Performing Department
Microbiology and Cell Science
Non Technical Summary
Plants employ sophisticated defense signaling networks to survive the challenge of pathogens with diverse lifestyles. These defense signal networks are mostly regulated by a group of signal molecules including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). Depending upon the lifestyle of the invader, plants synthesize one or more of these primary defense signal molecules to turn on the signaling pathways that are the most effective for fending off the invading pathogens. After being synthesized, SA, JA, and ET are perceived by their receptors and the signals are transduced by components in their respective signaling pathways, ultimately leading to defense-associated transcriptional responses and pathogen resistance. We have shown that the Elongator complex is an important regulator of SA-mediate resistance to bacterial pathogens, but its role in JA/ET-mediated resistance to fungal pathogens is unknown. It is also unclear how exactly Elongator regulates plant defense responses. The proposed work will determine the function of Elongator in JA/ET-mediated resistance, study the function of Elongator in regulating the chromatin structure of defense genes, and establish the relationship between Elongator and other defense regulators. Other signaling components in Elongator-mediated resistance will also be identified. The rationale for the proposed research is that, once how Elongator regulates plant defense responses is clear, a better understanding of plant defense mechanisms will be achieved, which will undoubtedly help design new strategies for making crop plants more resistant to microbial attack.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Goals / Objectives
The objective of this project is to elucidate the mechanisms through which the Elongator complex regulates plant immune responses and to identify Elongator-related plant defense signaling network.
Project Methods
Specific Aim #1: Determine the function of Elongator in the JA- and ET-mediated defense pathways. We will first test JA-induced root hair promotion and ET-induced triple response. For JA-induced root hair promotion, elp seedlings will be grown on ½ MS medium for four days, and then transferred to fresh ½ MS medium with or without 10 mM of the JA derivative methyl jasmonate (MeJA). Root hair densities will be determined after 60 hours. For ET-induced triple response, elp seedlings will be grown on ½ MS medium supplemented with 10 mM of the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the dark for three days, and then morphology, hypocotyl length, and root length will be determined. For JA- and/or ET-induced defense gene expression, 10-day-old seedlings grown on ½ MS medium will be transplanted onto ½ MS medium or ½ MS medium supplemented with 0.1 mM ACC, 0.1 mM MeJA, or both. Two days later, plant tissues except root will be collected. Total RNA will be extracted from the tissues and subjected to RNA gel blot analysis or real-time qPCR analysis of the JA/ET-responsive genes PLANT DEFENSIN1.2 (PDF1.2), BASIC CHITINASE (CHIB), and HEVEIN-LIKE (HEL). The UBQ5 gene will be used as a loading or internal control. B. cinerea-induced expression of PDF1.2, CHIB, and HEL in elp mutants will also be tested. Briefly, four-week-old soil-grown plants will be inoculated with B. cinerea spores, and the inoculated leaves will be collected four days later. Total RNA will be extracted and subjected to RNA gel blot analysis or real-time qPCR analysis. For susceptibility of elp mutants to B. cinerea, sizes of the necrotic lesions on at least 24 leaves for each genotype will be measured. For B. cinerea growth on elp mutants, inoculated leaves will be collected and total RNA will be extracted. The B. cinerea ActinA DNA will be analyzed by real-time qPCR. The Arabidopsis Actin2 gene will be used as the internal control. We will also perform microarray experiment to monitor B. cinerea-induced transcriptome changes in one of the elp mutants (elp2), which will identify more JA/ET pathway genes that are regulated by Elongator. Four-week-old soil-grown plants will be inoculated with B. cinerea, and total RNA samples will be extracted from the inoculated leaves collected at different time points and subjected to microarray analysis. We have performed multiple microarray experiments and will follow the same protocol for experiment and data analysis. Additionally, we will test the susceptibility of elp mutants to other necrotrophic fungal pathogens such as Alternaria brassicicola and Sclerotinia sclerotiorum. Defense responses induced by these pathogens in elp mutants will be analyzed as described for B. cinerea. Growth of the pathogens on elp mutants will be estimated by lesion sizes. For A. brassicicola, the