Performing Department
(N/A)
Non Technical Summary
Citrus is one of the most important fruit crops in the U.S. and in the world with many cultivated varieties. The production of citrus in the U.S. has been devasted by the bacterial diseases Huanglongbing (HLB), and to some extent by citrus canker. We have generated multiple citrus genome-edited lines targeting six selected host plant susceptibility (S) genes to achieve resistance against these devastating bacterial diseases. Because commercial citrus has to be clonally propagated, gene-edited citrus plants will have stably incorporated CRISPR reagents in their genomes through generations. This indicates that there is a possibility of continued CRISPR activity affecting both genotype and phenotype of gene-edited citrus lines. Therefore, risk-assessment of gene-editing technology for engineering citrus species is imperative to meet the urgent need from the U.S. citrus industry. We propose to perform comprehensive evaluation of genome-wide off target effects and associated phenotypes in these citrus gene-edited lines compared to non-modified controls in field studies. Further, we will also evaluate the stability of edits and phenotypes over multiple years under field conditions. The results from this project will provide citrus germplasm to combat bacterial diseases and assist regulatory agencies to effectively and comprehensively assess the risks of gene-editing in citrus at both genotypic and phenotypic levels.
Animal Health Component
85%
Research Effort Categories
Basic
15%
Applied
85%
Developmental
(N/A)
Goals / Objectives
Citrus is one of the most important fruit crops in the U.S. and in the world with many cultivated varieties. The production of citrus in the U.S. has been devasted by the bacterial diseases Huanglongbing (HLB), and to some extent by citrus canker. We have generated multiple citrus genome-edited lines targeting six selected host plant susceptibility (S) genes to achieve resistance against these devastating bacterial diseases. Because commercial citrus has to be clonally propagated, gene-edited citrus plants will have stably incorporated CRISPR reagents in their genomes through generations. This indicates that there is a possibility of continued CRISPR activity affecting both genotype and phenotype of gene-edited citrus lines. Therefore, risk-assessment of gene-editing technology for engineering citrus species is imperative to meet the urgent need from the U.S. citrus industry. We propose to perform comprehensive evaluation of genome-wide off target effects and associated phenotypes in these citrus gene-edited lines compared to non-modified controls in field studies. Further, we will also evaluate the stability of edits and phenotypes over multiple years under field conditions. The results from this project will assist regulatory agencies to effectively and comprehensively assess the risks of gene-editing in citrus at both genotypic and phenotypic levels.
Project Methods
Obj 1: Generate high quality chromosome scale and haplotype resolved reference genomes for 'Duncan' grapefruit, 'Carrizo' and 'Kuharske' citrange varieties. Genomic DNA will be isolated and PacBio HiFi SMRTbell Library with 15 kb DNA fragment will be constructed and sequenced by Pacbio Sequel II platform at CD genomics. For Hi-C, sequencing will be used to generate chromosome scale and haplotype resolved citrus reference genomes.Generation of whole genome Illumina short reads for 'Duncan', 'Carrizo' and 'Kuharske', gene-edited events, tissue culture and transformation controls. WGS; 50x coverage will be generated from gene-edited lines, wild type controls, transformation controls and tissue culture regeneration controls by Illumina WGS on Illumina Novaseq 6000 platform in paired-end mode. The WGS reads for wild type controls including, 'Duncan' grapefruit, rootstocks varieties 'Carrizo' and 'Kuharske' will be used to correct the PacBio Hifi reads to further improve the genome assemblies by filling any gaps using the GapCloser package as previously done in citrus. Identification of genome-wide variants to quantify off-target effects of gene-edited lines compared to controls. We will align the whole genome sequencing reads of the genome-edited lines and controls to their respective wildtype reference genomes generated to identify genome-wide variants or off-targets. Single nucleotide variants, presence-absence variants, structural variants (>10 bp; deletions and insertions) for all the genome assemblies will be characterized using the smartie-sv pipeline, which aligns, compares, and calls insertions, deletions, and inversions. We will catalog all variants induced by tissue culture induced somaclonal variation, genetic transformation induced variation and finally gene-editing reagents induced variation.Obj 2: Evaluate the effects of targeted mutations in gene-edited citrus lines on bacterial disease resistance, vegetative growth and flowering compared to controls. All the selected citrus mutants were generated for improving resistance to HLB. We will assess the effects of edited genes on resistance to Xcc, and HLB in addition to general fitness of the trees. Artificial inoculation has proven to be a reliable, efficient approach for assessing citrus resistance to Xcc. On the other hand, CLas, the causal pathogen of HLB, has become endemic in Florida citrus groves, and the culturing of CLas has not been fully