Osmotic Stress Signaling Research Articles

Time-course analysis of the transcriptome of Arabidopsis thaliana leaves under high-concentration ammonium sulfate treatment

ABSTRACT Ammonium fertilization is of great interest for future agriculture, as unlike nitrate, ammonium use efficiency does not decrease in C3 plants, even in environments with elevated CO2 concentrations. However, excess ammonium often results in toxic effects such as growth suppression and chlorosis, i.e. ammonium toxicity. The addition of nitrate, a major source of nitrogen commonly found in soils, has been shown to alleviate these toxic effects. Understanding the mechanisms of ammonium toxicity in the presence of nitrate is crucial for the effective use of ammonium fertilizers in crop cultivation. To elucidate these responses, a time-course analysis of the transcriptome was performed on A. thaliana leaves treated with high concentrations of ammonium sulfate in the presence of sufficient nitrate. The expression of nitrate-inducible genes tended to be downregulated by the treatment. The expression of genes relating to abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and membrane trafficking was upregulated, whereas that of photosynthesis-, auxin-, and cytokinin-related genes involved in growth and development was downregulated. The induction of many osmotic stress-responsive genes and nitric oxide (NO)-inducible genes suggests the involvement of osmotic stress and NO signaling in the response. This study provides a novel and comprehensive overview of transcriptional changes occurring in response to high ammonium sulfate concentrations with sufficient nitrate, and sheds light on potential pathways involved in ammonium toxicity under these conditions.

Osmotic signaling releases PP2C-mediated inhibition of Arabidopsis SnRK2s via the receptor-like cytoplasmic kinase BIK1.

Osmotic stress and abscisic acid (ABA) signaling are important for plant growth and abiotic stress resistance. Activation of osmotic and ABA signaling downstream of the PYL-type ABA receptors requires the release of SnRK2 protein kinases from the inhibition imposed by PP2Cs. PP2Cs are core negative regulators that constantly interact with and inhibit SnRK2s, but how osmotic signaling breaks the PP2C inhibition of SnRK2s remains unclear. Here, we report that an Arabidopsis receptor-like cytoplasmic kinase, BIK1, releases PP2C-mediated inhibition of SnRK2.6 via phosphorylation regulation. The dominant abi1-1 ABA-signaling mutation (G180D) disrupts PYL-PP2C interactions and disables PYL-initiated release of SnRK2s; in contrast, BIK1 releases abi1-1-mediated inhibition of SnRK2.6. BIK1 interacts with and phosphorylates SnRK2.6 at two tyrosine residues, which are critical for SnRK2.6 activation and function. Phosphorylation of the two tyrosine residues may affect the docking of the tryptophan "lock" of PP2C into SnRK2.6. Moreover, the bik1 mutant is defective in SnRK2 activation, stress-responsive gene expression, ABA accumulation, growth maintenance, and water loss under osmotic stress. Our findings uncover the critical role of BIK1 in releasing PP2C-mediated inhibition of SnRK2s under osmotic stress.

ROLE OF BIOTECHNOLOGY IN ABIOTIC STRESS TOLERANCE IN PLANTS

As population grows, abiotic stresses reduce plant growth and agricultural output, affecting global food security. Heat, cold, drought, salinity, and heavy metals affect crops. Heat stress from climate change disrupts cellular metabolism and lowers productivity. Tropical and subtropical crops suffer physiologically from cold stress. Climate variability affects drought, reducing crop output and germination. Salinity stunts growth and causes nutritional imbalances in big agricultural areas. Heavy metal buildup in soils threatens plant health. To overcome these problems, plants use osmotic adjustment, antioxidant defenses, and stress signaling pathways. CRISPR/Cas9 and other genetic engineering and molecular breeding methods may improve crop stress tolerance. Using omics technologies (genomics, transcriptomic, proteomics, and metabolomics) in breeding programs helps us understand stress tolerance processes and generate resilient crop types. This research is crucial for sustainable agriculture and global food security.

