Novel MAP kinase substrates identified by solid-phase phosphorylation screening in Arabidopsis thaliana

박형철,Xuan Canh Nguyen,Sunghwa Bahk,Byung Ouk Park,김호수,Min-Chul Kim,Hans J. Bohnert,정우식 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.6

Phosphorylation of substrate proteins by mitogen-activated protein kinases (MPKs) determines the specific cellular responses elicited by a particular extracellular stimulus. However, downstream targets of plant MPKs remain poorly characterized. In this study, 29 putative substrates of AtMPK3, AtMPK4 and AtMPK6 were identified by solid-phase phosphorylation screening of a k phage expression library constructed from combined mRNAs from salt-treated, pathogen-treated and mechanically wounded Arabidopsis seedlings. To test the efficiency of this screening, we performed in vitro kinase assay with 10 recombinant fusion proteins. All proteins were phosphorylated by AtMPK3, AtMPK4 and AtMPK6, indicating the efficiency of this screening procedure. To confirm phosphorylation of isolated substrates by plant MPKs, we performed in-gel kinase assays. All test substrates were strongly phosphorylated by wounding or H2O2-activated AtMPK3 and AtMPK6. Three substrates, encoded by genes At2g41430, At2g41900, and At3g16770, were strongly phosphorylated, suggesting a function as AtMPK substrates. The type of screening provides a powerful way for identifying potential substrates of MAP kinases responsive to biotic and abiotic stresses.

Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in <i>Arabidopsis</i>

Kim, Sun Ho,Kim, Ho Soo,Bahk, Sunghwa,An, Jonguk,Yoo, Yeji,Kim, Jae-Yean,Chung, Woo Sik Oxford University Press 2017 Nucleic acids research Vol.45 No.11

<P><B>Abstract</B></P><P>The expression of <I>CBF</I> (C-repeat-binding factor) genes is required for freezing tolerance in <I>Arabidopsis thaliana. CBFs</I> are positively regulated by INDUCER OF CBF EXPRESSION1 (ICE1) and negatively regulated by MYB15. These transcription factors directly interact with specific elements in the <I>CBF</I> promoters. Mitogen-activated protein kinase (MAPK/MPK) cascades function upstream to regulate <I>CBFs</I>. However, the mechanism by which MPKs control <I>CBF</I> expression during cold stress signaling remains unknown. This study showed that the activity of MYB15, a transcriptional repressor of cold signaling, is regulated by MPK6-mediated phosphorylation. MYB15 specifically interacts with MPK6, and MPK6 phosphorylates MYB15 on Ser168. MPK6-induced phosphorylation reduced the affinity of MYB15 binding to the <I>CBF3</I> promoter and mutation of its phosphorylation site (MYB15<SUP>S168A</SUP>) enhanced the transcriptional repression of <I>CBF3</I> by MYB15. Furthermore, transgenic plants overexpressing MYB15<SUP>S168A</SUP> showed significantly reduced <I>CBF</I> transcript levels in response to cold stress, compared with plants overexpressing MYB15. The <I>MYB15<SUP>S168A</SUP></I>-overexpressing plants were also more sensitive to freezing than <I>MYB15</I>-overexpressing plants. These results suggest that MPK6-mediated regulation of MYB15 plays an important role in cold stress signaling in <I>Arabidopsis</I>.</P>

Ca2+/CaM increases the necrotrophic pathogen resistance through the inhibition of a CaM-regulated dual-specificity protein phosphatase 1 in Arabidopsis

Nguyen Nhan Thi,김선호,김경은,Bahk Sunghwa,Nguyen Xuan Canh,김민갑,홍종찬,정우식 한국식물생명공학회 2022 Plant biotechnology reports Vol.16 No.1

Jasmonic acid (JA) is a phytohormone that plays a central role in plant defense against necrotrophic pathogens. JA signal- ing stimulates the increase of cytosolic calcium ion (Ca2+) and implicates the activity of mitogen-activated protein kinases (MPKs). We previously characterized that Ca2+/calmodulin (CaM) activates MPKs by inhibiting a CaM-regulated dual- specificity protein phosphatase1 (DsPTP1) at the biochemical level. In this study, we reported that Ca2+/CaM-mediated DsPTP1 negatively regulates the resistance to necrotrophic pathogens through the inhibition of JA-responsive MPK6. To elucidate the physiological function of inhibiting DsPTP1 activity by Ca2+/CaM, we constructed transgenic plants over- expressing DsPTP1 wild type (DsPTP1WT OX) and CaM deregulated mutant (DsPTP1K166E OX). Interestingly, the MPK6 activity was significantly reduced in DsPTP1K166E OX plants in response to JA compared to DsPTP1WT OX plants. Moreover, transcript levels of JA-responsive gene PDF1.2 and VSP1 were also highly decreased in DsPTP1K166E OX plants compared to DsPTP1WT OX plants. Furthermore, DsPTP1K166E OX plants showed more susceptibility to necrotrophic pathogens than DsPTP1WT OX plants. Conclusively, these results suggest that Ca2+/CaM activates the JA-responsive MPKs by inhibiting DsPTP1 for the resistance to the necrotrophic pathogen

