Progress 08/01/19 to 07/31/24
Outputs
Target Audience:The results of this project were presented primarily to academic and scientific audiences through peer-reviewed publications and presentations at national and international conferences. This group included faculty, students, postdoctoral trainees, professionals in the plant biotechnology industry, and plant breeders. Some of our presentations were also attended by commodity group leaders, representing groups like the United Soybean Board and the Iowa Soybean Association in the USA, and Donau Soja (European Union). Results from this project were also incorporated in graduate level courses at Iowa State University. Communications at this level were focused on technical aspects of our research and potential applications. Finally, direct communication to soybean farmers was achieved through activities organized by the Iowa Soybean Research Center. When talking to farmers, the main focus was on the long-term significance of our research, emphasizing that the knowledge obtained would inform companies and breeders to develop better technologies that may improve yield and provide better management practices. Changes/Problems:As explained in previous reports, the COVID pandemic caused disruptions in our ability to work in the lab, hire some personnel, and pursue collaborations with other laboratories. While the majority of the proposed work was accomplished and expanded in several areas, the analysis of trans-kingdom RNA signaling was not completed. The datasets for this analysis have been produced, and the analysis will continue through collaborations with experts in the field. What opportunities for training and professional development has the project provided?One graduate student was the main contributor to this project and generated a large proportion of the data, which were used as her PhD thesis. The graduate student was interested in exploring careers in Industry, the PI encouraged her to join the Bayer mentorship program. This student completed her PhD program in the last year of the grant and is currently a postdoctoral fellow at the Donald Danforth Plant Science Center. Two other graduate students participated in the project in a collaborative effort and were trained during the project. They also served as mentors for undergraduate students engaged in the project, developing mentorship and communication skills in the process. Four undergraduates participated in this project. Each student developed independent research projects in the framework of the NIFA project, with mentoring provided by the PI and graduate students. The PI also worked with each student to develop a career plan, identifying long-term goals, and career development opportunities. Undergraduate students were encouraged to apply for department and college level scholarships and all received at least one scholarship, some multiple. In addition, one undergraduate was awarded a fellowship from the American Society of Plant Biologists to do research full time in the PD laboratory during the summer. Two of the undergraduates are now pursuing PhD degrees (Biochemistry and Plant Biology at Washington University in St Louis). The other two are still working on their BS degree. The laboratory also hosted a summer intern, as part of the IINSPIRE LSAMP Summer Rise Up Program. The student continued in contact with the Project Director after the internship. These interactions led the student to pursue a PhD degree (current) at Iowa State University. Graduate and undergraduate students were encouraged and prepared to present their research at local, regional and national conferences, which included the Stupka symposium at ISU, the annual meeting of the American Society of Plant Biologists and the meeting of the Midwest section of society, and the Molecular and Cellular Biology of the Soybean Conference. How have the results been disseminated to communities of interest?Results have been included in two peer-reviewed publications, and four others are in preparation. In addition, as direct result of the activities of this project, the PD was invited to chair the Biotic Interactions section of the workshop organized by the Soybean Genomics Executive Committee (SoyGEC) to develop a 5 year "soybean genomics research community strategic plan" that resulted in a peer-reviewed publication (see other products section). The research was also disseminated through presentations at national conferences like the annual meeting of the American Society of Plant Biologists and the meeting of the Midwest section of society, and the Molecular and Cellular Biology of the Soybean Conference, and international conferences such as the World Soybean Research Conference (Austria) and the annual meeting of the Argentinian Society of Biochemistry. The PD was also invited to present seminars at several universities and research centers, including a webinar organized by the American Society of Plant Biologists that was globally broadcasted. Commodity groups and farmers were reached through presentations at the Soybean Breeders Workshop and presentations organized by the Iowa Soybean Research Center. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In order to manage crop pests, it is necessary to understand the virulence mechanism used by these organisms to increase plant susceptibility and favor colonization. Aphids use salivary proteins and other compounds, collectively known as effectors, to hijack plant metabolism and suppress defenses. However, the arsenal of effectors used by the soybean aphid is not known. The activities carried out during this project to understand soybean aphid's ability to colonize plants are: 1) identification of aphid effectors, and 2) characterization of molecular changes induced in the plant that result in reduce defense outputs. Aim 1: Aphid effectors Our proposal described a strategy to identify aphid effectors that was not implemented due to unexpected technical challenges that reduced its effectiveness. In that approach, we relied on ABA transcriptional outputs; however, further characterization showed that most of the regulation exerted by ABA was done at the posttranscriptional level. A new strategy was developed to identify soybean aphid effectors. We used transcriptomics to identify aphid genes with preferential expression in head/salivary gland. We then evaluated ability of EffectorP, a program originally developed for fugal effector prediction, to identify aphid effectors. Good sensitivity and specificity allowed us to include it in our