Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
(N/A)
Non Technical Summary
Researching plant immunity is crucial for global food security, as plant pathogens are responsible for yearly billion-dollar yield losses. This project will gain mechanistic insights into how lysine acetylation mediates plant-pathogen interactions. Pathogen infection alters acetylation, a major post-translational modification affecting many cellular processes, yet little is known on how this impacts plant immune response. My research will test the hypothesis that protein acetylation impacts immune signaling in corn, and this immune signaling is mediated bythe transcriptional corepressor RAMOSA ENHANCER LOCUS2 (REL2). This work will uncover novel molecular mechanisms underpinning plant immunity in a key agricultural crop by leveraging large-scale "omics" data via proteomics, and high-throughput RNA sequencing, in combination with biochemical and genetic experiments. These experiments will generate large-scale sequencing data related to immune responses, the identification of candidate genes involved in immunity and hormone responses, a model of how plant pathogens are transcriptionally regulated in corn, and ultimately will lead to a deeper understanding of molecular plant immunity. Additionally, this work will enhance our understanding of how protein acetylation impacts biological functions and processes and how immune signaling in corn is regulated. Gaining deeper insight into the function of acetylation in plant-pathogen interactions is vital for elucidating mechanisms of disease susceptibility and can lead to the development of disease-resistant crops, which will ultimately improve the global food supply. In addition to supporting agricultural workforce training, this research also aligns with the USDA AFRI NIFA program priority area of "plant health and production and plant products."
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Plant pathogens are a devastating agricultural stressor that annually costs billions of dollars in yield loss. Protein acetylation is a significant post-translational modification that affects numerous molecular functions, such as stress and immune responses. However, it is not well understood how acetylation affects plant-pathogen interactions. In this study, the hypothesis that the transcriptional corepressor RAMOSA ENHANCER LOCUS2 (REL2) mediates immune signaling in maize and that protein acetylation affects this immune signaling will be tested. Through the use of large-scale "omics," genetic, and molecular techniques, this work aims to identify novel molecular processes underlying plant immunity in a key agricultural crop. This will allow us to construct a model of REL2 transcriptional regulation during pathogen and hormone response and gain a deeper molecular understanding of plant immunity.Objective 1: Determine REL2-associated gene expressionDetermine genes directly and indirectly regulated by REL2Determine the direct connection between transcript and phenotype via integrating differentially expressed mRNA and proteinsDetermine novel REL2-dependent gene expression regulation associated with pathogen response to HC-toxin in maizeDetermine predictive targets of REL2Objective 2: Elucidate how REL2 acetylation state impacts maize immunityDetermine the impact of K307 acetylation on REL2-mediated defense responseDetermine the impact of K307 acetylation on REL2 protein:protein interactionsDetermine novel REL2 protein interactorsDetermine the interplay between jasmonic acid, auxin, and REL2-mediated response to C. carbonum
Project Methods
Objective 1: Determine REL2-associated gene expression Gene expression changes modulated by REL2 during pathogen infection will be determined via integrated transcriptome and proteome profiling. Total RNA and protein from B73 (WT control) and rel2-ref (mutant) 12-day-old leaves treated with and without HCtoxin will be extracted at 22 and 44 hours to capture different signaling events. RNA-seq libraries will be made in-house and sequenced on an Illumina NovaSeq platform. After FastQC, reads will be trimmed, mapped, and normalized using bbduk, STAR, and HTSeq. Differential gene expression analysis will be performed by comparing rel2 samples to B73 using PoissonSeq. Total protein extracts will be digested with trypsin and LysC, and peptides will be labeled using Tandem Mass Tags (TMT) and proteome profiling of multiplexed samples will be performed via liquid chromatography/mass spectrometry (2D-LC-MS/MS). Spectra will be identified and quantified, and differential protein abundance will be determined by comparing rel2 to B73 using TMTNeat. GO Term analysis will be done with the DE gene products to identify biological processes impacted by REL2 at both the mRNA and protein levels. Differential gene expression data will be utilized for gene network analysis via the spatiotemporal clustering and inference of omics networks (SC-ION) tool. Objective 2: Elucidate how REL2 acetylation state impacts maize immunity Genetic and molecular techniques will be utilized to determine the impact of acetylation on REL2-mediated defense response and protein:protein interactions in maize. Disease assays with C. carbonum will be performed with four genotypes: B73, rel2-ref (mutant), and transgenic tagged REL2 lines: ProUBIL:REL2-GSYellow (overexpressor) and ProUBIL:REL2-K307Q-GSYellow (acetyl-mimic). 12-day old leaves will be inoculated with C. carbonum supplemented with solvent control and/or the defense hormone jasmonic acid (JA) or IAA (auxin). The disease response will be quantified via time-coursed lesion measurements obtained from manual annotation, and the statistical significance of phenotypes will be determined using a generalized linear mixed model (GLMM). JA and auxin responses of the different genotypes will be determined using primary root length assays on 5-day-old seedlings. The statistical significance of phenotypes will be determined by comparing rel2-ref root lengths +/- hormone to B73 via GLMM. To determine if REL2 interacts with genes in MYC2-JA and Aux-IAA hormone complexes and if these interactions depend on K307 acetylation, I will perform affinity purification followed by mass spectrometry (AP-MS) with the B73, REL2-GSY, and REL2-K307Q-GSY transgenic lines and anti-GFP antibodies using 12-day-old leaves. Immunoprecipitations will be performed on each genotype. AP peptides from each sample will be TMT labeled, pooled, and analyzed by LC-MS/MS. Statical determination of enrichment will be done to identify REL2 interactors as well as identify how K307 acetylation impacts these interactions. Efforts to deliver knowledge will be implemented via annual presentations at scientific conferences, and this work will culminate in peer-reviewed journal publications.