SummaryInfection of a plant by a pathogen induces a variety of defense responses that imply the action of several signaling molecules, including salicylic acid (SA), jasmonic acid (JA) and ethylene (E). Here we describe the role of ETHYLENE-RESPONSE-FACTOR1 (ERF1) as a regulator of ethylene responses after pathogen attack in Arabidopsis. The ERF1 transcript is induced on infection by Botrytis cinerea, and overexpression of ERF1 in Arabidopsis is suf®cient to confer resistance to necrotrophic fungi such as B. cinerea and Plectosphaerella cucumerina. A positive co-operation between E and SA pathways was observed in the plant response to P. cucumerina. Infection by Pseudomonas syringae tomato DC3000, however, does not affect ERF1 expression, and activation of ethylene responses by ERF1 overexpression in Arabidopsis plants reduces tolerance against this pathogen, suggesting negative crosstalk between E and SA signaling pathways, and demonstrating that positive and negative interactions between both pathways can be established depending on the type of pathogen.

Cellulose is synthesized by cellulose synthases (CESAs) contained in plasma membrane-localized complexes. In Arabidopsis thaliana, three types of CESA subunits (CESA4/IRREGULAR XYLEM5 [IRX5], CESA7/IRX3, and CESA8/IRX1) are required for secondary cell wall formation. We report that mutations in these proteins conferred enhanced resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. By contrast, susceptibility to these pathogens was not altered in cell wall mutants of primary wall CESA subunits (CESA1, CESA3/ISOXABEN RESISTANT1 [IXR1], and CESA6/IXR2) or POWDERY MILDEW-RESISTANT5 (PMR5) and PMR6 genes. Double mutants indicated that irx-mediated resistance was independent of salicylic acid, ethylene, and jasmonate signaling. Comparative transcriptomic analyses identified a set of common irx upregulated genes, including a number of abscisic acid (ABA)-responsive, defenserelated genes encoding antibiotic peptides and enzymes involved in the synthesis and activation of antimicrobial secondary metabolites. These data as well as the increased susceptibility of ABA mutants (abi1-1, abi2-1, and aba1-6) to R. solanacearum support a direct role of ABA in resistance to this pathogen. Our results also indicate that alteration of secondary cell wall integrity by inhibiting cellulose synthesis leads to specific activation of novel defense pathways that contribute to the generation of an antimicrobial-enriched environment hostile to pathogens.

The peptide snakin-2 (StSN2) has been isolated from potato (Solanum tuberosum cv Jaerla) tubers and found to be active (EC 50 ϭ 1-20 m) against fungal and bacterial plant pathogens. It causes a rapid aggregation of both Gram-positive and Gram-negative bacteria. The corresponding StSN2 cDNA encodes a signal sequence followed by a 15-residue acidic sequence that precedes the mature StSN2 peptide, which is basic (isoelectric point ϭ 9.16) and 66 amino acid residues long (molecular weight of 7,025). The StSN2 gene is developmentally expressed in tubers, stems, flowers, shoot apex, and leaves, but not in roots, or stolons, and is locally up-regulated by wounding and by abscisic acid treatment. Expression of this gene is also up-regulated after infection of potato tubers with the compatible fungus Botritys cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum and Erwinia chrysanthemi. These observations are congruent with the hypothesis that the StSN2 is a component of both constitutive and inducible defense barriers.An important component of plant defense is a diverse set of constitutive and pathogen-inducible antimicrobial compounds that includes the so-called pathogenesis-related proteins, several families of antimicrobial peptides, a variety of chemically diverse organic compounds classified as phytoalexins and phytoanticipins, and certain active oxygen and nitrogen species (Osbourn, 1996(Osbourn, , 1999 Broekaert et al., 1997;Kombrink and Somssich, 1997; García-Olmedo et al., 1998). Accumulation of these compounds and the ability of a given pathogen to deal with them may be decisive for the outcome of the interaction (Titarenko et al., 1997a;Ló pez-Solanilla et al., 1998Miguel et al., 2000; Alamillo and García-Olmedo, 2001;García-Olmedo et al., 2001). Thus, it has been shown that increased levels of certain antimicrobial peptides, either through overexpression of the corresponding genes or by appropriate exogenous treatments, result in enhanced tolerance to particular pathogens (Carmona et al., 1993; Terras et al., 1995; Epple et al., 1997;Molina and García-Olmedo, 1997; Holtorf et al., 1998; Thomma et al., 1998 Thomma et al., , 1999.Furthermore, pathogen mutants that are sensitive to antimicrobial peptides show decreased virulence when inoculated in the plant (Titarenko et al., 1997a; Ló pez-Solanilla et al., 1998).Several families of antimicrobial peptides have been characterized in plants ( García-Olmedo et al., 1992, 1995 Broekaert et al., 1997). The majority of them are Cys-rich and their globular structure is stabilized by disulphide bridges, although linear Gly-/His-rich and macrocyclic Cysknot peptides have also been recently identified (Tam et al., 1999;Park et al., 2000). The peptides are generally encoded by multigenic families in which some genes are developmentally regulated in certain tissues, whereas others are pathogen inducible, and a number of them show both constitutive and pathogen-inducible expression (García-Olmedo et al., 1995 Broekaert et al., 1997).In a previous...

