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High-resolution mapping of Rym14Hb, a wild relative resistance gene to barley yellow mosaic disease

Barley yellow mosaic disease is caused by Barley yellow mosaic virus and Barley mild mosaic virus, and leads to severe yield losses in barley (Hordeum vulgare) in Central Europe and East-Asia. Several resistance loci are used in barley breeding. However, cases of resistance-breaking viral strains are known, raising concerns about the durability of those genes. Rym14Hb is a dominant major resistance gene on chromosome 6HS, originating from barleys secondary genepool wild relative Hordeum bulbosum. As such, the resistance mechanism may represent a case of non-host resistance, which could enhance its durability. A susceptible barley variety and a resistant H. bulbosum introgression line were crossed to produce a large F2 mapping population (n=7,500), to compensate for a ten-fold reduction in recombination rate compared to intraspecific barley crosses. After high-throughput genotyping, the Rym14Hb locus was assigned to a 2Mbp telomeric interval on chromosome 6HS. The co-segregating markers developed in this study can be used for marker-assisted introgression of this locus into barley elite germplasm with a minimum of linkage drag.

plant biology↗

Opposing functions of the plant TOPLESS gene family during SNC1-mediated autoimmunity

Regulation of the plant immune system is important for controlling the specificity and amplitude of responses to pathogens and in preventing growth-inhibiting autoimmunity that leads to reductions in plant fitness. In previous work, we reported that SRFR1, a negative regulator of effector-triggered immunity, interacts with SNC1 and EDS1. When SRFR1 is non-functional in the Arabidopsis accession Col-0, SNC1 levels increase, causing a cascade of events that lead to autoimmunity phenotypes. Previous work showed that some members of the transcriptional co-repressor family TOPLESS interact with SNC1 to repress negative regulators of immunity. Therefore, to explore potential connections between SRFR1 and TOPLESS family members, we took a genetic approach that examined the effect of each TOPLESS member in the srfr1 mutant background. The data indicated that an additive genetic interaction exists between SRFR1 and two members of the TOPLESS family, TPR2 and TPR3, as demonstrated by increased stunting and elevated PR2 expression in srfr1 tpr2 and srfr1 tpr2 tpr3 mutants. Furthermore, the tpr2 mutation intensifies autoimmunity in the auto-active snc1-1 mutant, indicating a novel role of these TOPLESS family members in negatively regulating SNC1-dependent phenotypes. This negative regulation can also be reversed by overexpressing TPR2 in the srfr1 tpr2 background. Thus, this work uncovers diverse functions of individual members of the TOPLESS family in Arabidopsis and provides evidence for the additive effect of transcriptional and post-transcriptional regulation of SNC1. Author SummaryThe immune system is a double-edged sword that affords organisms with protection against infectious diseases but can also lead to negative effects if not properly controlled. Plants only possess an innate antimicrobial immune system that relies on rapid upregulation of defenses once immune receptors detect the presence of microbes. Plant immune receptors known as resistance proteins play a key role in rapidly triggering defenses if pathogens breach other defenses. A common model of unregulated immunity in the reference Arabidopsis variety Columbia-0 involves a resistance gene called SNC1. When the SNC1 protein accumulates to unnaturally high levels or possesses auto-activating mutations, the visible manifestations of immune overactivity include stunted growth and low biomass and seedset. Consequently, expression of this gene and accumulation of the encoded protein are tightly regulated on multiple levels. Despite careful study the mechanisms of SNC1 gene regulation are not fully understood. Here we present data on members of the well-known TOPLESS family of transcriptional repressors. While previously characterized members were shown to function in indirect activation of defenses, TPR2 and TPR3 are shown here to function in preventing high defense activity. This study therefore contributes to the understanding of complex regulatory processes in plant immunity.

