A study of mutual antagonism between EDS1 and transcription factor MYC2 in Arabidopsis immunity

Intracellular immune signaling plays an important role in modulating plant defense responses against pathogens. Arabidopsis nucleo-cytoplasmic protein EDS1 (Enhanced Disease Susceptibility1), together with its sequence-related signaling partners, PAD4 (Phytoalexin-Deficient4) and SAG101 (Senescence-Associated Gene101), is essential for transcriptional reprogramming during intracellular signaling in basal and TNL (TIR-NB-LRR) receptor-triggered immunity. EDS1 regulates both SA (salicylic acid)-dependent and SA-independent pathways in immune responses. Interactions between TNLs and EDS1 place EDS1 as a bridge between TNLs and induced transcriptional reprogramming in cells. How EDS1 signaling is regulated and which molecular events connect EDS1 to transcriptional defense reprogramming are still unclear. A genetic screen was used to identify suppressors of Arabidopsis eds1-2 hypersusceptibility to Pst (Pseudomonas syringae pv. tomato) DC3000 avrRps4 with the purpose to identify potential components of EDS1 signaling in immunity. Bacterial effector avrRps4 is recognized by the paired Arabidopsis TNL receptors RRS1 (Resistance to Ralstonia solanacearum1)/RPS4 (Resistance to Pseudomonas syringae4). I identified seven mutants with restored resistance to Pst DC3000 avrRps4. Among these, four mutants contain different mutations in COI1 (Coronatine-Insensitive1). Further analysis of several mutants suggests that four different signaling pathways can compensate for defects of eds1-2 in defense responses in Arabidopsis. Because COI1 is essential for activating JA (jasmonic acid) signaling which antagonizes SA (salicylic acid), I hypothesized that EDS1 negatively regulates JA signaling in order to promote SA resistance. In transient expression assays, EDS1, PAD4 and SAG101 formed complexes with MYC2-family transcription factors (TFs) which regulate an important JA signaling branch. EDS1-MYC2 association was found to interfere with MYC2 transcriptional activity in transient expression assays. This is the first evidence that EDS1 regulates transcriptional reprogramming through association with TFs such as MYC2. Because EDS1 interferes with MYC2 transcriptional activity, I tested whether MYC2 reciprocally affects EDS1 protein and/or gene expression. MYC2 specifically suppressed EDS1 promoter activity independently of MYC2-binding G-box and G-box-related motifs but, surprisingly, requiring MYC2 bHLH domain DNA-binding activity. After exogenous application of a bacterial mimic of bioactive JA, coronatine (COR), MYC2-family TFs were found to act redundantly to repress EDS1 expression. This repressive function on EDS1 was manifested in Arabidopsis protoplasts transient assays and at an early stage of Pst DC3000 infection and was not detectable at the late infection stage, probably due to activated EDS1 signaling. In summary, results presented in this thesis reveal a new and important level of regulation of immunity and SA-JA pathway balance by mutual antagonism between EDS1- and MYC2-dependent processes

Chemically induced dimerization modules as a platform for plant biosensor engineering