Multi-omics analysis of green lineage osmotic stress pathways unveils crucial roles of different cellular compartments

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

Transcriptomic and metabolomic insights into drought response strategies of two Astragalus species

Drought is a major environmental constraint to plant productivity, and plants cope with water limitation using drought avoidance and/or tolerance strategies. Mesoxerophyte Astragalus mongholicus displayed better drought resistance than the mesophyte A. membranaceus. However, the underlying response mechanisms are still elusive. In this study, we characterized the morphological, physiological, molecular, and metabolic responses to drought stress to reveal the different response strategies in the two Astragalus species. The susceptible A. membranaceus, with a larger leaf area, showed significant stomatal closure at moderate drought stress, indicating drought avoidance strategy to minimize transpiration. A. mongholicus, with a smaller leaf area, was better in maintaining photosynthesis and activating antioxidant enzymes than A. membranaceus towards the drought tolerance strategy. Drought has greater disturbance on gene expression in A. membranaceus, compared to A. mongholicus, indicating that A. membranaceus was more sensitive in the face of drought. Meanwhile, highly conserved osmotic stress signal transduction pathways were discovered in two Astragalus species under drought treatment. The up-regulation of dehydration-responsive element-binding gene (DREB) expression in A. mongholicus, initiating a series of stress response pathways to help plants adapt and cope with water limitation, showed better “tolerance” than in A. membranaceus. An increase of compatible solutes in A. membranaceus indicates a positive response in regulating osmotic homeostasis in response to drought. The greater investment in antioxidant supplementation phenolic compounds of A. mongholicus suggests the “Tolerance” strategy. Furthermore, the greater antioxidant investment of A. mongholicus is evidenced by higher antioxidant efficacies in terms of antiradical activity. In summary, we conclude that the “Tolerance” strategy of A. mongholicus and the “Avoidance” strategy of A. membranaceus in the face of drought stress can be established.

High-Dose Fenofibrate Stimulates Multiple Cellular Stress Pathways in the Kidney of Old Rats.

We investigated the age-related effects of the lipid-lowering drug fenofibrate on renal stress-associated effectors. Young and old rats were fed standard chow with 0.1% or 0.5% fenofibrate. The kidney cortex tissue structure showed typical aging-related changes. In old rats, 0.1% fenofibrate reduced the thickening of basement membranes, but 0.5% fenofibrate exacerbated interstitial fibrosis. The PCR array for stress and toxicity-related targets showed that 0.1% fenofibrate mildly downregulated, whereas 0.5% upregulated multiple genes. In young rats, 0.1% fenofibrate increased some antioxidant genes' expression and decreased the immunoreactivity of oxidative stress marker 4-HNE. However, the activation of cellular antioxidant defenses was impaired in old rats. Fenofibrate modulated the expression of factors involved in hypoxia and osmotic stress signaling similarly in both age groups. Inflammatory response genes were variably modulated in the young rats, whereas old animals presented elevated expression of proinflammatory genes and TNFα immunoreactivity after 0.5% fenofibrate. In old rats, 0.1% fenofibrate more prominently than in young animals induced phospho-AMPK and PGC1α levels, and upregulated fatty acid oxidation genes. Our results show divergent effects of fenofibrate in young and old rat kidneys. The activation of multiple stress-associated effectors by high-dose fenofibrate in the aged kidney warrants caution when applying fenofibrate therapy to the elderly.

SCAB1 coordinates sequential Ca2+ and ABA signals during osmotic stress induced stomatal closure in Arabidopsis.

Hyperosmotic stress caused by drought is a detrimental threat to plant growth and agricultural productivity due to limited water availability. Stomata are gateways of transpiration and gas exchange, the swift adjustment of stomatal aperture has a strong influence on plant drought resistance. Despite intensive investigations of stomatal closure during drought stress in past decades, little is known about how sequential signals are integrated during complete processes. Here, we discovered that the rapid Ca2+ signaling and subsequent abscisic acid (ABA) signaling contribute to the kinetics of both F-actin reorganizations and stomatal closure in Arabidopsis thaliana, while STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1) is the molecular switch for this entire process. During the early stage of osmotic shock responses, swift elevated calcium signaling promotes SCAB1 phosphorylation through calcium sensors CALCIUM DEPENDENT PROTEIN KINASE3 (CPK3) and CPK6. The phosphorylation restrained the microfilament binding affinity of SCAB1, which bring about the F-actin disassembly and stomatal closure initiation. As the osmotic stress signal continued, both the kinase activity of CPK3 and the phosphorylation level of SCAB1 attenuated significantly. We further found that ABA signaling is indispensable for these attenuations, which presumably contributed to the actin filament reassembly process as well as completion of stomatal closure. Notably, the dynamic changes of SCAB1 phosphorylation status are crucial for the kinetics of stomatal closure. Taken together, our results support a model in which SCAB1 works as a molecular switch, and directs the microfilament rearrangement through integrating the sequentially generated Ca2+ and ABA signals during osmotic stress induced stomatal closure.