A. brassicicola Cutinase DNA will be analyzed by real-time qPCR as described for B. cinerea. Specific Aim #2: Determine the function of Elongator in regulating the chromatin structure of defense genes. We will first test whether Elongator is required for maintaining normal (basal) histone acetylation levels in some JA/ET pathway genes. Our preliminary data have shown that, in elp2, B. cinerea-induced expression of WRKY33, OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59), and PDF1.2 is reduced. We will therefore focus on the acetylation status of histone H3 in WRKY33, ORA59, and PDF1.2. Histone H3 acetylation levels in WRKY33, ORA59, and PDF1.2 will be assessed by ChIP. Briefly, after formaldehyde crosslinking and cell lysis of elp2 (or other elp mutants) and wild-type leaf tissues, histone-DNA complexes will be immunoprecipitated using an antibody specific for histone H3 acetylated at lysines 9 and 14 (H3K9/14ac). Precipitated DNA will be quantified using real-time qPCR to estimate the levels of histone H3K9/14ac. We have previously performed anti-H3K9/14ac antibody ChIP experiment and will follow the published protocol. We will further test whether Elongator is associated with the chromatin of WRKY33, ORA59, and PDF1.2. ChIP assays will be performed with chromatin isolated from transgenic plants containing the 35S:ELP2-GFP transgene in the elp2 mutant background using an anti-GFP antibody. The ELP2-GFP fusion protein has previously been shown to be functional, since it complements all the elp2 mutant phenotypes. Wild-type Columbia (Col-0) plants will be used as negative controls, as the elp2 mutant is in the Col-0 genetic background. WRKY33, ORA59, and PDF1.2 chromatin enrichment will be estimated by real-time qPCR and determined as percentage of input DNA. We have previously done anti-Myc antibody ChIP experiment and will use a similar experimental procedure. We will also carry out DNA methylation analysis to determine whether Elongator regulates DNA methylation in some JA/ET pathway genes. We will use bisulfite sequencing for this purpose. We will first identify JA/ET pathway genes whose DNA methylation changes during pathogen infection, and then define whether Elongator plays a role in this process. We have done DNA bisulfite sequencing and do not expect major technical problems for this analysis. In addition, we will study the relationship between histone acetylation and DNA methylation in plant immunity. We will use the elp mutants as genetic tools to help dissect this complicated relationship. We will generate double mutants between elp and other epigenetic mutants. Further experiments will be designed based on results obtained from these double mutants. Specific Aim #3: Determine the relationship between Elongator and the JA signaling component COI1 and the ET signaling component EIN2. We will first compare the B. cinerea susceptibility of elp mutants with that of coi1 and ein2, two mutants in which the JA and ET signaling are almost completely blocked, respectively. We will then generate the double mutants elp2 coi1 and elp2 ein2. After confirming their genotypes, these double mutants will be tested for susceptibility to B. cinerea. Wild type, elp2, and coi1 or ein2 will be included in the experiments. Results from the experiments will be analyzed using the linear mixed-effects model to identify the genetic relationship between elp2 and coi1 or ein2. We will also study the relationship between elp2 and jasmonate-insensitive1 (jin1), a mutation in the transcription factor MYC2, which negatively regulates resistance to necrotrophic fungal pathogens. We will generate the elp2 jin1 double mutant and analyze its defense response to test whether MYC2 plays a role in elp2-mediated susceptibility to B. cinerea. Furthermore, 35S:WRKY33 and 35S:ORA59 will be transformed into the elp2 mutant. After identifying single insertion homozygous lines, expression levels of the transgenes will be analyzed, and defense gene expression and resistance of the transgenic lines to B. cinerea will be tested. If overexpression of WRKY33 or ORA59 restores defense gene expression and resistance to B. cinerea in elp2, Elongator should function upstream of WRKY33 or ORA59. Conversely, if overexpression of WRKY33 or ORA59 is not able to restore defense gene expression and resistance in elp2, Elongator should function downstream or independently of WRKY33 and ORA59. Finally, we will perform a genetic screen for suppressors of elp mutants to identify new components of plant defense pathways. We will first characterize the defense phenotypes of the suppressors and then cloned the mutation loci in important suppressors. The relationship between the suppressor genes and Elongator as well as other major defense components such as NPR1, COI1, and EIN2 will be analyzed.