Performing Department
(N/A)
Non Technical Summary
Crop plants respond to pathogen challenge by activating their immune systems. Plant pathogens, in turn, have developed ways to counter these defenses. Proteins called "effectors" are one such counter-defense. These proteins work by shutting down, or suppressing, immune responses in plants, effectively enabling the pathogen invasion. When we learn about how effectors work, and which plant immunity components they target, we can use this information to develop new sustainable ways to manage disease in crops. This project will produce this knowledge for a unique, and economically damaging group of plant pathogens called Liberibacters. Liberibacters are transmitted by insects called psyllids and are causal agents of disease in citrus, tomato, potato, carrots, and celery. They invade cells of both plant and psyllid hosts. In plant hosts, Liberibacters cause many unique symptoms, which we hypothesize are due to the activity of effectors. We will carry out experiments to understand how effectors cause disease, how disease symptoms may encourage attraction of psyllid vectors, and how effectors are altering the plant immune system. Once we know how effectors alter plant immunity and plant interactions with psyllid vectors, we can use this information to improve Liberibacter and psyllid management. Examples of relevant applications include: improved crop breeding to create varieties with immune system components that cannot be manipulated by effectors, and therapeutics (low-, or no-toxicity chemical treatments) that bind and disable effectors during infections in plants. Our project will also generate knowledge of Liberibacter genetics and biology that will help us understand how and why these pathogens emerge as causal agents of disease in crops.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Psyllid-transmitted bacterial pathogens in the Candidatus Liberibacter taxon cause billions of dollars in crop losses annually. Novel Ca. Liberibacter variants are emerging as causal agents of disease in different plants at an alarming rate and many are threats to US agriculture. Despite stakeholders identifying Ca. Liberibacter and psyllid vector control as key priorities, few control options are available. Insecticides for vector management remain the primary control tactic. The goal of the proposed project is to contribute to development of more sustainable management tools by understanding of how Ca. Liberibacter manipulates hosts to establish infections, cause disease, and facilitate spread by vectors.Candidatus Liberibacter plant pathogens have evolved ways to interrupt the activation and propagation of defense responses in plant hosts through production of effectors, which are broadly defined as pathogen-secreted proteins that interact with host plant cellular components to favor pathogen establishment. Discovery and characterization of effectors, and their targets in plant hosts, is essential for reducing yield losses due to plant pathogens in agriculture. For example, effector presence has been used for disease detection and identification and deployment of host resistance genes, and effector targets can subsequently be edited to induce disease tolerance and even resistance. We propose to use computational approaches to study effector diversity and structural features across the entire Ca. Liberibacter taxon. We will also conduct focused molecular and organismal-level studies using one pathosystem: Ca. Liberibacter solanacearum (Lso), which is a causal agent of disease in solanaceous and apiaceous crops. Our work will encompass the following objectives:Objective 1. Investigate the ability of Lso haplotypes to alter host traits involved in defense responses and host-psyllid interactions.Objective 2. Identify Ca. Liberibacter effectors that regulate modification of host physiology and plant defense.Objective 3. Determine effector targets in plant hosts.
Project Methods
Objective 1. Investigate the ability of Lso haplotypes to alter host traits involved in defense responses and host-psyllid interactions.1A: Quantify changes in phytohormones and gene expression in host plants challenged with Lso haplotypes A, B, and G. We will conduct time-course measurements of primary metabolites, phytohormones, and gene expression in potato, tomato, and wild nightshade hosts in response to challenge with three Lso haplotypes. Metabolites and phytohormones will be extracted from leaf tissues, isolated from contaminating leaf components, derivatized, and analyzed using gas chromatography and mass spectrometry. Gene expression will be measured using RNAseq.1B: Quantify effects of Lso inoculation and infection progression on host plant traits important for psyllid vector foraging. We will quantify effects of infection with each Lso haplotype on plant traits that mediate interactions with psyllid vectors and that relate to yield/fitness (survival, plant size, color profiles, and odor profiles). Each trait will be measured at several time points during disease progression. Color profiles will be measured using imaging to quantify individual color components. Odor profiles will be analyzed using headspace collections followed by gas chromatography and mass spectrometry analysis. 1C: Quantify psyllid behavioral responses to plants challenged with Lso throughout changes in disease progression. For compatible host-Lso interactions, we will measure psyllid behavioral responses to plant phenotypic changes at 4 and 6 weeks post inoculation. Psyllid preferences for plant odors will be assessed using two-way and four-way choice test arenas that allow orientation to odors from live plants by flying and walking. For the same comparisons, will also quantify psyllid foraging preferences in the presence of multiple cues (odor, color, and contact). To quantify effects of Lso infection on feeding behaviors involved in transmission, we will use the EPG technique to record in-plant probing and feeding behaviors.Objective 2. Identify Ca. Liberibacter effectors that regulate modification of host physiology and plant defense.2A: Computational and structural prediction of Liberibacter effectors. We will use our recently generated and publicly available Ca. Liberibacter genomic information to predict effectors using bioinformatic tools, such as signalP, TMHMM, and BLASTP. We will explore putative functions of effectors using protein prediction tools, including PFAM, Superfamily, and InterProScan. We will then explore structural features of putative effectors using AlphaFold.2B: Profiling Lso effector expression in psyllid hosts. We will use RNAseq to profile expression of putative Lso effectors in psyllid host environments.2C: Identify Lso effectors capable of altering plant morphology. We will use a potato virus X (PVX) expression system to explore the phenotypic effects of putative effectors in different host environments (tomato, potato, and wild nightshade).2D: Investigate the role of Lso effectors in modifying psyllid host selection and feeding behaviors. We will use choice assays and EPG recordings (as in 1C) to evaluate how psyllids respond to phenotypic modifications induced by expression of putative effectors.Objective 3. Determine effector targets in plant hosts.3A: Identify effector targets using proximity labeling. Proximity labeling enables identification of protein targets in their native environments. We will use this technique, along with the PVX expression system, to study the interactions of at least five putative effectors with native proteins.3B: Validate effector targets. We will use Nicotiana benthamiana to visualize effector-target interactions using confocal microscopy and expression of effectors and targets fused fo fluorophores (expressed using Agrobacterium). Promising candidate effector targets in tomato will be overexpressed or silenced using PVX or tobacco rattle virus, then subjected to inoculation with Lso to determine if targets are susceptibility factors.