Performing Department
Plant Science & Landscape Architecture
Non Technical Summary
Understanding plant immunity mechanisms is critical for breeding disease-resistant crops. While the main structure of plant immunity consisting of immune receptors recognizing pathogen molecules has been well established, understanding how signals for defense and growth are generated, integrated and communicated to allow plants under attack to achieve optimum growth remains elusive. The plant hormone ethylene (ET) plays a role in immune signaling leading to disease resistance but a deeper mechanistic characterization is lacking. Additionally, recent studies have shown that 1-amino-cyclopropane-1-carboxylic acid (ACC), the precursor of ET, can itself serve as a novel signaling molecule that induces responses distinct from those triggered by ET. Mutants with reduced ACC show altered levels of antimicrobial glucosinolates, unlike mutants specifically lacking ET production, thus raising the question of whether ACC itself plays a role in pathogen resistance. Preliminary infection tests of Arabidopsis acs mutants defective in ACC (and ET) biosynthesis suggest a role of ACC in disease resistance against powdery mildew. The proposed collaborative project will combine expertise and resources from two laboratories to test whether ACC / ET signaling contributes to plant immunity against filamentous pathogen such as powdery mildew, and if so, whether ACC acts independently of ET in boosting plant immunity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Understanding plant immunity mechanisms is critical for breeding disease-resistant crops. While the main structure of plant immunity consisting of immune receptors recognizing pathogen molecules has been well established, understanding how signals for defense and growth are generated, integrated and communicated to allow plants under attack to achieve optimum growth remains elusive. The plant hormone ethylene (ET) plays a role in immune signaling leading to disease resistance but a deeper mechanistic characterization is lacking. Additionally, recent studies have shown that 1-amino-cyclopropane-1-carboxylic acid (ACC), the precursor of ET, can itself serve as a novel signaling molecule that induces responses distinct from those triggered by ET. Mutants with reduced ACC show altered levels of antimicrobial glucosinolates, unlike mutants specifically lacking ET production, thus raising the question of whether ACC itself plays a role in pathogen resistance. Preliminary infection tests of Arabidopsis acs mutants defective in ACC (and ET) biosynthesis suggest a role of ACC in disease resistance against powdery mildew. The proposed collaborative project will combine expertise and resources from two laboratories to test whether ACC / ET signaling contributes to plant immunity against filamentous pathogen such as powdery mildew, and if so, whether ACC acts independently of ET in boosting plant immunity.This seed grant proposal has the following two objectives:Objective 1: Characterize the roles of ET-signaling in immunity against PM and other biotrophic filamentous pathogens. PM fungi are important obligate biotrophic pathogens. Plant resistance against PM has clear spatiotemporal characteristics, namely penetration resistance and post-penetration resistance. Penetration resistance is thought to be primarily attributable to PAMP-triggered immunity (PTI) during which cell-surface (the plasma membrane or cell wall-localized) immune receptors recognize pathogen-associated molecular patterns (PAMP) (such as chitin from fungal cell walls) and induce cell wall-based defense. By contrast, post-penetration resistance is often elicited upon recognition of fungal effector proteins in the host cell by intracellular immune receptors, hence it is also referred to as effector-triggered immunity (ETI). The possession of both adapted (aggressive), poorly-adapted (weak) and non-adapted (nonpathogenic) powdery mildew species in the Xiao lab, coupled with a broad collection of ET/ACC biosynthesis and ET signaling mutants available in the Chang lab, will allow for dissection of the role of ET/ACC-signaling in penetration resistance (mostly PTI) and post-penetration resistance (mostly ETI) using various mutants defective in ET/ACC biosynthesis and in ET signaling. Similar experiments will be conducted using obligate biotrophic oomycete pathogens.Objective 2: Determine if ACC modulates immunity in an ET-dependent or -independent manner. The Chang lab's findings on the roles of ACC in several plant species, including Arabidopsis [8-11], together with the preliminary data presented above, raise the possibility of a distinct role for ACC in plant immunity. While the experiments in Objective 1 use ET and ACC mutants to examine the role of ET, Objective 2 focuses on the role of ACC. Since ACC is the immediate precursor of ET in seed plants, dissecting the role of ACC independent of ET is intrinsically challenging. However, available mutants, including new unpublished acs mutants in the Chang lab, can allow for the specific discrimination of responses that are dependent on ACC vs. ET. We will thus determine whether ACC has a role in immunity in Arabidopsis independent of ET that is beyond its role as the ET precursor by comparing immunity responses in the newly-generated acs oct mutants (three independent alleles available to date), in which all eight Arabidopsis ACC synthase genes have been knocked out by CRISPR, with those of the ET-free mutants (two independent alleles), in which all five Arabidopsis ACC oxidase (ACO) genes have been knocked out by CRISPR [13]. We will be able to discriminate ACC response from ET response by using the acs mutants that cannot produce ACC, and the ET-free mutants that cannot convert ACC to ET, in combination with rescue experiments by treating the mutants with ACC vs. ET.In addition, results from this seed grant project will provide critical evidence to determine if ACC functions as a signaling molecule distinct from ET to promote plant immunity. If so, the CoPIs will be in a stronger position to compete for a major NSF or USDA award to identify the ACC receptor and its signaling pathway in plants and explore potential translational application in tomato.
Project Methods
We will employ the following methods for this project:1) We will useCRISPR-targeted mutagenesis to create single and higher-order mutants in which ACC/ET signaling components are mutated;2) We will conduct infection assays of various ACC/ET mutants using a standard powdery mildew inoculation method established in the PD's lab;3) We will also use quantitative RT-PCR to measure expression of defense-related genes in wild-type and ACC/ET mutants;4) We will measure the levels of relevant glucosinolate metabolites to see if there is a correlation between increased susceptibility and decreased glucosinolate levels.?