Project Details:

Title:
VIGS enabled interrogation of Soybean R Genes

Parent Project: VIGS-enabled interrogation of SB R Genes
Checkoff Organization:Ohio Soybean Council
Categories:Breeding & genetics, Soybean diseases, Insects and pests
Organization Project Code:19-R-03
Project Year:2019
Lead Principal Investigator:Feng Qu (Ohio Agricultural Research and Development Center)
Co-Principal Investigators:
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

Ohio soybean is threatened by pests and pathogens that cause serious yield losses, among them soybean aphid, and seedling rot pathogens including Phytophthora sojae and Fusarium graminearum. As a collaborative team of OARDC scientists, we strive to help soybean growers to combat diseases by searching for resistant varieties, and identifying the underlying R genes. We previously mapped Rag5 and Rps8 that confer resistance to soybean aphid and P.sojae, and quantitative resistance loci (QRLs) that confer partial resistance to both P. sojae and F. graminearum. The current project aims to identify the actual gene(s) within each of the multi-gene R/QRL loci. Use of a virus-induced gene silencing (VIGS) approach recently allowed us to pinpoint the Rag5 gene. Continuation of these investigations will enable the mobilization of these R genes to susceptible varieties, providing Ohio soybean growers with superior disease management tools, assuring the long-term viability of Ohio soybean farmers.

Project Objectives

1. Verify the function of the aphid-antagonizing resistance gene Rag5 in transgenic soybean.
2. Identify soybean resistance genes and quantitative resistance loci that subdue seedling rot pathogens using VIGS.
3. Create a new VIGS vector based on Alfalfa mosaic virus.

Project Deliverables

First quarter (October 1 - December 31, 2018):
1. Characterization of transgenic soybean plants expressing the Rag5 gene.
2. Generation of ALSV-based VIGS constructs for Rps8 candidate genes.
3. Initiation of VIGS-assisted QRL interrogation.
4. Generation of AMV cDNAs in preparation for VIGS vector construction.
Second quarter (January 1 - March 31, 2019):
1. Functional evaluation of the Rag5-transgenic plants with aphid-feeding experiments.
2. Continued interrogation of the Rps8 locus with BPMV and ALSV VIGS vectors.
3. Initiation of functional analysis of significant genes within two QRLs.
4. Modification of AMV cDNAs to allow for incorporation of soybean gene fragments, prepare for VIGS testing.
Third quarter (April 1 - June 30, 2019):
Continue working on the investigations initiated during the first six months.
Fourth quarter (July 1 - September 30, 2019):
1. Complete the testing of the 16 candidate genes within the Rps8 locus.
2. Complete the characterization of at least two genes within the two QRLs.
3. Conclude the research on Rag5 by completing the characterization of transgenic soybean, publish the study.
4. Complete the development of the AMV-based VIGS system.

