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.

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.

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.

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.

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.

Salicylic Acid and Jasmonic Acid Pathways are Activated in Spatially Different Domains Around the Infection Site During Effector-Triggered Immunity in Arabidopsis thaliana.

The innate immune response is, in the first place, elicited at the site of infection. Thus, the host response can be different among the infected cells and the cells surrounding them. Effector-triggered immunity (ETI), a form of innate immunity in plants, is triggered by specific recognition between pathogen effectors and their corresponding plant cytosolic immune receptors, resulting in rapid localized cell death known as hypersensitive response (HR). HR cell death is usually limited to a few cells at the infection site, and is surrounded by a few layers of cells massively expressing defense genes such as Pathogenesis-Related Gene 1 (PR1). This virtually concentric pattern of the cellular responses in ETI is proposed to be regulated by a concentration gradient of salicylic acid (SA), a phytohormone accumulated around the infection site. Recent studies demonstrated that jasmonic acid (JA), another phytohormone known to be mutually antagonistic to SA in many cases, is also accumulated in and required for ETI, suggesting that ETI is a unique case. However, the molecular basis for this uniqueness remained largely to be solved. Here, we found that, using intravital time-lapse imaging, the JA signaling pathway is activated in the cells surrounding the central SA-active cells around the infection sites in Arabidopsis thaliana. This distinct spatial organization explains how these two phythormone pathways in a mutually antagonistic relationship can be activated simultaneously during ETI. Our results re-emphasize that the spatial consideration is a key strategy to gain mechanistic insights into the apparently complex signaling cross-talk in immunity.

Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.

SUPPRESSOR OF LLP1 1-mediated C-terminal processing is critical for CLE19 peptide activity.

Cell-to-cell communication is essential for the coordinated development of multicellular organisms. Members of the CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED (CLE) family, a group of small secretory peptides, are involved in these processes in plants. Although post-translational modifications are considered to be indispensable for their activity, the detailed mechanisms governing these modifications are not well understood. Here, we report that SUPPRESSOR OF LLP1 1 (SOL1), a putative Zn²âº carboxypeptidase previously isolated as a suppressor of the CLE19 over-expression phenotype, functions in C-terminal processing of the CLE19 proprotein to produce the functional CLE19 peptide. Newly isolated sol1 mutants are resistant to CLE19 over-expression, consistent with the previous report (Casamitjana-Martinez, E., Hofhuis, H.F., Xu, J., Liu, C.M., Heidstra, R. and Scheres, B. (2003) Curr. Biol. 13, 1435-1441). As expected, our experiment using synthetic CLE19 peptide revealed that the sol1 mutation does not compromise CLE signal transduction pathways per se. SOL1 possesses enzymatic activity to remove the C-terminal arginine residue of CLE19 proprotein in vitro, and SOL1-dependent cleavage of the C-terminal arginine residue is necessary for CLE19 activity in vivo. Additionally, the endosomal localization of SOL1 suggests that this processing occurs in endosomes in the secretory pathway. Thus, our data indicate the importance of C-terminal processing of CLE proproteins to ensure CLE activities.

Differential effects of the peptides Stomagen, EPF1 and EPF2 on activation of MAP kinase MPK6 and the SPCH protein level.

The positioning and density of leaf stomata are regulated by three secretory peptides, EPIDERMAL PATTERNING FACTOR 1 (EPF1), EPF2 and stomagen. Several lines of published evidence have suggested a regulatory pathway as follows. EPF1 and EPF2 are perceived by receptor complexes consisting of a receptor-like protein, TOO MANY MOUTHS (TMM), and receptor kinases, ERECTA (ER), ERECTA-LIKE (ERL) 1 and ERL2. These receptors activate a mitogen-activated protein (MAP) kinase module. MAP kinases phosphorylate and destabilize the transcription factor SPEECHLESS (SPCH), resulting in a decrease in the number of stomatal lineage cells. Stomagen acts antagonistically to EPF1 and EPF2. However, there is no direct evidence that EPF1 and EPF2 activate or that stomagen inactivates the MAP kinase cascade, through which they might regulate the SPCH level. Experimental modulation of these peptides in Arabidopsis thaliana would change the number of stomatal lineage cells in developing leaves, which in turn would change the expression of SPCH, making the interpretation difficult. Here we reconstructed this signaling pathway in differentiated leaf cells of Nicotiana benthamiana to examine signaling without the confounding effect of cell type change. We show that EPF1 and EPF2 are able to activate the MAP kinase MPK6, and that both EPF1 and EPF2 are able to decrease the SPCH level, whereas stomagen is able to increase it. Our data also suggest that EPF1 can be recognized by TMM together with any ER family receptor kinase, whereas EPF2 can be recognized by TMM together with ERL1 or ERL2, but not by TMM together with ER.

RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis.

The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. In this study, we used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), also known as TOADSTOOL 2 (TOAD2), is another key regulator of meristem maintenance. Mutations in the RPK2 gene result in stem cell expansion and increased number of floral organs, as seen in the other clv mutants. These phenotypes are additive with both clv1 and clv2 mutations. Moreover, our biochemical analyses using Nicotiana benthamiana revealed that RPK2 forms homo-oligomers but does not associate with CLV1 or CLV2. These genetic and biochemical findings suggest that three major receptor complexes, RPK2 homomers, CLV1 homomers and CLV2-CRN/SOL2 heteromers, are likely to mediate three signalling pathways, mainly in parallel but with potential crosstalk, to regulate the SAM homeostasis.

The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus.

In legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulating phenotype. Here, we report the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which mediates the systemic negative regulation of nodulation in Lotus japonicus. In leaf, KLV is predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. A double-mutant analysis indicated that KLV and HAR1 function in the same genetic pathway that governs the negative regulation of nodulation. LjCLE-RS1 and LjCLE-RS2 represent potential root-derived mobile signals for the HAR1-mediated systemic regulation of nodulation. Overexpression of LjCLE-RS1 or LjCLE-RS2 did not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required and acts downstream of LjCLE-RS1 and LjCLE-RS2. In addition to the role of KLV in symbiosis, complementation tests and expression analyses indicated that KLV plays multiple roles in shoot development, including maintenance of shoot apical meristem, vascular continuity, shoot growth and promotion of flowering. Biochemical analyses using transient expression in Nicotiana benthamiana revealed that KLV has the ability to interact with HAR1 and with itself. Together, these results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodule development and that KLV is also a key component in other signal transduction pathways that mediate non-symbiotic shoot development.

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.

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.

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.

Mitogen-activated protein kinase regulated by the CLAVATA receptors contributes to shoot apical meristem homeostasis.

In Arabidopsis, the CLAVATA (CLV) pathway operates in the regulation of the size of the stem cell population in the shoot apical meristem (SAM). CLV3 functions as a small peptide ligand to negatively regulate the expression of the WUSCHEL (WUS) transcription factor through three major receptor kinase complexes of CLV1, CLV2-SUPPRESSOR OF LLP1-2 (SOL2)/CORYNE (CRN) and recently identified RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOADSTOOL 2 (TOAD2). Aiming to understand the precise molecular details of CLV3 signaling, we investigated the contribution of phospho-signaling, potentially regulated by these kinase complexes, to the CLV pathway. We detected CLV3-triggered CLV1 phosphorylation, which is also conditioned by the rest of the CLV receptors, presumably by their direct association. Our comprehensive analysis of the activities of the respective CLV receptors on mitogen-activated protein kinases (MAPKs) suggested that the precise balanced regulation of MAPK activity by the CLV receptors is likely to be key for SAM homeostasis.