Progress 05/01/15 to 04/30/19
Outputs
Target Audience:The target audience reached included members of the scientific community that research plant pathology, nematology, and the plant sciences as well as stakeholders in the agricultural community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant has made it possible for us to train graduate students as well as scientists. This funding has also made it possible for us to send these future scientists to conferences to present their research in form of posters or oral presentations. Without this grant, it would not have been possible for us to train these individuals in the fields of molecular biology, molecular plant pathology and bioinformatics essential for their professional development. How have the results been disseminated to communities of interest?Results are disseminated to the broader communities via oral as well as poster presentations at scientific conferences as well as via publishing research articles in peer-reviewed, widely circulated scientific journals. What do you plan to do during the next reporting period to accomplish the goals?This is the final report of this grant funding.
Impacts
What was accomplished under these goals?
Overall impact statement: SCN is the most devastating soybean pathogen that ravages soybean industry in the USA each year. The most prevalent method to control this pest is to use SCN resistant soybean cultivars. However, sustained use of such cultivars has stimulated SCN to develop the ability to overcome resistance.Such newly virulent SCN populations are spreading and pose significant economic uncertainty for the US soybean industry. To safeguard US soybean industry and to limit the economic losses due to SCN infection, it is essential to develop novel soybean cultivars that can resist the most virulent SCN populations. Our project has taken the most important first step towards that goal by generating scientific knowledge about the molecular foundation of SCN virulence.Our results identified specific SCN genetic elements that are required to overcome soybean resistance. Identification and functional characterization of virulence-determining genetic elements will pave way to identify vulnerable points in soybean defenses that can be plugged using transgenic or breeding approaches in the future. The results obtained in this project are key enabling discoveries in developing novel management tools against the #1 soybean disease in the US. Objective #1: Effectors are nematode proteins delivered into the soybean plant to enable disease development. To explore the genotypic variability of effector arsenals in SCN populations differing in virulence, we developed transcriptomic resources from a susceptible as and a virulent SCN population, in the form of esophageal gland cell cDNA libraries. This group has isolated esophageal gland cells from each of these populations and has constructed RNA-seq libraries from pools of approximately 150 gland cells from each population. These RNA-seq libraries are constructed using exceptionally low initial quantities (approximately 1-2 ng). In order to conduct robust statistical analysis, the goal was to construct three independent biological replicates of each population. We have successfully constructed three replications from the avirulent populations and the quality analysis of these libraries shows that these are ready for the further analysis while the virulent SCN populations await additional work. Simultaneously, we already have sequenced and analyzed some of these libraries and used them to validate recently discovered novel effector candidates. Objective #2: We have successfully assembled the SCN parasitic transcriptome from whole nematodes. Using this transcriptome, we have developed and utilized two effector prediction pipelines, one based on traditional effector identification criteria and the other utilizing N-Preffector. In this algorithm, the candidate peptides were run through a machine-learning algorithm trained on known SCN effector sequences. For each protein sequence, N-Preffector calculates a vector of length-invariant features; the feature vector is then used as an input for a classification model. N-Preffector's goal was to minimize the number of proteins erroneously misclassified as effectors, i.e., false positives, while trying to maximize the number of predicted real effectors. We then employed a dual effector prediction strategy coupling N-Preffector with a traditional secreted protein prediction pipeline to uncover a suite of novel effector candidates. To determine which effectors may play a role in modulation of host defense responses we examined sequence variation both within a single SCN population and across multiple populations that differ in their infective capabilities. New variants of known effector sequences have been identified. These novel sequences demonstrate different forms of variation, including single polymorphisms, intron retention, and large-scale deletions. Expression patterns of these variants are being examined across populations to determine if there are correlations with virulence. The transcriptome assembly and effector identification pipeline was published. The transcriptome was made available to the SCN community. A test of an in silico subtraction in situ hybridization was performed using sequence data obtained from gland cells from the three biological replicates of gland sequences identified from the avirulent and virulent SCN populations. These gland cell sequences were used to identify overlapping