEGFP). Here, we present a genetically encoded calcium sensor that employs green apple (GA) Matryoshka technology by incorporating a newly designed red LSSmApple fluorophore. LSSmApple matures faster and provides an optimized excitation spectrum overlap with cpEGFP, allowing for monochromatic coexcitation with blue light. The LSS of LSSmApple results in improved emission spectrum separation from cpEGFP, thereby minimizing fluorophore bleed-through and facilitating imaging using standard dichroic and red FP (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new FP, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.
Subject(s)
Calcium , Saccharomyces cerevisiae , Luminescent Proteins/chemistry , Calcium/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Red Fluorescent Protein , Fluorescent DyesABSTRACT
Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated complete continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic African Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and iTALes suppressing Xa1. Phylogenomics clustered these strains with Xoo from Southern-China. African rice varieties do not carry effective resistance. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit all known TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. The strategy could help to protect global rice crops from BB pandemics.
Subject(s)
Oryza , Xanthomonas , Gene Editing , Oryza/genetics , Transcription Activator-Like Effectors , Xanthomonas/genetics , Tanzania , Plant Diseases/microbiology , Disease Resistance/geneticsABSTRACT
Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.
Subject(s)
Plasmodesmata , Proteome , Proteome/metabolism , Plasmodesmata/metabolism , Phylogeny , Reproducibility of Results , Cell Wall/metabolismABSTRACT
We report the realization of an advanced technique for measuring relative length changes ΔL/L of mm-sized samples under the control of temperature (T) and helium-gas pressure (P). The system, which is an extension of the apparatus described in the work of Manna et al. [Rev. Sci. Instrum. 83, 085111 (2012)], consists of two 4He-bath cryostats, each of which houses a pressure cell and a capacitive dilatometer. The interconnection of the pressure cells, the temperature of which can be controlled individually, opens up various modes of operation to perform measurements of ΔL/L under the variation of temperature and pressure. Special features of this apparatus include the possibility (1) to increase the pressure to values far in excess of the external pressure reservoir, (2) to substantially improve the pressure stability during temperature sweeps, (3) to enable continuous pressure sweeps with both decreasing and increasing pressure, and (4) to simultaneously measure the dielectric constant of the pressure-transmitting medium, viz., helium, εr He(T,P), along the same T-P trajectory as that used for taking the ΔL(T, P)/L data. The performance of the setup is demonstrated by measurements of relative length changes (ΔL/L)T at T = 180 K of single crystalline NaCl upon continuously varying the pressure in the range 6 ≤ P ≤ 40 MPa.
ABSTRACT
SWEET sucrose transporters play important roles in the allocation of sucrose in plants. Some SWEETs were shown to also mediate transport of the plant growth regulator gibberellin (GA). The close physiological relationship between sucrose and GA raised the questions of whether there is a functional connection and whether one or both of the substrates are physiologically relevant. To dissect these two activities, molecular dynamics were used to map the binding sites of sucrose and GA in the pore of SWEET13 and predicted binding interactions that might be selective for sucrose or GA. Transport assays confirmed these predictions. In transport assays, the N76Q mutant had 7x higher relative GA3 activity, and the S142N mutant only transported sucrose. The impaired pollen viability and germination in sweet13;14 double mutants were complemented by the sucrose-selective SWEET13S142N, but not by the SWEET13N76Q mutant, indicating that sucrose is the physiologically relevant substrate and that GA transport capacity is dispensable in the context of male fertility. Therefore, GA supplementation to counter male sterility may act indirectly via stimulating sucrose supply in male sterile mutants. These findings are also relevant in the context of the role of SWEETs in pathogen susceptibility.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Fertility/genetics , Gene Expression Regulation, Plant , Gibberellins/metabolism , Monosaccharide Transport Proteins , Plant Growth Regulators/metabolism , Sucrose/metabolismABSTRACT
Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, the OzTrac sensors reported osmolality changes as a proportional change in the emission ratio of the two fluorescent protein domains. Live-cell imaging revealed an accumulation of fluorescent sensors in internal aggregates, presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.
Subject(s)
Arabidopsis Proteins , Escherichia coli Proteins , Ion Channels , Membrane Proteins , Arabidopsis , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Ion Channels/genetics , Ion Channels/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Osmolar Concentration , Osmotic PressureABSTRACT
SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator-Like effectors (TALe) of Xanthomonas ssp. is key for virulence in rice, cassava and cotton. We identified OsSWEET11b with roles in male fertility and potential bacterial blight (BB) susceptibility in rice. While single ossweet11a or 11b mutants were fertile, double mutants were sterile. As clade III SWEETs can transport gibberellin (GA), a key hormone for spikelet fertility, sterility and BB susceptibility might be explained by GA transport deficiencies. However, in contrast with the Arabidopsis homologues, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility therefore are likely to depend on sucrose transport activity. Ectopic induction of OsSWEET11b by designer TALe enabled TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b is relevant for fertility and for protecting rice against emerging Xoo strains that target OsSWEET11b.
