C-Type LECTIN receptor-like kinase 1 and ACTIN DEPOLYMERIZING FACTOR 3 are key components of plasmodesmata callose modulation.

Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C-type lectin receptor-like kinase 1 (CLRLK1) in the aspect of PD callose-modulated symplasmic continuity. Here, we found that loss-of-function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine-tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis.

Biochemical Analysis of the Role of Leucine-Rich Repeat Receptor-Like Kinases and the Carboxy-Terminus of Receptor Kinases in Regulating Kinase Activity in Arabidopsis thaliana and Brassica oleracea.

Protein post-translational modification by phosphorylation is essential for the activity and stability of proteins in higher plants and underlies their responses to diverse stimuli. There are more than 300 leucine-rich repeat receptor-like kinases (LRR-RLKs), a major group of receptor-like kinases (RLKs) that plays an important role in growth, development, and biotic stress responses in higher plants. To analyze auto- and transphosphorylation patterns and kinase activities in vitro, 43 full-length complementary DNA (cDNA) sequences were cloned from genes encoding LRR-RLKs. Autophosphorylation activity was found in the cytoplasmic domains (CDs) of 18 LRR-RLKs; 13 of these LRR-RLKs with autophosphorylation activity showed transphosphorylation in Escherichiacoli. BRI1-Associated Receptor Kinase (BAK1), which is critically involved in the brassinosteroid and plant innate immunity signal transduction pathways, showed strong auto- and transphosphorylation with multi-specific kinase activity within 2 h of induction of Brassica oleraceae BAK1-CD (BoBAK1-CD) in E. coli; moreover, the carboxy-terminus of LRR-RLKs regulated phosphorylation and kinase activity in Arabidopsis thaliana and vegetative crops.

Comparison of Root Transcriptomes against Clubroot Disease Pathogens in a Resistant Chinese Cabbage Cultivar (Brassica rapa cv. 'Akimeki').

Clubroot, caused by Plasmodiophora brassicae, is one of the diseases that causes major economic losses in cruciferous crops worldwide. Although prevention strategies, including soil pH adjustment and crop rotation, have been used, the disease's long persistence and devastating impact continuously remain in the soil. CR varieties were developed for clubroot-resistant (CR) Chinese cabbage, and 'Akimeki' is one of the clubroot disease-resistant cultivars. However, recent studies have reported susceptibility to several Korean pathotypes in Akimeki and the destruction of the resistance to P. brassicae in many Brassica species against CR varieties, requiring the understanding of more fine-tuned plant signaling by fungal pathogens. In this study, we focused on the early molecular responses of Akimeki during infection with two P. brassicae strains, Seosan (SS) and Hoengseong2 (HS2), using RNA sequencing (RNA-seq). Among a total of 2358 DEGs, 2037 DEGs were differentially expressed following SS and HS2 infection. Gene ontology (GO) showed that 1524 and 513 genes were up-regulated following SS and HS2 inoculations, respectively. Notably, the genes of defense response and jasmonic acid regulations were enriched in the SS inoculation condition, and the genes of water transport and light intensity response were enriched in the HS2 inoculation condition. Moreover, KEGG pathways revealed that the gene expression set were related to pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) mechanisms. The results will provide valuable information for developing CR cultivars in Brassica plants.

Role of tyrosine autophosphorylation and methionine residues in BRI1 function in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BRs), a group of plant growth hormones, control biomass accumulation and biotic and abiotic stress tolerance, and therefore are highly relevant to agriculture. BRs bind to the BR receptor protein, brassinosteroid insensitive 1 (BRI1), which is classified as a serine/threonine (Ser/Thr) protein kinase. Recently, we reported that BRI1 acts as a dual-specificity kinase both in vitro and in vivo by undergoing autophosphorylation at tyrosine (Tyr) residues. OBJECTIVE: In this study, we characterized the increased leaf growth and early flowering phenotypes of transgenic lines expressing the mutated recombinant protein, BRI1(Y831F)-Flag, compared with those expressing BRI1-Flag. BRI1(Y831F)-Flag transgenic plants showed a reduction in hypocotyl and petiole length compared with BRI1-Flag seedlings. Transcriptome analysis revealed differential expression of flowering time-associated genes (AP1, AP2, AG, FLC, and SMZ) between BRI1(Y831F)-Flag and BRI1-Flag transgenic seedlings. We also performed site-directed mutagenesis of the BRI1 gene, and investigated the effect of methionine (Met) substitution in the extracellular domain (ECD) of BRI1 on plant growth and BR sensitivity by evaluating hypocotyl elongation and root growth inhibition. METHODS: The pBIB-Hyg+-pBR-BRI1-Flag construct(Li et al. 2002) was used as the template for SDM with QuickChange XL Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) to make the SDM mutants. After PCR with SDM kit, add 1 µl of Dpn1 to PCR reaction. Incubate at 37 °C for 2 h to digest parental DNA and then transformed into XL10-gold competent cells. Transcriptome analysis was carried out at the University of Illinois (Urbana-Champaign, Illinois, USA). RNA was prepared and hybridized to the Affymetrix GeneChip Arabidopsis ATH1 Genome Array using the Gene Chip Express Kit (Ambion, Austin, TX, USA). RESULTS: Tyrosine 831 autophosphorylation of BRI1 regulates Arabidopsis flowering time, and mutation of methionine residues in the extracellular domain of BRI1 affects hypocotyl and root length. BRI1(M656Q)-Flag, BRI1(M657Q)-Flag, and BRI1(M661Q)-Flag seedlings were insensitive to the BL treatment and showed no inhibition of root elongation. However, BRI1(M665Q)-Flag and BRI1(M671Q)-Flag seedlings were sensitive to the BL treatment, and exhibited root elongation inhibition. the early flowering phenotype of BRI1(Y831F)-Flag transgenic plants is consistent with the expression levels of key flowering-related genes, including those promoting flowering (AP1, AP2, and AG) and repressing flowering (FLC and SMZ).