Plant salinity stress, sensing, and its mitigation through WRKY.

Salinity or salt stress has deleterious effects on plant growth and development. It imposes osmotic, ionic, and secondary stresses, including oxidative stress on the plants and is responsible for the reduction of overall crop productivity and therefore challenges global food security. Plants respond to salinity, by triggering homoeostatic mechanisms that counter salt-triggered disturbances in the physiology and biochemistry of plants. This involves the activation of many signaling components such as SOS pathway, ABA pathway, and ROS and osmotic stress signaling. These biochemical responses are accompanied by transcriptional modulation of stress-responsive genes, which is mostly mediated by salt-induced transcription factor (TF) activity. Among the TFs, the multifaceted significance of WRKY proteins has been realized in many diverse avenues of plants' life including regulation of plant stress response. Therefore, in this review, we aimed to highlight the significance of salinity in a global perspective, the mechanism of salt sensing in plants, and the contribution of WRKYs in the modulation of plants' response to salinity stress. This review will be a substantial tool to investigate this problem in different perspectives, targeting WRKY and offering directions to better manage salinity stress in the field to ensure food security.

The osmotic stress-activated receptor-like kinase DPY1 mediates SnRK2 kinase activation and drought tolerance in Setaria.

Plant genomes encode many receptor-like kinases (RLKs) that localize to the cell surface and perceive a wide variety of environmental cues to initiate downstream signaling cascades. Whether these RLKs participate in dehydration stress signaling in plants is largely unknown. DROOPY LEAF1 (DPY1), a leucine-rich repeat (LRR)-RLK, was recently shown to regulate plant architecture by orchestrating early brassinosteroid signaling in foxtail millet (Setaria italica). Here, we show that DPY1 is essential for the acclimation of foxtail millet to drought stress. DPY1 can be phosphorylated and activated in response to osmotic stress and is required for more than half of osmotic stress-induced global phosphorylation events, including the phosphorylation of sucrose nonfermenting kinase 2s (SnRK2s), the central kinases involved in osmotic stress. DPY1 acts upstream of STRESS-ACTIVATED PROTEIN KINASE 6 (SAPK6, a subclass I SnRK2) and is required for full SAPK6 activation, thereby allowing regulation of downstream genes to mount a response against drought stress. These signaling events are largely independent of DPY1-mediated brassinosteroid signaling. The DPY1-SAPK6 module is specific to seed plants and is absent in ancestral nonseed plants. Our findings reveal a dehydration stress-activated RLK that plays an indispensable role in osmotic stress signaling and mediates SnRK2 activation at the cell surface.

Arabidopsis PLANT U-BOX44 down-regulates osmotic stress signaling by mediating Ca2+-DEPENDENT PROTEIN KINASE4 degradation.

Calcium (Ca2+)-dependent protein kinases (CPKs) are essential regulators of plant responses to diverse environmental stressors, including osmotic stress. CPKs are activated by an increase in intracellular Ca2+ levels triggered by osmotic stress. However, how the levels of active CPK protein are dynamically and precisely regulated has yet to be determined. Here, we demonstrate that NaCl/mannitol-induced osmotic stress promoted the accumulation of CPK4 protein by disrupting its 26S proteasome-mediated CPK4 degradation in Arabidopsis (Arabidopsis thaliana). We isolated PLANT U-BOX44 (PUB44), a U-box type E3 ubiquitin ligase that ubiquitinates CPK4 and triggers its degradation. A calcium-free or kinase-inactive CPK4 variant was preferentially degraded compared to the Ca2+-bound active form of CPK4. Furthermore, PUB44 exhibited a CPK4-dependent negative role in the response of plants to osmotic stress. Osmotic stress induced the accumulation of CPK4 protein by inhibiting PUB44-mediated CPK4 degradation. The present findings reveal a mechanism for regulating CPK protein levels and establish the relevance of PUB44-dependent CPK4 regulation in modulating plant osmotic stress responses, providing insights into osmotic stress signal transduction mechanisms.

