Plants

Discovery of compounds that suppress plant immune responses | RIKEN

December 15, 2021

Riken
Okayama University

– A new tool for elucidating plant immune responses –

Ken Shirasu, head of the Plant Immunology Research Group, RIKEN Center for Sustainable Resource Science, International Collaborative Research Group, Nobuaki Ishihama, and Yoshiteru Nonen, Research Professor, Department of Environmental and Life Sciences, Okayama University Academic Institute Human NSAIDs[1]We found that tenoxicam inhibits plant immune responses.

The results of this study are expected to help understand the plant’s immune system and develop techniques for controlling disease resistance.

Salicylic acid, known as an NSAID, is an endogenous signaling molecule that enhances disease resistance in plants. However, the mechanism by which salicylic acid transmits signals in plant cells remains unclear.

This time, the International Collaborative Research Group has added a new compound library.[2]We have identified three oxicam-based NSAIDs (tenoxicam, meloxicam, and piroxicam) that are similar in chemical structure to compounds that inhibit plant immune responses. In addition, we found that tenoxicam tilted the intracellular redox state to the oxidative side and broadly inhibited the gene cluster with increased expression of salicylic acid, elucidating part of the signal transduction mechanism of salicylic acid.

This research is based on the online scientific journal “Nature Communications‘ (December 15) will be published.

Schematic diagram of tenoxicam inhibiting salicylic acid-dependent plant immune response

Inhibitory effect of tenoxicam on salicylic acid-dependent plant immune response

background

When a plant recognizes the infectious behavior of a pathogen, it induces a biodefense response to prevent the infection and growth of the pathogen. This defense system is called “plant immunity” because of its many similarities to the innate immune system of animal cells.

Salicylic acid, known as a human non-steroidal anti-inflammatory drug, has been used for over 2,000 years as an antipyretic analgesic derived from willow bark. On the other hand, in plants, salicylic acid is an endogenous signaling molecule and a transcriptional cofactor.[3]It has the function of activating plant immune responses through NPR1. However, the role of NPR1 in salicylic acid-dependent signaling remains unclear. If compounds that interfere with this signaling pathway can be identified, it could serve as a key to advancing the understanding of plant immune systems and developing technologies to control disease resistance in crops.

Research Methods and Results

In 2012, an international collaborative research team added drugs and a pathogenic bacteria (patella variegata) to suspension culture cells of the model plant Arabidopsis thaliana.[4]), a high-throughput assay that quantitatively tests programmed cell death, one of the resistance responses exhibited by cultured cells.[5]developedNote 1).

This assay system is used to search for bioactive substances that disrupt plant immune responses from a compound library of approximately 2,800 small molecule compounds. The selected compounds and foliar fungi were then inoculated into the plants to study the effect on bacterial growth. Therefore, we identified three NSAIDs, the oxicam compounds (tenoxicam, meloxicam, and piroxicam), that promote bacterial growth and suppress plant immune responses (Figure 1).

Chemical structures of xicam compounds and their effects on plant immune responses

Figure 1 Chemical structures of oxicam compounds and their effects on plant immune responses

  • (A sort of)Chemical structures of salicylic acid and oxycam compounds.
  • (two)Bacterial fungi of tomato spotted leaves were infected with wild-type Arabidopsis thaliana in the presence of the xicam compound shown in a, and the bacterial counts in the plants were examined 3 days later.Asterisk
  • Indicates a statistically significant difference compared to the control. The oxicam compound increases the growth of pathogenic bacteria.

Salicylic acid is a defense-related gene after infection with plant pathogensPR 1Induced expression.Tenoxicam induces salicylic acidPR 1

Tenoxicam may inhibit salicylic acid signaling due to suppressed gene expression (Figure 2a). A comprehensive analysis of the effect of tenoxicam on salicylic acid-induced fluctuations in gene expression revealed that, of 631 NPR1-dependent genes, 313 (49.6%) of the genes whose expression was increased by salicylic acid were affected by tenoxicam. Kang inhibited (Fig. 2b).

Schematic diagram of tenoxicam inhibiting salicylic acid-dependent gene expression

    Figure 2 Inhibitory effect of tenoxicam on salicylic acid-dependent gene expression


  • (A sort of)Infection of wild-type Arabidopsis thaliana in the presence of tenoxicam results in tomato variegated bacterial disease in infected tissues 1 day laterPR 1Check gene expression levels. Groups a and b showed no statistically significant difference.Add TenoxicamPR 1
  • The expression level of this gene was found to be reduced.


  • (two)
  • Comprehensive analysis of Arabidopsis genes whose expression levels fluctuated by salicylic acid and/or tenoxicam treatment. Of the 631 NPR1-dependent genes whose expression was increased by salicylic acid, 313 genes were found to be repressed by tenoxicam.

