Location: Physiology and Pathology of Tree Fruits Research
2023 Annual Report
Objectives
The long-term objective of this program is to formulate effective and economically sustainable methods for management of tree fruit diseases in high value production systems. Utilization of host resistance presents an economically effective, ecologically desirable, and durable disease control strategy. Additionally, horticultural traits that allow for disease avoidance, such as dwarfing of the scion that allows for better spray and light penetration of canopies, have yet to be widely incorporated into pear rootstocks. For this purpose, it is a necessary first step to understand the molecular mechanisms underlying resistance/avoidance traits in apple and pear roots by identifying key genes regulating root resistance responses and other relevant physiological processes. This research plan will make it possible to develop molecular tools for accurately and efficiently incorporating resistance traits into new apple and pear rootstocks.
Objective 1: Discover rootstock resistance traits and architectural features in deciduous tree fruits involved in disease avoidance.
Sub-objective 1.A: Characterize genotype-specific variations in biochemical and metabolic features linked to apple root resistance to P. ultimum.
Sub-objective 1.B: Determine the connection between pear root architecture, disease resistance, and dwarfing.
Sub-objective 1.C: Identify genetic components involved in rootstock-mediated dwarfing of pear scions.
Sub-objective 1.D: Develop a rapid-cycle breeding tool for use in breeding pear rootstocks.
Objective 2: Identify genotype-specific expression patterns of candidate genes underlying disease resistance and disease avoidance traits.
Sub-objective 2.A: Conduct bioinformatic analysis of the sequence features of selected candidate genes identified in previous transcriptome analyses.
Sub-objective 2.B: Characterize genotype-specific expression patterns for selected candidate genes between resistant and susceptible genotype groups.
Sub-objective 2.C: Transgenic manipulation of selected apple candidate genes using CRISPR/Cas9 tool and in planta expression analysis.
Approach
A combination of current and emerging genetic and genomic techniques will be applied in this research plan. Various methodologies in phenotyping and molecular analysis including genomics, transcriptomics genetics, biochemistry, breeding and microscopy will be utilized to identify the genetic elements and unravel the regulation mechanisms underlying apple root disease resistance and pear architectural features. Such studies will improve knowledge of molecular mechanisms underlying important traits and genome annotation, facilitate the development of germplasm and establishment of a rapid-cycle breeding tool to enhance our understanding of how rootstocks confer traits to scions.
Experiments will be conducted using previously identified apple rootstock germplasm pairs with contrasting resistant versus susceptible phenotypes, such as O3R5-#161 vs # 132; #58 vs #47 and/or #164 vs #1, respectively. Expression analysis of selected candidate genes and biochemical or enzymatic assays will provide experimental evidence connecting genes and trait during apple root response to P. ultimum infection. De novo sequencing of specific genomic fragments containing the genes of interest will identify variations at gene structure and sequences between resistant and susceptible O3R5 genotype groups, which may provide valuable reference for their functional roles in defense activation. The knockout (KO) transgenic line will be generated using established in-house protocols including plasmid construction, transformation of E. coli and Agrobacterium, subsequent callus induction and individual transgenic line establishment/propagation by tissue culture.
For research related to pear architectural traits, parental genotypes will be established in tissue culture, rooted, acclimated, and grown to similar sizes before moving to different phenotyping methods during the first year. Data analysis will compare differential gene expression between the most and least dwarfing individuals across tissue type and time, allowing us to understand more about dynamic gene activity on the scion and rootstock sides of the graft union, as well as in the root system and how the rootstock is affecting scion leaf tissue. A major limitation for breeding new rootstocks is the long juvenility period of pear (reaching up to ~10 years in European pear), especially when stacking multiple traits is the desired goal. DNA-informed breeding speeds the selection process, but juvenility periods remain long, meaning variety releases are 20-40 years from initial crosses, depending on species and breeding scheme. To shorten this process, development of a rapid-cycle breeding tools by modifying flowering gene expression can greatly reduce amount of time allows for stacking multiple loci or traits.
Progress Report
This report documents fiscal year (FY) 2023 progress for project 2094-21220-003-000D, titled, “Uncovering Rootstock Disease Resistance Mechanisms in Deciduous Tree Fruit Crops and Development of Genetics-Informed Breeding Tools for Resistant Germplasm”.
