Responses of Arabidopsis thaliana to Challenge by Pseudomonas syringae

김민갑,Sun Young Kim,Woe Yeon Kim,David Mackey,이상열 한국분자세포생물학회 2008 Molecules and cells Vol.25 No.3

Plants are continually exposed to a variety of potentially pathogenic microbes, and the interactions between plants and pathogenic invaders determine the outcome, disease or disease resistance. To defend themselves, plants have developed a sophisticated immune system. Unlike animals, however, they do not have specialized immune cells and, thus all plant cells appear to have the innate ability to recognize pathogens and turn on an appropriate defense response. Using genetic, genomic and biochemical methods, tremendous advances have been made in understanding how plants recognize pathogens and mount effective defenses. The primary immune response is induced by microbe-associated molecular patterns (MAMPs). MAMP receptors recognize the presence of probable pathogens and evoke defense. In the co-evolution of plant-microbe interactions, pathogens gained the ability to make and deliver effector proteins to suppress MAMP-induced defense responses. In response to effector proteins, plants acquired R-proteins to directly or indirectly monitor the presence of effector proteins and activate an effective defense response. In this review we will describe and discuss the plant immune responses induced by two types of elicitors, PAMPs and effector proteins.

Mutational Analysis of the Effector Domain of Brassica Sar1 Protein

김민갑,Jung Ro Lee,Hye Song Lim,Mi Rim Shin,Min Gyeong Cheon,Deok Ho Lee,Woe Yeon Kim,이상열 한국응용생명화학회 2007 Journal of Applied Biological Chemistry (J. Appl. Vol.50 No.3

Sar1p is a ras-related GTP-binding protein that functions in intracellu lar protein transport between the endoplasmic reticulum (ER) and the Golgi complex. The effector domain of Ras family proteins is highly conserved and this domain is functionally interchangeable in plant, yeast and mammalian Sar1. Using a recombinant Brassica sar1 protein (Bsar1p) harboring point mutations in its effector domain, we here investigated the ability of Sar1p to bind and hydrolyze GTP and to interact with the two sar1-specific regu lators, GTPase activating protein (GAP) and guanine exchange factor (GEF). The T51A and T55A mutations impaired Bsar1p intrinsic GTPbinding and GDP-dissociation activity. In contrast, mutations in the switch domain of Bsar1 did not affect its intrinsic GTPase activity. Moreover, the P50A, P54A, and S56A mutations affected the interaction between Bsar1p and GAP. P54A mutant protein did not interact with two regulating proteins, GEF and GAP, even though the mutation didn’t affect the intrinsic GTP-binding, nucleotide exchange or GTPase activity of Bsar1p.

Working Mechanism of Peroxiredoxins (Prxs) and Sulphiredoxin1 (Srx1) in Arabidopsis thaliana

Min Gab Kim(김민갑),Mukhamad Su’udi(수디 무하마드),Sang Ryeol Park(박상렬),Duk-Ju Hwang(황덕주),Shin Chul Bae(배신철) 한국생명과학회 2010 생명과학회지 Vol.20 No.12