pipeline. Filtering using these 2 criteria identified 575 putative effectors, including 85 with a secretion signal. From this set, we identified fast-evolving candidates (dN/dS ratio) and found 94 proteins (26 with secretion signal) showing evidence of positive selection. AlphaFold was used for protein structure prediction and identification of effectors with structural similarity. We found effectors belonging to 8 structural families, suggesting similar function for its members. Our prediction pipeline provided a well-defined and curated list of putative effectors that can be used to prioritize those that should be functionally characterized. To validate our prediction and functionally characterize aphid effectors, it was necessary to express individual effectors in soybean plants, which is technically challenging. We optimized a virus-vectored expression system to transiently express candidate effectors in soybean. Defense outputs in response to biotic challenges and aphid performance were evaluated using these plants. To assess the effectiveness of this approach, we functionally characterized the soybean aphid orthologs of two aphid effectors, C002 and Mp10, that were previously shown to increase aphid performance in other systems (see results in Aim 3). Metabolic and gene expression analyses suggested that aphids modify plant cuticle. A detailed characterization of soybean cuticle is missing in published literature. Thus, we first characterized soybean cuticle, and then analyzed changes induced by aphids. Our results showed that aphids secrete triacyclglycerols and fatty acids on the plant surface, indicating a novel class of effectors. These secretions appear to have a dispersion effect on aphid populations, and we have some evidence that these compounds may also have a role controlling the growth of microorganisms. Aim 1 produced a large dataset of soybean aphid gene expression, a list of predicted effectors, a validation system for aphid effectors, a detailed characterization of soybean cuticle, and the identification of a new class of aphid effectors. Two manuscripts are in preparation. Our results increased knowledge of soybean and insect biology, and provided tools to design management strategies such as in planta RNAi to target aphid effectors and reduce the insect's ability to colonize plants. Aim 2: RNA signals To identify RNA signals that can be transferred between plant and insect, we generated RNA transcriptome datasets of control and aphid-infested plants, and RNA transcriptome of aphids feeding on soybean. We characterized plant miRNAs differentially expressed in response to aphid infestation and identified targets. Our plant transcriptome data has been used extensively to generate hypotheses (for example see Aim 3). However, problems to secure personnel to complete the more sophisticated portion of the study arose mainly due to the COVID pandemic. Although we were able to secure no-cost extensions, limitation in funds available further complicated this process. While the analysis of trans-kingdom RNA signals is still underway, it was not completed during period covered by the grant. However, we have established a new collaboration with Sayma Sahib, an expert on this field, and we expect to finish the analysis in the near future. This portion of the project generated significant datasets that have been extremely useful for our own research, and will also provide important information for other researchers in the field of plant-insect interactions. Aim 3: Resistance to aphids and suppression of defenses by aphid effectors We characterized the plant defense mechanisms used by soybean plants to limit aphid colonization, comparing the transcriptional response of susceptible and resistant plants. We identified physical (cell wall strengthening) and chemical (isoflavone accumulation) mechanisms of defense and hormonal signals and transcriptional networks that mediate the resistance response. We also characterized pattern-triggered immunity signals that are induced by aphids in the early stages of colonization. Aphid feeding suppressed the ability of soybean plants to respond to JA, and our transcriptional data showed that this effect is mediated by induction of the catabolism of JA-Ile, the active form of the hormone. We also showed that ABA signals mediate this block in JA. Reducing the expression of ABA biosynthetic genes in soybean using virus-induced gene silencing (VIGS) caused an increase in plant resistance to soybean aphids. In addition, aphids lost the ability to block JA signaling on plants deficient in ABA. Using similar approached we identified the transcriptional activator SCOF-1 as a key mediator of the ABA effect on JA. Interestingly, we observed that ABA-deficient plants free of aphids had an elevate level of JA response under stress-free conditions, indicating that soybean plants use an ABA-dependent mechanism to control basal levels of JA and that aphids are exploiting this mechanism during colonization. In parallel, we observed that aphids induce activation of MAP Kinases. Biochemical and proteomic approaches allowed us to identify MPKs differentially regulated by aphid feeding. We selected two of these kinases, MPK4 and MPK6, for further analysis. Using VIGS, we showed that they participate in the suppression of soybean defenses. Plants with knockdown expression of MPK4 and MPK6 are more resistant to aphids. Our results suggest that these kinases block salicylate (SA)signaling. Using VIGS we silenced the SA pathway and demonstrated that this hormone has a positive effect on defenses against aphids. Using the system developed in Aim 1 we characterized C002 and Mp10 orthologs. Unlike results in other systems, Mp10 alone causes primer of soybean defenses. On the other hand, expression of C002 resulted in compromised ability of plants to respond to bacterial and fungal effectors, specifically by blocking the ROS burst. We found that C002 forms aggregates in the cytoplasm, likely the result of phase-separation. Plants expressing C002 are more susceptible to soybean aphids. Our results identified novel mechanisms of suppression of defenses triggered by aphid effectors. We also identified key differences in hormonal crosstalk in soybean defenses compared with model systems such as Arabidopsis. These advances increase our knowledge of plant-insect interactions and highlight the need to carry out studies using crop plants, as the advances made in model systems are not always translatable to crops. We published 2 manuscripts and 2 more are in preparation.
