Source: WASHINGTON STATE UNIVERSITY submitted to NRP
SYSTEMS VIEW OF PLANT DEFENSE RESPONSE TO PROTIST AND VIRAL PATHOGENS IN POTATO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030835
Grant No.
2023-67011-40505
Cumulative Award Amt.
$116,867.00
Proposal No.
2022-11477
Multistate No.
(N/A)
Project Start Date
Jun 1, 2023
Project End Date
May 31, 2026
Grant Year
2023
Program Code
[A7101]- AFRI Predoctoral Fellowships
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
(N/A)
Non Technical Summary
Potato is the third highest consumed staple crop in the world. One of the significant threats to potato crop production is microorganisms that cause plant diseases. Such organisms are generally named plant pathogens. There are around 75 potato pathogens that have been recognized in the US. The proposed research focuses on two potato pathogens, Spongospora subterranea f. sp. subterranea (Sss) and potato mop-top virus (PMTV). These pathogens cause powdery scab and potato mop-top diseases, respectively. Sss causes scabby lesions on potato tuber skin and galling on potato roots. Severe root gall formation can reduce the plant uptake of nutrients and water leading to stunted growth. Sss also transmits PMTV, facilitating its survival in the soil and entry into the plant. The survival spores (cystosori) produced by Sss can survive in the soil for over ten years and carry PMTV. PMTV infection can also damage the potato tuber, causing necrosis in the tuber flesh. The tuber damage caused by Sss and PMTV makes the tuber unsuitable for fresh market and processing. Unfortunately, there are no successful chemical control methods available for Sss or PMTV. Therefore, disease management is mainly preventative via pathogen-free seed tubers and non-contaminated fields. Alternatively, potato cultivars with partial and sporadic resistance could be used to minimize disease impact. While the mechanism of potato disease resistance to Sss and PMTV is unknown, varying resistance among different potato cultivars has been observed. However, the lack of complete resistance in commercially favorable cultivars can make suppressing powdery scab and mop-top diseases using resistant cultivars challenging.In every living organism, including plants, genes determine the inherent traits. While some genes in an organism are expressed at all times, other genes are expressed as responses to external stimuli. Plant pathogen infection is one such stimulus that affects gene expression in plants. Some genes expressed after a pathogen infection are directly associated with resistance or susceptibility of the plant to the respective pathogen. Both prior studies and our data have shown that the different potato cultivars with different Sss resistances show differing expression of genes when challenged with Sss.Thus, the main goal of the proposed project is to identify and characterize the function of genes involved in the interaction among potato, Sss, and PMTV. We hypothesize that by identifying the differentially expressed genes in cultivars with different resistances, we can identify potato genes that originate resistance against Sss and PMTV. Also, comparing gene expressions in Sss before and during interaction can reveal the genes involved in the interaction between Sss and PMTV.To achieve this goal, we propose the following methods. First, three selected potato cultivars with varying resistance to Sss/PMTV will be inoculated with Sss with and without PMTV. Next, the gene expression of each sample will be read using established sequencing technologies. Then, using multiple bioinformatics tools, the gene expression of each cultivar under each infection condition with compared with one another to identify genes that are unique to each host-pathogen interaction. Finally, we would confirm the effect of candidate genes identified through the above process using genetic engineering methods. All work will be done in laboratory and greenhouse settings, and genetically modified materials will not be released into the external environment.The resistance and pathogenicity genes discovered from this study can be used directly to control Sss and PMTV in multiple ways. Phenotype-based screening for resistance in conventional breeding programs could be time-consuming and misleading, as disease symptom expression depends heavily on potato cultivars and the environment. This project will boost the efficiency of breeding programs by identifying genes that can be used as selection markers for resistance cultivar screening. Also, resistance genes identified in the proposed work can be used as a target for genetic modification in breeding programs in the future, according to proper state and federal regulations and guidelines. Information gathered on pathogen virulence genes could contribute to disease control via host genome modification-based pathogen control methods. Overall, the proposed work will lead to the development of plant resistance against powdery scab and potato mop-top diseases, reducing economic losses and contributing to food security. Boosting plant resistance will reduce the use of partially effective fungicides, easing detrimental effects on the environment and human health.Genetic mechanisms of plant virus transmission by protists remain largely unknown. The proposed study will generate novel knowledge on the genomic basis of such tripartite interactions and contribute to building a pathogenesis model of plant-protist interactions. Such a model can help understand and control similar host-protist interactions. Therefore, the proposed work significantly advances the scientific understanding of plant host-pathogen interactions.
Animal Health Component
(N/A)
Research Effort Categories
Basic
90%
Applied
(N/A)
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121310116050%
2011310104050%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1310 - Potato;

Field Of Science
1040 - Molecular biology; 1160 - Pathology;
Goals / Objectives
Powdery scab disease of potato (Solanum tuberosum) is caused by protist soilborne pathogen, Spongospora subterranea f.sp. subterranea (Sss). Sss is the only known vector for potato mop-top virus (PMTV), which cause tuber necrosis. Sss causes root galls and skin lesions on tubers, producing aggregates of resting spores called cystosori. Sss cystosori can survive in the soil for over ten years without a host and carry PMTV, and release infectious zoospores when conditions are favorable.Effective chemical control is currently unavailable, and crop rotation is ineffective as Sss has many alternative hosts. Therefore, disease management is mainly preventative via pathogen-free seed tubers and non-contaminated fields. Alternatively, potato cultivars with partial and sporadic resistance could be used to minimize disease impact. While the mechanism of resistance is currently unknown, there is varying resistance among different potato cultivars. In addition, differential regulations of potato genes upon Sss infection have been shown in our previous workand prior literature. Thus, we hypothesize that the resistance originates from a genomic basis during the host-pathogen interaction. The main goal of the proposed project is to identify and characterize the function of genes or gene modules involved in the interaction among potato, Sss, and PMTV. Identification of the genes involved in the plant -vector-pathogen interaction will lead to the development andenhancement of plant genetic resistance against Sss and PMTV, via conventional breeding and genetic manipulation. Furthermore, the genetic mechanisms of plant virus transmission by protists remain largely unknown. The proposed study will also generate novel knowledge on the genomic basis of such tripartite interactions and contribute to building a pathogenesis model of plant-protist interactions. Such a model can help understand and control similar host-protist interactions, such as Polymyxa graminis and BaYMV/SBWMV vector-pathogen interaction and Plasmodiophora brassicae pathosystem.To test the hypothesis and attain the goal, we propose the following specific objectives.(1) Identifying regulatory genes involved in potato-Sss-PMTV interaction via system-level analysis: under this first objectivewe would use a novel hairy roots system to collect plant tissue post-infection to collect transcriptomic data. Wewould then analyze the post-infection gene expression data collectedusing bioinformatic tools includingdifferential gene expression analysis (DGE) and gene co-expression network analysis (GCN). These analyses will reveal genes that may be involved in resistance or susceptibility against Sss and PMTV.(2) Functional validation of candidate genes conferring plant resistance/susceptibility, pathogen virulence, and viral transmission: Under thissecond objective we wouldvalidate candidate genes identified through DGE and GCN using transgenic engineering, i.e., RNA interference (RNAi), gene editing, and overexpression. Such gene engineering will be first performed in potato hairy roots. Then, the genes that show an effect on Sss and PMTV pathogenicity will be tested in greenhouse assays to confirm the effect in full plants.
Project Methods
Objective 1. Identification of regulatory genes involved in the potato-Sss-PMTV interaction.Hairy roots of three potato cultivars (Shepody, Russet Burbank, and Premier Russet) with differing resistance will be inoculated with viruliferous and non-viruliferous Sss. Samples will be collected from inoculated and control roots at four time points post-infection (3, 6, 48, and 72 hours). Four replicates will be collected per treatment condition. Total RNA from samples will be sequenced using the 2 × 150 bp Illumina NovaSeq platform.Data analysis: RNA-seq data will be aligned to the genome of S. tuberosum Group Phureja DM1-3 (v6.1). Then, correlation coefficients will be calculated between each pair of genes using GEMmaker workflow, and DGE analysis will be done using the DEseq2 package in R. Comparing the untreated control and the Sss inoculated resistant samples may reveal plant defense genes responding to Sss infection. Comparison between viruliferous Sss and non-viruliferous Sss inoculated samples may reveal plant genes related to PMTV resistance. Correlation coefficients calculated with GEMmaker will also be used to construct GCN using KINC and KINC-R packages. Genes that interact and contribute to the same process, i.e., gene modules in the network, will be identified using the R linkcomm package. A functional enrichment using FUNC-E software will assign functional categories to genes discovered from DGE and GCN. Genes and modules associated with plant defense or physiology will be considered candidates for resistance. RNA-seq data will also be aligned to the Sss genome to identify Sss genes. DGE and GCN will be done as described above. In DGE, a comparison between Sss inoculated susceptible cultivar, and Sss inoculated resistant cultivar will reveal putative Sss virulence genes. Comparing viruliferous Sss-inoculated susceptible cultivarto non-viruliferous Sss-inoculated susceptible cultivarwill reveal Sss genes related to PMTV transmission. GCN will reveal Sss genes that interact under each condition. Following functional annotation, modules or genes associated with pathogen virulence-related functional categories will be selected as candidates for validation.Objective 2. Functional validation of regulatory genesFirst, RT-qPCR will be done to validate the differential expression of candidate genes identified from DGE and GCN. For potato candidates, each gene will be overexpressed, knocked out via CRISPR/Cas9, or knocked down via RNAi in the hairy roots of resistant and susceptible cultivars. RNAi vectors will be created for HIGS targeting each candidate Sss gene, and hairy roots will be induced from a susceptible cultivar using R. rhizogenes carrying the vector. Sss candidate genes will also be overexpressed in the resistant cultivar. All transgenic hairy roots will be challenged with viruliferous and non-viruliferous Sss. The Sss propagation rates and expression of defense marker genes will be quantified. Finally, selected transgenics will be regenerated as whole plants and assessed the same as above in greenhouse trials.If knockdown or knockout of a potato candidate gene from a resistant cultivar makes it susceptible to Sss or PMTV, and overexpression of the same gene in a susceptible cultivar makes it resistant, then such gene can be predicted to be involved in plant defense against the pathogen. If the HIGS-based silencing of a putative Sss pathogenicity gene in a susceptible cultivar or overexpression in a resistant cultivar would increase pathogen growth, it indicates that the gene is a virulence factor of the Sss. If a putative candidate related to Sss-PMTV interaction silenced via HIGS causes a short-term reduction of PMTV infection post-inoculation, it suggests that the candidate gene is involved in PMTV transmission by Sss. This experimental verification of gene candidates will accelerate downstream applications.

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The results obtained through the overall project isare targeted toward three groups: the scientific community, the potato industry, and growers. The information gathered via the experiments conducted during the reporting period was disseminated to the scientific community and potato industry representativesvia three conference presentations ( 2 oral presentations and one poster). The specific groups reached include: The worldwide group of Spongospora researchers: Oral presentation at3rdinternational Spongospora workshop, Oslo, Norway Potato research community and industry: Oral presentation at 22nd European Association of Potato Research Triennial Conference: Oslo, Norway. : oral presentation Plant pathology research community and industry: Poster presentation atAmerican Phytopathological Society Annual Conference - Plant Health 2024 Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI presented the knowledgegenerated from the project and three different conferences: 1. 3rd international Spongospora workshop, Oslo, Norway : oral presentation 2. 22ndEuropean association of potato research triennial conference: Oslo, Norway.: oral presentation 3. American Phytopathological Society Annual ConferencePlant Health 2024: poster presentation How have the results been disseminated to communities of interest?The generated data and knowledge have been disseminated via conference and poster presentations to the scientific community, including graduate students and other researchers. Additionally, amanuscript is in preparation to share the work related to SA-mediated defense against Sss, which is expected to be submitted within 2 months. What do you plan to do during the next reporting period to accomplish the goals?To accomplish the specific goals, the following activities will be completed within the next reporting period: 1)Identifying regulatory genes involved in potato-Sss-PMTV interaction via system-level analysis: Analysis of transcriptomic data will be completed within the next three months to identify candidates of resistance/ susceptibility in potatoes against Sss and PMTV. Additionally, a spatial transcriptomic study will be conducted using the curio-seeker platform to study gene expression at different cellular localities. The data generated from this study will also contribute to the identification of candidates for resistance or susceptibility in potatoto Sss and PMTV. Additionally, we will also investigate genes of Sss involved in the interaction with potato. 2)Functional validation of candidate genes conferring plant resistance/susceptibility, pathogen virulence, and viral transmission. The top ten candidate genes identified in specific objectiveone will validated via molecular genetic manipulations. For this, trangenic hairy root lines will be generated with eitheroverexpression or RNAi-mediated knockdown of each gene. Pathogenicity assays will be conducted to test the impact of each gene on pathogen propagation in roots. Whole plants will be regenerated from the hairy roots, and pathogenicity assays for each trangenic line will be conducted under greenhouse conditions to quantify the impact of each gene on disease development.

Impacts
What was accomplished under these goals? The main goal of the proposed project is to identify and characterize genes or gene modules involved in the interaction among potatoes, Spongospora subterraneaf.sp subterranea(Sss), and potato mop-top virus (PMTV). Specifically, we aim to : 1)Identifying regulatory genes involved in potato-Sss-PMTV interaction via system-level analysis 2)Functional validation of candidate genes conferring plant resistance/susceptibility, pathogen virulence, and viral transmission. - To reach specific objective 1, RNA sequencing data was collected from three potato cultivars, with and without Sss and PMTV pathogen inoculation, at six different time points, with 3-4 replicated for each combination. - Data analysis to identify the specific genes or gene modules involved in the defense against Sss and PMTV is underway. The techniques used include differential gene expression analysis, gene co-expression network analysis, and machine learning techniques. - Additionally, experimentshave been conducted to understand the involvement of salicylic acid (SA)-mediated defense response toSss. The data generated points towards the involvement of SA to regulate defense against Sss in potato. This work included: The quantification of SA in potatocv. Shepody hairy roots post sss inoculation: The SA levels in roots increased significantly 4 weeks post pathogen inoculation, while Jasmonic acid levels did not change significantly. Results indicate a potential involvement of SA in defense response against Sss. Quantify the impact ofSA, methyl jasmonate,and 1-aminocyclopropane-1-carboxylic acidpre-treatmenton Sss propagation in potato cv. Shepody hairy roots. Pathogen quantification via qPCR indicated that the pathogen levels in SA pre-treated roots were significantly reduced one week after inoculation.External application of a plant hormone can boost plant defense against a pathogen if the said hormone pathway is involved in defense. Therefore, reduction in Sss levels in roots post-SAtreatment indicates a potential involvement of SA in defense response to Sss. Targeting the downstream regulatory genes of SA-mediated defense response pathway. NPR1 is a downstream positive regulator of SA-mediated defense.SlNPR1 overexpression hairy root lines were generated using cv. Shepody. The initial selection of transgenic lines was based on GFP expression and kanamycin resistance.SlNPR1 expression levels were quantified using RT-qPCR. Four selected transgenic lines were inoculated with Sss cystosori, and the Sss levels post-inoculation were quantified using qPCR. Results indicated that the Sss propagation level in NPR1 overexpression lines was significantly lower compared to the empty vector control one week after pathogen inoculation. The pathogen level remainedsignificantly lower in twotransgenic lines 4 weeks post-inoculation. Reduction in Sss levels in the presence of overexpression of SA positive regulator indicates involvement of SA in defense response to Sss. NPR3is a downstream positive regulator ofSA-mediated defense.StNPR3knockdown hairy root lines were generated from cv. Shepody, using CRISPR-cas9-basedediting technique. The initial selection of transgenic lines wasbased on GFP expression and kanamycin resistance. The knockdown efficiency of transgenic lines was quantified using amplicon sequencing. A selected knockdown line with 78% knockdown efficiency was inoculated with Sss cystosori.Results indicated that the Sss propagation level inNPR3 knockdown linewas significantly lower compared to the empty vector control fourweeks after pathogen inoculation. Reduction in Sss levels in the presence of knockdownof SA negative regulator indicates involvement of SA in defense response to Sss. Overall, based on our results, we conclude thatSA-mediated defense plays a role against the control of Spongospora subterranea f. sp. subterranea in potato. Further experiments are currently being conducted to verify the involvement of SA, including nahGoverexpression lines, which have reduced SA levels, and NPR3 knockdown lines via RNAi, which will have higher SA levels compared to wild type.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Moroz, N., Colvin, B., Jayasinghe, S., Gleason, C., & Tanaka, K. (2024). Phytocytokine StPep1-secreting bacteria suppress potato powdery scab disease. Phytopathology�. https://doi.org/10.1094/PHYTO-01-24-0019-R
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jayasinghe, S.,Moroz, N., and Tanaka, K., (2024) Salicylic-acid mediated defense against the powdery scab disease. 22nd EAPR Triennial conference, Oslo, Norway.
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Jayasinghe,S. K.; Moroz, N.; Tanaka, K., Involvement of salicylic acid in the interaction betweenpotato and Spongospora subterranea: insights from a hairy root system. MPMI
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Jayasinghe, S.,Moroz, N., Pappu, H.,Ficklin, S., and Tanaka, K., (2024) Salicylic acid-mediated defense against powdery scab disease in potato, Plant Health 2024: American Phytopathological society annual meeting.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Jayasinghe, S.,Moroz, N., and Tanaka, K., (2024) Salicylic-acid mediated defense against the powdery scab disease. 3rd international Spongospora workshop, Oslo, Norway