Progress of Work

Updated October 31, 2019:
Objectives:
1. Verify the function of the aphid-antagonizing resistance gene Rag5 in transgenic soybean.
2. Identify soybean resistance genes and quantitative resistance loci that subdue seedling rot pathogens using VIGS.
3. Create a new VIGS vector based on Alfalfa mosaic virus.
Progresses:
This project is a continuation of 18-R-03. Much of the progresses documented below represents advancements extending from that project. However, we are also testing novel procedures that could potentially improve efficiency and save time and money, allowing us to satisfy growers’ needs in a more cost-conscious manner.
- Rag5: We described in the last report that at least five lines of Rag5-transgenic plants, plus five control transgenic plants expressing an unrelated transgene (green fluorescent protein or GFP), were being reared in greenhouse, waiting for seed setting. We have now harvested the Rag5-transgenic seed from two of the Rag5 lines and two control GFP lines. The transgenic seeds have been sowed during the October to December quarter of 2018. The seedlings are now been tested to verify the presence of the Rag5 transgene. Once these testing are complete, the positively transgenic plants will be subjected to feeding with soybean aphids to determine whether they indeed become resistant to the soybean aphids. As noted earlier, the putative Rag5 gene was identified through virus-induced gene silencing (VIGS) of seven different candidate genes. Soybeans that are naturally susceptible to aphids, should become resistant if transformed with the correct Rag5 gene.
- Rps8: We have decided recently to use the PI line #399073 as the target plant for VIGS-enabled interrogation of Rps8 gene candidates. This is because (i) the Rps8 gene was originally isolated in PI 399073; and (ii) compared to the breeding lines that acquired the Rps8 gene from PI399073, the PI 399073 itself is substantially more responsible to ALSV VIGS. We are currently carrying out two parallel sets of experiments under this objective. (a) we are further calibrating the condition for ALSV VIGS in the PI399073 in order to maximizing the efficiency of VIGS. (b) we are busy cloning the candidate gene fragments into the ALSV VIGS vector.
- QTLs on Chromosome 19: We are using two different approaches to attack the candidate genes mapped to the QTLs. (i) To assess the candidate genes that potentially confer resistance to Fusarium graminearum in the seed coat, we are using ALSV VIGS to target the candidate genes CG1, CG2, CG3, CG4, and CG5 in the PI line #567301B resistant to F. graminearum. Our preliminary testing showed that we probably will succeed in silencing these genes in the seed coat, so that their expression would be diminished in the seed coat. The seed harvested from these plants will then be tested for loss of resistance to F. graminearum. (ii) In order to investigate a new gene found in the Conrad/Sloan population that was implicated in resistance to the root rot pathogen P. sojae, we used a hairy root procedure to successfully silence this gene, named as CG6. The resulting hairy roots are being assessed for changes in resistance to P. sojae.
- Using the virus BPMV to interrogate avr proteins encoded by P. sojae. We described in last quarter’s report that we sought to screen for the functionality of Rps genes function in various soybean cultivars or PI (plant introduction) by expressing their corresponding Avr proteins using a virus (Bean pod mottle virus, BPMV). This idea has now been tested with two Avr proteins, Avr1a and 1c, and revealed a high correlation between development of rusty leaf spots and the presence of the corresponding Rps genes in ten different soybean cultivars. We further found that some cultivars previously thought to have Rps1a or 1c weakly responded to Avr1a or 1c expression, indicating that the Rps gene expression could be influenced by other genes present in the cultivars. These results are being repeated in order to finalize a publication.
- A new VIGS vector based on Alfalfa mosaic virus (AMV): The two VIGS vector we have been using, based on BPMV and ALSV respectively, have both advantages and drawbacks. BPMV is a robust VIGS vector applicable in multiple soybean cultivars. However soybean infected by the BPMV vector rendered plants more resistant to P. sojae. On the other hand, although ALSV causes milder infection, it can only infect a few soybean varieties. During the last quarter we have begun to explore the possibility to develop AMV into an alternative VIGS vector for use in soybean. Progresses on this front will be reported next quarter.
Activities planned for the next quarter:
- Continue the pursuit of the Rag5, Rps8 genes and disease resistance QTLs.
- Repeat and complete the study of using BPMV to express Avr proteins as a way to screen for Rps gene functionality.
- Continue the development of the AMV VIGS vector.