Publications
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Progress 08/01/22 to 07/31/23
Outputs
Target Audience:Our work was disseminated through presentations (seminars and talks at the local and international level). The audience is mainly scientists in the Plant Sciences and Agriculture areas, including faculty and people working in Industry, students (undergraduates and graduates) and postdoctoral trainees. We also presented our work for commodity groups that include farmers. Changes/Problems:As explained in previous reports, the COVID pandemic caused disruptions in our ability to work in the lab, hire some personnel, and pursue collaborations with other laboratories. While a significant portion of the proposed work was accomplished, the analysis of trans-kingdom RNA signaling is not complete, and will likely not be finished before the end of the grant. However, the main datasets needed for the analysis have been collected, and we hope to complete the bioinfimatics pipeline in the near future. The PI became president of the American Socity of Plant Biologists during the course of this grant, and this obligation slowed down the submission of manuscripts that are in preparation. Four manuscripts should be submitted in the next few months. What opportunities for training and professional development has the project provided?One graduate student and two undergraduate students participated in this project during the reporting period. As part of their career development plan, all presented their work at regional and local symposia. The graduate student was interested in exploring careers in Industry, so the PI encouraged her to join an industry mentorship program. How have the results been disseminated to communities of interest?The PI presented this work at the World Soybean Research Conference (Austria) and the annual meeting of the Argentinian Society of Biochemistry, in addition to informal presentations to commodity groups through the Iowa Soybean Research Center. The graduate student and one undergraduate student presented a talk and poster, respectively, at the Midwest meeting of the American Society of Plant Biologists. What do you plan to do during the next reporting period to accomplish the goals?After the period included in this report there were only a few extra months before the end of the grant. We completed a few experiments to be able to publish the main discoveries already described in this and previous reports.
Impacts
What was accomplished under these goals?
In previous periods, we used a combination of transcriptomics and computational pipeline to predict soybean aphid salivary proteins that function as effectors during plant colonization. In the current period we performed additional bioinformatics analyses to include proteins that were not correctly annotated in the soybean aphid genome. For example, a novel protein family that is present exclusively in aphids, "bicycle" proteins, was characterized in other aphids after the release of the soybean aphid genome. These proteins act as effectors in other aphid species, but their short size and lack of homology to other proteins normally escape annotation by standard pipelines. We identified ~30 putative orthologs of bicycle proteins in the soybean aphid genome, and these have been incorporated in our effector set. During this period, we also optimized the use of viral vectors to express candidate aphid effectors in planta. The system allowed the characterization of two effectors. AgMP10 primes plant defenses when it is expressed in soybean. On the other hand, AgC002 suppresses soybean defenses by blocking the production of reactive oxygen species during the pattern-triggered immunity (PTI) response. Using the same viral-vector approach, we were able to show that expression of AgC002 in soybean causes an increase in aphid susceptibility, resulting in larger aphid populations when compared to controls. Protein structure analysis indicated that AgC002 contains disordered regions commonly associated with liquid-liquid phase separation, a process that allows for the formation of intracellular foci with specific functions. Expression of full-length and truncated versions of AgC002 in Arabidopsis protoplasts showed that when these regions are present in the protein, the protein aggregates in puncta that resemble stress granules. We generated transcriptome data for soybean leaves and soybean aphids. These datasets have been extremely useful to characterize the changes in plant defense triggered by aphid colonization, and for the identification of putative effectors. Our plan included using these datasets to identify mobile RNAs that could be transferred from the aphid to the plant and vice versa. However, lack of a dedicate bioinformatician for this project has made it difficult to achieve this portion of the project so far. Previous analyses of our transcriptome datasets and evidence from our analysis of aphid effectors suggested that soybean aphids can block PTI-type of responses in soybean. These responses are characterized by a reactive oxygen species burst and activation of MPK cascades that can control defense responses. We found that aphid infestation causes dynamic changes in MPK activation in soybean, and use of proteomics approaches allowed us to identify several MPK orthologs differentially regulated by aphids at specific times during colonization. We focused on MPK4 and MPK6 for further characterization, because the role of these proteins in defense has been well-characterized in other plant species. Using virus-induced gene silencing we reduced the expression of these genes in soybean. Surprisingly, both MPK4 and MPK6 knockdown plants were more resistant to aphid infestation than the vector controls or mock-treated plants. Plants defective in MPK4 and MPK6 expression had elevated levels of salicylate signaling. Our results suggest that aphids may activate these proteins to exploit their negative effect on SA signaling and thus increase plant susceptibility to colonization.
