Location: Subtropical Horticulture Research
Project Number: 6038-21000-026-015-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2024
End Date: Aug 31, 2025
Objective:
The causative pathogen of Avocado Laurel wilt disease is Harringtonia lauricola, a fungal pathogen transmitted by insect vector ambrosia beetle. H. lauricola obtains nutrients from living cells in the sapwood, and propagates in the xylem, a specialized tissue responsible for water and nutrient transport in vascular plants. Xylem has four main cell types, tracheids, vessels, living fibers and parenchyma. As a part of plant response to pathogen invasion, H. lauricola infection in avocado triggers tyloses in the xylem. Tyloses is initiated through the out-growth of parenchyma cell pit membranes into the lumen of xylem vessels. Early stages of tyloses are characterized by active synthesis of pectin rich materials, as well as the production of gels that have a high abundance of antimicrobial secondary metabolites, including catechol, ¿avonoids and coumarins. Once the out-growth within the xylem lumen reaches a certain volume and comes into contact with each other, the cell walls undergo a maturation process that involves deposition of lignin, phenolic, and most notably, a hydrophobic polymer, suberin. Production of suberin is a unique and prominent metabolic feature of tyloses because suberin is normally absent in the xylem, and suberization blocks the apoplastic pathways, and hence the hydraulic transport of water and solutes. While at the early stage of pathogen infection tyloses contribute to preventing pathogen spread in the contiguous tissues and vessels of the xylem, hyper-response in tyloses also results in reduction in the transport function of the xylem, hindering water flow to stems and leaves, and limiting the uptake of minerals, micro elements and other nutrients. Ploetz et al (2014) highlighted a possible relationship between progress of Laurel wilt disease symptom and the reduction in xylem sap flow rate in Avocado inoculated with H. lauricola. This is important because the major cause of death of H. lauricola diseased plants is the wilt and dessication of the upper branches. It is hypothesized that a fine-tuned suberin biosynthesis may delay the progression of late stage tyloses, because a reduced suberin in tyloses may render it partially permeable to water and hydrophilic movement of nutrients, thereby alleviating the negative impact on xylem flow. Study on pathogen induced suberin biosynthesis in avocado thus presents a unique and so far unexplored angle to understand H. lauricola and plant host interactions. The overarching goal of this collaboration is to identify genes and gene alleles associated with laurel wilt disease resistance by focusing on the following three objectives:
1. Comparative RNAseq analysis of laurel wilt diseased and disease-free avocado trees to evaluate transcriptome features associated with laurel wilt disease resistance.
2. Anatomical dissection of suberin deposition in infected avocado stems to seek mechanistic understanding on the pathogenesis and disease progression of laurel wilt in avocado.
3. Metabolic network analysis to uncover candidate genes involved in laurel wilt induced suberin deposition and tyloses development in the diseased xylem.
Approach:
1. Activity 1: RNAseq analysis-Avocado growers in SHRS, Miami mostly plant West Indian (WI) and Guatemalan x West Indian (G x WI) hybrid cultivars, which, while adapting well to local climate, are unfortunately highly vulnerable to laurel wilt. The scientists at SHRS has developed Avocado breeding populations and is cultivating them at the location. These populations, when challenged with H. lauricola, display a varying degree of laurel wilt disease susceptibility. The ARS PI will sample tissues from both diseased and disease-free avocado lines and conduct RNAseq analysis. The ARS PI will be responsible for ensuring RNAseq data quality and bioinformatics workflow, including sequence assembly and normalization of the RNAseq results, transcript annotation, Gene Ontology (GO) analysis, and classification of GO term enrichment, and analysis of differentially expressed genes (DEG). The cooperator has expertise and work on KEGG pathway annotation to uncover gene co-expression hubs pertinent to metabolic networks that are potentially related to plant defence and disease tolerance. Most of the objectives under Activity 1 will be performed during year one.
2. Activity 2: Anatomy of suberin deposition in xylem- The Penn State University has a fee -for-service Laser Ablation Tomography (LAT) platform that employs pulsed laser to ablate the surface of the cut end of plant tissues, typically stem or root, and generates images of consecutive tissue sections. This unique technology platform is ideally suited to dissecting the tyloses development in the avocado population displaying variation in laurel wild disease susceptibility. The stem tissues can also be stained with berberine aniline blue or Sudan III to aid the detection of suberin. The stems will then be subjected to laser ablation and under excitation at 390-420 nm for 3D digital images of anatomical features at the subcellular level. The cooperator will organize efforts to conduct Laser Ablation Tomography (LAT) on avocado stems that exhibit Laurel wilt disease to seek structural insights in tyloses deposition during H. lauricola infection. The ARS will be responsible for fees and service charges incurred for utilizing the LAT platform.Most of the objectives under Activity 2 will be performed during years 2-4.
3. Activity 3: Suberin metabolism and candidate gene analysis-The Cooperator will conduct GC-MS analysis of suberin monomers to investigate the compositional profiles of tyloses suberins. Combining with data expected from Activity 1, 2, this activity will generate valuable information on enzymes and associated genes participating in suberin biosynthesis during tyloses. Most of the objectives under Activity 3 will be performed during years 2-4.