Updated October 31, 2019:
Objectives:
1. Verify the function of the aphid-antagonizing resistance gene Rag5 in transgenic soybean.
2. Identify soybean resistance genes and quantitative resistance loci that subdue seedling rot pathogens using VIGS.
3. Create a new VIGS vector based on Alfalfa mosaic virus.
Progresses:
- Rag5: We reported earlier that at least five lines of Rag5-transgenic plants, plus five control transgenic plants expressing an unrelated transgene (green fluorescent protein or GFP), were being reared in greenhouse, waiting for seed setting. We have now harvested the Rag5-transgenic seed from all of the transgenic lines. Furthermore, we have tested all of the lines for aphid resistance during the last three months. The biggest disappointment is that these transgenic plants did not become substantially more resistant to aphid infestation. Many factors could have contributed the negative outcome. It could be that more than one genes are needed for efficient resistance to aphids. It may also be possible that the gene we retrieved from the resistant variety (PI567301B) is regulated by other genes that are not yet known. However, considering the amount of additional efforts involved to analyze these factors, we feel it is time for us to halt the further pursuit of this objective.
- Rps8: We are still in the process of optimizing the conditions for carrying out VIGS in the PI line #399073, which is the variety from which the Rps8 gene was isolated. We have passaged the ALSV VIGS vector in soybean for eight generations, and obtained an ALSV mutant virus that appears to be more potent at inducing VIGS. We have begun the process of anaylyze the new ALSV mutant in order to identify the specific changes that made the virus more robust. We will then use the more robust virus as the vector to interrogate the Rps8 candidate genes through VIGS.
- QTLs on Chromosome 19: We continue to use two different approaches to attack the candidate genes mapped to the QTLs. (i) To assess the candidate genes that potentially confer resistance to Fusarium graminearum in the seed coat, we are using ALSV VIGS to target the candidate genes CG1, CG2, CG3, CG4, and CG5 in the PI line #567301B resistant to F. graminearum. Our preliminary testing showed that we probably will succeed in silencing these genes in the seed coat, so that their expression would be diminished in the seed coat. The seed harvested from these plants will then be tested for loss of resistance to F. graminearum. (ii) In order to investigate a new gene found in the Conrad/Sloan population that was implicated in resistance to the root rot pathogen P. sojae, we used a hairy root procedure to successfully silence this gene, named as CG6. The resulting hairy roots are being assessed for changes in resistance to P. sojae.
- Using BPMV to interrogate avr proteins encoded by P. sojae. We have been screening for the functionality of Rps genes function in various soybean cultivars or PI (plant introduction) by expressing their corresponding Avr proteins using Bean pod mottle virus (BPMV). This idea has now been tested with two Avr proteins, Avr1a and 1c, and revealed a high correlation between development of rusty leaf spots and the presence of the corresponding Rps genes in ten
different soybean cultivars. We further found that some cultivars previously thought to have Rps1a or 1c weakly responded to Avr1a or 1c expression, indicating that the Rps gene expression could be influenced by other genes present in the cultivars. These results are being repeated in order to finalize a publication.
- A new VIGS vector based on Alfalfa mosaic virus (AMV): The two VIGS vector we have been using, based on BPMV and ALSV respectively, have both advantages and drawbacks. BPMV is a robust VIGS vector applicable in multiple soybean cultivars. However soybean infected by the BPMV vector rendered plants more resistant to P. sojae. On the other hand, although ALSV causes milder infection, it can only infect a few soybean varieties. During the last quarter we have begun to explore the possibility to develop AMV into an alternative VIGS vector for use in soybean. Progresses on this front will be reported next quarter.
Activities planned for the next quarter:
- Continue the pursuit of the Rps8 genes and disease resistance QTLs.
- Repeat and complete the study of using BPMV to express Avr proteins as a way to screen for Rps gene functionality.
- Continue the development of the AMV VIGS vector.

Updated October 31, 2019:
Objectives:
1. Verify the function of the aphid-antagonizing resistance gene Rag5 in transgenic soybean.
2. Identify soybean resistance genes and quantitative resistance loci that subdue seedling rot pathogens using VIGS.
3. Create a new VIGS vector based on Alfalfa mosaic virus.
Progresses:
- Rag5: Due to the reasons stated in the last quarterly report, the pursuit of the Rag5 gene has now been halted.
- Rps8: We continue to optimize the conditions for carrying out VIGS in the PI line #399073, which is the variety from which the Rps8 gene was isolated. We have passaged the ALSV VIGS vector in soybean for eight generations, and obtained an ALSV mutant virus that appears to be more potent at inducing VIGS. We are now analyzing the new ALSV mutant in order to identify the specific changes that made the virus more robust. We will then use the more robust virus as the vector to interrogate the Rps8 candidate genes through VIGS.
- QTLs on Chromosome 19: We are using two different approaches to attack the candidate genes mapped to the QTLs. (i) To assess the candidate genes that potentially confer resistance to Fusarium graminearum in the seed coat, we are using ALSV VIGS to target the candidate genes CG1, CG2, CG3, CG4, and CG5 in the PI line #567301B resistant to F. graminearum. Our preliminary testing showed that we probably will succeed in silencing these genes in the seed coat, so that their expression would be diminished in the seed coat. The seed harvested from these plants will then be tested for loss of resistance to F. graminearum. (ii) In order to investigate a new gene found in the Conrad/Sloan population that was implicated in resistance to the root rot pathogen P. sojae, we used a hairy root procedure to successfully silence this gene, named as CG6. The resulting hairy roots are being assessed for changes in resistance to P. sojae.
- Using BPMV to interrogate avr proteins encoded by P. sojae. We have been screening for the functionality of Rps genes function in various soybean cultivars or PI (plant introduction) by expressing their corresponding Avr proteins using Bean pod mottle virus (BPMV). This idea has now been tested with two Avr proteins, Avr1a and 1c, and revealed a high correlation between development of rusty leaf spots and the presence of the corresponding Rps genes in ten different soybean cultivars. We further found that some cultivars previously thought to have Rps1a or 1c weakly responded to Avr1a or 1c expression, indicating that the Rps gene expression could be influenced by other genes present in the cultivars. These results are being repeated in order to finalize a publication.
- A new VIGS vector based on Alfalfa mosaic virus (AMV): The two VIGS vector we have been using, based on BPMV and ALSV respectively, have both advantages and drawbacks. BPMV is a robust VIGS vector applicable in multiple soybean cultivars. However soybean infected by the BPMV vector rendered plants more resistant to P. sojae. On the other hand, although ALSV causes milder infection, it can only infect a few soybean varieties. During the last quarter we
have begun to explore the possibility to develop AMV into an alternative VIGS vector for use in soybean. Progresses on this front will be reported next quarter.
Activities planned for the next quarter:
- Continue the pursuit of the Rps8 genes and disease resistance QTLs.
- Repeat and complete the study of using BPMV to express Avr proteins as a way to screen for Rps gene functionality.
- Continue the development of the AMV VIGS vector.

