Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Botany and Plant Sciences
Non Technical Summary
The global apple industry, and especially the U.S. organic sector, depend on apple rootstocks that are resistant to diseases and insects, especially the woolly apple aphid (WAA). The main resistance factor used against WAA has remained viable for decades; however, an emerging ecotype was found in a global hub for rootstock export to overcome this resistance, threatening the viability of apple production. Large-scale replanting, reduced yields, and additional barriers to market entry alongside increased management costs will occur should this aphid invade without knowing how to manage it. Our international team has confirmed the breakdown of resistance in the field, identified what genes in rootstock provide resistance to typical WAA, and developed a plan to assess the threat of this ecotype and other WAA populations to current apple production while characterizing how resistance failed. Specifically, we will identify what genes allow this new ecotype to overcome rootstock resistance, determine if this ecotype or others with the same capacity to adapt occur across North American growing regions, and incorporate this knowledge into screens of current and breeding of new rootstocks to advance pest management. These results will provide growers with regionally specific risk assessment, the means to manage emerging and longstanding WAA genotypes, and novel understanding of how aphids adapt to host plants. This project addresses the legislatively mandated focus areas of Research in plant breeding, genetics, and genomics, Efforts to identify and address threats from pests, and New innovations and technology.
Animal Health Component
45%
Research Effort Categories
Basic
45%
Applied
45%
Developmental
10%
Goals / Objectives
Our preliminary results show that an emerging ecotype of WAA can overcome R5+ rootstocks, the main WAA management tool used solely by organic producers and employed throughout the US by conventional producers. This ecotype emerged in a hub of global export, suggesting invasion and resistance failure is imminent. Our previous work on R5+ resistance has identified the chromosomal region for both major and minor resistance genes, and we now have candidate genes underlying R5- resistance. However, our data also indicate a gene-for-gene interaction may play a role in resistance, setting the stage for regional management challenges. We have identified plant immune (resistance) genes in both R5+ and wild relatives and have unreleased genotypes that vary in these genes but remain untested against WAA. We have also identified other plant physiological and developmental processes that play a role in the resistance phenotype, possibly providing a means for enhanced (cross) resistance against other aphids and pathogens. We have predicted and begun characterization of WAA effectors shared with other aphids and have candidates that likely underlie the emerging ecotype's ability to overcome resistance. Until now, WAA performance assays have been the only means to assess rootstock resistance, with few studies attempting characterize resistance phenotypes or the molecular mechanisms underlying resistance for any Er loci. Understanding the fundamental processes that underlie WAA colonization and host immune responses are crucial for predicting the efficacy and durability of resistance in the field. Thus, we propose to:• Identify gene presence and expression that allow a new WAA ecotype to overcome rootstock resistance in Europe• Assess presence of the emerging ecotype (Obj. 1) and overall WAA genetic diversity across North American growing regions.• Identify plant genes that interact with WAA genes of interest from the emerging ecotype (Obj. 1 and 2) to advance rootstock development.
Project Methods
To reveal what new (or missing) genes and enhanced gene expression enable resistance to be overcome, we will use a combination of DNA and RNA sequencing of plants and insects across a resistance spectrum. For gene discovery and sequence polymorphism analysis, individuals with a reciprocal resistance phenotype will be collected and allowed to feed on R5+ and R5- plants to confirm performance. Two pools of each genotype will be collected (one for archive) and DNA will be extracted and shipped to UCR as part of the international survey of WAA genetics. To resolve the plant molecules linked to the resistance phenotype and test if overexpression enables performance, individuals will be colonized on a R5+ rootstock and a R5- rootstock alongside a local genotype that performs opposite on these genotypes. Age synchronized aphids (n=25 per plant) will be allowed to feed over time with both aphid and plant tissues collected for transcriptome profiling at 2 and 7d. Aphid feeding sites will be quickly excised and flash frozen and aphids will be collected and pooled per plant making four biological replicates/pools per genotype x time designation. All samples will be flash frozen and stored at -80C prior to extraction. RNA will be extracted following manufactured protocols. Extracted samples will be sent to a fee-based sequencing