plant biology↗

Regulatory signatures of drought response in stress resilient Sorghum bicolor

The effects of drought stress can be devastating to crop production worldwide. A grand challenge facing agriculture is development of crop varieties with improved drought resilience through breeding or biotechnology. To accelerate this, a mechanistic understanding is needed of the regulatory networks underlying drought response in crop genomes and the genetic elements that modulate them. Here, we explore the regulatory landscape of sorghum [Sorghum bicolor (L.) Moench] in response to controlled-environment drought stress. Sorghum is a C4 cereal crop with innate drought resilience. To define molecular signatures of drought response, we mapped genome-wide chromatin accessibility using an Assay for Transposase Accessible Chromatin by sequencing and analyzed parallel transcriptional profiles in drought-stressed sorghum shoot and root tissues. Drought-responsive changes in accessibility were largely in proximal promoters of differentially expressed genes and also in distal regions. Data were integrated to infer gene network connections and cis-regulatory modules underlying drought response and the transcription factors that control them. Inspection of pan-genomic data and phenotyping across sorghum diversity revealed variation in genomic signatures that associated with water use efficiency. Our analyses provide drought-inducible regulatory modules in sorghum that can be leveraged for fine-tuning responses to stress, mining for advantageous alleles, and translating across species.

plant biology↗

AtHDA15 attenuates COP1 via transcriptional quiescence, direct binding, and sub-compartmentalization during photomorphogenesis

Light is an essential environmental cue that determines the overall growth and development of plants. However, the molecular mechanisms underpinning the light signaling network are obscured by the epigenetic machinery where reversible acetylation and deacetylation play crucial roles in modulating light-regulated gene expression. In this paper, we demonstrate that HDA15 represses COP1, the master switch in the light signaling network, by deacetylation, protein interaction, and sub-compartmentalization. hda15 T-DNA mutant lines exhibited light hyposensitivity with significantly reduced HY5 and PIF3 transcript levels leading to long-hypocotyl phenotypes in the dark while its overexpression exhibited elevated HY5 transcripts and short hypocotyl phenotypes. In vivo and in vitro binding assays further show that HDA15 directly interacts with COP1 inside the nucleus modulating COP1s repressive activities. Crossing hda15-t27 with cop1-4 mutants resulted in short-hypocotyl and dwarfed phenotypes, reminiscent of cop1-4 mutants suggesting COP1 is epistatic to HDA15. Although light signals the nucleocytoplasmic shuttling of HDA15, the presence of COP1 triggers its nuclear localization. A working model is presented elucidating the concerted interplay between HDA15 and COP1 under light and dark conditions.

plant biology↗

Identification of QTLs for dynamic and steady state photosynthetic traits in a barley mapping population

Enhancing the photosynthetic induction response to fluctuating light has been suggested as a key target for improvement in crop breeding programs, with the potential to substantially increase whole canopy carbon assimilation and contribute to crop yield potential. Rubisco activation may be the main physiological process that will allow us to achieve such a goal. In this study, we phenotypically assessed the rubisco activation rate in a doubled haploid (DH) barley mapping population [131 lines from a Yerong/Franklin (Y/F) cross] after a switch from moderate to saturating light. Rates of rubisco activation were found to be highly variable across the mapping population, with a median activation rate of 0.1 min-1 in the slowest genotype and 0.74 min-1 in the fastest genotype. A QTL for rubisco activation rate was identified on chromosome 7H. This is the first report on the identification of a QTL for rubisco activation rate in planta and the discovery opens the door to marker assisted breeding to improve whole canopy photosynthesis of barley. Further strength is given to this finding as this QTL colocalised with QTLs identified for steady state photosynthesis and stomatal conductance. Several other distinct QTLs were identified for these steady state traits, with a common overlapping QTL on chromosome 2H, and distinct QTLs for photosynthesis and stomatal conductance identified on chromosomes 4H and 5H respectively. Future work should aim to validate these QTLs under field conditions so that they can be used to aid plant breeding efforts. HighlightSignificant variation exists in the photosynthetic induction response after a switch from moderate to saturating light across a barley doubled haploid population. A QTL for rubisco activation rate was identified on chromosome 7H, as well as overlapping QTLs for steady state photosynthesis and stomatal conductance.