In support of Objective 1, ARS researchers in Wenatchee, Washington continued to identify apple and pear rootstock resistance traits and architectural features in deciduous tree fruits involved in disease avoidance. Characterization of the selected cellular changes and biochemical reactions is part of the continuing in-depth phenotyping of apple root resistance responses under pathogenic pressure from Pythium ultimum. Several biochemical assays to assess the activated defense response under pathogenic pressure, including those of measuring ((phenylalanine ammonia-lyase (PAL), catalyzing the first committed step of phenylpropanoid pathway)) activity and determining superoxide generation rate, were tested using a Tecan Infinite 200 Pro microplate reader. In addition, these evaluated assays can be utilized for detecting the potentially altered resistance responses from planned transgenic manipulation of selected candidate genes based on our previous transcriptome datasets (Sub-objective 1A). Experiments designed to explore the expression of select biosynthetic genes associated with tolerance to replant disease in apple roots (Sub-objective 1A) have been delayed due to staffing challenges/shortages, however, a highly qualified candidate is expected to start in July. Due to the lack of technical support, ARS researchers in Wenatchee, Washinton, conducted a reassessment of the research goals and objectives and specifically refocused efforts to ensure progress was made on NP Action Plan Problem Statement 1A (Characterize and Integrate Computational and Culture Resources, Genomics, and Systemics of Plant Pathogens). These actions included the establishment of a collaboration with researchers at the University of Turin, Italy, to explore potential similarities in Kiwivine decline syndrome (KVDS) and apple replant disease (ARD) pathosystems, disease systems which are similar in temporal and biological attributes. For example, in both systems, Phytopythium and Pythium spp. are key causative agents of the disease syndrome. To this end, a PhD student (University of Turin) has been approved to work at the ARS research lab in Wenatchee, Washgington, through the Foreign Visitor Fasttrack Program. By focusing on plausible resistance genes previously identified in apple, she will examine kiwifruit gene expression patterns in response to Phytopythium root infection. Initial experiments examining the effect of Phytopythium root infection on gene expression in kiwi are currently underway. Exploring potential similarities in KVDS and ARD pathosystems is expected to provide new information that will improve disease-management in both systems. To address pear architectural traits that are important for disease avoidance, SY received additional pear germplasm and expanded micropropagation to include 20 cultivars. Development and optimization of rooting protocols for these cultivars has continued, as a prerequisite for successful growth and testing with different phenotyping technologies and analysis programs. Growth responses to different agar-based media have been tested and optimal media has been determined for 19 cultivars. Due to unexpected loss of personnel, assessment of phenotyping platforms was temporarily delayed. Work has begun on building rhizotrons, aeroponic chambers, and minirhizotron arrays for phenotyping pear RSA (root system architecture) (Sub-objective 1B). Arbuscular mycorrhizal fungi (AMF) have been shown to play a role in providing tolerance/resistance to replant pathogens in apple. Research assessing functional outcomes resulting from specific apple rootstock/AMF associations continues. Experiments designed to assess the effects of the interaction between apple rootstock genotype and AMF species on root mycorrhization were conducted. AMF species identity was found to be a significant source of variation affecting the percentage of mycorrhizal colonization. The highest levels of colonization were obtained by G.41 x S. deserticola and G.890 x C. claroideum, although percent colonization did not reflect growth outcomes or nutritional status. In G.210, AMF species identity was associated with leaf nitrogen status as well as the plant growth response. These findings suggest that matching host genetics with compatible AMF species has the potential to enhance agricultural practices in nursery and orchard systems (Subobjective 1B). Pear rootstocks (87 and 97; ungrafted) arrived from the nursery in Spring of 2023. Experiments designed to characterize the effect of pear rootstock genotype on composition of the rhizosphere microbiome when cultivated in orchard replant soils are being planted during the current summer season. Rootstock sampling and analysis of the rhizosphere microbiome will be conducted in the autumn of 2023. To investigate transcriptional changes associated with dwarfing, an architectural trait important for minimizing disease pressure, sibling germplasm from a population segregating for a dwarfing trait has been transitioned to soil and will be grown for an additional year before grafting ‘Bartlett’ scions. To aid in analyzing transcriptional data, a second year of tissue collection from Bartlett and Anjou was completed for development of an expression atlas. Tissue is currently being processed and RNA being extracted from it, which will be sequenced in the coming year. For complementation experiments of key RSA-related proteins using Arabidopsis, seed has been obtained and cloning of appropriate constructs is in progress. Related to understanding dwarfing traits, SY has additionally worked with the Washington State Universtiy breeding program to narrow down potentially important genes within a Quantitative Trait Locus (QTL) for dwarfing they recently identified. This work has included identification of functional annotations within the QTL region and preparing samples for a QTL-Seq analysis to determine genomic changes associated with the dwarfing phenotype (Sub-objective 1C). In the development of a rapid-cycle breeding (RCB) tool, the first transformant has recently recovered from transformation attempts, and transformation protocols continue to be optimized. Efficiency of all established and published protocols is quite low with the cultivars of interest. However, the RCB construct being used has a red fluorescent marker integrated, thus it shows that cells within the callus are indeed becoming transformed, demonstrating that transformation is successful. This has helped identify that regeneration of shoot tissue from these cells is the experimental bottleneck and has led to dedication of more experimental time to optimizing regeneration. Additionally, a version of the RCB construct with a different antibiotic selection marker (Hygromycin instead of Kanamycin) has been developed to test for effects on efficiency (Sub-objective 1D).