[6]The intracellular redox state of plants was temporarily tilted to the oxidized side after salicylic acid treatment, and then significantly shifted to the reducing side. At this time, glutathione, the main antioxidant component in cells,

As the total amount of glutathione increased, the proportion of reduced glutathione increased. However, when tenoxicam was added, the increase in the amount of total glutathione and the increase in the proportion of reduced glutathione in response to salicylic acid treatment were suppressed (Fig. 3).

The effect of tenoxicam on the amount of intracellular glutathione

    Figure 3 The effect of tenoxicam on the level of intracellular glutathione


  • (A sort of)
  • Wild-type Arabidopsis was treated with salicylic acid and/or tenoxicam, and total intracellular glutathione levels were measured 1 day later. The addition of tenoxicam was found to inhibit the increase in total glutathione amount in response to salicylic acid treatment. NT stands for unhandled control.Asterisk

  • Indicates that there is a statistically significant difference between the samples.
  • (two)

Wild-type Arabidopsis was treated with salicylic acid and/or tenoxicam and the ratio of reduced to oxidized forms of intracellular glutathione was examined after 1 day. The addition of tenoxicam was found to inhibit the increase in the ratio of reduced glutathione in response to salicylic acid treatment.[7]
[8]NPR1 is a protein that contains a large number of cysteine ​​residues that form disulfide bonds depending on the redox state within the cell.

The possibility of forming has always been advocated. This time, whether the redox state of cysteine ​​residues in NPR1 is altered depends on treatment with salicylic acid or tenoxicam, making new free cysteine ​​residues into high-molecular-weight maleyl imine.

It was investigated in detail using the labelled method (Fig. 4a). It was found that the cysteine ​​residue in NPR1 was always in the reduced form with or without salicylic acid or tenoxicam treatment (Fig. 4b). Therefore, the effect of tenoxicam is not related to the redox state of cysteine ​​residues in NPR1.

    Effect of salicylic acid and tenoxicam on the redox state of cysteine ​​residues in NPR1

  • Figure 4 Effects of salicylic acid and tenoxicam on the redox state of cysteine ​​residues in NPR1
  • (A sort of)

  • Polyethylene glycol (PEG) is added to cysteine ​​residues of proteins to increase molecular weight and alter mobility in gels when separated by electrophoresis. By comparing the mobilities of sample 3) labeled with only free cysteine ​​residues and sample 4) labeled with all cysteine ​​residues after reduction, it can be seen whether disulfide bonds are present in proteins in vivo.
  • (two)

Arabidopsis thaliana expressing the fusion protein of green fluorescent protein and NPR1 was treated with salicylic acid and/or tenoxicam, and the protein was extracted from plant tissue 1 day later. The labeled samples were prepared according to the protocol of (a). After separation by SDS-PAGE gel, NPR1 protein was detected using anti-NPR1 antibody. The mobility of PEG-labeled NPR1 in the gel was constant with or without salicylic acid or tenoxicam treatment, indicating that the cysteine ​​residues in NPR1 are always in reduced form.

[9]future expectations

The NSAIDs salicylic acid and tenoxicam are both human cyclooxygenases.[10]Although they show antipyretic and analgesic effects, this study shows that they have opposite positive and negative effects on plant immunity, respectively. Since plants do not have cyclooxygenase, tenoxicam is expected to target different proteins in plants than in humans. In the future, by identifying the targets of tenoxicam, we are expected to discover new targets of salicylic acid or new components of salicylic acid signal transduction.[11]In addition, the phytoimmunosuppressive effect of tenoxicam is Agrobacterium.

Efficiency of gene transfer into plants can be improved through legal, genome editing[12]It is expected to accelerate the development of functional crops.

The results of this research are the 17 “Sustainable Development Goals (SDGs)” announced by the United Nations in 2016.

    It can be expected to make a great contribution to “2. Zero Hunger” and “15. Protecting the Land of Abundance”.

  • Supplementary Instructions

    1.
  • NSAIDs

  • A drug that inhibits the production of prostaglandins, which act as signal transduction substances for human inflammation and have anti-inflammatory, antipyretic and analgesic effects.

    2.
  • compound library

  • A group of compounds of interest when searching for a specific biologically active substance.

    3.
  • transcription cofactor

  • A protein that binds to specific transcription factors and activates or represses gene transcription.

    4.
  • variegated bacteria

  • A pathogen that mainly causes spots on tomato fruit, petioles, petioles and leaves. Since it also infects Arabidopsis, it was used as a model pathogen in the study.

    5.
  • high-throughput assay

  • An experimental system that can evaluate the physiological effects of a large number of compounds in a short period of time.

    6.
  • glutathione

  • A tripeptide composed of three amino acids glutamic acid, cysteine ​​and glycine. It is the main antioxidant component in cells, with a reduced form with a free thiol group and an oxidized form with two molecules of glutathione linked by disulfide bonds. The cytoplasm usually contains high concentrations of reduced glutathione and is maintained in a reducing environment.