Role of bZIP Transcription Factors in Plant Salt Stress.

Soil salinity has become an increasingly serious problem worldwide, greatly limiting crop development and yield, and posing a major challenge to plant breeding. Basic leucine zipper (bZIP) transcription factors are the most widely distributed and conserved transcription factors and are the main regulators controlling various plant response processes against external stimuli. The bZIP protein contains two domains: a highly conserved, DNA-binding alkaline region, and a diverse leucine zipper, which is one of the largest transcription factor families in plants. Plant bZIP is involved in many biological processes, such as flower development, seed maturation, dormancy, and senescence, and plays an important role in abiotic stresses such as salt damage, drought, cold damage, osmotic stress, mechanical damage, and ABA signal response. In addition, bZIP is involved in the regulation of plant response to biological stresses such as insect pests and pathogen infection through salicylic acid, jasmonic acid, and ABA signal transduction pathways. This review summarizes and discusses the structural characteristics and functional characterization of the bZIP transcription factor group, the bZIP transcription factor complex and its molecular regulation mechanisms related to salt stress resistance, and the regulation of transcription factors in plant salt stress resistance. This review provides a theoretical basis and research ideas for further exploration of the salt stress-related functions of bZIP transcription factors. It also provides a theoretical basis for crop genetic improvement and green production in agriculture.

Rate thresholds in cell signaling have functional and phenotypic consequences in non-linear time-dependent environments.

All cells employ signal transduction pathways to respond to physiologically relevant extracellular cytokines, stressors, nutrient levels, hormones, morphogens, and other stimuli that vary in concentration and rate in healthy and diseased states. A central unsolved fundamental question in cell signaling is whether and how cells sense and integrate information conveyed by changes in the rate of extracellular stimuli concentrations, in addition to the absolute difference in concentration. We propose that different environmental changes over time influence cell behavior in addition to different signaling molecules or different genetic backgrounds. However, most current biomedical research focuses on acute environmental changes and does not consider how cells respond to environments that change slowly over time. As an example of such environmental change, we review cell sensitivity to environmental rate changes, including the novel mechanism of rate threshold. A rate threshold is defined as a threshold in the rate of change in the environment in which a rate value below the threshold does not activate signaling and a rate value above the threshold leads to signal activation. We reviewed p38/Hog1 osmotic stress signaling in yeast, chemotaxis and stress response in bacteria, cyclic adenosine monophosphate signaling in Amoebae, growth factors signaling in mammalian cells, morphogen dynamics during development, temporal dynamics of glucose and insulin signaling, and spatio-temproral stressors in the kidney. These reviewed examples from the literature indicate that rate thresholds are widespread and an underappreciated fundamental property of cell signaling. Finally, by studying cells in non-linear environments, we outline future directions to understand cell physiology better in normal and pathophysiological conditions.

Osmotic stress responses and the biology of the second messenger c-di-AMP in Streptomyces.

Streptomyces are prolific antibiotic producers that thrive in soil, where they encounter diverse environmental cues, including osmotic challenges caused by rainfall and drought. Despite their enormous value in the biotechnology sector, which often relies on ideal growth conditions, how Streptomyces react and adapt to osmotic stress is heavily understudied. This is likely due to their complex developmental biology and an exceptionally broad number of signal transduction systems. With this review, we provide an overview of Streptomyces' responses to osmotic stress signals and draw attention to open questions in this research area. We discuss putative osmolyte transport systems that are likely involved in ion balance control and osmoadaptation and the role of alternative sigma factors and two-component systems (TCS) in osmoregulation. Finally, we highlight the current view on the role of the second messenger c-di-AMP in cell differentiation and the osmotic stress responses with specific emphasis on the two models, S. coelicolor and S. venezuelae.

Preliminary Expression Analysis of the OSCA Gene Family in Maize and Their Involvement in Temperature Stress.

