Targeted single-cell gene induction by optimizing the dually regulated CRE/loxP system by a newly defined heat-shock promoter and the steroid hormone in Arabidopsis thaliana.

Multicellular organisms rely on intercellular communication systems to organize their cellular functions. In studies focusing on intercellular communication, the key experimental techniques include the generation of chimeric tissue using transgenic DNA recombination systems represented by the CRE/loxP system. If an experimental system enables the induction of chimeras at highly targeted cell(s), it will facilitate the reproducibility and precision of experiments. However, multiple technical limitations have made this challenging. The stochastic nature of DNA recombination events, especially, hampers reproducible generation of intended chimeric patterns. Infrared laser-evoked gene operator (IR-LEGO), a microscopic system that irradiates targeted cells using an IR laser, can induce heat shock-mediated expression of transgenes, for example, CRE recombinase gene, in the cells. In this study, we developed a method that induces CRE/loxP recombination in the target cell(s) of plant roots and leaves in a highly specific manner. We combined IR-LEGO, an improved heat-shock-specific promoter, and dexamethasone-dependent regulation of CRE. The optimal IR-laser power and irradiation duration were estimated via exhaustive irradiation trials and subsequent statistical modeling. Under optimized conditions, CRE/loxP recombination was efficiently induced without cellular damage. We also found that the induction efficiency varied among tissue types and cellular sizes. The developed method offers an experimental system to generate a precisely designed chimeric tissue, and thus, will be useful for analyzing intercellular communication at high resolution in roots and leaves.

Local calcium signal transmission in mycelial network exhibits decentralized stress responses.

Many fungi live as mycelia, which are networks of hyphae. Mycelial networks are suited for the widespread distribution of nutrients and water. The logistical capabilities are critical for the extension of fungal survival areas, nutrient cycling in ecosystems, mycorrhizal symbioses, and virulence. In addition, signal transduction in mycelial networks is predicted to be vital for mycelial function and robustness. A lot of cell biological studies have elucidated protein and membrane trafficking and signal transduction in fungal hyphae; however, there are no reports visualizing signal transduction in mycelia. This paper, by using the fluorescent Ca2+ biosensor, visualized for the first time how calcium signaling is conducted inside the mycelial network in response to localized stimuli in the model fungus Aspergillus nidulans. The wavy propagation of the calcium signal inside the mycelium or the signal blinking in the hyphae varies depending on the type of stress and proximity to the stress. The signals, however, only extended around 1,500 µm, suggesting that the mycelium has a localized response. The mycelium showed growth delay only in the stressed areas. Local stress caused arrest and resumption of mycelial growth through reorganization of the actin cytoskeleton and membrane trafficking. To elucidate the downstream of calcium signaling, calmodulin, and calmodulin-dependent protein kinases, the principal intracellular Ca2+ receptors were immunoprecipitated and their downstream targets were identified by mass spectrometry analyses. Our data provide evidence that the mycelial network, which lacks a brain or nervous system, exhibits decentralized response through locally activated calcium signaling in response to local stress.

In vivo Imaging Enables Understanding of Seamless Plant Defense Responses to Wounding and Pathogen Attack.

Plants are exposed to varied biotic stresses, including sequential or simultaneous attack by insects and pathogens. To overcome these complex stresses, plants must perceive each of the stresses, then integrate and relay the information throughout the plant body and eventually activate local and systemic resistance responses. Previous molecular genetic studies identified jasmonic acid and salicylic acid as key plant hormones of wound and immune responses. These hormones, combined with their antagonistic interaction, play critical roles in the initiation and regulation of defense responses against insects and pathogens. Aside from molecular and genetic information, the latest in vivo imaging technology has revealed that plant defense responses are regulated spatially and temporally. In this review, we summarize the current knowledge of local and systemic defense responses against wounding and diseases with a focus on past and recent advances in imaging technologies. We discuss how imaging-based multiparametric analysis has improved our understanding of the spatiotemporal regulation of dynamic plant stress responses. We also emphasize the importance of compiling the knowledge generated from individual studies on plant wounding and immune responses for a more seamless understanding of plant defense responses in the natural environment.

DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate.