sequences present in the predicted effector candidate pool, resulting in a smaller set of confirmed high priority candidates. We initially focused on one class of 132 predicted novel effectors, which included 32 candidates containing a nuclear localization signal (NLS). The two classes of predicted effectors were further categorized into different tiers based on expression in the two nematode populations. Full-length cDNA sequences obtained using BLAST at SCN base were reanalyzed for signal peptide using SignalP-5.0 software and presence or absence of NLS using several nuclear localization prediction programs. Several of these newly discovered potential effectors have been confirmed for nuclear localization and are being tested for gland expression. We identified a member of the Bcl-2 associated family of proteins in soybean (GmBAG6) expressed during the hypersensitive response (HR)-like programmed cell death (PCD) in soybean plants resistant to SCN. We demonstrated that GmBAG6-1 induces cell death in yeast and soybean same as its Arabidopsis homolog. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean resistance. Thus, we utilized a yeast viability assay to screen SCN effectors for their ability to specifically suppress GmBAG6-1 induced cell death. We screened forty-seven SCN effectors originally isolated from a virulent SCN population for suppression of GmBAG6-1, as well as BAX (a mammalian pro-apoptosis regulator of the Bcl-2 family) -induced cell death as a means to identify effectors that may be targeting regulatory mechanisms underlying PCD. We identified several effectors that can function as anti-PCD proteins, including two effectors interacting with with GmBAG6-1 in yeast two-hybrid screens. This work has been finalized and a manuscript is ready for publication. We also established a Nicotiana benthamiana system using the microbe strains Pseudomonas syringae pv tomato (Pst DC3000) and Pseudomonas fluorescens strain EtHAn (Pf EtHAn) for ETI and PTI triggered immunity suppression screening. 21 SCN effector proteins were screened at the University of Missouri for ETI-triggered suppression of by Pst DC3000. Compared to the negative control, which showed less than 10% ETI suppression, 4 SCN effectors showed more than 25% suppression, and 1 effector showed more than 30% suppression. Simultaneously, a group at Iowa State University screened a total of 54 effectors to identify those inhibiting ETI and/or PTI. Three effectors were identified and confirmed to be strong ETI suppressors while seven effectors were identified and confirmed as PTI suppressors. For all ETI-suppressing effectors, quantitative analysis of Pseudomonas bacterial growth rate was performed. In addition, for all PTI and ETI suppressors, qPCR assays of plant immune marker genes were also performed. A manuscript detailing these findings is ready for publication. Objective #3: We created a mini yeast two-hybrid library of SCN effectors and screened this library for interaction with the soybean protein BAG6. We identified and confirmed two effectors that directly interact with BAG6. The group at Iowa State University conducted functional characterization of the immune suppressing effectors. Yeast 2-hybird screens for 6 of the effectors that show most potent host immunity modulation have been performed and further analyses to confirm these results are ongoing. Simultaneously, we have developed transgenic Arabidopsis lines expressing these effectors constitutively for further analyses.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Mitchum, MG. The 17th Biennial Conference on the Molecular & Cellular Biology of the Soybean, August 26-29th, 2018, Athens, GA The Complex Interplay between Soybean Resistance and Nematode Virulence . Invited Speaker.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Mitchum, MG. European Society of Nematologists, September 9-13th, 2018, Ghent, Belgium, Keynote Speaker Progress and Current Challenges in our Understanding of the Soybean Cyst Nematode-Soybean Interaction
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Verma A, Lin M, Smith D, Chronis D, Gardner M, Lin Y, Hewezi T, Baum TJ, Mitchum MG. Functional characterization of a highly expanded superfamily of dorsal gland effector proteins in cyst nematodes. European Society of Nematology, September 9-14, 2018, Ghent, Belgium
|
Progress 05/01/17 to 04/30/18
Outputs
Target Audience:The target audience reached included members of the scientific community that research plant pathology, nematology, and the plant sciences as well as stakeholders in the agricultural community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant has given invaluable support to the graduate students as well as scientists. Without this grant, it would not have been possible for us to train these individuals in the fields of molecular biology, molecular plant pathology and bioinformatics essential for their professional development. How have the results been disseminated to communities of interest?Results are disseminated to the communities of interest via oral as well as poster presentations at scientific conferences as well as via publishing research articles in peer-reviewed, widely circulated scientific journals. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will continue our diligent efforts to make progress for all the listed objectives. The specific plan for each objective is listed above. Along with the experiments, we will plan on presenting our work in oral or poster presentations, as well as publications in peer reviewed journals.