Hyperosmolality-gated calcium-permeable channels (OSCA) are characterized as an osmosensor in plants; they are able to recognize and respond to exogenous and endogenous osmotic changes, and play a vital role in plant growth and adaptability to environmental stress. To explore the potential biological functions of OSCAs in maize, we performed a bioinformatics and expression analysis of the ZmOSCA gene family. Using bioinformatics methods, we identified twelve OSCA genes from the genome database of maize. According to their sequence composition and phylogenetic relationship, the maize OSCA family was classified into four groups (Ⅰ, Ⅱ, Ⅲ, and Ⅳ). Multiple sequence alignment analysis revealed a conserved DUF221 domain in these members. We modeled the calcium binding sites of four OSCA families using the autodocking technique. The expression profiles of ZmOSCA genes were analyzed in different tissues and under diverse abiotic stresses such as drought, salt, high temperature, and chilling using quantitative real-time PCR (qRT-PCR). We found that the expression of twelve ZmOSCA genes is variant in different tissues of maize. Furthermore, abiotic stresses such as drought, salt, high temperature, and chilling differentially induced the expression of twelve ZmOSCA genes. We chose ZmOSCA2.2 and ZmOSCA2.3, which responded most strongly to temperature stress, for prediction of protein interactions. We modeled the calcium binding sites of four OSCA families using autodocking tools, obtaining a number of new results. These results are helpful in understanding the function of the plant OSCA gene family for study of the molecular mechanism of plant osmotic stress and response, as well as exploration of the interaction between osmotic stress, high-temperature stress, and low-temperature stress signal transduction mechanisms. As such, they can provide a theoretical basis for crop breeding.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Affinity Purification Followed by Liquid Chromatography-Tandem Mass Spectrometry to Identify Proteins Interacting with ABA Signaling Components.

Abscisic acid (ABA) is a key phytohormone involved in plant development, seed germination and responses to osmotic stresses, such as drought and high salinity. SNF1-related protein kinases (SnRK2s) play important roles in ABA-dependent and ABA-independent osmotic stress signaling. SnRK2s phosphorylate transcription factors and ion channels in response to ABA or osmotic stress to induce the expression of stress-responsive genes and stomatal closure, respectively, to confer osmotic stress tolerance. The activity of SnRK2s is directly or indirectly regulated by several protein factors. Identification of downstream substrates or upstream regulators of SnRK2s is very useful for elucidating protein components that regulate ABA and osmotic stress signaling. Here, we describe the use of affinity purification by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry to identify protein complexes involved in ABA and osmotic stress signaling in plants. We previously identified several protein factors that regulate ABA and osmotic stress signaling by using this method.

Overview of the roles of calcium sensors in plants’ response to osmotic stress signalling.

Calcium signals serve an important function as secondary messengers between cells in various biological processes due to their robust homeostatic mechanism, maintaining an intracellular free Ca2+ concentration. Plant growth, development, and biotic and abiotic stress are all regulated by Ca2+ signals. Ca2+ binding proteins decode and convey the messages encoded by Ca2+ ions. In the presence of high quantities of Mg2+ and monovalent cations, such sensors bind to Ca2+ ions and modify their conformation in a Ca2+ -dependent manner. Calcium-dependent protein kinases (CPKs), calmodulins (CaMs), and calcineurin B-like proteins are all calcium sensors (CBLs). To transmit Ca2+ signals, CPKs, CBLs, and CaMs interact with target proteins and regulate the expression of their genes. These target proteins may be protein kinases, metabolic enzymes, or cytoskeletal-associated proteins. Beyond its role in plant nutrition as a macroelement and its involvement in the plant cell wall structure, calcium modulates many aspects of development, growth and adaptation to environmental constraints such as drought, salinity and osmotic stresses. This review summarises current knowledge on calcium sensors in plant responses to osmotic stress signalling.

A PIP-mediated osmotic stress signaling cascade plays a positive role in the salt tolerance of sugarcane