BACKGROUND: Quantification of gene expression such as RNA-Seq is a popular approach to study various biological phenomena. Despite the development of RNA-Seq library preparation methods and sequencing platforms in the last decade, RNA extraction remains the most laborious and costly step in RNA-Seq of tissue samples of various organisms. Thus, it is still difficult to examine gene expression in thousands of samples. RESULTS: Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. The DTT concentration in Direct-RT buffer prevented RNA degradation but not RT in the lysates of several plant tissues, yeast, and zebrafish larvae. Direct reverse transcription on these lysates in Direct-RT buffer produced comparable amounts of cDNA to those synthesized from purified RNA. To maximize the advantage of the Direct-RT buffer, we integrated Direct-RT and targeted RNA-Seq to develop a cost-effective, high-throughput quantification method for the expressions of hundreds of genes: DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq). The DeLTa-Seq method could drastically improve the efficiency and accuracy of gene expression analysis. DeLTa-Seq analysis of 1056 samples revealed the temperature-dependent effects of jasmonic acid and salicylic acid in Arabidopsis thaliana. CONCLUSIONS: The DeLTa-Seq method can realize large-scale studies using thousands of animal, plant, and microorganism samples, such as chemical screening, field experiments, and studies focusing on individual variability. In addition, Direct-RT is also beneficial for gene expression analysis in small tissues from which it is difficult to purify enough RNA for the experiments.

Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in Arabidopsis thaliana.

Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces, including raindrops, to anticipate pathogen infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) and mitogen-activated protein kinases. We propose an early layer of plant immunity in which trichomes function as mechanosensory cells that detect potential risks.

Root-specific CLE3 expression is required for WRKY33 activation in Arabidopsis shoots.

KEY MESSAGE: This study focused on the role of CLE1-7 peptides as defense mediators, and showed that root-expressed CLE3 functions as a systemic signal to regulate defense-related gene expression in shoots. In the natural environment, plants employ diverse signaling molecules including peptides to defend themselves against various pathogen attacks. In this study, we investigated whether CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes (CLE1-7) respond to biotic stimuli. CLE3 showed significant up-regulation upon treatment with flg22, Pep2, and salicylic acid (SA). Quantitative real-time PCR (qRT-PCR) analysis revealed that CLE3 expression is regulated by the NON-EXPRESSOR OF PR GENES1 (NPR1)-dependent SA signaling and flg22-FLAGELLIN-SENSITIVE 2 (FLS2) signaling pathways. We demonstrated that SA-induced up-regulation of CLE3 in roots was required for activation of WRKY33, a gene involved in the regulation of systemic acquired resistance (SAR), in shoots, suggesting that CLE3 functions as a root-derived signal that regulates the expression of defense-related genes in shoots. Microarray analysis of transgenic Arabidopsis lines overexpressing CLE3 under the control of a ß-estradiol-inducible promoter revealed that root-confined CLE3 overexpression affected gene expression in both roots and shoots. Comparison of CLE2- and CLE3-induced genes indicated that CLE2 and CLE3 peptides target a few common but largely distinct downstream genes. These results suggest that root-derived CLE3 is involved in the regulation of systemic rather than local immune responses. Our study also sheds light on the potential role of CLE peptides in long-distance regulation of plant immunity.

An Evolutionarily Conserved Coreceptor Gene Is Essential for CLAVATA Signaling in Marchantia polymorpha.

Growth and development of land plants are controlled by CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) family of peptide hormones. In contrast to the genetic diversity of CLE family in flowering plants, the liverwort Marchantia polymorpha possesses a minimal set of CLE, MpCLE1(TDIF homolog), and MpCLE2 (CLV3 homolog). MpCLE1 and MpCLE2 peptides exert distinct function at the apical meristem of M. polymorpha gametophyte via specific receptors, MpTDIF RECEPTOR (MpTDR) and MpCLAVATA1 (MpCLV1), respectively, both belonging to the subclass XI of leucine-rich repeat receptor-like kinases (LRR-RLKs). Biochemical and genetic studies in Arabidopsis have shown that TDR/PXY family and CLV1/BAM family recognize the CLE peptide ligand in a heterodimeric complex with a member of subclass-II coreceptors. Here we show that three LRR-RLK genes of M. polymorpha are classified into subclass II, representing three distinct subgroups evolutionarily conserved in land plants. To address the involvement of subclass-II coreceptors in M. polymorpha CLE signaling, we performed molecular genetic analysis on one of them, MpCLAVATA3 INSENSITIVE RECEPTOR KINASE (MpCIK). Two knockout alleles for MpCIK formed narrow apical meristems marked by prom MpYUC2:GUS marker, which were not expanded by MpCLE2 peptide treatment, phenocopying Mpclv1. Loss of sensitivity to MpCLE2 peptide was also observed in gemma cup formation in both Mpclv1 and Mpcik. Biochemical analysis using a Nicotiana benthamiana transient expression system revealed weak association between MpCIK and MpCLV1, as well as MpCIK and MpTDR. While MpCIK may also participate in MpCLE1 signaling, our data show that the conserved CLV3-CLV1-CIK module functions in M. polymorpha, controlling meristem activity for development and organ formation for asexual reproduction.

CLE2 regulates light-dependent carbohydrate metabolism in Arabidopsis shoots.