식물체는 대사과정의 부산물로서 또는 생물학적으로 피해를 줄 수 있는 다양한 종류의 외부 스트레스에 직면했을 활성산소(Reactive Oxygen Species, ROS)를 생산한다. 이러한 oxidative 스트레스로부터 자신들을 보호하기 위하여 식물세포들은 다양한 종류의 항산화 단백질들을 보유하고 있다. 하지만 이들의 작용기작은 여전히 자세히 밝혀지지 않았다. Peroxiredoxins (Prxs) 은 식물체에 광범위하게 존재하는 thiol-을 함유한 항산화 단백질로 N-말단에 존재하는 cysteine 잔기를 이용하여 hydrogen peroxide를 환원한다. 이러한 과정에서 peroxiredoxins의 활성부위인 cysteine 잔기는 선택적으로 cysteine sulfinic acid로 산화됨으로써 peroxidase activity의 불활성화를 일으킨다. 이러한 산화과정은 비가역적으로 일어난다. 최근 발견된 진핵생물들에 잘 보존된 sulphiredoxin (Srx1)이라 불리는 단백질은 cysteine-sulphinic acid를 환원시키는 기능을 지닌다. 본 논문에서는 애기장대에 존재하는 Prxs와 Srx의 기능에 대하여 서술할 예정이다. Plants generate reactive oxygen species (ROS) as a by-product of normal aerobic metabolism or when exposed to a variety of stress conditions, which can cause widespread damage to biological macromolecules. To protect themselves from oxidative stress, plant cells are equipped with a wide range of antioxidant proteins. However, the detailed reaction mechanisms of these are still unknown. Peroxiredoxins (Prxs) are ubiquitous thiol-containing antioxidants that reduce hydrogen peroxide with an N-terminal cysteine. The active-site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid during catalysis, which leads to inactivation of peroxidase activity. This oxidation was thought to be irreversible. Recently identified small protein sulphiredoxin (Srx1), which is conserved in higher eukaryotes, reduces cysteine?sulphinic acid in yeast peroxiredoxin. Srx1 is highly induced by H₂O₂-treatment and the deletion of its gene causes decreased yeast tolerance to H₂O₂, which suggest its involvement in the metabolism of oxidants. Moreover, Srx1 is required for heat shock and oxidative stress induced functional, as well as conformational switch of yeast cytosolic peroxiredoxins. This change enhances protein stability and peroxidase activity, indicating that Srx1 plays a crucial role in peroxiredoxin stability and its regulation mechanism. Thus, the understanding of the molecular basis of Srx1 and its regulation is critical for revealing the mechanism of peroxiredoxin action. We postulate here that Srx1 is involved in dealing with oxidative stress via controlling peroxiredoxin recycling in Arabidopsis. This review article thus will be describing the functions of Prxs and Srx in Arabidopsis thaliana. There will be a special focus on the possible role of Srx1 in interacting with and reducing hyperoxidized Cys-sulphenic acid of Prxs.

무름병에 감수성인 애기장대 돌연변이체 Atstp1 선발

최창현,황덕주,김민갑,안일평,박상렬,배신철 한국식물병리학회 2010 식물병연구 Vol.16 No.3

Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft rot disease in various plants. Although many studies about Pcc have been going on, little is known yet about the defense genes from plants. To identify defense associated genes in response to Pcc, we screened about 20 thousand Arabidopsis T-DNA knock out lines by inoculation with Pcc. We obtained a line (Atspt1) showing more susceptible symptom compared to WT (Col-0) on 1 day after the inoculation of Pcc on leaves of Arabidopsis with toothpicks. In this study, we optimized the system to select resistant and susceptible lines to Pcc from T-DNA inserted pool of Arabidopsis and expect the system and Atspt1 might be used for molecular breeding to produce resistant vegetables against Pcc. 본 연구는 애기장대에서 무름병에 대한 저항성 유전자를 탐색하고자 2만여개의 T-DNA 삽입 돌연변이군을 이용하여 Pcc에 대한 스크리닝을 수행하고 이 방법을 소개한 연구다. 1차 선발을 통하여 15개의 저항성 line과 20개의 감수성 line을 선발하였으며, 이로부터 2차 선발하여 3개의 저항성 line과 4개의 감수성 line을 선발하였고, 최종적으로 3차 선발을 통하여 1개의 감수성 line (Atstp1)을 선발할 수 있었다. 현재 Atstp1을 이용해 flanking sequencing 하여 유전자를 탐색하고 있으며, 앞으로 클로닝을 통하여 다양한 무름병 저항성 식물 개발에 유용하게 이용될 것으로 기대한다.