BackgroundPlasma membrane intrinsic proteins (PIPs) are plant channel proteins involved in water deficit and salinity tolerance. PIPs play a major role in plant cell water balance and responses to salt stress. Although sugarcane is prone to high salt stress, there is no report on PIPs in sugarcane.ResultsIn the present study, eight PIP family genes, termed ScPIP1–1, ScPIP1–2, ScPIP1–3, ScPIP1–4, ScPIP2–1, ScPIP2–2, ScPIP2–4 and ScPIP2–5, were obtained based on the sugarcane transcriptome database. Then, ScPIP2–1 in sugarcane was cloned and characterized. Confocal microscopy observation indicated that ScPIP2–1 was located in the plasma membrane and cytoplasm. A yeast two-hybridization experiment revealed that ScPIP2–1 does not have transcriptional activity. Real time quantitative PCR (RT-qPCR) analysis showed that ScPIP2–1 was mainly expressed in the leaf, root and bud, and its expression levels in both below- and aboveground tissues of ROC22 were up-regulated by abscisic acid (ABA), polyethylene glycol (PEG) 6000 and sodium chloride (NaCl) stresses. The chlorophyll content and ion leakage measurement suggested that ScPIP2–1 played a significant role in salt stress resistance in Nicotiana benthamiana through the transient expression test. Overexpression of ScPIP2–1 in Arabidopsis thaliana proved that this gene enhanced the salt tolerance of transgenic plants at the phenotypic (healthier state, more stable relative water content and longer root length), physiologic (more stable ion leakage, lower malondialdehyde content, higher proline content and superoxide dismutase activity) and molecular levels (higher expression levels of AtKIN2, AtP5CS1, AtP5CS2, AtDREB2, AtRD29A, AtNHX1, AtSOS1 and AtHKT1 genes and a lower expression level of the AtTRX5 gene).ConclusionsThis study revealed that the ScPIP2–1-mediated osmotic stress signaling cascade played a positive role in plant response to salt stress.

Evaluation of type-B RR dimerization in poplar: A mechanism to preserve signaling specificity?

Plants possess specific signaling pathways, such as the MultiStep Phosphorelay (MSP), which is involved in cytokinin and ethylene sensing, and light, drought or osmotic stress sensing. These MSP comprise histidine-aspartate kinases (HKs) as receptors, histidine phosphotransfer (HPts) proteins acting as phosphorelay proteins, and response regulators (RRs), some of which act as transcription factors (type-B RRs). In previous studies, we identified partners of the poplar osmosensing signaling pathway, composed of two HKs, three main HPts, and six type-B RRs. To date, it is unresolved as to how cytokinin or osmotic stress signal specificity is achieved in the MSP in order to generate specific responses. Here, we present a large-scale interaction study of poplar type-B RR dimerization. Using the two-hybrid assay, we were able to show the homodimerization of type-B RRs, the heterodimerization of duplicated type-B RRs, and surprisingly, a lack of interaction between some type-B RRs belonging to different duplicates. The lack of interaction of the duplicates RR12-14 and RR18-19, which are involved in the osmosensing pathway has been confirmed by BiFC experiments. This study reveals, for the first time, an overview of type-B RR dimerization in poplar and makes way for the hypothesis that signal specificity for cytokinin or osmotic stress could be in part due to the fact that it is impossible for specific type-B RRs to heterodimerize.

Identification of microRNA-like RNAs from Trichoderma asperellum DQ-1 during its interaction with tomato roots using bioinformatic analysis and high-throughput sequencing

MicroRNA-like small RNAs (milRNAs) and their regulatory roles in the interaction between plant and fungus have recently aroused keen interest of plant pathologists. Trichoderma spp., one of the widespread biocontrol fungi, can promote plant growth and induce plant disease resistance. To investigate milRNAs potentially involved in the interaction between Trichoderma and tomato roots, a small RNA (sRNA) library expressed during the interaction of T. asperellum DQ-1 and tomato roots was constructed and sequenced using the Illumina HiSeqTM 2500 sequencing platform. From 13,464,142 sRNA reads, we identified 21 milRNA candidates that were similar to other known microRNAs in the miRBase database and 22 novel milRNA candidates that possessed a stable microRNA precursor hairpin structure. Among them, three milRNA candidates showed different expression level in the interaction according to the result of stem-loop RT-PCR indicating that these milRNAs may play a distinct regulatory role in the interaction between Trichoderma and tomato roots. The potential transboundary milRNAs from T. asperellum and their target genes in tomato were predicted by bioinformatics analysis. The results revealed that several interesting proteins involved in plant growth and development, disease resistance, seed maturation, and osmotic stress signal transduction might be regulated by the transboundary milRNAs. To our knowledge, this is the first report of milRNAs taking part in the process of interaction of T. asperellum and tomato roots and associated with plant promotion and disease resistance. The results might be useful to unravel the mechanism of interaction between Trichoderma and tomato.