Ethylene response factor 1 (ERF1) is a transcriptional factor from Arabidopsis thaliana that regulates plant resistance to the necrotrophic fungi Botrytis cinerea and Plectosphaerella cucumerina and whose overexpression enhances resistance to these fungi. Here, we show that ERF1 also mediates Arabidopsis resistance to the soilborne fungi Fusarium oxysporum sp. conglutinans and F. oxysporum f. sp. lycopersici, because its constitutive expression in Arabidopsis confers enhanced resistance to these pathogens. Expression of ERF1 was upregulated after inoculation with F. oxysporum f. sp. conglutinans, and this response was blocked in ein2-5 and coi1-1 mutants, impaired in the ethylene (ET) and jasmonic acid (JA) signal pathways, respectively, which further indicates that ERF1 is a downstream component of ET and JA defense responses. The signal transduction network controlling resistance to F. oxysporum fungi was explored using signaling-defective mutants in ET (ein2-5), JA (jar1-1), and salicylic acid (SA) (NahG, sid2-1, eds5-1, npr1-1, pad4-1, eds1-1, and pad2-1) transduction pathways. This analysis revealed that Arabidopsis resistance to F. oxysporum requires the ET, JA, and SA signaling pathways and the NPR1 gene, although it is independent of the PAD4 and EDS1 functions.

Chitin is a major component of fungal walls and insect exoskeletons. Plants produce chitinases upon pathogen attack and chito-oligomers induce defense responses in plants, though the exact mechanism behind this response is unknown. Using the ATH1 Affymetrix microarrays consisting of about 23,000 genes, we examined the response of Arabidopsis (Arabidopsis thaliana) seedlings to chito-octamers and hydrolyzed chitin after 30 min of treatment. The expression patterns elicited by the chitooctamer and hydrolyzed chitin were similar. Microarray expression profiles for several genes were verified via northern analysis or quantitative reverse transcription-PCR. We characterized T-DNA insertion mutants for nine chito-oligomer responsive genes. Three of the mutants were more susceptible to the fungal pathogen, powdery mildew, than wild type as measured by conidiophore production. These three mutants included mutants of genes for two disease resistance-like proteins and a putative E3 ligase. The isolation of loss-of-function mutants with enhanced disease susceptibility provides direct evidence that the chito-octamer is an important oligosaccharide elicitor of plant defenses. Also, this study demonstrates the value of microarray data for identifying new components of uncharacterized signaling pathways.Plants in the environment are constantly under siege by a multitude of disease-causing organisms including bacteria, fungi, viruses, and nematodes. Plants may resist pathogen attack using both preformed defenses (e.g. antimicrobial compounds) and inducible defense responses (for review, see HammondKosack and Jones, 2000;Heath, 2000). Inducible defenses can be activated upon recognition of general elicitors such as bacterial flagellin (Gomez-Gomez and Boller, 2002), the polypeptide systemin (Ryan and Pearce, 1998), and multiple host or pathogen cell wall fragments released during pathogen attack (Nü rnberger et al., 2004). Recently, oligosaccharide elicitors such as chito-oligomers and oligogalacturonides have received renewed attention as important signals in plant defense responses. The activation of defense genes by these elicitors is thought to be receptor-mediated though little is known about the initial perception and consequent signaling pathways involved in plant cells.Chito-oligosaccharides can be generated from the cell walls of pathogenic fungi by the action of endochitinases and were shown to elicit strong defense responses in many plant species (Stacey and Shibuya, 1997;Shibuya and Minami, 2001). In a previous study, we showed that transcript levels for 71 expressed sequence tags, representing 61 genes, were altered more than 3-fold in chito-oligomer treated Arabidopsis (Arabidopsis thaliana) seedlings, demonstrating the usefulness of Arabidopsis as a model for studying chitin signaling in plants . Further experimentation by Zhang et al. (2002) showed that this response was not mediated by the well-characterized salicylic acid, jasmonic acid, or ethylene-responsive plant defense pathways. More recently, Wan et al. (...

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Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst.

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