Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
The project is designed to understand HLB tolerance by identifying the genetic pathways/components regulating CLas-induced callose and ROS accumulation and to identify HLB tolerance in citrus and its close relatives. The proposed research is based on our recent discovery of the HLB tolerance mechanisms in transgenic citrus trees over-expressing the Arabidopsis thaliana NPR1 (AtNPR1) gene and in RNAi trees in which the citrus NPR3 gene is silenced. We found that both over-expression of AtNPR1 and silencing of citrus NPR3 elevate basal callose levels and inhibit CLas-induced callose and ROS accumulation. These results suggest that (1) the basal callose level is correlated with HLB tolerance; (2) AtNPR1 and citrus NPR3 oppositely regulate HLB tolerance in citrus; and (3) the citrus NPR1 genes in commercial varieties are unable to prevent CLas-induced over-accumulation of callose and ROS. In this project, we will use basal callose as a selection marker to identify HLB tolerance in large citrus collections. Moreover, both NPR1 and NPR3 are regulators of the salicylic acid (SA) signaling pathway, indicating that the SA pathway regulates callose and ROS accumulation as well as HLB tolerance. We will investigate the functionalities of the SA pathway signaling components of various citrus varieties and relatives. We hypothesize that the past citrus breeding activities resulted in the development of elite cultivars with high yield and fruit quality, but a weak immune system, especially SA pathway, that cannot prevent CLas-induced over-accumulation of callose and ROS.
Animal Health Component
30%
Research Effort Categories
Basic
55%
Applied
30%
Developmental
15%
Goals / Objectives
Theprimary aim of this projectis to comprehend HLB susceptibility in commercial citrus and the tolerance seen in citrus relatives by identifying the genetic pathways and components that regulateCLas-induced callose and ROS accumulation. Ourcentral hypothesisposits that historical citrus breeding activities have led to the development of elite cultivars that, despite high yield and fruit quality, possess a weakened immune system, particularly within the SA pathway, incapable of preventingCLas-induced over-accumulation of callose and ROS. To achieve this goal, we have outlinedfour specific objectives: (1) Identify HLB tolerance in citrus collections (2) Investigate the immune systems of susceptible citrus varieties and tolerant relatives (3) Evaluate the SA pathway genes with high activities for HLB tolerance through CTV-mediated transient expression (4) Engage stakeholders in project development and execution processRecognizing the urgency of developing HLB tolerance in commercial varieties, we will leverage our discovery of the correlation between basal callose levels and HLB tolerance to identify HLB tolerance within citrus collections. We will expedite the translation of our findings in HLB tolerance mechanisms into practical applications within conventional and biotechnological breeding programs using the CTV-mediated transient expression system. These proposed research activities will thus help reduce the time and effort required to create HLB tolerance in commercial varieties.Objective 1.Identify HLB tolerance in citrus collectionsWe will screen uninfected citrus germplasm collections in California and Florida for varieties exhibiting elevated basal callose levels. Varieties with elevated basal callose will undergo further testing forCLas-induced callose and ROS accumulation, and ultimately for HLB tolerance. Concurrently, we will conduct genome-wide association mapping of basal callose levels to identify genetic variants that underlie differences in callose deposition. This will provide potential markers for breeding and targets for gene editing.Objective 2.Investigate the immune systems of susceptible citrus varieties and tolerant relativesThe SA signaling pathway is crucial for defense against HLB because the causal pathogen,CLas, is an obligate biotroph. Therefore, this objective will focus on investigating the SA signaling pathway. We will clone genes encoding the key signaling components of the SA pathway from elite citrus varieties and their relatives. Their activity will be tested in appropriate Arabidopsis mutants, as this provides the fastest approach for assessing citrus gene activity. The results from this objective will help understand the citrus immune system and guide genetic engineering efforts, including gene editing, silencing, and overexpression.Objective 3. Evaluate the SA pathway genes with high activities for HLB tolerance through CTV-mediated transient expressionAlthough transgenic approaches can be used to test if overexpression of a gene creates HLB tolerance in susceptible varieties, this method is time-consuming. To expedite the evaluation process, we will transiently overexpress the candidate genes using the CTV vector. This approach allows for rapid introduction and expression of genes within the citrus plants, providing a faster means to assess whether overexpression leads to HLB tolerance. By using the CTV vector, we can bypass the lengthy stages of stable transformation and quickly screen for effective gene candidates, accelerating the overall research and development timeline.Objective 4.Engage stakeholders in project development and execution processWe will actively engage stakeholders throughout the project and keep citrus communities informed through diverse outreach and communication strategies. To expedite this process, we will establish a Stakeholder Advisory Board. This board will facilitate the exchange of information and ensure that the findings are effectively communicated to all relevant parties, thereby enhancing the practical application of our research.