Phosphorylation of BIK1 is critical for interaction with downstream signaling components.

BACKGROUND: Botrytis-induced Kinase 1 (BIK1) is a receptor-like cytoplasmic kinase (RLCK) involved in the defense, growth, and development of higher plants. It interacts with various receptor-like kinases (RLKs) such as Brassinosteroid Insensitive 1 (BRI1), Flagellin Sensitive 2 (FLS2), and Perception of the Arabidopsis Danger Signal Peptide 1 (PEPR1), but little is known about signaling downstream of BIK1. OBJECTIVE: In this study, we aimed to identify Arabidopsis thaliana BIK1 (AtBIK1) and Brassica rapa BIK1 (BrBIK1) interacting proteins, which is downstream signaling components in Arabidopsis. In addition, the effect of BIK1 phosphorylation on their interaction were examined. METHODS: For yeast two hybrid (Y2H) screening, a B. rapa cDNA activation domain (AD) library and an A. thaliana cDNA library were used. Reverse reaction (LR) recombinations of appropriate open reading frames (AtBIK1, BrBIK1, AtRGP2, AtPATL2, AtPP7) in either pDONR207 or pDONR/zeo were performed with the split-YFP destination vectors pDEST-GWVYNE and pDEST-GWVYCE to generate N- or C-terminal fusions with the N- and C-terminal yellow fluorescent protein (YFP) moieties, respectively. Recombined vectors were transformed into Agrobacterium strain GV3101. The described GST-AtBIK1, Flag-AtBIK1, and Flag-BrBIK1 constructs were used as templates for site-directed mutagenesis with a QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). RESULTS: In results, A. thaliana BIK1 (AtBIK1) displays strong autophosphorylation kinase activity on tyrosine and threonine residues, whereas B. rapa BIK1 (BrBIK1) does not exhibit autophosphorylation kinase activity in vitro. Herein, we demonstrated that four proteins (RGP2, PATL2, PP7, and SULTR4.1) interact with BrBIK1 but not AtBIK1 in a Y2H system. To confirm interactions between BIK1 and protein candidates in Nicotiana benthamiana, BiFC analysis was performed and it was found that only BrBIK1 bound the three proteins tested. Three phosphosites, T90, T362, and T368, based on amino acid sequence alignment between AtBIK1 and BrBIK1, and performed site-directed mutagenesis (SDM) on AtBIK1 and BrBIK. S90T, P362T, and A369T mutations in BrBIK1 restored autophosphorylation kinase activity on threonine residues comparable to AtBIK1. However, T90A, T362P, and T368A mutations in AtBIK1 did not alter autophosphorylation kinase activity on threonine residues compared with wild-type AtBIK1. BiFC results showed that BIK1 mutations restored kinase activity led to the loss of the binding activity to RGP2, PATL2, or PP7 proteins. CONCLUSION: Phospho-BIK1 might be involved in plant innate immunity, while non-phospho BIK1 may regulate plant growth and development through interactions with RGP2, PATL2, and PP7.

Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants.

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.

Predictors of intentional intoxication using decision tree modeling analysis: a retrospective study.

OBJECTIVE: The suicide rate in South Korea is very high and is expected to increase in coming years. Intoxication is the most common suicide attempt method as well as one of the common reason for presenting to an emergency medical center. We used decision tree modeling analysis to identify predictors of risk for suicide by intentional intoxication. METHODS: A single-center, retrospective study was conducted at our hospital using a 4-year registry of the institute from January 1, 2013 to December 31, 2016. Demographic factors, such as sex, age, intentionality, therapeutic adherence, alcohol consumption, smoking status, physical disease, cancer, psychiatric disease, and toxicological factors, such as type of intoxicant and poisoning severity score were collected. Candidate risk factors based on the decision tree were used to select variables for multiple logistic regression analysis. RESULTS: In total, 4,023 patients with intoxication were enrolled as study participants, with 2,247 (55.9%) identified as cases of intentional intoxication. Reported annual percentages of intentional intoxication among patients were 628/937 (67.0%), 608/1,082 (56.2%), 536/1,017 (52.7), 475/987 (48.1%) from 2013 to 2016. Significant predictors identified based on decision tree analysis were alcohol consumption, old age, psychiatric disease, smoking, and male sex; those identified based on multiple regression analysis were alcohol consumption, smoking, male sex, psychiatric disease, old age, poor therapeutic adherence, and physical disease. CONCLUSION: We identified important predictors of suicide risk by intentional intoxication. A specific and realistic approach to analysis using the decision tree modeling technique is an effective method to determine those groups at risk of suicide by intentional intoxication.