    7.
  • disulfide bond

  • A covalent bond formed by oxidation between the thiol groups of cysteine. It is known to contribute to the stability and function of the three-dimensional structure of proteins. It is cleaved by reductions such as reduced glutathione.

    8.
  • polymer maleimide

  • A labeling reagent in which maleimide, which selectively reacts with thiol groups under neutral conditions, is added to a high molecular weight polymer such as polyethylene glycol. Depending on the number of free thiol groups in the protein, the molecular weight of the protein increases.

    9.
  • cyclooxygenase

  • It is an enzyme complex involved in prostaglandin biosynthesis with two distinct enzymatic activities, cyclooxygenase activity and peroxidase activity.

    10.
  • Agrobacterium

  • It is a soil bacterium belonging to the genus Rhizobium that has the ability to infect plant cells and insert a DNA segment called T-DNA into the plant genome. In the field of biotechnology, it is used to insert arbitrary DNA fragments into the genome of plants.

    11.
  • genome editing

  • Any technique that modifies the DNA sequence of an organism’s genome.

    12.
  • Sustainable Development Goals (SDGs)

International goals for the period 2016 to 2030 are set out in the 2030 Agenda for Sustainable Development, adopted at the United Nations summit in September 2015. In a sustainable world composed of 17 goals and 169 goals, not only developing countries, but also developed countries themselves are working hard, and Japan is also actively working hard. This is a common thing. (Reproduced from the website of the Ministry of Foreign Affairs, with minor modifications)


International Cooperation Research Group
RIKEN Sustainable Resources Science Center
Plant Immunology Research Group
Group Director Ken Shirasu
(Deputy Director, Center for Sustainable Resources Science, Institute of Physics and Chemistry)
Researcher Nobuaki Ishihama
Researcher (at the time of research) Cui Keji
Researcher (while researching) Ivana Saska
Senior Researcher Shuta Asai
Technician II Kaori Takizawa

Chemical Biology Research Group
Group Chairman Tian Bozhi

Okayama University Department of Environmental and Life Sciences (Agriculture)
Photo of research professor Neng Nian
Michigan State University

Professor (at the time of research) He Shengyang

(Howard Hughes Medical Institute Investigator)

research support

    This research was funded by the Japan Society for the Promotion of Science (JSPS) Scientific Research Fundamental Research (S) “Molecular Mechanisms of Offensive and Defense of Plants and Pathogens (Research Leader: Ken Shirasu)” and Basic Research (S) “Plant Immune System” (Research Representative : Ken Shirasu) “, ​​Ministry of Education, Culture, Sports, Science and Technology New Academic Field Research (Type of Research Field Proposal)” Molecular Mechanism “Independent and Decentralized Control System for Environmental Recognition and Memory Supporting Plant Growth Plasticity (Representative in Research Field: Kinoshita) Toshinori)” plan Research “Elucidation of Canal Bundle Information Hijacking Mechanisms of Parasitic Plants (Research Representative: Ken Shirasu)”, Research Translation in Academic Field (A) “Multi-level Information Program Research Supporting Plant Resilience to Heterogeneous Environmental Changes” Molecules Controlled Mechanisms (representative of research: Tomohiro Matsushita)” “Mechanisms of infection control of parasitic plants in response to heterogeneous soil environments (representative of research: Satoko Yoshida)”, research in the same specific field “Plant environment” Organella differentiation as an adaptation strategy (research on Field representative: Mikio Nishimura)”, open call for research “Elucidation of Plant Immunosignal crosstalk in chloroplasts by Chemicalgenetics (Research representative: Ken Shirasu)” I am.

  • Base paper information
    Nobuaki Ishihama, Seung-won Choi, Yoshiteru Nooutoshi, Ivana Saska, Shuta Asai, Kaori Takizawa, Sheng Yang He, Hiroyuki Osada, Ken Shirasu, “Oxicam-type NSAIDs inhibit NPR1-mediated salicylic acid pathway”,
  • Nature Communications

10.1038/s41467-021-27489-w


host
Riken
RIKEN Sustainable Resource Science Center Plant Immunology Research Group
Group Director Ken Shirasu

(Deputy Director, Center for Sustainable Resources Science, Institute of Physics and Chemistry)
Researcher Nobuaki Ishihama

Okayama University Department of Environmental and Life Sciences (Agriculture)
Photo of research professor Neng Nian
Photo of Ken Shirasu Group Director
white beard
Photo of Researcher Nobuaki Ishihama
Ishihama Nobuaki

Photo of Professor Nai Nian Fanghui

Noh Yoshiki
according to

Riken Public Relations Press
Questionnaire
Publicity Division, General Affairs Planning Department, Okayama University [at] Tel: 086-251-8415 / Fax: 086-251-7294

Email: www-adm

adm.okayama-u.ac.jp



Industrial Use Consulting Questionnaire

About the author

Nkinfoweb

Leave a Comment