Impacts
What was accomplished under these goals?
IMPACT:The soybean cyst nematode(SCN) is a pest that causes severe economic losses each year worldwide. Our research increased knowledge of plant defense suppression by soybean cyst nematode (SCN) effectors. Specific Objective #1: Explore the genotypic variability of effector arsenals of multiple SCN populations differing in virulence. 1.2.1 Nematode populations: Three biological replicates each of the avirulent SCN population on susceptible soybeans as well as the highly virulent SCN population on resistant soybeans is complete. 1.2.2 Gland cell purification and RNA preparation: Three biological replicates each of both the avirulent and virulent SCN population have been received by Iowa State University, and nematodes from each replicate extracted.Subsequently, gland cells from each replicate were extracted, and pools of approximately 100 gland cells from each replicate were microaspirated and collected for total RNA isolation.RNA extraction and rna-seq library construction was performed for each of the six biological replicates. Illumina Nextseq next generation sequencingwas performed and high quality rna-seq datawas generated from each of the six biological replicates.This data is ready for analysis. 1.2.3 Sequence analyses: See below Specific Objective #2: Comprehensive identification of SCN effectors that modulate host defense responses. 2.1.2.1 In silico identification of the novel effectors: In the absence of a gland specific transcriptome, we have assembled the SCN parasitic transcriptome from whole nematodes.Using this transcriptome we have developed and utilized two effector prediction pipelines, one based on traditional effector identification criteria and the other utilizing N-preffector, a novel prediction tool.In this algorithm, the candidate peptides were run through a machine-learning algorithm trained on 72 known H. glycines effector sequences and 150 known non-effector sequences from H. glycines were added to the set of sequences (both effectors and non-effectors) from Gram negative bacteria. For each protein sequence, N-Preffector calculates a vector of length-invariant features; the feature vector is then used as an input for the classification model. Feature categories that were considered are: residue composition, sequence/structure information, and physico-chemical properties of proteins. N-Preffector's goal was to minimize the number of proteins erroneously misclassified as effectors, i.e., false positives, while trying to maximize the number of predicted real effectors. We then employed a dual effector prediction strategy coupling N-Preffector with a traditional secreted protein prediction pipeline to uncover a suite of novel effector candidates. The transcriptome assembly and effector identification pipelines have been summarized in a manuscript that was accepted for publication (see Gardner et al. below).The transcriptome was made available to the SCN community to aid in other research efforts, including assembly and annotation of the SCN genome. To determine which effectors may play a role in modulation of host defense responses we continue to examine sequence variation both within a single SCN population and across multiple populations that differ in their infective capabilities. The identification of new variants of known effector sequences that may play a role in virulence have been identified.These novel sequences demonstrate several different forms of variation, including single polymorphisms, intron retention, and large-scale deletions. Currently expression patterns of these variants are being examined across populations to determine if there are correlations with virulence that may be pursued at the functional level. 2.1.2.2 In situ hybridization to confirm gland specific expression of the newly discovered potential effectors: A preliminary test of the in silico subtraction was performed using sequence data obtained from gland cells from a single replicate of the avirulent SCN population on a susceptible host.These gland cell sequences were used to identify overlapping sequences present in the effector candidate pool, resulting in a smaller set of confirmed high priority candidates.Once additional gland cell sequence data from more biological replicates is available this analysis can be performed again to increase the confidence of these predictions and when additional populations are sequenced this analysis can be expanded to identify effectors that may be missing in the single population sequenced. Specific Objective 2.2: Screen all available and newly discovered SCN effectors from in silico analysis for PTI and ETI suppression. We identified a member of the Bcl-2 associated athanogene family of proteins in soybean (GmBAG6) expressed during the hypersensitive response (HR)-like programmed cell death (PCD) cell death in resistant soybean to SCN. We demonstrated that GmBAG6-1 induces cell death in yeast like its Arabidopsis homolog AtBAG6 and also in soybean. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean resistance. Thus, we utilized a yeast viability assay to screen SCN stylet-secreted effector (SSE) candidates for their ability to specifically suppress GmBAG6-1 induced cell death. We screened forty-seven SCN SSEs originally isolated from a virulent SCN population for suppression of GmBAG6-1, as well as BAX (a mammalian pro-apoptosis regulator of the Bcl-2 family)-induced cell death as a means to identify effectors that may be targeting conserved regulatory mechanisms underlying PCD. We identified several SSEs that can function as anti-PCD proteins, including two effectors that showed direct interaction with GmBAG6-1 in yeast two-hybrid experiments, suggesting that these effectors likely play a central role in plant immune suppression and nematode virulence. This work has been finalized and a manuscript "Suppression of soybean BAG6 induced cell death by soybean cyst nematode Heterodera glycines effectors" has been revised and will be resubmitted for publication. We also established the Nicotiana benthamiana system and the microbe strains Pseudomonas syringae pv tomato (Pst DC3000) and Pseudomonas fluorescens strain EtHAn (Pf EtHAn) for ETI and PTI screening, respectively. Twenty-one SCN effector proteins were screened at the University of Missouri for suppression of ETI-triggered by Pst DC3000. Compared to the negative control, which showed less than 10% ETI suppression, 4 SCN effectors showed more than 25% suppression, and one showed more than 30% suppression. Simultaneously, a group at Iowa State University screened total of 54 effectors in order to identify those inhibiting ETI and/or PTI. Three effectors were identified and confirmed to be strong ETI suppressors while seven effectors were identified and confirmed as PTI suppressors. For all the ETI suppressing effectors, quantitative analysis of Pseudomonas bacterial growth rate was performed. In addition, for all PTI and ETI suppressors, qPCR assays of plant immune marker genes were also performed. Currently we are working on finalizing a manuscript describing the above data. Specific Objective #3: Functional characterization of effector proteins determined to modulate host immunity. We created a mini yeast two-hybrid library of SCN effectors and screened this for interaction with the soybean protein BAG6. We identified and confirmed two effectors that directly interact with BAG6 and each other. We continue to study these interactions. The group at Iowa State University has performed yeast 2-hybird screens for six of the effectors that show host immunity modulation. Currently we are in a process of confirming these results. The transgenic Arabidopsis lines expressing these effectors constitutively have been generated and we are currently working to identify homozygous lines. Once we generate homozygous lines, we will examine their potential differential nematode susceptibility.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Gardner M, Dhroso A, Johnson N, Davis EL, Baum TJ, Korkin D, Mitchum, MG. Novel global effector mining from the transcriptome of early life stages of the soybean cyst nematode Heterodera glycines. Scientific Reports 2018; 8:2505
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Jones J, and Mitchum MG. Biology of effectors. Chapter 5 IN: P Rolo and J Jones (Eds.) Cyst Nematodes CABI; 2018
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Mitchum, MG. American Phytopathological Society Meeting, San Antonio, TX, August 5-9, 2017, Nematode Effector Proteins: A Look to the Past with an Eye to the Future . Invited Speaker.
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Identification and Characterization of Soybean Cyst Nematode Effectors Suppressing Cell Death
Jianying Wang1, Greg Yeckel1, Gennady Pogorelko2, Parijat S. Juvale2, Thomas J. Baum2, Eric L. Davis3, Melissa G. Mitchum1
1Division of Plant Sciences and Bond Life Sciences Center, University of Missouri, Columbia, MO, USA.; 2Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, U.S.A.; 3Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Dhroso, Andi, Samantha Eidson, and Dmitry Korkin. "Genome-wide prediction of bacterial effectors across six secretion system types using a feature-based supervised learning framework." bioRxiv (2018): 255604.
|
Progress 05/01/16 to 04/30/17
Outputs
Target Audience:The target audience reached included members of the scientific community that research plant pathology, nematology, and the plant sciences as well as stakeholders in the agricultural community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant has given invaluable support to the graduate students as well asscientists. Without this grant, it would not have been possible for us to train these individuals in the fields of molecular biology, molecular plant pathology and bioinformatics essential for their professional development. How have the results been disseminated to communities of interest?Results are disseminated to the communities of interest via oral as well as poster presentations at scientific conferences as well as via publishing research articles in peer-reviewed, widely circulated scientific journals. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will continue our diligent efforts to makeprogress for all the listed objectives. The specific plan for each objective is listed above. Along with the experiments, we will plan on presenting our work in oral or poster presentations, as well as publications in peer reviewed journals.