facility and raw reads will be cleaned and aligned each to their respective genome to generate gene counts.Differential expression between genotypes will be assessed using EdgeR with statistical contrasts made for general patterns (e.g., time, aphid, genotype) and genotype specific patterns. We will also construct a baseline constitutive R5+ gene expression profile and assess interactions with time to detect genes linked to constitutive and induced resistance in both R5+ and R5- plants and to normalize for changes in time. We will also compare plant gene expression to an unpublished Robusta 5 genome to manually curate novel genes not found in M. domestica genomes.To identify what gene absence or presence may underlie its resistance phenotype, DNA (collected in Obj 1) will be compared to WAA populations across the major growing regions from both cultivated and wild hosts. To leverage a whole genome resequencing approach, insect samples will be collected from commercial and research orchards and nurseries throughout Europe (n≥15), Canada (n≥15), and across the major growing regions in the U.S. (n≥300). Insects will also be sampled from wild hosts near orchards when present, and chosen on the basis of apple production density and degree of geographic isolation, with a goal to sample the widest possible area. Samples will be both targeted and opportunistic. Field collections will occur over two years. Samples will be collected, maintained in refrigeration (on ice), and then preserved in 95% alcohol back at the lab. Samples will be stored at -20C prior to shipping to UCR and then DNA extracted and prepped for Illumina NovaSeq at the UCR core facilities. Sequencing will target >30x coverage for all samples and nucleotide polymorphisms (SNPs) will be identified using standard bioinformatics analyses.To identify the specific plant genes that interact with WAA genes of interest we will employ two complementary protein interaction assays and screen newly developed rootstocks for resistance. For the Y2H-BiFC protein interaction pipeline candidate effectors defined in Obj. 1 and 2 will be screened against protein products of an apple cDNA library in a yeast-two hybrid assay as in with modifications. Here, WAA effector genes (excluding signal peptide) will be PCR amplified from cDNA and the amplified genes will be cloned into a gateway cloning vector and then recombined and transformed into yeast. Next, plant candidate genes will be PCR amplified from apple cDNA and cloned into a complementary vector using the gateway recombination technique, and subsequently screened for potential interactions. To validate the apple resistance-related proteins that interact with WAA effector proteins during the Y2H, a Bimolecular Fluorescence Complementation (BiFC) approach for direct protein-protein interaction will be used. The genes will be transferred from the previously prepared entry clones into gateway compatible BiFC destination vectors. Both insect and plant vectors (plasmids) containing target candidate genes will be transformed into apple protoplasts and visualized for YFP signals to detect protein interaction under confocal laser scanning microscope. For additional validation, the same vectors will be transformed into agrobacterium strain GV3101 followed by inoculation into Nicotiana benthamiana leaves. After incubation in dark for 2-3 days, transformed leaves from N. benthamiana plants will be observed for YFP signals under confocal microscope.Protoplasts from young apple leaves will be harvested where 20-25 days old leaves from in-vitro cultivated apple plants will be collected and cut into small slices of 1 mm. Leaf slices will be digested in Cell-wall digestion enzyme solution (macerozyme R-10 (0.15% (wt/vol)), cellulase R-10 (1% (wt/vol)), and hemicellulose (1.5% (wt/vol)) in 20 mM MES, supplemented with 0.5 M mannitol, 20 mM KCl, and 10 mM CaCl2, and vacuum infiltrated for 20-30 min followed by gentle shaking (40 rpm) in dark at room temperature overnight. Samples will be washed and protoplasts will be filtered through nylon mesh before centrifugation. Following resuspension in a sucrose solution. The supernatant will be discarded. and protoplasts will be washed again. The viability and density of apple protoplasts will be determined using a haemocytometer. Isolated protoplasts used in interaction assays will be incubated at room temperature overnight and examined under fluorescence microscope for YFP signals.To increase short-term tangible results from this proposal, we will also perform enhanced WAA-rootstock screens each summer. Unreleased rootstocks derived from parents Robusta 5, G.16, and several wild species that feature resistance to WAA have been or will be genotyped with high resolution SNP chips for discovery of the physical location of additional genetic effectors that modulate resistance to WAA. Diverse regional collections of WAA from the Northeast U.S. (where diversity is predicted to be highest) will be allowed to freely colonize rootstocks used in performance trials with successful genotypes sequenced for DNA (as in Obj. 2) to link insect genetic patterns with performance and diverse host plant genotypes.