In support of Objective 2, several groups of apple candidate genes, including four transcription factor (TF) families, were selected for continuing bioinformatics analyses. The selection of these candidate genes is based on our previous transcriptome analyses of apple root defense response to P. ultimum infection. The candidate genes for this batch include apple NAC (NAM, ATAF and CUC), ethylene response factor, auxin response factor and myeloblastosis related proteins transcription factors, which were implicated in the direct regulation of phenylpropanoid biosynthesis pathway in response to P. ultimum infection based on our recent study on microRNA profiling and degradome sequencing. Sequences were retrieved from publicly available apple genome sequences, and their sequence features relevant to their proposed functions were analyzed using various bioinformatics tools. The presence of specific sequence domains and motifs in the promoter of these genes provided the potential biological context and new insights of these genes during apple resistance response to P. ultimum infection such as certain hormonal regulation. The primers were designed for cloning the genomic copies of O3R5 (‘Ottawa 3’ x ‘Robusta 5’) genotypes. This is because the exact genomic sequences are essential for designing “guide sequences” for subsequent transgenic study using genome editing tools such as CRISPR (Sub-objective 2A). The primers were also designed to evaluate their expression patterns using a quantitative reverse transcription PCR method. Genotype-specific mRNA levels for a specific candidate gene can be an important indication of its potential roles during defense activation in apple root under the challenge by P. ultimum between resistant versus susceptible genotypes (Sub-objective 2B).
Accomplishments
1. Enhancement of pear genetics research through development of molecular biological and genomics tools. Among tree crops, resources for pear genetics and genomics research are relatively few and underdeveloped, resulting in limited research competitiveness. ARS researchers in Wenatchee, Washington, identified the need for enhanced breeding tools and began development of a rapid-cycle breeding tool for pears, which has resulted in improved pear transformation protocols and knowledge of cultivar-specific differences in transformation, regeneration, and rooting. ARS researchers identified major issues with pear genomics resources and built a collaboration to address them. Through this collaboration, they polished the ‘Bartlett’ genome and developed a novel bioinformatic workflow to improve gene annotations. Further, they built a collaboration with UC Davis researchers and began development of a transgene-free gene-editing systems for pears, initially leading to improved pear regeneration protocols benefitting the pear research community. Once established, these tools will allow for introduction of genetic changes linked to key traits into existing germplasm, which adds an additional route for genetic improvements benefitting pear growers.
Review Publications
Zhu, Y. 2022. The feasibility of using autofluorescence to detect lignin deposition pattern during defense response in apple roots to Pythium ultimum infection. Horticulturae. 8(11). Article 1085. https://doi.org/10.3390/horticulturae8111085.
Fazio, G., Mazzola, M., Zhu, Y. 2023. Genetic analysis of resistance to Pythium ultimum a major component of replant disease in apple rootstocks. Journal of the American Pomological Society. 77(1)28-37.
Chen, L., Cochran, A.M., Waite, J.M., Shirasu, K., Bemis, S.M., Torii, K.U. 2022. Direct attenuation of Arabidopsis ERECTA signalling by a pair of U-box E3 ligases. Nature Plants. 9:112-127. https://doi.org/10.1038/s41477-022-01303-x.
Berihu, M., Somera, T.S., Malik, A., Medina, S., Piombo, E., Tal, O., Cohen, M., Ginat, A., Ofek-Lalzar, M., Doron-Faigenboim, A., Mazzola, M., Freilich, S. 2023. A framework for the targeted recruitment of crop-beneficial soil taxa based on network analysis of metagenomics data. Microbiome. 11. Article 8. https://doi.org/10.1186/s40168-022-01438-1.
Waite, J.M., Kelly, E., Zhang, H., Hargarten, H.L., Waliullah, S., Altman, N.S., dePamphilis, C., Honaas, L.A., Kalcsits, L. 2023. Transcriptomic approach to uncover dynamic events in the development of mid-season sunburn in apple fruit. G3, Genes/Genomes/Genetics. 13(8). Article jkad120. https://doi.org/10.1093/g3journal/jkad120.
