Necrosis- and Ethylene-Inducing Peptide from Fusarium oxysporum Induces a Complex Cascade of Transcripts Associated with Signal Transduction and Cell Death in Arabidopsis[W] Hanhong Bae*, Moon S. Kim, Richard C. Sicher, Hyeun-Jong Bae, and Bryan A. Bailey U.S. Department of Agriculture/Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland 20705 (H.B., M.S.K., R.C.S., B.A.B.); and Department of Wood Science and Engineering, Chonnam University, Gwangju, Korea 500–757 (H.-J.B.) Treatment of Arabidopsis (Arabidopsis thaliana) with a necrosis- and ethylene-inducing peptide (Nep1) from Fusarium oxysporum inhibited both root and cotyledon growth and triggered cell death, thereby generating necrotic spots. Nep1-like proteins are produced by divergent microbes, many of which are plant pathogens. Nep1 in the plant was localized to the cell wall and cytosol based on immunolocalization results. The ratio of chlorophyll a fluorescence (F685 nm/F730 nm) significantly decreased after 75-min treatment with Nep1 in comparison to the control. This suggested that a short-term compensation of photosynthesis occurred in response to localized damage to cells. The concentrations of most water-soluble metabolites ana- lyzed were reduced in Arabidopsis seedlings after 6 h of Nep1 treatment, indicating that the integrity of cellular membranes had failed. Microarray results showed that short-term treatment with Nep1 altered expression of numerous genes encoding proteins putatively localized to organelles, especially the chloroplast and mitochondria. Short-term treatment with Nep1 induced multiple classes of genes involved in reactive oxygen species production, signal transduction, ethylene biosynthesis, membrane modification, apoptosis, and stress. Quantitative PCR was used to confirm the induction of genes localized in the chloroplast, mitochondria, and plasma membrane, and genes responsive to calcium/calmodulin complexes, ethylene, jasmonate, ethylene biosynthesis, WRKY, and cell death. The majority of Nep1-induced genes has been associated with general stress responses but has not been critically linked to resistance to plant disease. These results are consistent with Nep1 facilitating cell death as a component of diseases caused by necrotrophic plant pathogens. The fungal plant pathogen Fusarium oxysporum pro- Plant responses to Nep1 include induction of pathogen- duces necrosis- and ethylene-inducing peptide (Nep1), related (PR) genes, changes in K1 and H1 channel a 24-kD necrosis and ethylene-inducing peptide (Bailey, fluxes, callose apposition, accumulation of reactive 1995; Bailey et al., 1997). Nep1 and Nep1-like proteins oxygen species (ROS) and ethylene, altered cell respi- (NLPs) from other microbes cause necrosis and induce ration, the hypersensitive response, and localized cell ethylene production in dicots but are inactive in mono- death (Jennings et al., 2001; Fellbrich et al., 2002; Keates cots (Bailey, 1995; Veit et al., 2001). NLPs have been et al., 2003). In Arabidopsis (Arabidopsis thaliana), spotted identified in many different microorganisms includ- knapweed (Centaurea maculosa), and dandelion (Tarax- ing fungi and bacteria (for review, see Pemberton and acum officinale), Nep1 caused the breakdown of the Salmond, 2004). Bae et al. (2005a) identified complex cuticle layer and internal chloroplast membrane struc- multiple copies of NEP1 orthologs in five Phytoph- tures 1 to 4 h after treatment began (Keates et al., 2003). thora species. Phytopthera sojae necrosis-inducing pro- Various genes involved in plant stress responses in- tein was produced during the necrotrophic phase of cluding wounding, drought, senescence, and disease infection in soybean (Glycine max; Qutob et al., 2002). resistance, were also induced (Keates et al., 2003). A The presence of multiple NLPs in various plant path- related peptide, NPP1, induced rapid accumulation of ogenic microorganisms suggests a significant role for the salicylic acid (SA)-dependent PR1 transcript in these genes in pathogenicity. Arabidopsis (Fellbrich et al., 2002). The objective of this study was to characterize the responses of Arabidopsis to Nep1 with respect to * Corresponding author; e-mail [email protected]; fax 301– changes in plant growth, ultrastructural modifications, 504–1998. metabolite levels, and transcription profiles. Only a The author responsible for the distribution of materials integral to handful of genes that respond to Nep1 treatment have the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: been identified to date, even though the effects of Nep1 Hanhong Bae ([email protected]). on plants can be dramatic. In this investigation, high- [W] The online version of this article contains Web-only