plant biology↗

Arabidopsis thaliana Zn2+-efflux ATPases HMA2 and HMA4 are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina BMM

O_LIZinc is an essential nutrient at low concentrations, but toxic at slightly higher ones. This could be used by plants to fight pathogens colonization. C_LIO_LIElemental distribution in Arabidopsis thaliana leaves inoculated with the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM) was determined and compared to mock-inoculated ones. Infection assays were carried out in wild type and long-distance zinc trafficking double mutant hma2hma4, defective in root-to-shoot zinc partitioning. Expression levels of genes involved in zinc homeostasis or in defence phytohormone-mediated pathways were determined. C_LIO_LIZinc and manganese levels increased at the infection site. Zinc accumulation was absent in hma2hma4. HMA2 and HMA4 transcription levels were upregulated upon PcBMM inoculation. Consistent with a role of these genes in plant immunity, hma2hma4 mutants were more susceptible to PcBMM infection, phenotype rescued upon zinc supplementation. Transcript levels of genes involved in the salicylic acid, ethylene and jasmonate pathways were constitutively upregulated in hma2hma4 plants. C_LIO_LIThese data are consistent with a role of zinc in plant immunity not only of hyperaccumulator plants, but also of plants containing ordinary levels of zinc. This new layer of immunity seems to run in parallel to the already characterized defence pathways, and its removal has a direct effect on pathogen resistance. C_LI

plant biology↗

Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves

The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth and that transient depletion of a host immune gene and transient expression of a T3 effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers reliable opportunity for further comprehensive analysis of plant immunity. One sentence summaryAgromonas is a rapid and robust disease assay to monitor Pseudomonas syringae growth in agroinfiltrated leaves expressing immune components and their suppressors.

plant biology↗

Regulation of CYP94B1 by WRKY33 controls apoplastic barrier formation in the roots leading to salt tolerance

Salinity is an environmental stress that causes decline in crop yield. Avicennia officinalis and other mangroves have adaptations such as ultrafiltration at the roots aided by apoplastic cell-wall barriers to thrive in saline conditions. We studied a Cytochrome P450 gene, AoCYP94B1 from A. officinalis and its Arabidopsis ortholog AtCYP94B1 that are involved in apoplastic barrier formation, and are induced by 30 minutes of salt treatment in the roots. Heterologous expression of AoCYP94B1 in atcyp94b1 Arabidopsis mutant and wild-type rice conferred increased NaCl tolerance to seedlings by enhancing root suberin deposition. Histochemical staining and GC-MS/MS quantification of suberin precursors confirmed the role of CYP94B1 in suberin biosynthesis. Using chromatin immunoprecipitation, yeast one-hybrid and luciferase assays, we identified AtWRKY33 as the upstream regulator of AtCYP94B1 in Arabidopsis. In addition, atwrky33 mutants exhibited reduced suberin and salt sensitive phenotypes, which were rescued by expressing 35S::AtCYP94B1 in atwrky33 mutant. This further confirms that the regulation of AtCYP94B1 by AtWRKY33 is part of the salt tolerance mechanism, and our findings can help in generating salt tolerant crops. One sentence summaryAtWRKY33 transcription factor regulates AtCYP94B1 to increase plant salt tolerance by enhanced suberin deposition in the endodermal cells of Arabidopsis roots

plant biology↗

The conserved plant PM19 protein functions as an osmosensor and regulator of germination