Impacts
What was accomplished under these goals?
Overall impact statement: The soybean cyst nematode(SCN) is the most destructive pathogen of soybeans grown in the United States with estimated losses of over $1 billion annually. As virulent SCN populations,that can survive on some of the most resistant soybean cultivars, are spreading and environmentally hazardous chemical nematicides are being phased out, there is an immediate need to discover novel sources of resistance against this pathogen. Our experimental plan follows comprehensive molecular strategies to identify vulnerable points in the parasitic process to devise novel control strategies against the SCN. Objective 1. Explore the genotypic variability of effector arsenals of multiple SCN populations differing in virulence. The inoculation of the avirulent SCN population on susceptible soybeans in three biological replicates is complete. Two biological replicates of the highly virulent SCN population have been inoculated, and shipped to Baum laboratory for gland cell isolation. An additional third biological replicate has been scheduled for shipment in the next month. Pools of at least 100 gland cells have been purified for each of three biological replicates from both the virulent (Pa3) and avirulent (MM10) SCN populations. These six pools are awaiting total RNA isolation and subsequent low-input RNA-seq library construction. ­­­­We estimate that this will be completed within the next month, at which time, subject to passing quality control, the libraries will be submitted for sequencing. Objective 2. Comprehensive identification of SCN effectors that modulate host defense responses. In the absence of a gland specific transcriptome, we have assembled the SCN parasitic transcriptome from whole nematodes. Using this transcriptome we have developed and utilized two effector prediction pipelines, one based on traditional, signal-based, effector identification criteria and the other utilizing N-preffector, a novel machine learning prediction tool. N-preffector was trained on 71 known SCN secreted effectors and 250 non-effectors to look for additional criteria beyond a signal peptide that may characterize an effector. These two pipelines were then compared and overlapping candidates were identified as high priority candidates to pursue for in situ hybridization and functional characterization. The transcriptome assembly and effector identification pipelines have been summarized in a manuscript that is currently being prepared for publication (see Gardner et al. below). To determine which effectors may play a role in modulation of host defense responses sequence variation was examined both within a single SCN population and across multiple populations that differ in their infective capabilities. For this analysis initially the known SCN effectors were examined, resulting in the identification of new variants of these sequences that may play a role in virulence. These novel sequences demonstrate several different forms of variation, including single polymorphisms, intron retention, and large-scale deletions. Currently expression patterns of these variants are being examined across populations to determine if there are correlations with virulence. Once the known SCN effectors are exhausted this analysis will be expanded to high priority effector candidates identified by the effector mining. 2.1.2.2 In situ hybridization to confirm gland specific expression of the newly discovered potential effectors: A preliminary test of the in silico subtraction was performed using sequence data obtained from gland cells from a single replicate of the avirulent SCN population on a susceptible host. These gland cell sequences were used to identify overlapping sequences present in the effector candidate pool, resulting in a smaller set of confirmed high priority candidates. Once additional gland cell sequence data from more biological replicates is available this analysis can be performed again to increase the confidence of these predictions and when additional populations are sequenced this analysiswill be expanded to identify effectors that may be missing in the single population sequenced. Objective 2.2: Screen all available and newly discovered SCN effectors from in silico analysis for PTI and ETI suppression. We identified a member of the Bcl-2 associated athanogene family of proteins in soybean (GmBAG6) expressed during the hypersensitive response (HR)-like programmed cell death (PCD) in resistant soybean. We demonstrated that GmBAG6-1 induces cell death in yeast like its Arabidopsis homolog AtBAG6 and also in soybean. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean resistance. Thus, we utilized a yeast viability assay to screen SCN stylet-secreted effector (SSE) candidates for their ability tosuppress GmBAG6-1 induced cell death. We screened forty-seven SCN SSEs originally isolated from a virulent SCN population for suppression of GmBAG6-1, as well as BAX (a mammalian pro-apoptosis regulator of the Bcl-2 family)-induced cell death as a means to identify effectors that may be targeting conserved regulatory mechanisms underlying PCD. From this analysis, we identified several SSEs that can function as anti-PCD proteins, including two effectors that showed direct interaction with GmBAG6-1 in yeast two-hybrid experiments, suggesting that these effectors likely play a central role in plant immune suppression and nematode virulence. A manuscriptdescribing the above resultswas submitted to the journal Molecular Plant-Microbe Interactions. The manuscript was reviewed favorably, however, it was requested that we test for cell death suppression by SCN effectors in planta. To address this, we have set up an in planta cell death suppression assay by using Nicotiana benthamiana transient expression system. We are trying to test if some our effector candidates are able to suppress the cell death triggered by BAX in N. benthamiana leaves like they did in yeast cells. We also established the Nicotiana benthamiana system and the microbe strains Pseudomonas syringae pv tomato (Pst DC3000) and Pseudomonas fluorescens strain EtHAn (Pf EtHAn) for ETI and PTI screening, respectively. Both the groups at Iowa State University as well as at the University of Missouri are working simultaneously to identify SCN effectors that can suppress host immunity. Twenty-one SCN effector proteins were screened at the University of Missouri for suppression of ETI-triggered by Pst DC3000. Compared to the negative control, which showed less than 10% ETI suppression, 4 SCN effectors showed more than 25% suppression, and one showed more than 30% suppression. Simultaneously, the group at Iowa State University has completed screening 53 effectors for both ETI and PTI suppression assays. While 6 effectors were identified to be ETI suppressors, 14 effectors were identified to be PTI suppressors. For all the confirmed ETI and PTI suppressing effectors the quantitative analysis of bacterial growth rate as well as qPCR assay to test expression of plant immune marker genes will be performed. Objective 3. Functional characterization of effector proteins determined to modulate host immunity. We created a mini yeast two hybrid library of SCN effectors and screened this for interaction with the soybean protein BAG6. We identified 3 effectors that directly interact with BAG6 and each other. We are following up with co-IP analyses. The group at Iowa State University has performed yeast 2-hybird screen for 6 of the effectors that show host immunity modulation. Currently we are in a process of confirming these results by co-transformation. The transgenic Arabidopsis lines expressing these effectors constitutively have been generated and we are currently working to identify homozygous lines. Once we generate homozygous lines, we will examine their potential differential nematode susceptibility.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Dhroso A, Warren S, and Korkin D. 2017. Genome-wide prediction of bacterial effectors across six secretion system types using a feature-based statistical framework. Sci. Reports. Under Review.
- Type:
Journal Articles
Status:
Other
Year Published:
2017
Citation:
Gardner, M., Dhroso, A., Johnson, N., Davis, E., Baum, T., Korkin, D., and Mitchum, M. 2017. Novel effector mining from a de novo transcriptome assembly of the early parasitic stages of the soybean cyst nematode Heterodera glycines. In preparation.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
SUPPRESSION OF SOYBEAN BAG6 INDUCED PROGRAMMED CELL DEATH BY SOYBEAN CYST NEMATODE HETERODERA GLYCINES EFFECTORS.
Wang Jianying 1, G. Yeckel1, P. K. Kandoth1, L.Wasala1, R. S. Hussey2, E. L. Davis3, T. J. Baum4 and M. G. Mitchum1.
