Genetic Dissection of Salicylic Acid-Mediated Defense Signaling Networks in Arabidopsis

Abstract

Properly coordinated defense signaling networks are critical for the fitness of plants. One hub of the defense networks is centered on salicylic acid (SA), which plays a key role in activating disease resistance in plants. However, while a number of genes are known to affect SA-mediated defense, relatively little is known about how these gene interact genetically with each other. Here we exploited the unique defense-sensitized Arabidopsis mutant accelerated cell death (acd) 6-1 to dissect functional relationships among key components in the SA hub. We show that while enhanced disease susceptibility (eds) 1-2 and phytoalexin deficient (pad) 4-1 suppressed acd6-1-conferred small size, cell death, and defense phenotypes, a combination of these two mutations did not incur additive suppression. This suggests that EDS1 and PAD4 act in the same signaling pathway. To further evaluate genetic interactions among SA regulators, we constructed 10 pairwise crosses in the acd6-1 background among mutants defective in: SA INDUCTION-DEFICIENT 2 for SA biosynthesis; AGD2-LIKE DEFENSE 1, EDS5, and PAD4 for SA accumulation; and NONEXPRESSOR OF PR GENES 1 for SA signaling. Systematic analysis of the triple mutants based on their suppression of acd6-1-conferred phenotypes revealed complex and interactive genetic relationships among the tested SA genes. Our results suggest a more comprehensive view of the gene networks governing SA function and provide a framework for further interrogation of the important roles of SA and possibly other signaling molecules in regulating plant disease resistance. IN response to pathogen infection, plants can activate different layers of defense responses and undergo global gene expression reprogramming (Maleck et al. 2000;Tao et al. 2003;Katagiri 2004). A major challenge of the postgenomic era is to identify genes that control plant innate immunity and to elucidate how they are organized into networks to orchestrate host defense responses.One key hub in plant defense signaling networks is centered on the small phenolic molecule salicylic acid (SA). SA is important for basal defense, resistance proteinmediated defense, and systemic acquired resistance (HammondKosack and Jones 1996;Ryals et al. 1996;Tsuda et al. 2008). The SA hub of Arabidopsis includes many genes, which can be further grouped into three types on the basis of how they affect SA-mediated defense (Lu 2009). Type I SA genes encode enzymes that are directly involved in SA biosynthesis. One example is SA INDUCTION-DEFICIENT 2/ENHANCED DISEASE SUSCEPTIBILITY 16 (SID2/EDS16), which encodes isochorismate synthase contributing to bulk SA biosynthesis (Wildermuth et al. 2001). Type II SA genes encode proteins that do not act directly as SA biosynthetic enzymes. Mutations in these genes lead to partially compromised SA accumulation and enhanced disease susceptibility to pathogen infection, which can be rescued by exogenous SA treatment. The precise mechanism of action for each type II SA gene, however, still remains to be resolved. Example...

Early Senescence and Cell Death in Arabidopsis saul1 Mutants Involves the PAD4-Dependent Salicylic Acid Pathway

“…It has been shown that PAD4 and EDS1 participate not only in resistance to pathogens but also in the transduction of photooxidative stress signals resulting in cell death and growth inhibition. Cell death and growth inhibition phenotypes of lsd1, acd6-1, cpr1, and cpr6 mutants require the presence of PAD4 and EDS1, as demonstrated by the rescue of mutant phenotypes in double mutants with pad4 or eds1 (Jirage et al, 2001;Rustérucci et al, 2001;Mateo et al, 2004;Ochsenbein et al, 2006;Mühlenbock et al, 2008;Ng et al, 2011). The SA amplification loop is also crucial for cell death and senescence, increased SA content, and gene expression changes in saul1 mutants challenged by low light (Fig.…”

Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis

“…Thus, the change of acd6-1 dwarfism induced by second-site mutations could be conveniently used as a visual readout to provide a rapid assessment of the effect of these second-site mutations in some genes on defense responses. acd6-1 has been successfully used in genetic analyses to reveal epistatic relationships between defense mutants and in a genetic screen to identify, to our knowledge, novel defense genes (Song et al, 2004;Lu et al, 2009;Ng et al, 2011;Wang et al, 2011aWang et al, , 2011bWang et al, , 2014a. We found that while acd6-1 sim-1 resembled acd6-1 in plant morphology, acd6-1 smr1-1 and acd6-1 sim-1 smr1-1 were significantly larger than acd6-1 plants (Fig.…”

“…These results prompted us to ask whether SA could affect cell-cycle progression as part of downstream signaling of some cell-cycle genes. To test this, we analyzed cell ploidy in the major SA biosynthetic mutant sid2-1 (Wildermuth et al, 2001;Ng et al, 2011) and the SA signaling mutant npr1-1 (Dong, 2004;Lu, 2009;Fu and Dong, 2013). Our results show that both sid2-1 and npr1-1 had significantly increased numbers of 4C cells but reduced numbers of 8C and 16C cells (Fig.…”

“…Free and total (glucosylated) SA were extracted from plants and quantified with an HPLC instrument as previously described (Ng et al, 2011;Wang et al, 2011a).…”

Roles of Defense Hormones in the Regulation of Ozone-Induced Changes in Gene Expression and Cell Death

“…EDS1 and PAD4 are interacting proteins that regulate pathogen signaling from TIR-NB-LRR receptors (Rusté rucci et al, 2001;Heidrich et al, 2011;Rietz et al, 2011). Furthermore, EDS1 and/or PAD4 regulates cell death in a variety of contexts, including light signaling (Mü hlenbock et al, 2008), treatments with the chloroplastic-ROS-generating compound methyl viologen (Straus et al, 2010), and suppression of cell death in lesion mimic mutants (Rusté rucci et al, 2001;Ng et al, 2011;. The molecular mechanisms The indicated genotypes were exposed to O 3 for 6 h (350 nl l À1 related to the regulation of cell death via EDS1 are still unknown.…”