Publications
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Progress 08/01/21 to 07/31/22
Outputs
Target Audience:Our work was disseminated through presentations (seminars and talks at the local and international level). The audience is mainly scientists in the Plant Sciences and Agriculture areas, including faculty and people working in Industry, students (undergraduates and graduates) and postdoctoral trainees. Changes/Problems:The university policies regarding lab access after the start of the COVID-19 pandemic caused an unexpected slow down of our research. While students were able to stay engaged through literature reviews, writing manuscript sections, and other activities that could be performed remotely, experiments were stopped for a few months. When laboratories were able to reopen, we established a protocol for social distancing and reduced exposure using lab shifts that reduced the time each student was able to be at the lab. Access to facilities was also completely stopped or severely reduced, and some facilities are still working with limited access. The pandemic also made it difficult to identify a second graduate student to work on this project. Instead, we are using the genome bioinformatics facility at Iowa State University for our prediction pipeline and RNA analyses. We also had to delay our collaboration with Dr. Meyers (Danforth), but we are now planning a visit to his institute to complete our analyses. While we expect that our objectives will be achieved, our proposed timeline had to be revised. What opportunities for training and professional development has the project provided?One graduate student and one undergraduate student worked on the project. Each student was in charge of an independent research projects in the framework of this NIFA project, with mentoring provided by the PI. The PI also worked with each student to develop a career plan, identifying long-term goals, and career development opportunities. Both students will present their research at the annual meeting of the American Society of Plant Biologists in July. The undergraduate student also presented her research at a local symsposium. The undergraduate student applied and was accepted in a Plant Biology Graduate Program to pursue her PhD (starting August). Another undergraduet student that was onvolved in the project in the previous periods was also recently accepted in a Biochemistry Graduate Program to pursue her PhD (starting August). How have the results been disseminated to communities of interest?The PI presented the research at the Soybean Breeders Workshop meeting and at the Annual Meeting of the Entomological Society of America. These meeting serve different demographics, soybean breeders and molecular biologists the first and entomologists and plant pathologists the second, and both included members of academia and industry. One journal article was published (open access). What do you plan to do during the next reporting period to accomplish the goals?We expect to continue the plan described in the original proposal.
Impacts
What was accomplished under these goals?