1Division of Plant Sciences and Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA;
2Department of Plant Pathology, University of Georgia, Athens, GA; 3Department of Plant Pathology, North Carolina State University, Raleigh, NC; 4Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA. Submitted to MPMI
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Mitchum, MG. SCN National Conference, Coral Gables, FL, December 13-15, 2016, The Nematode is Talking, But Are We Listening? Plenary Speaker
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Mitchum, MG. Missouri Soybean Center Symposium, University of Missouri, April 28, 2016, Soybean Cyst Nematode: Shifting Populations Call for Strategic Measures
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Novel effector mining from a de novo assembly of the early parasitic stages of the soybean cyst nematode Heterodera glycines
Michael N. Gardner1, Andi Dhroso2, Eric Davis3, Dmitry Korkin2, Thomas Baum4, and Melissa G. Mitchum1
1Division of Plant Sciences and Bond Life Sciences Center, University of Missouri; 2Department of Computer Science, Worcester Polytechnic Institute; 3Department of Plant Pathology, North Carolina State University; 4Department of Plant Pathology and Microbiology, Iowa State University
2017 MU Interdisciplinary Plant Group Symposium on Root Biology
|
Progress 05/01/15 to 04/30/16
Outputs
Target Audience:The target audience reached included members of the scientific community that research plant pathology, nematology, and the plant sciences as well as stakeholders in the agricultural community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant has given invaluable support to the graduate students as well as post-doctoral scientists and scientists. Without this grant, it would not have been possible for us to train these individuals in the fields of molecular biology, molecular plant pathology and bioinformatics essential for their professional development. How have the results been disseminated to communities of interest?Results are disseminated to the communities of interest via oral as well as poster presentations at scientific conferences as well as via publishing research articles in peer-reviewed, widely circulated scientific journals. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will continue our efforts to make meaningful progress for all the listed objectives in the grant. The specific plan for each objective is listed above. Along with the experiments, we will plan on presenting our work in oral or poster presentations, as well as publications in peer reviewed journals.
Impacts
What was accomplished under these goals?
The soybean cyst nematode(SCN) is one of the most damaging pests that causes severe economic losses each year worldwide. As virulent SCN populations, which can survive on some of the most resistant soybean cultivars, are spreading and environmentally hazardous chemical nematicides are being phased out, there is an immediate need to discover novel sources of resistance against this pathogen. Our experimental plan follows comprehensive molecular strategies to identify vulnerable points in the parasitic process to devise novel control strategies against the SCN. Objective 1. Explore the genotypic variability of effector arsenals of multiple SCN populations differing in virulence. Three biological replicates of the avirulent SCN population have been inoculated on susceptible soybeans and have been shipped to ISU for parasitic life stage extraction and gland cell purification. Three biological replicates of the highly virulent SCN population are scheduled for shipment during the next two months. Each of the three biological replications of the avirulent SCN population were allowed to grow five days post inoculation and then the parasitic stages of these nematodes were extracted from infected soybean roots, purified and processed to release intact gland cells, using established methods. At least 100 gland cells were isolated for each biological replication. Total RNA was isolated from each pool of 100 cells and quantified via Agilent Bioanalyzer. Sufficient quantities of total RNA were isolated from each pool of 100 cells to proceed with RNA-seq library construction via a Clontech SMARTer Stranded Low Input RNA-seq kit. Three libraries, representing the three biological replications of the avirulent SCN population, were constructed. These libraries are now being tested for quality before being submitted for next generation sequencing. Objective 2. Comprehensive identification of SCN effectors that modulate host defense responses. As a starting place, prior to having a gland specific transcriptome, we have begun developing computational methods to identify nematode effectors from the assembled SCN parasitic transcriptome from whole nematodes. To do so we have adopted PrEffector, our recently developed automated approach for genome-wide de novo prediction of effectors in Gram-negative bacteria. Originally, PrEffector was carefully evaluated on a dataset of known bacterial effectors from 6 types of secretion systems revealing the accuracy of nearly 90% and applied to the genomes of five major pathogens, revealing common features of pathogenicity islands and enrichment/depletion of the same functions shared between the predicted effectors of all five bacteria. Preliminary results on a set of putative nematode effectors identified from microaspirated gland cell contents, confirmed 50 out of 61 (~82%, assuming that all putative effectors are true positives). To improve the accuracy, we are investigating several methodological alternatives. One such alternative is the enrichment