An endogenous peptide signal in Arabidopsis activates components of the innate immune response

Abstract

Innate immunity is initiated in animals and plants through the recognition of a variety of pathogen-associated molecules that in animals are called pathogen-associated molecular patterns and in plants are called elicitors. Some plant pathogen-derived elicitors have been identified as peptides, but peptide elicitors derived from the plant itself that activate defensive genes against pathogens have not been previously identified. Here, we report the isolation and characterization of a 23-aa peptide from Arabidopsis, called AtPep1, which activates transcription of the defensive gene defensin (PDF1.2) and activates the synthesis of H(2)O(2), both being components of the innate immune response. The peptide is derived from a 92-aa precursor encoded within a small gene that is inducible by wounding, methyl jasmonate, and ethylene. Constitutive expression of the AtPep1 precursor gene PROPEP1 in transgenic Arabidopsis plants causes a constitutive transcription of PDF1.2. When grown in soil, the transgenic plants exhibited an increased root development compared with WT plants and an enhanced resistance toward the root pathogen Pythium irregulare. Six paralogs of PROPEP1 are present in Arabidopsis, and orthologs have been identified in species of several agriculturally important plant families, where they are of interest for their possible use in crop improvement.

Fig. 1.

Isolation of AtPep1. (A) Peptides present in an 1% trifluoroacetic acid/water extract of Arabidopsis tissues were passed through a reverse-phase semipreparative C18 flash chromatography column and separated on a G-25 Sepharose column as described in Materials and Methods. The breakthrough peak was applied to a C18 HPLC column, and 10 μl from 2-ml fractions from the column was assayed for alkalinization activity. (B) The peak identified in A as AtPep1 was further purified through two additional chromatography steps and finally purified by narrow-bore HPLC as described in Materials and Methods. Fractions were assayed as in A. The active peak is identified with arrows. (C) Analysis of the biologically active peak by MALDI-MS. (D) The amino acid sequence of the purified peptide, determined by Edman degradation. The daltons calculated from the amino acid sequence matched that determined by MS.

Fig. 2.

AtPep1 precursor gene expression. (A) The amino acid sequence of the AtPep1 precursor protein PROPEP1 was encoded by the annotated gene At5g64900. The AtPep1 sequence at the carboxyl terminus of the precursor protein is underlined. (B) Semiquantitative RT-PCR analysis of PROPEP1 expression in response to wounding and treatment of leaves with MeJA, ethephon, and AtPep1. Relative abundance of the PROPEP1 transcript was estimated from the expression of the β-tubulin gene as a control. Leaves were wounded by crushing once across the midvein with a hemostat. Plants were sprayed with a 250 μM solution of MeJA in 0.1% Triton X-100, sprayed with a 7 mM solution of ethephon in 0.1% Triton X-100, or supplied throughout petioles with 10 nM AtPep1 in water.

Fig. 3.

AtPep1 regulates defense gene expression. (A) Fold induction of expression of PROPEP1 and PDF1.2 in excised Arabidopsis leaves supplied for 2 h with 10 nM AtPep1 through their cut petioles. Transcript levels were analyzed for expression levels of the two genes relative to their expression in excised leaves supplied with water. Expression was determined by semiquantitative RT-PCR with a β-tubulin gene as a control. (B) AtPep1-induced expression of PDF1.2 and AtproPep1 in leaves of WT plants, jasmonate-deficient fad3,7,8 triple mutant plants, and ethylene-insensitive ein2-1 mutant plants. AtPep1 was supplied for 2 h at 10 nM, and RNA was isolated and assayed as above. (C) Accumulation of H₂O₂ in leaves supplied for 2 h with water, 10 nM AtPep1, or 10 nM AtPep1 plus 100 μM DPI, an inhibitor of NADPH oxidase. Each treatment contained 1 mg/ml of diaminobenzidine (DAB) to visualize H₂O₂ accumulation. Leaves treated with AtPep1 and DAB were cosupplied with 100 μM DPI. (D) Expression of AtproPep1 and PDF1.2 in excised leaves of WT plants in response to supplying 10 nM AtPep1 in the presence or absence of DPI. The expression of each gene was analyzed by semiquantative RT-PCR.

Fig. 4.

Arabidopsis plants constitutively overexpressing PROPEP1 and PROPEP2 exhibit increased root and aerial growth over WT plants grown in potting soil with and without inoculation with the pathogen P. irregulare. (A) Root masses of WT (WT Col) and lines 1–3 of plants transformed with a CaMV 35S:PROPEP1 chimeric gene. Four plants were grown per pot in soil for 21 days, and the soil was removed by rinsing in a water bath until soil no longer could be washed from the combined root mass. The aerial portions of the plants were excised before photographing them. (B) (Upper) Rosettes of WT plants and transformed plants as in A 3.5 weeks after inoculation with P. irregulare strain 110305 or water. (Lower) Roots from plants treated as in Upper 3.5 weeks after inoculation. Soil was washed from the total root mass, and then the roots of each plant were carefully separated while immersed in water and photographed. (C) WT plants (Left), transgenic line 3 (Center), and transgenic line 8 (Right) of plants transformed with CaMV 35:PROPEP2. Four plants were grown per pot in soil for 4 weeks, and the soil was gently washed from the total root mass as in A before photographing them.

Fig. 5.

A cladogram showing the relationships of PROPEP1 (At5g64900) paralogs and orthologs estimated from their amino acid identities and similarities. GenBank accession numbers are as follows: for dicot genes, canola (Brassica napus) CD816645; potato (Solanum tuberosum) CV505388; poplar (Populus balsamifera) CV23975; medicago (Medicago sativa) BI311441; soybean (Glycine max) CD401281; and grape (Vitis vinifera) CF604664; for monocot genes, rice1 (Oryza sativa) CF333408; rice2 AK111113, wheat1 (Triticum aestivum) AL809059; wheat2 BF201609, maize (Zea mays) DN215793; and barley (Hordeum vulgare) BQ763246.