Subject(s)
Membrane Transport Proteins/metabolism , Oryza , Plant Proteins/metabolism , Xanthomonas , Bacterial Proteins/metabolism , Disease Resistance/genetics , Fertility , Gene Expression Regulation, Plant , Membrane Transport Proteins/genetics , Oryza/metabolism , Plant Diseases/microbiology , Plant Proteins/genetics , Sucrose , Xanthomonas/geneticsABSTRACT
During multicellularization, plants evolved unique cell-cell connections, the plasmodesmata (PD). PD of angiosperms are complex cellular domains, embedded in the cell wall and consisting of multiple membranes and a large number of proteins. From the beginning, it had been assumed that PD provide passage for a wide range of molecules, from ions to metabolites and hormones, to RNAs and even proteins. In the context of assimilate allocation, it has been hypothesized that sucrose produced in mesophyll cells is transported via PD from cell to cell down a concentration gradient towards the phloem. Entry into the sieve element companion cell complex (SECCC) is then mediated on three potential routes, depending on the species and conditions, - either via diffusion across PD, after conversion to raffinose via PD using a polymer trap mechanism, or via a set of transporters which secrete sucrose from one cell and secondary active uptake into the SECCC. Multiple loading mechanisms can likely coexist. We here review the current knowledge regarding photoassimilate transport across PD between cells as a prerequisite for translocation from leaves to recipient organs, in particular roots and developing seeds. We summarize the state-of-the-art in protein composition, structure, transport mechanism and regulation of PD to apprehend their functions in carbohydrate allocation. Since many aspects of PD biology remain elusive, we highlight areas that require new approaches and technologies to advance our understanding of these enigmatic and important cell-cell connections.
ABSTRACT
SWEETs are transporters with homologs in Archeae, plants, some fungi, and animals. As the only transporters known to facilitate the cellular release of sugars in plants, SWEETs play critical roles in the allocation of sugars from photosynthetic leaves to storage tissues in seeds, fruits, and tubers. Here, we report the design and use of genetically encoded biosensors to measure the activity of SWEETs. We created a SweetTrac1 sensor by inserting a circularly permutated green fluorescent protein into the Arabidopsis SWEET1, resulting in a chimera that translates substrate binding during the transport cycle into detectable changes in fluorescence intensity. We demonstrate that a combination of cell sorting and bioinformatics can accelerate the design of biosensors and formulate a mass action kinetics model to correlate the fluorescence response of SweetTrac1 with the transport of glucose. Our analysis suggests that SWEETs are low-affinity, symmetric transporters that can rapidly equilibrate intra- and extracellular concentrations of sugars. This approach can be extended to SWEET homologs and other transporters.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Biosensing Techniques , Monosaccharide Transport Proteins/metabolism , Sugars/metabolism , Arabidopsis Proteins/genetics , Biological Transport , Biosensing Techniques/methods , Monosaccharide Transport Proteins/geneticsABSTRACT
Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals, and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center, number of lateral roots, vegetative growth, and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes.
Subject(s)
Arabidopsis/growth & development , Arabidopsis/metabolism , Cell Membrane/metabolism , Glutamic Acid/genetics , Glutamic Acid/metabolism , Plant Development/genetics , Plant Leaves/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Plant Development/drug effects , Plant Leaves/geneticsABSTRACT
Glutamate has dual roles in metabolism and signaling; thus, signaling functions must be isolatable and distinct from metabolic fluctuations, as seen in low-glutamate domains at synapses. In plants, wounding triggers electrical and calcium (Ca2+) signaling, which involve homologs of mammalian glutamate receptors. The hydraulic dispersal and squeeze-cell hypotheses implicate pressure as a key component of systemic signaling. Here, we identify the stretch-activated anion channel MSL10 as necessary for proper wound-induced electrical and Ca2+ signaling. Wound gene induction, genetics, and Ca2+ imaging indicate that MSL10 acts in the same pathway as the glutamate receptorlike proteins (GLRs). Analogous to mammalian NMDA glutamate receptors, GLRs may serve as coincidence detectors gated by the combined requirement for ligand binding and membrane depolarization, here mediated by stretch activation of MSL10. This study provides a molecular genetic basis for a role of mechanical signal perception and the transmission of long-distance electrical and Ca2+ signals in plants.
ABSTRACT
Elucidating the transcriptional regulatory networks that underlie growth and development requires robust ways to define the complete set of transcription factor (TF) binding sites. Although TF-binding sites are known to be generally located within accessible chromatin regions (ACRs), pinpointing these DNA regulatory elements globally remains challenging. Current approaches primarily identify binding sites for a single TF (e.g. ChIP-seq), or globally detect ACRs but lack the resolution to consistently define TF-binding sites (e.g. DNAse-seq, ATAC-seq). To address this challenge, we developed MNase-defined cistrome-Occupancy Analysis (MOA-seq), a high-resolution (< 30 bp), high-throughput, and genome-wide strategy to globally identify putative TF-binding sites within ACRs. We used MOA-seq on developing maize ears as a proof of concept, able to define a cistrome of 145,000 MOA footprints (MFs). While a substantial majority (76%) of the known ATAC-seq ACRs intersected with the MFs, only a minority of MFs overlapped with the ATAC peaks, indicating that the majority of MFs were novel and not detected by ATAC-seq. MFs were associated with promoters and significantly enriched for TF-binding and long-range chromatin interaction sites, including for the well-characterized FASCIATED EAR4, KNOTTED1, and TEOSINTE BRANCHED1. Importantly, the MOA-seq strategy improved the spatial resolution of TF-binding prediction and allowed us to identify 215 motif families collectively distributed over more than 100,000 non-overlapping, putatively-occupied binding sites across the genome. Our study presents a simple, efficient, and high-resolution approach to identify putative TF footprints and binding motifs genome-wide, to ultimately define a native cistrome atlas.