Project Methods
?Objective 1: Identify HLB tolerance in citrus collectionsWe will leverage the genetic diversity in the UC Riverside Givaudan Citrus Variety Collection (GCVC) and Florida collections to identify citrus accessions or relatives with elevated basal levels of callose using the simple aniline blue staining method developed by Co-PD Levy. Accessions with elevated basal levels of callose will then be tested for CLas-induced callose and ROS accumulation following the protocol published by Co-PD Levy. Accessions with reduced induction of callose and ROS accumulation will finally be tested for HLB tolerance in the greenhouse.We will focus on a previously defined core set of 200 of the 1,084 accessions that represent a significant portion of the genetic variation in the complete collection. These core accessions include representatives of the three foundational citrus species (Citrus reticulata, Citrus grandis, and Citrus medica), as well as interspecific hybrids of these groups that include major citrus market types (mandarin, sweet orange, grapefruit, lemons, limes), and citrus relatives (papeda types, Poncirus trifoliata, and Australian citrus species). These core accessions will be analyzed for basal callose levels. Quantitative measurements of basal callose levels in each accession will be integrated with genetic information to pinpoint genomic regions linked to differential basal callose levels. This genome-wide association study will be conducted using GEMMA, with appropriate corrections for population structure.Objective 2: Investigate the immune systems of susceptible citrus varieties and tolerant relativesWe will characterize citrus homologs of Arabidopsis genes encoding the SA pathway key components. We will clone homologs from citrus rootstocks and relatives into the binary vector pCAMBIA1300S. After confirming the sequences by sequencing, these constructs will be transformed into the corresponding Arabidopsis mutants through Agrobacterium-mediated genetic transformation. Single insertion homozygous plants will be identified in T2 and T3 generations and tested for basal resistance against bacterial pathogens. Wild-type and mutant Arabidopsis plants will be included as controls. If the homologs from citrus rootstocks or relatives can complement the mutants, whereas those from sweet orange cannot, it suggests that the sweet orange homologs are defective, likely due to amino acid substitutions. If none of the homologs complement the mutants, we will use CRISPR/Cas9 to knock out the homologs in the rootstocks or relatives to determine their roles in HLB tolerance, given that rootstocks and citrus relatives are tolerant to HLB.Objective 3: Evaluate the SA pathway genes with high activities for HLB tolerance through CTV-mediated transient expressionWe will introduce the candidate gene CDS into the CTV vector through unique restriction sites. The Unique restriction site will be added to each of the forward and reverse primer sets, respectively. Ligation and transformations will be done according to standard molecular procedures established for CTV cloning in the El Mohtar Lab. All CTV vector plasmid clones will be checked for the right insert size by restriction digestion followed by sequencing.Recombinant virions of CTV vectors isolated from systemic leaves of N. benthamiana are concentrated by ultra centrifugation will be inoculated into 1-1.5-year-old C. macrophylla seedlings via bark flapping.After systemic infection is established in C. macrophylla and validated with ELISA results, the constructs will be graft-transmit into sweet orange or grapefruit seedlings for HLB phenotype screening. These sweet orange and grapefruit seedlings will be on their own rootstocks, since we recently found that sweet orange and grapefruit on their own roots show more pronounced HLB symptoms within 6-12 months after CLas infection. We will inoculate these trees with HLB by incubating them in a containment plant growth room with CLas-infected psyllids, usually for 2 - 4 weeks. The psyllids will be removed, and the plants will be moved to the greenhouse for growth and development of HLB symptoms.Objective 4: Engage stakeholders in project development and execution processWe have long collaboration with citrus growers and will leverage the collaboration to disseminate the new knowledge gained in this project. We will also assemble an Advisory Board primarily consisting of stakeholders to facilitate information exchanges and ensure delivery of the new knowledge to all relevant parties. Annual meetings will be arranged with the Advisory Board to present updates and receive feedback.Teaching materials, including PowerPoint slides, extension articles, and a website, will be created for broad audiences such as citrus growers, industry representatives, consumers, as well as state and federal regulators. These materials will provide the basics of the citrus immune system, the SA signaling pathway, and the key signaling components in the pathway.We will leverage established citrus grower and industry meetings in Florida and California to engage and educate stakeholders about the citrus immune system, the SA signaling pathway, callose and ROS accumulation and their relationship with HLB. Additionally, we will organize special workshops, seminars, meetings, and outreach efforts targeting interested parties to raise awareness and acceptance of the new knowledge created by this project.