Sun Jae Kwon

Secretome Analysis Reveals an Arabidopsis Lipase Involved in Defense against Alternaria brassicicola

The Arabidopsis thaliana secretome was analyzed by the proteomic approach, which led to the identification of secreted proteins implicated in many aspects of cell biology. We then investigated the change in the Arabidopsis secretome in response to salicylic acid and identified several proteins involved in pathogen response. One of these, a secreted lipase with a GDSL-like motif designated GDSL LIPASE1 (GLIP1), was further characterized for its function in disease resistance. glip1 plants were markedly more susceptible to infection by the necrotrophic fungus Alternaria brassicicola compared with the parental wild-type plants. The recombinant GLIP1 protein possessed lipase and antimicrobial activities that directly disrupt fungal spore integrity. Furthermore, GLIP1 appeared to trigger systemic resistance signaling in plants when challenged with A. brassicicola, because pretreatment of the glip1 mutant with recombinant GLIP1 protein inhibited A. brassicicola–induced cell death in both peripheral and distal leaves. Moreover, glip1 showed altered expression of defense- and ethylene-related genes. GLIP1 transcription was increased by ethephon, the ethylene releaser, but not by salicylic acid or jasmonic acid. These results suggest that GLIP1, in association with ethylene signaling, may be a critical component in plant resistance to A. brassicicola.

GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling in Arabidopsis

Systemic resistance is induced by necrotizing pathogenic microbes and non-pathogenic rhizobacteria and confers protection against a broad range of pathogens. Here we show that Arabidopsis GDSL LIPASE-LIKE 1 (GLIP1) plays an important role in plant immunity, eliciting both local and systemic resistance in plants. GLIP1 functions independently of salicylic acid but requires ethylene signaling. Enhancement of GLIP1 expression in plants increases resistance to pathogens including Alternaria brassicicola, Erwinia carotovora and Pseudomonas syringae, and limits their growth at the infection site. Furthermore, local treatment with GLIP1 proteins is sufficient for the activation of systemic resistance, inducing both resistance gene expression and pathogen resistance in systemic leaves. The PDF1.2-inducing activity accumulates in petiole exudates in a GLIP1-dependent manner and is fractionated in the size range of less than 10 kDa as determined by size exclusion chromatography. Our results demonstrate that GLIP1-elicited systemic resistance is dependent on ethylene signaling and provide evidence that GLIP1 may mediate the production of a systemic signaling molecule(s).

Arabidopsis GDSL lipase 2 plays a role in pathogen defense via negative regulation of auxin signaling.

GLIP1 was isolated previously from Arabidopsis, as a salicylic acid-responsive secreted GDSL lipase that functions in resistance to Alternaria brassicicola [I.S. Oh, A.R. Park, M.S. Bae, S.J. Kwon, Y.S. Kim, J.E. Lee, N.Y. Kang, S. Lee, H. Cheong, O.K. Park, Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola. Plant Cell 17 (2005) 2832-2847.]. To extend our knowledge of the roles played by GLIPs in Arabidopsis, we conducted functional studies of another family member, GLIP2. GLIP2 transcripts were expressed in young seedlings, as well as in the root and stem tissues of mature plants. GLIP2 transcript levels were elevated by treatment with salicylic acid, jasmonic acid and ethylene. Recombinant GLIP2 proteins possessed lipase and anti-microbial activities, inhibiting germination of fungal spores. In comparison to wild type plants, T-DNA insertion glip2 mutants exhibited enhanced auxin responses, including increased lateral root formation and elevated AUX/IAA gene expression. When challenged with the necrotropic bacteria Erwinia carotovora, glip2 mutants exhibited more susceptible phenotypes than wild type plants. These results suggest that GLIP2 plays a role in resistance to Erwinia carotovora via negative regulation of auxin signaling.

GDSL LIPASE1 Modulates Plant Immunity through Feedback Regulation of Ethylene Signaling

A GDSL lipase regulates ethylene-dependent pathogen resistance and ethylene-salicylic acid signaling interactions in plants. Ethylene is a key signal in the regulation of plant defense responses. It is required for the expression and function of GDSL LIPASE1 (GLIP1) in Arabidopsis (Arabidopsis thaliana), which plays an important role in plant immunity. Here, we explore molecular mechanisms underlying the relationship between GLIP1 and ethylene signaling by an epistatic analysis of ethylene response mutants and GLIP1-overexpressing (35S:GLIP1) plants. We show that GLIP1 expression is regulated by ethylene signaling components and, further, that GLIP1 expression or application of petiole exudates from 35S:GLIP1 plants affects ethylene signaling both positively and negatively, leading to ETHYLENE RESPONSE FACTOR1 activation and ETHYLENE INSENSITIVE3 (EIN3) down-regulation, respectively. Additionally, 35S:GLIP1 plants or their exudates increase the expression of the salicylic acid biosynthesis gene SALICYLIC ACID INDUCTION-DEFICIENT2, known to be inhibited by EIN3 and EIN3-LIKE1. These results suggest that GLIP1 regulates plant immunity through positive and negative feedback regulation of ethylene signaling, and this is mediated by its activity to accumulate a systemic signal(s) in the phloem. We propose a model explaining how GLIP1 regulates the fine-tuning of ethylene signaling and ethylene-salicylic acid cross talk.

GDSL lipase 1 regulates ethylene signaling and ethylene-associated systemic immunity in Arabidopsis

Arabidopsis GDSL lipase 1 (GLIP1) has been shown to modulate systemic immunity through the regulation of ethylene signaling components. Here we demonstrate that the constitutive triple response mutant ctr1‐1 requires GLIP1 for the ethylene response, gene expression, and pathogen resistance. The glip1‐1 mutant was defective in induced resistance following primary inoculation of necrotrophic pathogens, whereas GLIP1‐overexpressing plants showed resistance to multiple pathogens. Necrotrophic infection triggered the downregulation of EIN3 and the activation of ERF1 and SID2 in a GLIP1‐dependent manner. These results suggest that GLIP1 positively and negatively regulates ethylene signaling, resulting in an ethylene‐associated, necrotroph‐induced immune response.

The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1

Receptor-like kinases are important signaling components that regulate a variety of cellular processes. In this study, an Arabidopsis cDNA microarray analysis led to the identification of the cysteine-rich receptor-like kinase CRK36 responsive to the necrotrophic fungal pathogen, Alternaria brassicicola. To determine the function of CRK36 in plant immunity, T-DNA-insertion knockdown (crk36) and overexpressing (CRK36OE) plants were prepared. CRK36OE plants exhibited increased hypersensitive cell death and ROS burst in response to avirulent pathogens. Treatment with a typical pathogen-associated molecular pattern, flg22, markedly induced pattern-triggered immune responses, notably stomatal defense, in CRK36OE plants. The immune responses were weakened in crk36 plants. Protein-protein interaction assays revealed the in vivo association of CRK36, FLS2, and BIK1. CRK36 enhanced flg22-triggered BIK1 phosphorylation, which showed defects with Cys mutations in the DUF26 motifs of CRK36. Disruption of BIK1 and RbohD/RbohF genes further impaired CRK36-mediated stomatal defense. We propose that CRK36, together with BIK1 and NADPH oxidases, may form a positive activation loop that enhances ROS burst and leads to the promotion of stomatal immunity.