Kaempferol promotes bacterial pathogen resistance through the activation of NPR1 by both SA and MPK signaling pathways in Arabidopsis

안종욱,Nguyen Xuan Canh,김선호,Bahk Sunghwa,강호빈,Le Anh Pham Minh,박재민,RAMADANYZAKIYAH,김상희,Park Hyeong Cheol,정우식 한국식물생명공학회 2022 Plant biotechnology reports Vol.16 No.6

Flavonoids have a variety of biological functions including UV protectant, antioxidant, allelopathy and auxin transport inhibitor in plants. In addition, favonoids are implicated in defense response against pathogens. In this study, we found that kaempferol, a kind of favonol, induces bacterial pathogen resistance. In addition, we identifed the signaling pathway to explain how kaempferol can induce pathogen resistance in Arabidopsis. We showed that kaempferol upregulates the transcription of two pathogenesis-related (PR) genes. Interestingly, the monomerization and nuclear translocation of NPR1, a key regulator of PR gene expression, was induced by kaempferol through the accumulation of salicylic acid (SA). Furthermore, we found that the kaempferol-induced monomerization of NPR1 is mediated by the activation of MPK3 and MPK6. Taken together, this study suggests that kaempferol induces pathogen resistance by both SA and MPK-dependent signaling pathways in Arabidopsis.

Ca2+/Calmodulin Activates an MAP Kinase Through the Inhibition of a Protein Phosphatase (DsPTP1) in Arabidopsis

Kim Kyung Eun,Nguyen Nhan Thi,Kim Sun Ho,Bahk Sunghwa,Cheong Mi Sun,Park Hyeong Cheol,Lee Kyun Oh,Hong Jong Chan,Chung Woo Sik 한국식물학회 2022 Journal of Plant Biology Vol.65 No.1

Mitogen-activated protein kinases (MPKs) play roles as critical signal components in the environmental stress responses and developmental processes in plants. Calcium ion (Ca2+) is one of the most essential ubiquitous intracellular second messengers involved in many signal transduction pathways in plants. It was previously known that MPKs are activated by the increasing Ca2+ concentration. However, the mechanism of how Ca2+ activates MPKs is not elucidated yet. In this study, we revealed that Ca2+ could activate MPK signaling pathway via inhibiting the activity of a dual-specificity protein phosphatase1 (DsPTP1) by Ca2+/calmodulin (CaM). We showed that DsPTP1 directly interacts with MPK6 in vitro and in vivo. DsPTP1 was able to inactivate the active MPK6 by dephosphorylation. Interestingly, the DsPTP1-mediated dephosphorylation of MPK6 was strongly inhibited by Ca2+/CaM. Moreover, this inhibition was caused by the binding of CaM to the calmodulin-binding domain II (CaMBDII) of DsPTP1. This study implies that Ca2+/CaM is involved in the activation of MPKs through the inhibition of DsPTP1.

A NAC transcription factor and SNI1 cooperatively suppress basal pathogen resistance in <i>Arabidopsis thaliana</i>

Kim, Ho Soo,Park, Hyeong Cheol,Kim, Kyung Eun,Jung, Mi Soon,Han, Hay Ju,Kim, Sun Ho,Kwon, Young Sang,Bahk, Sunghwa,An, Jonguk,Bae, Dong Won,Yun, Dae-Jin,Kwak, Sang-Soo,Chung, Woo Sik Oxford University Press 2012 Nucleic acids research Vol.40 No.18

<P>Transcriptional repression of pathogen defense-related genes is essential for plant growth and development. Several proteins are known to be involved in the transcriptional regulation of plant defense responses. However, mechanisms by which expression of defense-related genes are regulated by repressor proteins are poorly characterized. Here, we describe the <I>in planta</I> function of CBNAC, a calmodulin-regulated NAC transcriptional repressor in <I>Arabidopsis</I>. A T-DNA insertional mutant (<I>cbnac1</I>) displayed enhanced resistance to a virulent strain of the bacterial pathogen <I>Pseudomonas syringae</I> DC3000 (<I>Pst</I>DC3000), whereas resistance was reduced in transgenic <I>CBNAC</I> overexpression lines. The observed changes in disease resistance were correlated with alterations in <I>pathogenesis-related protein 1</I> (<I>PR1</I>) gene expression. CBNAC bound directly to the <I>PR1</I> promoter. SNI1 (<I>suppressor of nonexpressor of PR genes1, inducible 1</I>) was identified as a CBNAC-binding protein. Basal resistance to <I>Pst</I>DC3000 and derepression of <I>PR1</I> expression was greater in the <I>cbnac1 sni1</I> double mutant than in either <I>cbnac1</I> or <I>sni1</I> mutants. SNI1 enhanced binding of CBNAC to its cognate <I>PR1</I> promoter element. CBNAC and SNI1 are hypothesized to work as repressor proteins in the cooperative suppression of plant basal defense.</P>