Molecular dissection of Arabidopsis RAR1 and SGT1 functions in plant immunity

Plants possess several layers of defence against pathogens. RAR1 (required for Ml-a12 conditioned resistance) and SGT1 (suppressor of G2 allele of skp1) are regulators of disease resistance conditioned by Resistance (R) proteins that recognise specific pathogen effectors. The model plant, Arabidopsis thaliana, has one copy of RAR1 (AtRAR1) and two recently duplicated copies of SGT1 (AtSGT1a and AtSGT1b). Despite their high sequence homology (78% identity at the amino acid level), AtSGT1b, but not AtSGT1a, is genetically recruited for resistance mediated by a subset of R proteins and for phytohormone signalling controlled by at least two plant SCF E3 ligases (SCF TIR1 and SCF COI1). AtRAR1, but not AtSGT1a or AtSGT1b, was also shown to contribute to plant basal defence against virulent pathogens, in which Arabidopsis EDS1 (Enhanced Disease Susceptibility 1) is an essential regulator. Recent studies revealed roles of RAR1 as co-chaperones of HSP90 to promote accumulation of pre-activated R proteins. SGT1 also shares molecular features of known cochaperones. SGT1 from plant, yeast and human interact with HSP90 and, in human and yeast, is an assembly factor in kinetocore complex formation. The precise role of SGT1 in plant defence was unclear. Recent biochemical experiments showed that SGT1 is required for Bs2 R protein folding that implies SGT1 activity in R protein complex assembly. However, recent genetic data in Arabidopsis suggested that SGT1 acts antagonistically with RAR1 in R protein accumulation, suggesting of a role of SGT1 in R protein degradation. The presence of an additional copy of SGT1 in Arabidopsis and lethality of the sgt1a/sgt1b double mutant complicates genetic interpretation using this system. This study aimed to characterize further the activities of RAR1 and SGT1 in plant immunity using various approaches. Several pieces of key data on the activities of RAR1 and SGT1 in plant immunity were generated in this study. AtRAR1, AtSGT1a and AtSGT1b proteins were expressed in all tissue tested and, although direct interaction between these proteins was not found, Hsc70 was identified as a potential interacting partner of AtRAR1. AtRAR1 regulates AtSGT1b accumulation in the nucleus. I established that both AtSGT1b and AtSGT1a are capable of functioning in R protein-mediated defence and phytohormone signalling in a dose-dependent manner. Lower levels of AtSGT1a in plant cells are likely insufficient to show a genetic effect on sgt1a mutants due to the presence of the more abundant AtSGT1b. The finding of AtSGT1a activity prompts us to reconsider the current model of RAR1/SGT1 antagonism in defence based on purely genetic data using Arabidopsis. I found that AtRAR1 and AtSGT1b contribute to basal defence. Intriguingly, the rar1 and sgt1b mutants lower EDS1 protein accumulation and change the molecular character of EDS1. The activities of AtRAR1 and AtSGT1b in basal defence may be through EDS1. EDS1 is an indispensable regulator of resistance conditioned by the TIR (Toll-Interleukin-1 Receptor) class of nucleotide-binding/leucine-rich-repeat (NB-LRR) R protein. These data therefore suggest a potential molecular link between EDS1 and TIR-NB-LRR via RAR and SGT1. My results highlight the need for further analysis to dissect mechanisms of TIR-NBLRR protein assembly and activation and their molecular connection with EDS1 and the chaperone/cochaperone machinery

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.A correction has been published:Plant and Cell Physiology, Volume 59, Issue 2, 1 February 2018, Pages 43

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

MRI for Advanced Gastric Cancer : Especially for Scirrhous Cancer of the Stomach

We conducted MRI examinations in 92 patients with advanced gastric cancer, and evaluated the clinical potential of MRI for diagnosis of scirrhous cancer of the stomach. The feature of scirrhous cancer of stomach by MRI are ; 1) thick-ened gastric wall, 2) shortening of T1 and T2 values ; and 3) clear contrast between the gastric mucosae and cancer areas found in the T1 and T2 weighted images (preservation of the mucosae). MRI for scirrhous cancer of the stomach is thought a useful image diagnosis as an adjunct method to gastric X-ray and gastric endoscopy

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

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.Video Articl

Focal Hepatic Tumors Using Inversion Recovery Sequence of 0.1-T MRI - Basic : Clinical Evaluation Gray Scale Vs T1 Values

Optimum conditions for image quality contrast were studied with phantom method by means of MRI system (of constant conduction type ; 0.1-T) in order to detect tumor lesions of liver that show no distinct contrast by usual roentgeno-graphic methods. Signal intensity of liver, fat and muscle were maximally suppressed at 1000 ms of TR and 100 ms of TI by the short inversion time inversion recovery (STIR) method, resulting in distinct visualization of liver tumor with extremely good contrast. Clinical investigation with the usual T1- and T2- weighted images under the same conditions identified hepatocellular carcinoma in 22 out of 31 patients (37 of 58 nodules, 64%), cholangiocellular carcinoma 3 of 5 (3 of 6 nodules, 50%), metastatic liver cancer in 55 out of 68 (111 of 143 nodules, 78%), hepatic hemangioma in 32 out of 36 (41 of 47 nodules, 87%) and liver cyst in 8 out of 8 (100%). In contrast, hepatocellular carcinoma was visualized in 30 out of 31 patients (54 of 58 nodules, 93%), cholangiocellular carcinoma in 5 out of 5 (6 of 6 nodules, 100%), metastatic liver cancer in 66 out of 68 (139 of 143 nod-ules, 97%), hepatic hemangioma in 36 out of 36 (47 of 47 nodules, 100%) and liver cyst in 8 out of 8 (100%). The results suggest that STIR (TR : 1000 ms, TI : 100 ms, TE : 18 ms) is extremely useful in screening tumor lesions of the liver

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 Receptor-Like Kinase SOL2 Mediates CLE Signaling in Arabidopsis

Arabidopsis sol2 mutants showed CLV3 peptide resistance. Twenty-six synthetic CLE peptides were examined in the clv1, clv2 and sol2 mutants. sol2 showed different levels of resistance to the various peptides, and the spectrum of peptide resistance was quite similar to that of clv2. SOL2 encoded a receptor-like kinase protein which is identical to CORYNE (CRN). GeneChip analysis revealed that the expression of several genes was altered in the sol2 root tip. Here, we suggest that SOL2, together with CLV2, plays an important role in the regulation of root meristem development through the CLE signaling pathway

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