The main objective of our project is to identify molecules (proteins, RNAs, and small molecules) produced by aphids and introduced in plants during feeding or while colonizing the plant. These molecules (effectors), are used by aphids to block plant defense responses. This strategy allows aphids to feed, reproduce, and form successful colonies on crops. We also want to understand the molecular mechanisms triggered by effectors that cause reduction in the plant's defense outputs. So far, we identified a new type of aphid effector, lipid molecules that are part of aphid secretions and accumulate on the leaf surface. These lipids could have an effect on plant defenses, or could act as general antimicrobial agents that prevent fungal infections of aphid colonies. We also developed a computational and experimental strategy to identify a new set of proteins that are putative aphid effectors, with expression enriched in head tissues, where the salivary glands are located. We have also started the functional characterization of some of these putative effectors. We have also identified plant biochemical pathways regulated by aphids that are likely involved in the suppression of soybean defenses. Our work has generated large datasets that will inform not only our research but also projects by other investigators. The effectors characterized by our project could be new targets for insecticidal efforts that will increase crop production and food security. Specific activities and achievements by Aim: Aim 1: The annotation of putative effectors in current releases of soybean aphid genomes is severely deficient and rely on initial prediction of secreted proteins. However, experiments in other laboratories have shown that only 40% of known salivary proteins contain a secretion signal. In the current reporting period, we refined our bioinformatics pipeline for identification of novel effector candidates. We used RNAseq to identify transcripts enriched in aphid head as a proxy for salivary gland expression. We then used EffectorP 2.0, validated in the previous period, to predict putative effectors among head-enriched transcripts. Finally, we used SignalP to predict proteins that are secreted among the candidate effectors. With this initial pipeline we identified 575 putative effectors, including 85 predicted to have a secretion signal. It has been previously shown that some effector families undergo positive selection (i.e. are fast evolving); and it is also known that fungal effectors can be grouped in families with conserved protein structure but little sequence identity. We identified orthologs of our effector candidates, and calculated dN/dS ratios to identify fast-evolving effectors. This analysis identified 94 proteins (26 with secretion signal) showing evidence of positive selection. Finally, AlphaFold was used for protein structure prediction and identification of effectors families with structural similarities. A manuscript describing our pipeline and soybean aphid effector prediction is currently in preparation. We optimized a viral vector system to express aphid effectors in soybean plants. We selected candidates for functional characterization using the viral system, and have so far identified two effectors that alter the plant response to elicitor treatments. One of the salivary effectors seems to prime plants, resulting in an increase in defenses after challenging with elicitors; while the second candidate strongly suppressed the production of reactive oxygen species normally triggered by chitin and flagellin treatments and is thus a prime candidate for a more detailed functional characterization. We are currently producing tagged versions of the effector to identify interactions with plant proteins. Aim 2: The identification of plant RNAs transported into aphids and aphid RNAs transported into soybean leaves is underway. Aim 3: We have previously shown that aphids are able to suppress JA responses in soybean. Analysis of phytohormone accumulation in response to 7 days of aphid feeding indicated that while jasmonate (JA) accumulates in infested soybean plants the active conjugate JA-Ile does not. One of our hypotheses is that the conjugating enzyme (JAR1) is posttransationally regulated in response to aphid, and we are working on testing it. During this period, we also tested another hypothesis, that aphids induce differential degradation of JA-Ile. We observed that 7 days of aphid feeding causes a massive induction (1000-2000 fold) of the two soybean genes encoding for CYP94C1, an enzyme that participates in the catabolism of JA-Ile. Comparison if aphid- versus wounding-induction of common JA markers and CYP94C1 showed that the latter is differentially regulated in response to aphids. We carried out a transcriptome analysis of resistant and susceptible plants infested with soybean aphids. This analysis identified important regulatory hubs that contribute to resistance and defense outputs (published, see products). MAP kinases (MPK) were differentially regulated by aphids in both compatible and incompatible interactions, suggesting that phosphorylation cascades are important components of the response to aphids. Using antibodies specific for phosphorylated forms of MPKs (the active form), we observed that different MPKs are activated early (24 h after colonization) when the plant is deploying defenses, and late (7 days after colonization) when the aphid is able to suppress defenses. Using a proteome approach, we were able to identify MPK4 as the protein kinase activated at day 7. We are currently generating virus-induced gene silencing (VIGS) constructs to characterize the role of MPK4 in suppression of defenses, and other kinases (MPK3, MPK6 and MPK20) as potential positive regulators of soybean immunity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Natukunda, M. I., Hohenstein, J. D., McCabe, C. E., Graham, M. A., Qi, Y., Singh, A. K., & MacIntosh, G. C. (2021). Interaction between Rag genes results in a unique synergistic transcriptional response that enhances soybean resistance to soybean aphids. BMC genomics, 22(1), 1-23.