How plants perceive water, especially during the critical stages of seed formation and germination, is key to their survival. During development and germination, seeds undergo large changes in water content, down to around 10% during maturation and up to 90% again within 24 hours of germination. We find the evolutionary conserved Arabidopsis plasma membrane protein PM19L1 to be an osmosensor, regulating dormancy and germination under osmotic stress. The PM19L1 protein structurally resembles the yeast osmosensor Sho1, with four transmembrane domains, and PM19L1 complements the osmosensitive sho1 mutant. Arabidopsis pm19l1 mutants have enhanced dormancy and reduced germination under salt and osmotic stress, and enhanced ABA levels. In a striking parallel to osmosensing in yeast, signalling downstream of PM19L1 involves a MAP kinase signal transduction pathway. PM19L1 is a positive regulator of ABI3, which promotes the late maturation of the seed, and negatively regulates the ABI4 and ABI5 dormancy- regulating transcription factors. These results have implications for the study of dormancy, drought, and salinity tolerance in crops, and may provide an insight into evolutionary adaptation of plants to a terrestrial environment.

plant biology↗

Crystal structures of Arabidopsis and Physcomitrella CR4 reveal the molecular architecture of CRINKLY4 receptor kinases.

Plant-unique receptor kinases harbor conserved cytoplasmic kinase domains and sequence-diverse ectodomains. Here we report crystal structures of CRINKLY4-type ectodomains from Arabidopsis ACR4 and Physcomitrella patens PpCR4 at 1.95 [A] and 2.70 [A] resolution, respectively. Monomeric CRINKLY4 ectodomains harbor a N-terminal WD40 domain and a cysteine-rich domain (CRD) connected by a short linker. The WD40 domain forms a seven-bladed {beta}-propeller with the N-terminal strand buried in its center. Each propeller blade is stabilized by a disulfide bond and contributes to the formation of a putative ligand binding groove. The CRD forms a {beta}-sandwich structure stabilized by six disulfide bonds and shares low structural homology with tumor necrosis factor receptor domains. Quantitative binding assays reveal that ACR4 is not a direct receptor for the peptide hormone CLE40. An ACR4 variant lacking the entire CRD can rescue the known acr4-2 mutant phenotype, as can expression of PpCR4. Together, an evolutionary conserved signaling function for CRINKLY4 receptor kinases is encoded in its WD40 domain.

plant biology↗

Autophagy is required for lipid homeostasis during dark-induced senescence in Arabidopsis

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) submitted to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming towards lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants. One-sentence summaryAutophagy is required for the mobilization of membrane lipid components and lipid droplet dynamics during extended darkness in Arabidopsis.

plant biology↗

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

Vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the rings lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

plant biology↗

Jasmonic acid coordinates with light to regulate branching angle of Arabidopsis lateral roots

The role of jasmonates (JAs) in primary root growth and development and in plant response to external stimuli is already known. However, its role in lateral root (LR) development remains to be explored. Our work identified methyl jasmonate (MeJA) as a key phytohormone in determining the branching angle of Arabidopsis LRs. MeJA inclines the LRs to a more vertical orientation, which was dependent on the canonical JAR1-COI1-MYC2, 3, 4 signalling. Our work also highlights the dual roles of light in governing LR angle. Light signalling enhances JA biosynthesis, leading to erect root architecture; whereas, glucose (Glc) induces wider branching angles. Combining physiological and molecular assays, we revealed that Glc antagonizes the MeJA response via TARGET OF RAPAMYCIN (TOR) signalling. Moreover, physiological assays using auxin mutants, MYC2-mediated transcriptional activation of LAZY2, LAZY4 and auxin biosynthetic gene CYP79B2, and asymmetric distribution of DR5::GFP and PIN2::GFP pinpointed the role of an intact auxin mechanism required by MeJA for vertical growth of LRs. We also demonstrated that light perception and signalling are indispensable for inducing vertical angles by MeJA. Thus, our investigation highlights antagonism between light and Glc signalling and how they interact with JA-auxin signals to optimize the branching angle of LRs.

plant biology↗

Effect of cutting depth during sugarcane harvest on root characteristics and yield