KEY MESSAGE: This study focused on the role of CLE1-CLE7 peptides as environmental mediators and indicated that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots. Plants sense environmental stimuli and convert them into cellular signals, which are transmitted to distinct cells and tissues to induce adequate responses. Plant hormones and small secretory peptides often function as environmental stress mediators. In this study, we investigated whether CLAVATA3/EMBRYO SURROUNDING REGION-RELATED proteins, CLE1-CLE7, which share closely related CLE domains, mediate environmental stimuli in Arabidopsis thaliana. Expression analysis of CLE1-CLE7 revealed that these genes respond to different environmental stimuli, such as nitrogen deprivation, nitrogen replenishment, cold, salt, dark, and sugar starvation, in a sophisticated manner. To further investigate the function of CLE2, we generated transgenic Arabidopsis lines expressing the ß-glucuronidase gene under the control of the CLE2 promoter or expressing the CLE2 gene under the control of an estradiol-inducible promoter. We also generated cle2-1 and cle2-2 mutants using the CRISPR/Cas9 technology. In these transgenic lines, dark induced the expression of CLE2 in the root vasculature. Additionally, induction of CLE2 in roots induced the expression of various genes not only in roots but also in shoots, and genes related to light-dependent carbohydrate metabolism were particularly induced in shoots. In addition, cle2 mutant plants showed chlorosis when subjected to a shade treatment. These results suggest that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots.

An Artificial Conversion of Roots into Organs with Shoot Stem Characteristics by Inducing Two Transcription Factors.

Somatic plant cells can regenerate shoots and/or roots or adventitious embryonic calluses, which may induce organ formation under certain conditions. Such regenerations occur via dedifferentiation of somatic cells, induction of organs, and their subsequent outgrowth. Despite recent advances in understanding of plant regeneration, many details of shoot induction remain unclear. Here, we artificially induced shoot stem-like green organs (SSOs) in Arabidopsis thaliana roots via simultaneous induction of two transcription factors (TFs), ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 25 (ATHB25, At5g65410) and the B3 family transcription factor REPRODUCTIVE MERISTEM 7 (REM7, At3g18960). The SSOs exhibited negative gravitropism and differentiated vascular bundle phenotypes. The ATHB25/REM7 induced the expression of genes controlling shoot stem characteristics by ectopic expression in roots. Intriguingly, the restoration of root growth was seen in the consecutive and adjacent parts of the SSOs under gene induction conditions. Our findings thus provide insights into the development and regeneration of plant shoot stems.

Automating measurements of fluorescent signals in freely moving plant leaf specimens.

Existing methods to quantify fluorescent signals are primarily limited to non-moving objects or tracking a limited number of cells. These techniques, however, are unsuitable for measuring fluorescent signals in time-lapse experiments using plant specimens that move naturally during a course of imaging. We developed an automated method to measure fluorescent signal intensities in transgenic Arabidopsis plants using a stereomicroscope with standard microscopy software. The features of our technique include: 1) recognizing the shape of plant specimens using autofluorescent signals; 2) merging targeted fluorescent signals to specimen outlines; 3) extracting signals within the shape of specimens from their background signals. Our method facilitates the measurement of fluorescent signals on freely moving plant leaves that are physically unrestrained. The method we developed addresses the challenge of recognizing plant shapes without relying on: a) manual definition which is prone to subjectivity and human error; b) introducing stable fluorescent markers to define plant shapes; c) recognizing plant shapes from bright field images which include a wide range of colors and background noise; d) unnecessarily stressing plants by immobilizing them; e) the use of multiple software packages or software development expertise.

Enrichment of Phosphatidylinositol 4,5-Bisphosphate in the Extra-Invasive Hyphal Membrane Promotes Colletotrichum Infection of Arabidopsis thaliana.

Pathogenic fungi from the genus Colletotrichum form invasive hyphae; the hyphae are surrounded by an extra-invasive hyphal membrane (EIHM), which is continuous with the plant plasma membrane. Although the EIHM plays a crucial role as the interface between plant and fungal cells, its precise function during Colletotrichum infection remains elusive. Here, we show that enrichment of phosphoinositides (PIs) has a crucial role in Colletotrichum infection. We observed the localization of PIs in Arabidopsis thaliana cells infected by A. thaliana-adapted Colletotrichum higginsianum (Ch), and found that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was extremely enriched in the EIHM during Ch infection. We also found that phosphatidylinositol 4-phosphate-5 kinase (PIP5K), which catalyzes production of PI(4,5)P2, also accumulated at the EIHM. The overexpression of PIP5K3 in A. thaliana increased hyphal invasion by Ch. An exocytic factor, EXO84b, was targeted to the EIHM during Ch infection, although endocytic factors such as CLATHRIN LIGHT CHAIN 2 and FLOTILLIN 1 did not. Intriguingly, the interfacial membranes between A. thaliana and powdery mildew- or downy mildew-causing pathogens did not accumulate PI(4,5)P2. These results suggest that Ch could modify the PI(4,5)P2 levels in the EIHM to increase the exocytic membrane/protein supply of the EIHM for successful infection. Our results also suggest that PI(4,5)P2 biosynthesis is a promising target for improved defense against Colletotrichum infection.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

Involvement of S-type anion channels in disease resistance against an oomycete pathogen in Arabidopsis seedlings.

