The secreted antifungal protein thionin 2.4 in Arabidopsis thaliana suppresses the toxicity of a fungal fruit body lectin from Fusarium graminearum

PLoS Pathog. 2013;9(8):e1003581. doi: 10.1371/journal.ppat.1003581. Epub 2013 Aug 22.

Abstract

Plants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4) antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimicrobial Cationic Peptides / biosynthesis*
  • Antimicrobial Cationic Peptides / genetics
  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis / microbiology
  • Arabidopsis Proteins / biosynthesis*
  • Arabidopsis Proteins / genetics
  • Cell Death
  • Flowers / genetics
  • Flowers / metabolism
  • Fruiting Bodies, Fungal
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Fusarium / metabolism*
  • Fusarium / pathogenicity
  • Gene Expression Regulation, Plant / genetics
  • Lectins / genetics
  • Lectins / metabolism*
  • Organ Specificity / genetics
  • Plant Diseases*
  • Plant Leaves / genetics
  • Plant Leaves / metabolism
  • Plant Leaves / microbiology
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Plants, Genetically Modified / microbiology

Substances

  • Antimicrobial Cationic Peptides
  • Arabidopsis Proteins
  • Fungal Proteins
  • Lectins

Grants and funding

This work was supported by Grants-in-Aid for Young Scientists (no. 21770038) and Scientific Research (no. 23580060) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also supported in part by Research for Promoting Technological Seeds (no. 07-070) and by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (AS232Z02753E and AS242Z03390N) from the Japan Science and Technology Agency (JST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.