Ratooning is an important cultivation practice in sugarcane production around the world, with underground buds on the remaining stalk acting as the source for establishment of a subsequent ratoon crop. However, the optimal depth of cutting during harvest in terms of yield and root growth remains unknown. We carried out a two-year field study to determine the effects of three cutting depths (0, 5 and 10 cm below the surface) ratoon cane root and yield. Results showed that cutting to a depth of 5 cm increased the root fresh weight and root volume by 32-40% and 49-85%, respectively, compared to cutting depths of 0 and 10 cm. Remarkably, cutting to a depth of 5 cm also had a significant effect on the development of fine roots, which is closely linked to cane yield. The effect was particularly noticeable in terms of two root traits, root volume and the surface area of roots with a diameter of 1.0-2.0mm, and root length and the number of root tips in roots with a diameter of 0-0.5mm. As a result, a cutting depth of 5 cm below the surface increased cane yield by 35 and 25% compared to depths of 0 and 10 cm below the surface, respectively. Overall, these findings suggest that a cutting depth of 5 cm is optimal in terms of sugarcane yield, largely due to the enhanced effect on root traits, especially the development of fine roots. These findings will help optimize sugarcane ratoon management and improve the ratoon cycle.

plant biology↗

Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

plant biology↗

Action potentials induce biomagnetic fields in Venus flytrap plants

Upon stimulation, plants elicit electrical signals that can travel within a cellular network analogous to the animal nervous system. It is well-known that in the human brain, voltage changes in certain regions result from concerted electrical activity which, in the form of action potentials (APs), travels within nerve-cell arrays. Electrophysiological techniques like electroencephalography1, magnetoencephalography2, and magnetic resonance imaging3,4 are used to record this activity and to diagnose disorders. In the plant kingdom, two types of electrical signals are observed: all-or-nothing APs of similar amplitudes to those seen in humans and animals, and slow-wave potentials of smaller amplitudes. Sharp APs appear restricted to unique plant species like the "sensitive plant", Mimosa pudica, and the carnivorous Venus flytrap, Dionaea muscipula5,6. Here we ask the question, is electrical activity in the Venus flytrap accompanied by distinct magnetic signals? Using atomic optically pumped magnetometers7,8, biomagnetism in AP-firing traps of the carnivorous plant was recorded. APs were induced by heat stimulation, and the thermal properties of ion channels underlying the AP were studied. The measured magnetic signals exhibit similar temporal behavior and shape to the fast de- and repolarization AP phases. Our findings pave the way to understanding the molecular basis of biomagnetism, which might be used to improve magnetometer-based noninvasive diagnostics of plant stress and disease.

plant biology↗

Diverse roles of MAX1 homologues in rice

Cytochrome P450 enzymes encoded by MORE AXILLARY GROWTH1 (MAX1)-like genes produce most of the structural diversity of strigolactones during the final steps of strigolactone biosynthesis. The diverse copies of MAX1 in Oryza sativa provide a resource to investigate why plants produce such a wide range of strigolactones. Here we performed in silico analyses of transcription factors and microRNAs that may regulate each rice MAX1, and compared the results with available data about MAX1 expression profiles and genes co-expressed with MAX1 genes. Data suggest that distinct mechanisms regulate the expression of each MAX1. Moreover, there may be novel functions for MAX1 homologues, such as the regulation of flower development or responses to heavy metals. In addition, individual MAX1s could be involved in specific functions, such as the regulation of seed development or wax synthesis in rice. Our analysis reveals potential new avenues of strigolactone research that may otherwise not be obvious.

plant biology↗

Structure of the activated Roq1 resistosome directly recognizing the pathogen effector XopQ

Plants and animals detect pathogen infection via intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Here we describe the 3.8 [A] resolution cryo-electron microscopy structure of the activated Roq1, an NLR native to Nicotiana benthamiana with a Toll-like interleukin-1 receptor (TIR) domain, bound to the Xanthomonas effector XopQ. Roq1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain. One Sentence SummaryVisualization of an activated plant immune receptor that triggers the immune response upon pathogen recognition.

plant biology↗