Protein biosensors for small molecules have important applications in agriculture, medicine, and security, but it remains difficult to rapidly produce a high-affinity sensor for a given ligand. This is partly due to two major challenges. First, most small molecule ligands have only a small number of residues with which a protein can make energetically favorable contacts, making it difficult to engineer high-affinity binding. Second, even if a high-affinity binding protein is engineered, it is difficult to transduce the binding event into an output. The majority of plant hormone perception occurs by chemically induced dimerization, where binding of the hormone to a soluble receptor causes a conformational change that allows the receptor to form a heterodimer with an interaction partner. These CID modules make an ideal platform for engineering small molecule biosensors because they naturally address the two primary challenges above: their unique architecture allows sensitive biosensors to be constructed from low-affinity receptors and protein dimerization provides a natural method of ligand binding transduction. The ability to engineer CID modules would lead directly to in planta biosensors and would also have broader applications to biosensor design in other biological systems. Here we describe the development of a general biosensor engineering platform using the abscisic acid receptor PYR1 of Arabidopsis thaliana, which was previously engineered to sense the agrochemical mandipropamid.1 We combine comprehensive mutagenesis2,3, high-throughput screening, deep sequencing, and machine learning to rapidly construct a model of the fitness landscape for binding of PYR1 to a specific ligand. We then use this model to design a targeted library to screen for higher affinity sensors. For high-throughput screening, we use both an established yeast two-hybrid (Y2H) screen and a novel yeast surface display (YSD) system. These techniques offer complementary advantages: Y2H is straightforward to implement and requires no purified protein, while YSD offers higher throughput and more stringent quantification of protein-protein interactions. Finally, we describe early development of two additional CID modules from the gibberellin and strigolactone sensing networks of A. thaliana. (1) Park, S.-Y.; Peterson, F. C.; Mosquna, A.; Yao, J.; Volkman, B. F.; Cutler, S. R. Agrochemical Control of Plant Water Use Using Engineered Abscisic Acid Receptors. Nature 2015, 520 (7548), 545–548. https://doi.org/10.1038/nature14123. (2) Wrenbeck, E. E.; Klesmith, J. R.; Stapleton, J. A.; Adeniran, A.; Tyo, K. E. J.; Whitehead, T. A. Plasmid-Based One-Pot Saturation Mutagenesis. Nat. Methods 2016, 13 (11), 928–930. https://doi.org/10.1038/nmeth.4029. (3) Medina-Cucurella, A. V.; Steiner, P. J.; Faber, M. S.; Beltrán, J.; Borelli, A. N.; Kirby, M. B.; Cutler, S. R.; Whitehead, T. A. User-Defined Single Pot Mutagenesis Using Unpurified Oligo Pools. Re

Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity

Transcriptional corepressors of the Topless family are important regulators of plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of transcriptional regulation in plant immune responses. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed on GFP-tagged Topless-related 1 (TPR1) expressed in Arabidopsis thaliana lines with and without constitutive immunity dependent on Enhanced Disease Susceptibility 1 (EDS1). RNA-seq profiling of pathogen-infected tpl/tpr mutants and assessments of growth and physiological parameters were employed to determine TPL/TPR roles in transcriptional immunity and defense homeostasis. TPR1 bound to promoter regions of ~1,400 genes and ~10% of the detected binding required EDS1 immunity signaling. A tpr1 tpl tpr4 (t3) mutant displayed mildly enhanced defense-related transcriptional reprogramming upon bacterial infection but not increased bacterial resistance. Bacteria or pep1 phytocytokine-challenged t3 plants exhibited, respectively, photosystem II dysfunction and exacerbated root growth inhibition. Transgenic expression of TPR1 restored the t3 physiological defects. We propose that TPR1 and TPL-family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity

Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity

Transcriptional corepressors of the Topless family are important regulators of plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of transcriptional regulation in plant immune responses. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed on GFP-tagged Topless-related 1 (TPR1) expressed in Arabidopsis thaliana lines with and without constitutive immunity dependent on Enhanced Disease Susceptibility 1 (EDS1). RNA-seq profiling of pathogen-infected tpl/tpr mutants and assessments of growth and physiological parameters were employed to determine TPL/TPR roles in transcriptional immunity and defense homeostasis. TPR1 bound to promoter regions of ~1,400 genes and ~10% of the detected binding required EDS1 immunity signaling. A tpr1 tpl tpr4 (t3) mutant displayed mildly enhanced defense-related transcriptional reprogramming upon bacterial infection but not increased bacterial resistance. Bacteria or pep1 phytocytokine-challenged t3 plants exhibited, respectively, photosystem II dysfunction and exacerbated root growth inhibition. Transgenic expression of TPR1 restored the t3 physiological defects. We propose that TPR1 and TPL-family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity