Project Details:

Title:
An integrated approach to enhance durability of SCN resistance for long-term, strategic SCN management (Phase II)

Parent Project: An integrated approach to enhance durability of SCN resistance for long-term strategic SCN management (Phase II)
Checkoff Organization:North Central Soybean Research Program
Categories:Soybean diseases, Nematodes, Breeding & genetics
Organization Project Code:00071398
Project Year:2021
Lead Principal Investigator:Andrew Scaboo (University of Missouri)
Co-Principal Investigators:
Thomas Baum (Iowa State University)
Andrew Severin (Iowa State University)
Gregory Tylka (Iowa State University)
Melissa Mitchum (University of Georgia)
Brian Diers (University of Illinois at Urbana-Champaign)
Matthew Hudson (University of Illinois at Urbana-Champaign)
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Keywords: Soybean Cyst Nematode

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

One of the major milestones in SCN biology is developing a completely annotated SCN reference genome, which we have recently accomplished for the TN10 SCN isolate (Masonbrink et al., 2019). Since then, we have made considerable progress and now have a chromosome level genome assembly ready and annotated. Simultaneously, we have developed a centralized, web-based repository called SCNBase (SCNBase.org). We have developed this web portal de novo and published it in November 2019 (https://doi.org/10.1093/database/baz111). This web portal now is the home to all bioinformatic, genetic, genomic, and molecular data generated for SCN, most of it through soybean check-off funding. Using this web portal, researchers and breeders from all over the world can access and analyze all public bioinformatic data generated in this proposal and curated from previous research at the nucleotide level. We are already seeing significant “web traffic” to this web portal suggesting that it is generating considerable interest in the SCN community from all over the world.

Our group is actively involved in developing, analyzing and comparing gene expression in virulent (i.e., able to infect resistant soybean cultivars) and avirulent (i.e., unable to infect resistant soybean cultivars) SCN populations with the goal to identify the genetic determinants of virulence. For that purpose, we are focusing on three individual gland cells, in which the nematode produces the effectors/tools required for infection and defense suppression, using a method we previously had developed. Toward that end, we have completed three independent biological replications of mRNA purification, library construction and sequencing (which is essentially an identification of all genes active in the gland cells at a given time) from each of the virulent (MM10) and avirulent (PA3) SCN populations. Furthermore, gene identities from all sequencing experiments have been generated utilizing the SCN TN10 genome produced by us (Masonbrink et al., 2019). We identified 13,617 unique transcripts from the PA3 population and 8,820 unique transcripts from the MM10 population, which represent a snapshot of gene activity during the early parasitic stage of SCN infection (parasitic J2). As a point of validation, we have identified all previously discovered SCN effectors expressed during this early parasitic stage within our gland cell-specific sequence data. Initial analysis of these sequence data has revealed intriguing gene expression differences between these virulent and avirulent SCN populations. Specifically, there are 32 genes upregulated in the SCN PA3 libraries versus the MM10 libraries. Protein products of 13 of these genes are predicted to be nuclear localized and likely have gene regulatory functions. This includes a diverse group of proteins involved in functions such as structural maintenance of chromosomes, splicing factors, a glycosyltransferase involved in increased virulence in other parasitic nematodes, DNA-binding proteins, and a ‘ran’-binding protein, among others. Conversely, we discovered 17 genes that are upregulated in SCN MM10 versus PA3. Protein products of eight of these genes are predicted to be nuclear localized. Among this group are previously reported effectors, a microtubule binding protein homolog from an animal-parasitic nematode, signal transduction proteins, and several unknown proteins. We are still at the initial stages of analysis but it is already clear that we have generated a rich source of critical data needed to understand SCN virulence. The MM26 parental population was prepared and sent for genome sequencing; the others will follow. RNA-seq data generated from early parasitic life stages of a virulent and avirulent population of SCN were used to conduct a reference-based transcriptomic analysis with the aid of the SCN genome. We identified 207 genes unique to the virulent SCN that could be turned on to overcome resistance mechanisms; conversely, 92 genes that could be turned off to evade triggering resistance mechanisms.

Being a sedentary endoparasite that relies exclusively on its host for survival, SCN has to suppress host defense responses for a significant duration in order to survive. The SCN achieves this by producing a large number of effector molecules and delivering them into the soybean cells via its mouth spear. These effectors specifically target host factors and modulate their functions, thereby also altering soybean defense responses. Generating an in-depth understanding of how individual effectors help SCN establish and maintain infection is a very difficult but necessary task that will reveal vulnerable “nodes” in host defense pathways that can be strengthened via either breeding or molecular approaches. As a part of this project, we are conducting an in-depth molecular characterization of SCN effectors specifically involved in host defense suppression. We are specifically focused on the effector named 28B03. We have identified that this particular effector is a robust host defense suppressor. We have observed that due to its function, plants become more susceptible to cyst nematodes. We have also identified that this effector specifically targets a previously uncharacterized plant protein kinase. By physically interacting with this novel protein kinase, the 28B03 effector suppresses phosphorylation of its substrate protein and completely alters the associated signal transduction pathway. This discovery and an in-depth molecular characterization of a novel defense response related kinase cascade in plants are breakthrough discoveries of this study. We are currently gearing up to conduct another high-impact protein interaction experiment in order to identify the complete ‘interactome’ of the proteins associated with this kinase cascade. As candidate SCN virulence genes are identified, gene function will be confirmed through biochemical and/or genetic analysis to better understand the mechanisms of virulence and to also evaluate these gene targets as vulnerable points of disruption in the SCN life cycle as a means to enhance resistance in soybean. We are focusing our efforts on validating the genes identified in 2.3 above to prioritize candidate virulence genes for further molecular functional studies.

Microplot field experiments were established in Illinois, Missouri, and Iowa during 2019 to evaluate how rotations of SCN resistance gene combinations affects SCN field population densities and virulence profiles. Treatments with resistance genes: rhg1-a, rhg1-b, Rhg4, cqSCN-006/cqSCN-007, and the chromosome 10 QTL, along with susceptible and resistant checks were designed to form 12 treatments with 3 replications in a randomized complete block design. The virulent SCN HG type 1.2.5.7 was selected for microplot inoculation at locations. At the end of the growing season, soil samples from all plots were obtained to determine egg density and SCN HG type at harvest. All resistance genotypes had a reduction in egg densities and the susceptible treatment had an significant increase in SCN egg density, while PI 90763 had the greatest percentage of egg count reduction followed by genotypes with resistance genes, rhg1-a + Rhg4. SCN HG type results at the end of the first growing season show no specific trend. These results are close to what we expected and show that the different sources influenced SCN reproduction in the field. During 2020, the second year of the rotation study will be grown and soil samples will be taken from each plot during the fall. The samples will be analyzed for SCN number and HG type to study the impact of rotations on these characteristics.

In 2020-2021, project co-PIs will continue to deliver farmer-friendly information about the ongoing research of the project through interviews requested by media and through interviews and other communications pieces developed by the SCN Coalition. Tylka and Mitchum were to organize, host, and lead in-person educational events for a group of select soybean farmers and national agriculture media near Ames, Iowa, on August 31st, 2020, and in Athens, Georgia, on January 26th, 2021. The research in our NCSRP-funded project was to be highlighted at both events. Many print stories and radio interviews were to emanate from the events as a means of informing growers of the research in the project. In addition, there was to be a National Soybean Nematode Conference held in Savannah, Georgia (immediately after the farmer-media event in Athens, Georgia) on January 28th, 2021. Results of the research in our NCSRP-funded project were to be highlighted at the conference as well. The COVID-19 pandemic and resultant restrictions on travel and large in-person events necessitated a change in these plans for the 3rd year of our project. The SCN Coalition and its communications firm redirected efforts in the spring of 2020 to making the planned educational events described above virtual by recording and distributing videos capturing much of the information that was to be delivered in the in-person events. Specifically, Tylka, Kaitlyn Bissonnette from the University of Missouri, and Sam Markell from North Dakota State University were interviewed while walking through demonstration plots by a video crew at an Iowa State University extension education farm on July 21, 2020. The video crew returned to that farm on August 31, 2020 to document via video Tylka plus in NCSRP executive director Ed Anderson and an Iowa farmer discussing additional aspects of SCN and its management. Plans currently are being made for the video crew and SCN Coalition communications staff to visit Georgia in early 2021 to collect similar video to represent virtually the information that Mitchum had originally planned to convey and demonstrate in her laboratory at the University of Georgia. All of this virtual educational content will be posted online at the SCN Coalition website, www.TheSCNCoalition.com, and the SCN Coalition YouTube channel, https://www.youtube.com/channel/UCaYpNqBx53-5MVmyYGHK7HQ. Some of the content already is available on the YouTube channel.

The 2020 tests have been organized and they include 194 entries from maturity group 00 to IV and will be grown in 32 environments in 10 states and one Canadian province. The seed for the tests has been shipped to collaborators and the tests are now being planted. During the growing season, soil samples will be taken from field sites and the samples will be evaluated for egg number and HG type of the SCN population at each location. The cooperators will maintain the plots, takes notes on agronomic traits and will harvest the plots to estimate yield. The data from the tests will be sent to a central location, the results will be analyzed and a summary will be reported to cooperators and other interested parties.

Project Objectives

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Past research efforts to determine the inheritance of SCN virulence (i.e., the ability of the nematode to reproduce on resistant varieties) led to the identification of the reproduction on resistant varieties, or ror genes, which were shown to be inherited in both dominant and recessive manners. However, since the initial discovery of these genes there has been no further information published concerning their sequence identity or mechanism in conferring SCN virulence. Genome and transcriptome comparisons of SCN populations that differ in virulence on resistant soybean have the potential to identify genes underlying virulence, determine the mechanism/s of virulence, and lead to the development of molecular diagnostic tools to assess for virulence in field populations.

1.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum)
1.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum)
1.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses (Severin, Hudson, Mitchum, Baum)
1.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum)

Objective 2. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.
As described above, experimental lines with resistance gene combinations developed in Objective 1 during Phase I of this project were tested in four different rotation schemes with experimental lines containing various resistance gene combinations in a greenhouse study. SCN population increase was measured after each of 8 generations. Following the eighth generation of selection, the HG type of each population was determined. From this, we identified alternative resistance gene combinations that when used in rotation reduce the selection pressure on the SCN population thereby slowing nematode adaptation to resistant varieties.

2.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum)

Objective 3. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.
This project and the researchers involved in it produce much of the newest information being generated about the biology and management of SCN in the United States. As primary financial supporters of the work, it is critical that soybean farmers understand the rationale for the research and be made aware of the activities of the project and the new information being discovered. It also is important that individuals involved in soybean breeding in seed companies be made aware of the research results so that they are ready to adopt the new genetic resources for incorporation into commercially available soybean varieties. In addition to working with traditional university extension, a very efficient means to convey the results of this research to the farming and soybean breeding communities is through communication with the agriculture media. Project co-PIs will work to engage agriculture media directly and through the SCN Coalition.

3.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum)

Objective 4. Coordinate the testing of publicly developed SCN resistant experimental lines.
During 2019, the testing of SCN resistant experimental lines developed by breeders in 11 north central US states and Ontario was coordinated. This test is important to help breeders develop high yielding SCN resistant varieties. The tests included SCN resistant experimental lines and varieties that were grown in maturity group specific trials across 39 locations. These lines were also tested for SCN resistance and the soil samples from the environments were be evaluated for SCN population density and HG type. An initial report of the results from the 2019 test was sent to collaborators on December 19th, 2019 and the final report was delivered on January 6th, 2020.

4.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario (Diers)

Project Deliverables

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.

1.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum)
1.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum)
1.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses (Severin, Hudson, Mitchum, Baum)
1.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum)

Objective 2. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.

2.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum)

Objective 3. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.

3.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum)

Objective 4. Coordinate the testing of publicly developed SCN resistant experimental lines.

4.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario (Diers)

Progress of Work

Final Project Results

Benefit to Soybean Farmers

This project will benefit soybean producers by creating a long term management strategy for SCN through knowledge and soybean germplasm development.

Performance Metrics

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Accomplishments: In Phase I of the project we used innovative methods and new sequencing technologies to enhance and complete the current SCN reference genome assembly for the TN10 population (HG type 0) and the sequence of its bacterial endosymbiont. Additionally, we used state of the art bioinformatics tools to make substantial progress to annotate and curate the SCN genome. We generated a comprehensive SCN gene expression atlas from whole nematodes, which was also used in the annotation of the SCN genome. We created a genomic toolbox for SCN (SCN-Base) that facilitates the integration of very large sequence data sets, molecular markers, QTL data and genetic maps into an easy-to-use web interface. This resource will soon be released as a website that includes a Genome Browser for easy visualization of the SCN genome. We also developed lower-cost methods to produce reference genomes of additional HG types.

Objective 2. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.
Accomplishments: As described above, experimental lines with resistance gene combinations developed in Objective 1 during Phase I of this project were tested in four different rotation schemes with experimental lines containing various resistance gene combinations in a greenhouse study. SCN population increase was measured after each generation for 8 generations. Following the eighth generation of selection, the HG type of each population was determined. From this, we identified alternative resistance gene combinations that when used in rotation reduce the selection pressure on the SCN population thereby slowing nematode adaptation to resistant varieties.

Objective 3. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.
Accomplishments: In Phase I of the project, an extension and outreach coordinator, advised by project Co-PI Dr. Tylka, was hired to provide farmer education and outreach for the project. A survey of extension and outreach educational materials about SCN biology and management in the NCSRP states was conducted by the coordinator. Materials from land-grant universities and private seed and chemical companies were gathered, analyzed, and compared.

Objective 4. Coordinate the testing of publicly developed SCN resistant experimental lines.
Accomplishments: During 2018, the testing of SCN resistant experimental lines developed by breeders in 11 north central US states and Ontario was coordinated. The tests include 182 SCN resistant experimental lines and varieties that are being grown in 104 maturity group specific trials across 39 locations. These lines are also being tested for SCN resistance and the soil samples from the environments will be evaluated for SCN population density and HG type.

Project Years