achieved. Natural inoculation of citrus by the Ascian citrus psyllids (ACPs) in citrus groves has proven effective and reliable. Thus, the resistance to Xcc will be assessed by artificial inoculation in the greenhouses, while the citrus resistance to HLB, plant growth, and flowering will be assessed in replicated field experiments. The selected citrus mutant plants are expected to flower and fruit in Year 3 and Year 4 after transplanting in Year 1 and growing in Year 2, as is typical for these citrus cultivars. Phenotyping of gene-edited citrus lines to assess resistance to bacterial pathogens Xcc and CLas. Mutant plants are being grown in the greenhouse to produce at least 20 fully expanded yet tender leaves per mutant plant. Xcc inoculum will be prepared by streaking stock culture of strain 2004-00059 onto nutrient agar plates. The diluted bacterial cell suspension [1 x 108 colony-forming units (CFU)/mL] will be used as inoculum for inoculating citrus mutant leaves. The inoculated plants will be maintained in a greenhouse and monitored daily for citrus canker lesion development. Each inoculated leaf will be documented in photos regularly for up to 20 days after inoculation and analyzed in ImageJ to measure the lesion size (area) on each inoculated leaf. To determine the Xcc bacterial population in inoculated citrus leaves, leaf discs will be sampled from inoculated sites at 4, 8 and 12 days after Xcc inoculation. The evaluation of citrus mutants for resistance to CLas will be conducted in the field studies.Obj 3: Phenotyping of gene-edited citrus lines in the field for multiple years. The gene-edited trees and controls will be planted in a randomized field block design in the field at Florida in the middle of year 2. Field set up will have 10 blocks with one clonal plant in each block. Five biological replicates for each gene-edited event and control will be planted. We have a total of 43 trees including three citrus wild type genotypes, tissue culture regenerated plants, transformation controls and gene-edited events. Morphological traits including tree height and growth rate, growth habit, size of leaves, trunk diameter, number of shoots, leaf SPAD, survival rate in the field, thorniness of leaf and stem and flower anatomy for trees that bloom in the field or greenhouse will be evaluated in year 2, 3 and 4. Phenotypic data will be collected for flower size and shape, number and color of petals, stamen, and style length. Any fruit that is produced by the data trees will be tested for total soluble solids (%), titratable acidity, fruit size, fruit length, fruit shape, fruit mass, number of seeds, and peel color. HLB visual grading will be done for each tree beginning 9 months post transplanting. Canopy color and canopy thickness will be evaluated with a rating scale with a scale of 1 to 5, with 1 representing the unhealthiest of the trees and 5 representing the healthiest of the trees. Foliar greening disease symptoms (called HLB disease index) will be rated on a scale of 1 to 5, with 1 representing the best (no foliar disease symptoms) and 5 representing the worst (75% to 100% of the canopy showing foliar disease symptoms); ratings of 2, 3, and 4 will represent 1% to 25%, 25% to 50%, and 50% to 75%, respectively, of the affected canopy. Leaves will be sampled from all four quarters of each plant to extract total DNA to conduct PCR detection of CLas by real-time qPCR assays as established to determine CLas-infection. Evaluate the risk of extended CRISPR activity on target and off-target sites over multiple years. To study the effects of continued expression of Cas9 over multiple years, we will utilize the citrus lines in field sites in year 4 to: i) evaluate if the CRISPR mutations due to Cas9 activity over time. We will re-sequence the respective target S-genes (and alleles) by amplicon-sequencing or clone individual alleles for sanger sequencing. We will also evaluate if the detected mutation site alterations (if any) are gene-edited event-specific or tree-specific. ii) In case we detect any genome-wide off target mutations in our gene-edited events from Obj 1, we will also perform targeted sequencing of those off-target sites as mentioned above. iii) We will confirm if Cas9 and other elements of T-DNA are present in all plants by PCR and investigate if Cas9 protein accumulation is variable in our gene-edited events before planting in the field in year 1 compared to field grown plants in year 4. Evaluate the stability of citrus gene-edited lines phenotype in the field. We will measure all the phenotypic parameters described in obj 2 every year to evaluate the growth and development of these trees. We will observe specifically for any aberrant phenotypes in the field due to pleiotropic phenotypes caused by knocking out S-genes or due to chimerism of gene-edited events. S-genes make the plants susceptible to pathogen infection, however they are also involved in several plant physiological functions which explains why they are evolutionarily conserved in plants. Therefore, there may be pleiotropic effects due to knocking out of S-genes and the fitness costs vary between species and must be evaluated in a species dependent manner. If we see any aberrant phenotypes, we will isolate genomic DNA from such aberrant trees in year 4 along with regular phenotype trees and perform targeted sequencing from both to compare if there are any new mutations.