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Progress 08/01/20 to 07/31/21
Outputs
Target Audience:Our work was disseminated through presentations (seminars and talks at the local and international level). The audience is mainly scientists in the Plant Sciences and Agriculture areas, including faculty and people working in Industry, students (undergraduates and graduates) and postdoctoral trainees. Changes/Problems:The university policies regarding lab access after the start of the COVID-19 pandemic caused an unexpected slow down of our research. While students were able to stay engaged through literature reviews, writing manuscript sections, and other activities that could be performed remotely, experiments were stopped for a few months. When laboratories were able to reopen, we established a protocol for social distancing and reduced exposure using lab shifts that reduced the time each student was able to be at the lab. Access to facilities was also completely stopped or severely reduced, and some facilities are still working with limited access. While we expect that our objectives will be achieved, our proposed timeline had to be revised. What opportunities for training and professional development has the project provided?One graduate student and two undergraduate students have worked in this project. Each student developed independent research projects in the framework of this NIFA project, with mentoring provided by the PI. The PI also worked with each student to develop a career plan, identifying long-term goals, and career development opportunities. Both undergraduate students were encouraged to apply for department and college level scholarships and both were successful. In addition, one of the undergraduates was awarded a fellowship from the American Society of Plant Biologists to do research full time in the PI laboratory during the summer. All students presented their research at local symposia (virtually). How have the results been disseminated to communities of interest?The PI presented a seminar for the "Plantae Presents" virtual seminar series organized by American Society of Plant Biologists. This series has worldwide reach, and a recording of the seminar is freely available on YouTube (https://youtu.be/gCONGzp6-LA). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The main objective of our project is to identify molecules (proteins, RNAs, and small molecules) produced by aphids and introduced in plants during feeding or while colonizing the plant. These molecules (effectors), are used by aphids to block plant defense responses. This strategy allows aphids to feed, reproduce, and form successful colonies on crops. So far, we identified a new type of aphid effector, lipid molecules. We refined the characterization of the lipids deposited by aphids on soybean leaf surfaces, and identified a specific triacylglycerol with a unique short chain fatty acid group that is part of aphid secretions. These lipids could have an effect on plant defenses, or could act as general antimicrobial agents that prevent fungal infections of aphid colonies. We also realized that the annotation of putative effectors in current releases of soybean aphid genomes is severely deficient. We developed a strategy that allowed us to identify a new set of proteins that are putative aphid effectors, with expression enriched in head tissues, where the salivary glands are located. We have also started the functional characterization of some of these putative effectors. Our work has generated large datasets that will inform not only our research but also projects by other investigators. The effectors characterized by our project could be new targets for insecticidal efforts that will increase crop production and food security. Specific activities and achievements by Aim: Aim 1: The current annotation of the aphid genome contains 94 genes identified as putative effectors. This annotation was produced by looking for homologs to putative effectors annotated in the genome of the pea aphid. We generated a list of effectors with known biological function from different aphids and determined that out of 16 different effectors, only four were annotated as effectors in the soybean aphid genome, despite all having a close homolog in this aphid. Thus, we created a pipeline to identify putative effectors and reanalyzed the soybean aphid genome. General criteria for salivary effector predictions include a signal peptide that directs the protein to the secretory pathway, and preferential expression in salivary glands. We identified all genes encoding secreted proteins, the secretome, using standard bioinformatic approaches. However, not all proteins with secretory signals are effectors, and recent work has shown that only 10-20% of proteins present in saliva have a precursor with signal peptide. Thus, we wondered whether machine learning programs trained to identify fungal effectors, such as EffectorP, would be able to identify aphid effectors. Using the same set of 16 known aphid effectors, we tested the performance of EffectorP. The software was able to identify 81% of these effectors (including homologs of each effector from different species), indicating that EffectorP could be used in our pipeline to reannotate effectors from the soybean aphid. To test differential expression in salivary glands, we performed a transcriptome (RNAseq) experiment comparing expression in soybean aphid head vs body. Head was used as proxy for salivary glands. Using our pipeline that included effector prediction by EffectorP and at least 2-fold enrichment in head vs body expression, we identified 625 genes that can encode putative effectors. This list includes most homologs of the 16-effector training set and ~30% of the genes currently annotated as putative effectors. We are currently refining our pipeline to include other criteria, including for example evolution rate, to genera a list of most likely effectors to continue our functional analysis. We have also developed two new approached for functional analyses. For the first, we