Plants possess inducible systemic defense responses when locally infected by pathogens. Bacterial infection results in the increased accumulation of the mobile metabolite azelaic acid, a nine-carbon dicarboxylic acid, in the vascular sap of Arabidopsis that confers local and systemic resistance against the pathogen Pseudomonas syringae. Azelaic acid primes plants to accumulate salicylic acid (SA), a known defense signal, upon infection. Mutation of the AZELAIC ACID INDUCED 1 (AZI1) gene, which is induced by azelaic acid, results in the specific loss of systemic immunity triggered by pathogen or azelaic acid and of the priming of SA induction in plants. Furthermore, the predicted secreted protein AZI1 is also important for generating vascular sap that confers disease resistance. Thus, azelaic acid and AZI1 are components of plant systemic immunity involved in priming defenses.

A novel pathogen-induced gene encoding the RAV (Related to ABI3/VP1) transcription factor, CARAV1, was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria. CARAV1 contains two distinct DNA-binding domains AP2 and B3 uniquely found in higher plants. Transient expression analysis of the smGFP:CARAV1 fusion construct in Arabidopsis protoplasts and pepper epidermal cells revealed the CARAV1 protein to be localized in the nucleus. The N-terminal region of CARAV1 fused to the GAL4 DNA-binding domain was required to activate transcription of reporter genes in yeast. In yeast one-hybrid, the recognition of CAACA and CACCTG motifs also were essential for the CARAV1 protein to bind to a specific target gene and activate the reporter gene. The expression of the CARAV1 gene was strongly induced early in pepper leaves during the pathogen infection, abiotic elicitors and environmental stresses. CARAV1 transcripts were localized in the phloem cells of leaf tissues during pathogen infection and ethylene treatment. Ectopic expression of the CARAV1 gene in transgenic Arabidopsis plants induced some PR genes and enhanced resistance against infection by Pseudomonas syringae pv. tomato DC3000 and osmotic stresses by high salinity and dehydration. The CARAV1 promoter activation was induced by P. syringae pv. tabaci, salicylic acid and abscisic acid. These data suggest that pathogen- and abiotic stress-inducible CARAV1 functions as a transcriptional activator triggering resistance to bacterial infection and tolerance to osmotic stresses.

The three cDNA clones, CALTPI , CALTPII , and CALT-PIII , corresponding to pepper lipid transfer protein (LTP) genes were isolated from a pepper ( Capsicum annuum ) cDNA library from hypersensitive response (HR) lesions of leaves infected with Xanthomonas campestris pv. vesicatoria . The CALTP genes are well conserved in their coding region with 57-72% identity at the amino acid level, but display 72-83% identity at the nucleotide sequence level. The transcripts of the three CALTP genes differentially accumulated in pepper leaf, stem, and fruit tissues infected by X. campestris pv. vesicatoria , Phytophthora capsici and Colletotrichum gloeosporioides . The CALTP genes were also strongly induced in the systemic, upper leaves after immunization on lower leaves by either pathogenic or nonpathogenic bacteria. In situ hybridization results showed that the CALTPI mRNA was localized in phloem cells of vascular tissues in pepper leaf, stem and fruit tissues after pathogen infection. CALTPI and CALTPIII genes were predominantly expressed in various pepper tissues infected by pathogens, while infection by P. capsici and C. gloeosporioides did not induce the transcription of the CALTPII gene. Ethylene, methyl jasmonate and abscisic acid induced CALTPI and III gene expression in pepper leaves. Drought, high salinity, low temperature and wounding stresses also induced the expression of the CALTPI and CALTPIII genes in a similar manner. In contrast, only high salinity induced the CALTPII expression that was not generally affected by abiotic and other environmental stimuli. When compared with each other and with LTPs from other plants, CALTPI is more distantly related than CALTPII and CALTPIII sequences, indicating that the three pepper CALTP genes represent two different classes. These results thus show that CALTPI and CALTPIII genes, although different in sequence structure, are transcriptionally activated in pepper tissues by pathogen infection as well as abiotic and environmental stresses.

Leucine-rich repeat proteins (LRRs) function in a number of signal transduction pathways via protein-protein interactions. The gene encoding a small protein of pepper, CaLRR1, is specifically induced upon pathogen challenge and treatment with pathogen-associated molecular patterns (PAMPs). We identified a pepper hypersensitive induced reaction (CaHIR1) protein that interacts with the LRR domain of the CaLRR1 protein using yeast two-hybrid screening. Ectopic expression of the pepper CaHIR1 gene induces cell death in tobacco and Arabidopsis, indicating that the CaHIR1 protein may be a positive regulator of HR-like cell death. Because transformation is very difficult in pepper plants, we over-expressed CaLRR1 and CaHIR1 in Arabidopsis to determine cellular functions of the two genes. The over-expression of the CaHIR1 gene, but not the CaLRR1 gene, in transgenic Arabidopsis confers disease resistance in response to Pseudomonas syringae infection, accompanied by the strong expression of PR genes, the accumulation of both salicylic acid and H(2)O(2), and K(+) efflux in plant cells. In Arabidopsis and tobacco plants over-expressing both CaHIR1 and CaLRR1, the CaLRR1 protein suppresses not only CaHIR1-induced cell death, but also PR gene expression elicited by CaHIR1 via its association with HIR protein. We propose that the CaLRR1 protein functions as a novel negative regulator of CaHIR1-mediated cell death responses in plants.