Updated October 31, 2019:
Objectives:
1. Verify the function of the aphid-antagonizing resistance gene Rag5 in transgenic soybean.
2. Identify soybean resistance genes and quantitative resistance loci that subdue seedling rot pathogens using VIGS.
3. Create a new VIGS vector based on Alfalfa mosaic virus.
Progresses:
- Rag5: Due to the reasons stated in the last quarterly report, the pursuit of the Rag5 gene has now been halted.
- Rps8: We continue to calibrate the conditions for carrying out VIGS in the PI line #399073, which is the variety from which the Rps8 gene was isolated. In order to make the ALSV vector more robust, we are now testing two strategies: (i) We have been testing whether shorter insert would enable the ALSV VIGS vector to infect the PI 399073 plants at higher rates. (ii) We also tried to isolate more potent ALSV mutants by passaging the ALSV VIGS vector in soybean for eight generations, and obtained an ALSV mutant virus that appears to be more potent at inducing VIGS. We are now analyzing the new ALSV mutant in order to identify the specific changes that made the virus more robust. We will then use the more robust virus as the vector to interrogate the Rps8 candidate genes through VIGS.
- QTLs on Chromosome 19: We are using two different approaches to attack the candidate genes mapped to the QTLs. (i) To assess the candidate genes that potentially confer resistance to Fusarium graminearum in the seed coat, we are using ALSV VIGS to target the candidate genes CG1, CG2, CG3, CG4, and CG5 in the PI line #567301B resistant to F. graminearum. Our preliminary testing showed that we probably will succeed in silencing these genes in the seed coat, so that their expression would be diminished in the seed coat. The seed harvested from these plants will then be tested for loss of resistance to F. graminearum. (ii) In order to investigate a new gene found in the Conrad/Sloan population that was implicated in resistance to the root rot pathogen P. sojae, we used a hairy root procedure to successfully silence this gene, named as CG6. The resulting hairy roots are being assessed for changes in resistance to P. sojae.
- Using BPMV to interrogate avr proteins encoded by P. sojae. We have been screening for the functionality of Rps genes function in various soybean cultivars or PI (plant introduction) by expressing their corresponding Avr proteins using Bean pod mottle virus (BPMV). This idea has now been tested with two Avr proteins, Avr1a and 1c, and revealed a high correlation between development of rusty leaf spots and the presence of the corresponding Rps genes in ten different soybean cultivars. We further found that some cultivars previously thought to have Rps1a or 1c weakly responded to Avr1a or 1c expression, indicating that the Rps gene expression could be influenced by other genes present in the cultivars. These results are being repeated in order to finalize a publication.
- A new VIGS vector based on Alfalfa mosaic virus (AMV): The two VIGS vector we have been using, based on BPMV and ALSV respectively, have both advantages and drawbacks. BPMV is a
robust VIGS vector applicable in multiple soybean cultivars. However soybean infected by the BPMV vector rendered plants more resistant to P. sojae. On the other hand, although ALSV causes milder infection, it can only infect a few soybean varieties. During the last quarter we have begun to explore the possibility to develop AMV into an alternative VIGS vector for use in soybean. Progresses on this front will be reported next quarter.
Activities planned for the next quarter:
- Continue the pursuit of the Rps8 genes and disease resistance QTLs.
- Repeat and complete the study of using BPMV to express Avr proteins as a way to screen for Rps gene functionality.
- Continue the development of the AMV VIGS vector.

Final Project Results

Benefit to Soybean Farmers

Through the research objectives described below, we strive to find solutions for disease problems prevalent in Ohio soybean fields through close collaborations with other scientists in Ohio and the Midwest region. These research efforts are expected to benefit Ohio soybean growers by developing superior seed stocks with enhanced disease resistance traits. Hence, our project is highly consistent with the vision of OSC to assure the long-term viability of Ohio soybean farmers.

Performance Metrics

Project Years