adapted protocols currently used to test bacterial effectors using transient expression in Nicotiana benthamiana. For the second, we used viral vectors to express candidate aphid effectors directly in soybean leaves, and we are testing their ability to block induction of defenses induced by chitin or fl22 treatments. Both approaches have generated promising preliminary results. We have a large collection of effectors already cloned in entry vectors. These will be used with either of the system to confirm their ability to suppress plant defenses. New candidates from our annotation pipeline will be added to the list of candidates to be tested. Aim 2: Due to the pandemic and our initial difficulty to identify a suitable bioinformatics student to work in this Aim, we decided to recruit a scientist from the ISU bioinformatics facility. With his help we developed the bioinformatics analysis pipeline described in Aim 1. The same scientist is helping us to analyze the soybean aphid transcriptome, combining sequences from head and body samples, to identify RNAs originated in the plant. This analysis will complement the small RNA transcriptome analysis already in progress. Aim 3: In the previous period, we reported the discovery of lipid secretions deposited by aphids on the surface of soybean leaves. Those secretion included palmitate and triacylglycerols, likely in the form of tripalmitate. We have now performed a more detailed analysis of the lipid depositions. While palmitate is still the most abundant secretion, the TAG previously identified potentially as tripalmitate had been misidentified. The main TAGs secreted by aphids mostly contain 2 palmitate groups but the third group is a short chain fatty acid of 6 carbons with two double bonds, sorbic acid. We have carried out bioassays to test aphid preference for surfaces covered with these secretions, but our results have been so far inconclusive. In addition, since sorbic acid has antimicrobial properties, we are testing whether these secretions could have a role helping aphids control fungal infections in their colonies by testing antifungal activity of the secretions purified from leaf surfaces. Based on our transcriptome analysis and phytohormone quantification of compatible soybean aphid-soybean interactions, we have hypothesized that aphids block the conversion of jasmonic acid to its active aminoacyl conjugate JA-Ile. This step is catalyzed by the amino acyl transferase JAR1. We are currently generating antibodies against soybean JAR1. These antibodies will be used to isolate the enzyme from mock-treated or aphid-infested plants. After purification, we will test enzymatic activity in vitro. Since enzymes in the JAR1 family can be phosphorylated, we will analyze the phosphorylation status of the purified enzyme using anti phospho-Ser/Thr antibodies. In addition, phosphatase treatments will be used to determine the effect of phosphorylation on enzymatic activity. We have also generated gene regulatory network models using transcriptome data from previous experiments. Our analysis indicated that a main hub involving WRKY and MYB transcription factors controls resistance to soybean aphids. We are currently developing functional analyses to test the role of the transcription factors identified.
Publications
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Progress 08/01/19 to 07/31/20
Outputs
Target Audience:Our work was disseminated through presentations (seminars and talks at national conferences) and through peer-reviewed publications. The audience is mainly scientists in the Plant Sciences and Agriculture areas, including faculty and people working in Industry, students (undergraduates and graduates) and postdoctoral trainees. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student and two undergraduate students were recruited to work on this project so far. Each student was allowed to develop an independent project in the framework of the overall NIFA project. Everyone worked one-on-one with the PI during the course of the project to develop hypotheses, test them, and analyze results. In addition, students worked on literature reviews and writing assignments to develop critical thinking and communication skills. Students presented their research periodically at group meetings. In addition, one of the undergraduate students prepared a proposal for a university scholarship and funding that were successful. She also presented her research as a poster at the American Society of Plant Biologists annual meeting (Plant Biology World Summit 2020). Other presentation plants for the graduate student and both undergrads were cancelled due to COVID19. The laboratory also hosted a summer intern, as part of the IINSPIRE LSAMP Summer Rise Up Program, which this year was conducted online as a result of COVID19 precautions. This student, a Latinx first-gen student, was able to work on a proposal based on aphid effectors that affect plant cell wall and other changes in plant cell wall induced by aphids. He then analyzed RNAseq data and was able to identify a significant number of cell-wall related processes affected by aphids. He delivered a successful presentation on his results for the LSAMP symposium that included students participating in the program at different Iowa universities. How have the results been disseminated to communities of interest?The results were presented at the American Society of Plant Biologists annual meeting (Plant Biology World Summit 2020), and in a peer reviewed publication recently accepted in Frontiers in Plant Sciences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the first year of the project we identified aphid molecules that can potentially affect the way plants respond to the insect attack, acting as "effectors". These effectors are lipids, a type of molecules that have not been associated with this function before, and thus open a completely new avenue of research. We have also made advances in the characterization of protein effectors. Together, these effectors could be new targets for insecticidal