Anti-Helicobacter pylori Compounds from Maackia amurensis

박우성,Ji-Yeong Bae,김민갑,이우곤,강형련,백승철,Kyung Mook Lim,이미경,안미정,김혜진 한국생약학회 2015 Natural Product Sciences Vol.21 No.1

Eight isoflavonoid compounds were isolated from the EtOAc fraction of Maackia amurensis which had shown the highest anti-Helicobacter pylori activity among the fractions, using medium pressure liquid chromatography and recrystallization. Based on the spectroscopic data including 1H-NMR, 13C-NMR, HMBC and MS data, the chemical structures of the isolates were determined to be (-)-medicarpin (1), afromosin (2), formononetin (3), tectorigenin (4), prunetin (5), wistin (6), tectoridin (7) and ononin (8). Anti-H. pylori activity of each compound was evaluated with broth dilution assay. As a result, (-)-medicarpin (1), tectorigenin (4) and wistin (6) showed anti-H. pylori activity. (-)-Medicarpin (1) exhibited the most potent growth inhibitory activity against H. pylori with the minimal inhibitory concentration (MIC)90 of 25 mM, and tectorigenin (4) with MIC90 of 100 mM ranked the second. This is the first study to show the anti-H. pylori activity of M. amurensis, and it is suggested that the stem bark of M. amurensis or the EtOAc fraction or the isolated compounds can be a new natural source for the treatment of H. pylori infection.

Arabidopsis Cell Death in Compatible and Incompatible Interactions with Alternaria brassicicola

Mukhamad Su’udi,김민갑,Sang-Ryeol Park,Duk-Ju Hwang,배신철,Il-Pyung Ahn 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.6

Two strains of necrotrophic Alternaria brassicicola, Ab40857 and Ab42464, are virulent on Korean cabbage and several wild types of Arabidopsis thaliana. Interaction between Ab42464 and Col-0 was compatible, whereas interaction between Ab40857 and Col-0 was incompatible. The loss of defense, no death (dnd) 1 function abrogated the compatibility between Ab42464 and Col-0, and the accelerated cell death (acd) 2 mutation attenuated the Col-0’s resistance against Ab40857. These two fungal strains induced PR1 transcription in Col-0. Ab40857 accelerated transcription of PDF1.2, THI2.1, CAT, and POX by 12 h compared to those challenged with Ab42464. More abundant cell death was observed in Col-0 infected with Ab42464, however, callose deposition was evident in the incompatible interaction. Remarkably, Ab40857-infected areas of acd2-2 underwent rampant cell death and Ab42464 triggered callose production in dnd1-1. Furthermore, the incompatibility between Ab40857 and Col-0 was nullified by the coronatine- insensitive 1 (coi1) and phytoalexin-deficient 3 (pad3) mutations but not by nonexpresser of PR genes (npr1) and pad4. Ab40857 induced abundant cell death in pad3. Taken together, cell death during the early infection stage is a key determinant that discriminates between a compatible interaction and an incompatible one, and the resistance within Col-0 against Ab40857 is dependent on a defense- signaling pathway mediated by jasmonic acid and PAD3.

Production and characterization of polyclonal antibody against Arabidopsis GIGANTEA, a circadian clock controlled flowering time regulator

Laila Khaleda,차준영,김민갑,김외연 한국식물학회 2017 Journal of Plant Biology Vol.60 No.6

Arabidopsis GIGANTEA (GI) is encoded by asingle gene and highly conserved among vascular plants andits mutants display pleiotropic phenotypes involved indiverse biological processes such as light signaling, circadianclock, and sucrose metabolism as well as abiotic stressresponses. However, molecular mechanisms of GI arelargely unknown due to the lack of useful antibody. To date,the epitope tags have been widely used to detect GI in plants,but it needs to generate the transgenic plants which take afew months. Here, we produced polyclonal α-GI antibodyusing truncated variants of GI having amino-terminal (1-858aa) and carboxyl-terminal (920-1173) regions as antigens. Both recombinant His-GI1-858 and His-GI920-1173 proteins wereindividually and successfully expressed in E. coli andimmunized into rabbit. Anti-serum was purified by antigenspecificaffinity purification method using both recombinantHis-GI1-858 and His-GI920-1173 proteins. Purified polyclonal α-GI antibody not only detected endogenous GI proteins inwild-type Arabidopsis plants, but also reenacted its dieloscillations. Furthermore, the antibody showed cross-reactivitywith the GI orthologs in other plants such as Chinesecabbage, rape and tomato. Our polyclonal GI antibody couldhelp to determine the molecular mechanisms of GI involvedin largely unknown pleiotropic responses in plants.