Effector proteins injected by the pathogenic bacteria Pseudomonas syringae into plants can have profound effects on the pathogen-host interaction due to their efficient recognition by plants and the subsequent triggering of defenses. The AvrRpt2 effector triggers strong local and systemic defense (called systemic acquired resistance [SAR]) responses in Arabidopsis thaliana plants that harbor a functional RPS2 gene that encodes an R protein in the coiled-coil, nucleotide-binding domain, leucine-rich repeat class. The newly identified win3-T mutant shows greatly reduced resistance to P syringae carrying avrRpt2. In win3-T plants, RIN4 cleavage, an early AvrRpt2-induced event, is normal. However, salicylic acid accumulation is compromised, as is SAR induction and the local hypersensitive cell death response after infection by P syringae carrying avrRpt2. WIN3 encodes a member of the firefly luciferase protein superfamily. Expression of WIN3 at an infection site partially requires PAD4, a protein known to play a quantitative role in RPS2-mediated signaling. WIN3 expression in tissue distal to an infection site requires multiple salicylic acid regulatory genes. Finally, win3-T plants show modestly increased susceptibility to virulent P syringae and modestly reduced SAR in response to P. syringae carrying avrRpm1. Thus, WIN3 is a key element of the RPS2 defense response pathway and a basal and systemic defense component.

Specific cDNAs showing differential expression in bacteria-infected pepper leaves as opposed to healthy leaves were isolated from a pepper cDNA library from hypersensitive response (HR) lesions of leaves infected with an avirulent strain of Xanthomonas campestris pv. vesicatoria. Among a total of 282 cDNA clones tested, 36 individual cDNA genes (13%) hybridized strongly or differentially to the cDNA probes from bacteria-infected leaves. Ten Capsicum Annuum-Induced (CAI) genes encoding putative thionin, lipid transfer protein I and II, osmotin (PR-5), class I chitinase, beta-1,3-glucanase, SAR 8.2, stellacyanin, leucine-rich repeat protein, and auxin-repressed protein were identified. Two CAI genes showed little or no sequence homology to the previously sequenced plant genes. Transcripts of the CAI genes were strongly or preferentially induced in pepper tissues by infection with X. campestris pv. vesicatoria or Phytophthora capsici, and by abiotic elicitor treatment. In particular, most of the CAI genes were strongly induced in pepper tissues by ethephon and methyl jasmonate.

Arabidopsis dnd1 and dnd2 mutants lack cyclic nucleotide-gated ion channel proteins and carry out avr/R-mediated defense with a greatly reduced hypersensitive response (HR). They also exhibit elevated broad-spectrum disease resistance and constitutively elevated salicylic acid (SA) levels. We examined the contributions of NPR1, SID2 (EDS16), NDR1 and EIN2 to dnd phenotypes. Mutations that affect SA accumulation or signaling (sid2, npr1 and ndr1) abolished the enhanced resistance of dnd mutants against Pseudomonas syringae pv. tomato and Hyaloperonospora parasitica but not Botrytis cinerea. When SA-associated pathways were disrupted, the constitutive activation of NPR1-dependent and NPR1-independent/SA-dependent pathways was redirected toward PDF1.2-associated pathways. This PDF1.2 over-expression was down-regulated after infection by P. syringae. Disruption of ethylene signaling abolished the enhanced resistance to B. cinerea but not P. syringae or H. parasitica. However, loss of NPR1, SID2, NDR1 or EIN2 did not detectably alter the reduced HR in dnd mutants. The susceptibility of dnd ein2 plants to B. cinerea despite their reduced-HR phenotype suggests that cell death repression is not the primary cause of dnd resistance to necrotrophic pathogens. The partial restoration of resistance to B. cinerea in dnd1 npr1 ein2 triple mutants indicated that this resistance is not entirely EIN2-dependent. The above findings indicate that the broad spectrum resistance of dnd mutants occurs due to activation and/or sensitization of multiple defense pathways, yet none of the investigated pathways are required for the reduced-HR phenotype.

Local interactions between individual plant organs and diverse microorganisms can lead to whole plant immunity via the mobilization of defense signals. One such signal is the plastid lipid-derived oxylipin azelaic acid (AZA). Arabidopsis lacking AZI1 or EARLI1, related lipid transfer family proteins, exhibit reduced AZA transport among leaves and cannot mount systemic immunity. AZA has been detected in roots as well as leaves. Therefore, the present study addresses the effects on plants of AZA application to roots. AZA but not the structurally related suberic acid inhibits root growth when directly in contact with roots. Treatment of roots with AZA also induces resistance to Pseudomonas syringae in aerial tissues. These effects of AZA on root growth and disease resistance depend, at least partially, on AZI1 and EARLI1. AZI1 in roots localizes to plastids, similar to its known location in leaves. Interestingly, kinases previously shown to modify AZI1 in vitro, MPK3 and MPK6, are also needed for AZA-induced root-growth inhibition and aboveground immunity. Finally, deuterium-labeled AZA applied to the roots does not move to aerial tissues. Thus, AZA application to roots triggers systemic immunity through an AZI1/EARLI1/MPK3/MPK6-dependent pathway and AZA effects may involve one or more additional mobile signals.