of the positive and negative training sets with known secreted effectors and non-effectors from SCN. Using this alternative, an updated version of our method, N-PrEffector, that looks for criteria beyond a signal peptide was developed by adding to the training set 71 known SCN secreted effectors and 250 non-effectors and retraining our top-performing classifier using the updated training set. This allows us to identify previously undiscovered effectors that may play a pivotal role in virulence. In order to investigate the repertoire of SCN stylet-secreted effectors, the parasitic transcriptome was mined using two different effector pipelines. One pipeline utilized traditional criteria such as the presence of a signal peptide, no predicted transmembrane domain, and up-regulation upon exposure to the host plant, whereas the other pipeline was through N-PrEffector. These two pipelines complement each other and allow for the complete mining of the transcriptome. Following mining of the transcriptome over 80% of known SCN effectors were re-discovered in the two pipelines. In addition to mining for new effector candidates, we have also examined the variation of known effector candidates within the transcriptome. New variants of several candidates were identified that may play a role in virulence. Other effectors were found to be highly conserved and may serve a core role in SCN parasitism. We will next apply this on a whole-genome scale using the recent draft SCN genome sequence that has been made available to the SCN community. 12 candidate effector sequences identified from in silico predictions have been selected for initial in situ hybridization testing. Seven of the corresponding cDNA sequences have been cloned for probe preparation and the others are in progress. Once the gland library is sequenced we will run an in silico subtraction to the current SCN parasitic transcriptome to identify those with gland-specific expression to provide global resolution. Specific Objective 2.2: Screen all available and newly discovered SCN effectors from in silico analysis for PTI and ETI suppression. We identified a member of the Bcl-2 associated athanogene family of proteins in soybean (GmBAG6) expressed during the hypersensitive response (HR)-like programmed cell death (PCD) in soybean resistant to SCN. We demonstrated that GmBAG6-1 induces cell death in yeast like its Arabidopsis homolog AtBAG6 and also in soybean. This led us to hypothesize that virulent SCN may target GmBAG6-1 as part of their strategy to overcome soybean resistance. Thus, we utilized a yeast viability assay to screen SCN stylet-secreted effector (SSE) candidates for their ability to specifically suppress GmBAG6-1-induced cell death. We screened forty-seven SCN SSEs originally isolated from a virulent SCN population for suppression of GmBAG6-1, as well as BAX (a mammalian pro-apoptosis regulator of the Bcl-2 family)-induced cell death as a means to identify effectors that may be targeting conserved regulatory mechanisms underlying PCD. This approach has been a powerful method for identifying bacterial and oomycete effectors with roles in cell death suppression. From this analysis, we identified several SSEs that can function as anti-PCD proteins, including two effectors that showed direct interaction with GmBAG6-1 in yeast two-hybrid experiments, suggesting that these effectors likely play a central role in plant immune suppression and nematode virulence. A manuscript "Suppression of soybean BAG6 induced cell death by soybean cyst nematode Heterodera glycines effectors" was submitted to the journal Molecular Plant-Microbe Interactions and will be included in a future report when published. In addition, we are using the already established system of the host Nicotiana benthamiana and the microbe strains Pseudomonas syringae pv tomato (Pst DC3000) and Pseudomonas fluorescens strain EtHAn (Pf EtHAn) for screening the effectors for their role in modulation of plant immune system. During this funding period, we tested total of 21 distinct effectors for both PAMP triggered immunity (PTI) and effector triggered immunity (ETI) suppression. We discovered that three effectors successfully suppressed PTI while six effectors suppressed ETI. Currently we are conducting further experiments to confirm these results. We will also continue testing new effectors for their potential role in immune suppression. Objective 3. Functional characterization of effector proteins determined to modulate host immunity. To identify if any of the SSEs directly interact with and suppress GmBAG6-1, we created a mini yeast two hybrid library of SCN SSEs and screened it for interaction with the soybean protein BAG6. We identified 3 effectors that directly interact with BAG6 and each other. We are following up with additional functional analyses.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Wang Jianying, G. Yeckel, P. K. Kandoth, L.Wasala, R. S. Hussey, E. L. Davis, T. J. Baum and M. G. Mitchum. 2016. SUPPRESSION OF SOYBEAN BAG6 INDUCED PROGRAMMED CELL DEATH BY SOYBEAN CYST NEMATODE HETERODERA GLYCINES EFFECTORS. The Joint meeting of Society of Nematologists and Organization of Nematologists of Tropical America. Montreal, Canada, July 17-21, 2016.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Michael N. Gardner, Andi Dhroso, Eric Davis, Dmitry Korkin, Thomas Baum, and Melissa G. Mitchum. 2016. De novo assembly of the early parasitic transcriptome and effector mining in the soybean cyst nematode Heterodera glycines. MU Life Science Week. University of Missouri, MO, April 18-23, 2016.
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