Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae

BACKGROUND: A common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant WRKY transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific WRKY DNA-binding transcription factors in plant defense. RESULTS: We analyzed the role of the WRKY25 transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. WRKY25 protein recognizes the TTGACC W-box sequences and its translational fusion with green fluorescent protein is localized to the nucleus. WRKY25 expression is responsive to general environmental stress. Analysis of stress-induced WRKY25 in the defense signaling mutants npr1, sid2, ein2 and coi1 further indicated that this gene is positively regulated by the salicylic acid (SA) signaling pathway and negatively regulated by the jasmonic acid signaling pathway. Two independent T-DNA insertion mutants for WRKY25 supported normal growth of a virulent strain of P. syringae but developed reduced disease symptoms after infection. By contrast, Arabidopsis constitutively overexpressing WRKY25 supported enhanced growth of P. syringae and displayed increased disease symptom severity as compared to wild-type plants. These WRKY25-overexpressing plants also displayed reduced expression of the SA-regulated PR1 gene after the pathogen infection, despite normal levels of free SA. CONCLUSION: The nuclear localization and sequence-specific DNA-binding activity support that WRKY25 functions as a transcription factor. Based on analysis of both T-DNA insertion mutants and transgenic overexpression lines, stress-induced WRKY25 functions as a negative regulator of SA-mediated defense responses to P. syringae. This proposed role is consistent with the recent finding that WRKY25 is a substrate of Arabidopsis MAP kinase 4, a repressor of SA-dependent defense responses

Conserved nematode signalling molecules elicit plant defenses and pathogen resistance

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture

Identification of multiple salicylic acid-binding proteins using two high throughput screens

Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays

The search for the salicylic acid receptor led to discovery of the SAR signal receptor

Systemic acquired resistance (SAR) is a state of heightened defense which is induced throughout a plant by an initial infection; it provides long-lasting, broad-spectrum resistance to subsequent pathogen challenge. Recently we identified a phloem-mobile signal for SAR which has been elusive for almost 30 years. It is methyl salicylate (MeSA), an inactive derivative of the defense hormone, salicylic acid (SA). This discovery resulted from extensive characterization of SA-binding protein 2 (SABP2), a protein whose high affinity for SA and extremely low abundance suggested that it might be the SA receptor. Instead we discovered that SABP2 is a MeSA esterase whose function is to convert biologically inactive MeSA in the systemic tissue to active SA. The accumulated SA then activates or primes defenses leading to SAR. SABP2's esterase activity is inhibited in the initially/primary infected tissue by SA binding in its active site; this facilitates accumulation of MeSA, which is then translocated through the phloem to systemic tissue for perception and processing by SABP2 to SA. Thus, while SABP2 is not the SA receptor, it can be considered the receptor for the SAR signal. This study of SABPs not only illustrates the unexpected nature of scientific discoveries, but also underscores the need to use biochemical approaches in addition to genetics to address complex biological processes, such as disease resistance

The Search for the Salicylic Acid Receptor LED to Discovery of the SAR Signal Receptor

Systemic acquired resistance (SAR) is a state of heightened defense which is induced throughout a plant by an initial infection; it provides long-lasting, broad-spectrum resistance to subsequent pathogen challenge. Recendy we identified a phloem-mobile signal for SAR which has been elusive for almost 30 years. It is methyl salicylate (MeSA), an inactive derivative of the defense hormone, salicylic acid (SA). This discovery resulted from extensive characterization of SA-binding protein 2 (SABP2), a protein whose high affinity for SA and extremely low abundance suggested that it might be the SA receptor. Instead we discovered that SABP2 is a MeSA esterase whose function is to convert biologically inactive MeSA in the systemic tissue to active SA. The accumulated SA then activates or primes defenses leading to SAR. SABP2\u27s esterase activity is inhibited in the initially/primary infected tissue by SA binding in its active site; this facilitates accumulation of MeSA, which is then translocated through the phloem to systemic tissue for perception and processing by SABP2 to SA. Thus, while SABP2 is not the SA receptor, it can be considered the receptor for the SAR signal. This study of SABPs not only illustrates the unexpected nature of scientific discoveries, but also underscores the need to use biochemical approaches in addition to genetics to address complex biological processes, such as disease resistance