Humic Acid Confers HIGH-AFFINITY K+ TRANSPORTER 1-Mediated Salinity Stress Tolerance in Arabidopsis

Laila Khaleda,박희진,윤대진,전종록,김민갑,차준영,김외연 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.12

Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to func-tion in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.

Priming by Rhizobacterium Protects Tomato Plants from Biotrophic and Necrotrophic Pathogen Infections through Multiple Defense Mechanisms

Il-Pyung Ahn,Sang-Woo Lee,김민갑,Sang-Ryeol Park,황덕주,배신철 한국분자세포생물학회 2011 Molecules and cells Vol.32 No.1

A selected strain of rhizobacterium, Pseudomonas putida strain LSW17S (LSW17S), protects tomato plants (Lyco-persicon esculentum L. cv. Seokwang) from bacterial speck by biotrophic Pseudomonas syringae pv. tomato strain DC3000 (DC3000) and bacterial wilt by necrotrophic Ralstonia solanacearum KACC 10703 (Rs10703). To investigate defense mechanisms induced by LSW17S in tomato plants, transcription patterns of pathogenesis-related (PR) genes and H2O2 production were analyzed in plants treated with LSW17S and subsequent pathogen inoculation. LSW17S alone did not induce transcriptions of employed PR genes in leaves and roots. DC3000 challenge following LSW17S triggered rapid transcriptions of PR genes and H_2O_2 production in leaves and roots. Catalase infiltration with DC3000 attenuated defense-related responses and resistance against DC3000 infection. Despite depriving H_2O_2 production and PR1b transcription by the same treatment, resistance against Rs10703 infection was not deterred significantly. H_2O_2 is indispensable for defense signaling and/or mechanisms primed by LSW17S and inhibition of bacterial speck, however, it is not involved in resistance against bacterial wilt.

Coronatine Induces Stomatal Reopening by Inhibiting Hormone Signaling Pathways

Uddin Shahab,Bae Dongryeoul,차준영,안경익,김외연,김민갑 한국식물학회 2022 Journal of Plant Biology Vol.65 No.5

Coronatine (COR), a phytotoxin, aids Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) invasion by suppressing stomatal immunity, thus promoting bacterial replication. COR also promotes disease development, increases disease resistance in non-infected plant parts, suppresses cell wall defenses, and delays the hypersensitive response. Moreover, COR inhibits stomatal closure by influencing reactive oxygen species (ROS) synthesis, by NADPH oxidases RBOHD/F, which highlights the role of NADPH oxidases in guard cell signaling. Plant resistance to Pst DC3000 is dependent on guard cell-specific mitogen-activated protein kinases (MAPKs) that function downstream of ROS in salicylic acid (SA) and abscisic acid (ABA)-mediated stomatal closure. COR inhibits the ABA signal transduction pathway in guard cells, which is linked to pathogen-associated molecular pattern (PAMP)-induced stomatal closure. Furthermore, COR uses the plant jasmonate (JA) receptor coronatine-insensitive 1 (COI1) to activate multiple NAC transcription factors, which then regulate SA metabolic genes, preventing the accumulation of SA. In this review, we highlight the negative impact of COR on ROS production, SA and ABA signaling transduction, and consequently stomatal immunity. We believe that the inhibitory effects of COR on SA and ABA-induced stomatal closure require substantially more research to fully understand the stress adaptation mechanisms of plants.