Pharmacological indications suggest that anion channel-mediated plasma membrane (PM) anion efflux is crucial in early defense signaling to induce immune responses and programmed cell death in plants. Arabidopsis SLAC1, an S-type anion channel required for stomatal closure, is involved in cryptogein-induced PM Cl- efflux to positively modulate the activation of other ion fluxes, production of reactive oxygen species and a wide range of defense responses including hypersensitive cell death in tobacco BY-2 cells. We here analyzed disease resistance against several pathogens in multiple mutants of the SLAC/SLAH channels of Arabidopsis. Resistance against a biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 was significantly enhanced in the SLAC1-overexpressing plants than in the wild-type, while that against a bacteria Pseudomonas syringae was not affected significantly. Possible regulatory roles of S-type anion channels in plant immunity and disease resistance against bacterial and oomycete pathogens is discussed.

A small peptide modulates stomatal control via abscisic acid in long-distance signalling.

Mammalian peptide hormones propagate extracellular stimuli from sensing tissues to appropriate targets to achieve optimal growth maintenance 1 . In land plants, root-to-shoot signalling is important to prevent water loss by transpiration and to adapt to water-deficient conditions 2, 3 . The phytohormone abscisic acid has a role in the regulation of stomatal movement to prevent water loss 4 . However, no mobile signalling molecules have yet been identified that can trigger abscisic acid accumulation in leaves. Here we show that the CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED 25 (CLE25) peptide transmits water-deficiency signals through vascular tissues in Arabidopsis, and affects abscisic acid biosynthesis and stomatal control of transpiration in association with BARELY ANY MERISTEM (BAM) receptors in leaves. The CLE25 gene is expressed in vascular tissues and enhanced in roots in response to dehydration stress. The root-derived CLE25 peptide moves from the roots to the leaves, where it induces stomatal closure by modulating abscisic acid accumulation and thereby enhances resistance to dehydration stress. BAM receptors are required for the CLE25 peptide-induced dehydration stress response in leaves, and the CLE25-BAM module therefore probably functions as one of the signalling molecules for long-distance signalling in the dehydration response.

A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus.

Legumes and rhizobia establish symbiosis in root nodules. To balance the gains and costs associated with the symbiosis, plants have developed two strategies for adapting to nitrogen availability in the soil: plants can regulate nodule number and/or stop the development or function of nodules. Although the former is accounted for by autoregulation of nodulation, a form of systemic long-range signaling, the latter strategy remains largely enigmatic. Here, we show that the Lotus japonicus NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1) gene encoding a NIN-LIKE PROTEIN transcription factor acts as a key regulator in the nitrate-induced pleiotropic control of root nodule symbiosis. NRSYM1 accumulates in the nucleus in response to nitrate and directly regulates the production of CLE-RS2, a root-derived mobile peptide that acts as a negative regulator of nodule number. Our data provide the genetic basis for how plants respond to the nitrogen environment and control symbiosis to achieve proper plant growth.

The effects of Lepidopteran oral secretion on plant wounds: A case study on the interaction between Spodoptera litura and Arabidopsis thaliana.

This paper is about the cellular responses of plants to chewing insect attacks. We deployed a recently developed experimental system to monitor the responsiveness of Arabidopsis thaliana (Arabidopsis) to the application of oral secretion (OS) from Lepidopteran generalist herbivore Spodoptera litura (S. litura). Oral secretion from S. litura contains gut regurgitant and saliva. We identified significant differences in the wound closure morphologies (e.g., dried and sealed tissue) between mechanically damaged leaves with and without an application of S. litura OS at the site-of-injury. Experimental controls were mechanically wounded leaves. Wounds were walled off by visible vertical cross sections. Cell death was restricted to the immediate areas of the wounds. In contrast, mechanically damaged leaves treated with S. litura OS did not display a clear sealing pattern due to an absence of a defined vertical cross section at the wound site. Notably, OS treated leaves exhibited a wider area of visible premature senescence (the declining of chlorophyll content caused by death of chloroplasts) around the injury than controls. More pronounced senescence was also observed around the injury in S. litura OS treated wounds than in controls. Heat inactivated S. litura OS elicited a similar response to non-heat inactivated samples. The causal compound is heat stable and thus not a protein. Our results suggest that S. litura OS: (1) inhibited wound recovery responses in leaves; (2) promoted senescence around injured areas. The function of senescence may be to relocate nutritional resources to support plant survival when attacked.