efforts that will increase crop production and food security. Specific activities and achievements by Aim: Aim 1: All vectors for the proposed screening were built. We tested the expression of reporters (expressed under the SCOF-1 promoter and under a constitutive promoter, 35S) using two different systems: Arabidopsis protoplast and Nicotiana benthamiana infiltration. After testing, we observed that the SCOF-1 promoter was not as responsive to abscisic acid (ABA) treatments as expected. Further analyses determined that SCOF-1 responsiveness to the hormone is primarily posttranscriptional. Thus, we are currently testing other promoters (based on genes that are differentially regulated by ABA and aphid feeding in susceptible plants at 7 h post infestation). We have cloned 5 candidate promoters and we are currently building the constructs to test their regulation in our systems. To avoid delays, we have also started the cloning of annotated soybean aphid effectors, starting with the top 11 effectors based on expression, putative function, conservation in other aphid species, and association with virulence. This list includes 4 genes with homology to proteins that have been shown to act as true effectors in other aphid species. These effectors were introduced in our original screening vector (with SCOF-1p-YFP reporter), but the screening will be performed directly by bioassay, instead of looking for expression of the reporter gene. For the bioassay, each effector will be expressed in hairy roots and aphid performance will be assayed on roots expressing the effector compared to roots expressing a YFP control. As we carry out the bioassays, the next 10-20 effectors will be cloned. Aim2: After several student rotations, we had some problems finding a suitable student for this portion of the project. However, we have secured a pilot soybean aphid small RNA dataset and a soybean aphid-infested soybean small RNA dataset and we are currently working with our collaborator, Dr. Meyers at the Danforth Center, to map the small RNA reads to the recently released soybean aphid biotype 1 genome and to the soybean reference genome. We expect to have a bioinformatics student in the lab soon to continue with the analysis of plant and aphid small RNAs. Aim 3: As part of our analysis of the Jasmonate pathway, we mined an extensive RNAseq dataset that includes the response of susceptible plants to aphids after 7 days of feeding, a time when we know that aphids suppress plant defense responses. One of the categories of interest was acyl-lipid metabolism, as fatty acids are precursor of JA and we have previously found that aphid feeding affects fatty acid accumulation in soybean. Our analysis determined that a large proportion of genes encoding proteins involved in lipid metabolism are differentially regulated by 7 days of aphid feeding. One of the main pathways was, as expected, the oxylipin pathway. The other pathway most affected by aphids was the cuticular lipids biosynthetic pathway. Given the importance of cuticle as first point of contact between plant and insect, and its known roles as physical barrier to biotic and abiotic stresses, we performed analyzed the changes in soybean cuticle composition after aphid feeding (7d). Surface lipids from control and aphid-infested plants were extracted and analyzed by GC-MS. We observed a significant increase (63%, p>0.001 t-test) in total lipids deposited in the cuticle in response to aphid feeding. The trend was also evident for all the main lipid categories identified, including long chain hydrocarbons, alcohols, aldehydes, and fatty acids. Among individual components, the largest increase was observed for palmitate (5-fold increase, p>0.001 t-test). We also identified a large accumulation of tripalmitin present only in aphid infested plants, which accounted for almost half of the increase in total lipids observed in infested plants. Triacylglycerols are not normally found in plant cuticle. On the other hand, triacylglycerols are common components of aphid secretions and cuticle. We hypothesized that the triacylglycerols observed in the leaf surface lipid extraction was not produced by the plant but rather deposited by aphids. To test this idea, we extracted surface lipids from aphids and analyzed it composition. We found that the main component of aphid surface lipids was fatty acids (75%) and that palmitate was the most abundant fatty acid (75% of all fatty acids quantified). Other major components included mono-, di-, and triacylglycerols and alcohols, while hydrocarbons were minor components. These results supported the idea that the main changes observed in leaf surface lipids are due to aphid secretions (TAG and palmitate) and not to lipids produced by the plant. We are currently conduction bioassays to test the effect of tripalmitate treatment ("leaf painting") on aphid behavior and aphid population growth, and testing different methods to obtains aphid cornicle secretions rather than total surface lipids to refine our hypothesis that aphids use lipid secretions as a novel type of effector to modify plant defense responses. While we propose that the main change in surface lipids is due to aphid secretions, we can see a change in leaf surface lipids even if we remove triacylglycerols (and/or palmitate) from our calculations. True plant surface lipids also increase in response to aphid feeding. To identify the function of these changes, we used network analyses to identify transcription factors that control cuticular lipid biosynthesis in our dataset and identified several TFs that may be acting as aphid-regulated nodes. We will target these TF for silencing using VIGS to test whether they control surface lipid production, and the consequence of modifying plant surface lipids on aphid performance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ibore M, MacIntosh GC (2020) The Resistant Soybean-Aphis glycines Interaction: Current Knowledge and Prospects. Front. Plant Sci. doi: 10.3389/fpls.2020.01223
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