data. throughput screening based on DNA microarray tech- Article, publication date, and citation information can be found at nology was used to identify Nep1 responsive genes in www.plantphysiol.org/cgi/doi/10.1104/pp.106.076869. Arabidopsis. In addition, chlorophyll a fluorescence 1056 Plant Physiology, July 2006, Vol. 141, pp. 1056–1067, www.plantphysiol.org Ó 2006 American Society of Plant Biologists Necrosis- and Ethylene-Inducing Peptide-Induced Cell Death imaging was employed as a nondestructive method seedlings, suggesting that callose formation had oc- to assess plant vigor subsequent to Nep1 treatment. curred (Fig. 1, E and L). Nep1 treatment altered the Molecular and biochemical analyses of the response of shape of cells on the root surfaces resulting in distorted Arabidopsis to Nep1 should enhance our understand- root growth. When grown under light, cotyledon de- ing of the signaling networks that are involved in plant velopment and hypocotyl elongation were inhibited responses to Nep1 and NLPs, a family of proteins of 79% and 55% by Nep1 treatment, respectively (Fig. 2). increasing importance in many different plant microbe The inhibitory effects of Nep1 on root growth were interactions. severe (92% inhibition), and the inhibition was greater in light-grown than in dark-grown seedlings (Fig. 1, A and H). However, there was negligible inhibition of cotyledon development and hypocotyl elongation in RESULTS AND DISCUSSION response to Nep1 using dark-grown seedlings (Fig. Nep1 Inhibits Seedling Growth 1H). Nep1 penetrates plant tissues via openings such as and Root Development stomata, hydathodes, or wounds (Bailey et al., 2000; Jennings et al., 2001). Developing roots have many To assess the effects of Nep1 on plant growth, Arabi- penetration points, including areas where lateral roots dopsis seedlings were grown for 5 d on agarose plates emerge and root tips that resemble wounded tissues containing 1 3 Murashige and Skoog (MS) basal salts (Scheres et al., 2001). Nep1 was able to damage the root supplemented with 1% Suc. When Arabidopsis seed- epidermis and cortex. However, Nep1 was unable to lings were treated with Nep1, the growth of roots and penetrate the casparian strip and enter the xylem of cotyledons was inhibited in comparison to control plants unwounded roots. Nep1 was shown to be transported (Figs. 1 and 2). Necrotic spots were visible on cotyledons through the xylem if access to this tissue was available of Nep1-treated seedlings (Fig. 1A, inset), particularly (Jennings et al., 2001). Etiolated Arabidopsis seedlings in association with stomata (Fig. 1C). The formation of have unexpanded cotyledons with minimal mature necrotic spots was also observed on cotyledons of dark- stomata, thereby limiting penetration of Nep1 through grown seedlings (Fig. 1J). Nep1 treatment inhibited the these points (Nadeau and Sack, 2001). The movement formation of root hairs and damaged root tips both of Nep1 from the root into the xylem and to the shoot under dark and light conditions (Fig. 1, E, G, and L). has not been demonstrated. This would require signif- White deposits were detected in roots of Nep1-treated icant movement across cell membranes and between Figure 1. Effect of F. oxysporum Nep1 on Arabidopsis growth. Nep1 treatment caused necrotic lesions and inhibited cotyledon and root growth. Sterilized Arabidopsis seeds were mixed with Nep1 (20 mgmL21), 0.001% (v/v) Silwet-L77, 0.3% agarose, 1% Suc in 1 3 MS media, and plated onto petri dishes. Controls were treated with 0.001% (v/v) Silwet-L77. Seeds were germinated and grown in growth cabinets at 22°C for 5 d under fluorescent lights providing 150 6 10 mmol m22 s21 PAR with 16 h light or under dark condition. A, Control seedling (left, no Nep1) and Nep1-treated seedling (right). Inset shows necrotic spots on cotyledons. B, Normal stomata. C, Necrotic region on Nep1-treated cotyledon. D, Normal roots with root hairs. E, Damaged root without root hairs from Nep1-treated seedling. F, Normal root tip. G, Abnormal root tip from Nep1-treated seedling. H, Control (left) and Nep1-treated (right), etiolated seedlings. I, Normal cotyledon of etiolated seedling. J, Cotyledon with necrotic regions in Nep1-treated, etiolated seedling. K, Normal root hairs from etiolated control seedling. L, Abnormal root without root hairs in Nep1-treated etiolated seedling. Growth conditions, A to G, 16 h light; H to L, dark. Plant Physiol. Vol. 141, 2006 1057 Bae et al. lum. This result indicates that Nep1 can penetrate through plant cells but may be not able to penetrate into organelles. Nep1 Causes Large-Scale Leakage of Cell Metabolites Changes of metabolites in Arabidopsis seedlings were analyzed with gas chromatography