Progress 04/01/19 to 12/31/22
Outputs
Target Audience:The target audience consists of researchers in the plant-insect interactions community, including molecular plant biologists, entomologists, and virologists studying plant viruses. During research activities, undergraduate researchers are also a target audience. Long-term, a target audience will be growers of specialty crops affected by whiteflies. The target audience was reached through publication of work in a peer reviewed journal, public deposition of raw data, and presentations at scientific conferences. Changes/Problems:Two main problems were encountered. The first is an error in the library preparation for RNA sequencing of single salivary glands from whiteflies (factorial experiment). The source of this error was determined and the experiment was repeated. This caused delays but we are still able to complete the proposed work. Data analysis will extend beyond the grant reporting period, but we already have pipelines in place for this effort. The second main problem is continued inconsistency in proteomic efforts. Given limited resources remaining on the grant, we opted to keep saliva samples already collected in long-term storage, and to focus on completing RNA sequencing experiments. The resources collected (saliva collections) during this grant period and prior periods will be leveraged to obtain additional funding. Since this is a seed grant, one of the intended outputs is resources that will assist with larger funding opportunities. We feel that we have been successful in generating these resources while identifying key areas in the proposed work that are more logistically difficult and/or require collaborator input. What opportunities for training and professional development has the project provided?The project scientist continued to receive training in bioinformatic analysis (RNAseq and associated tasks) from the UCR Bioinformatics Core Facility. One undergraduate assisted with various aspects of whitefly saliva collection experiments, learning more about whitefly biology. How have the results been disseminated to communities of interest?Results were presented at the 2022 Entomological Society of America Pacific Branch meeting and the 2022 Entomological Society of America joint meeting with the Entomological Society of Canada and Entomological Society of British Columbia. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT STATEMENT: The goal of this project is to identify and characterize the molecules that enable an invasive pest (sweet potato whitefly) to exploit crop plants for its own survival. In doing so, this project directly addresses the goals of the USDA AFRI-NIFA funding programs to "elucidate fundamental molecular processes affecting plant-associated pests, including vectors of diseases and microbes." During the current project period, we dissected the organs from the insect that produce proteins that help them feed on plants (salivary glands) and measured the levels of RNA in these tissues. RNA is the instructions for proteins, and the levels of RNA give us information about what is being produced in the insect. From these efforts, we have prepared material for RNA sequencing, a process that reads all the RNA instructions present in salivary gland cells at one time. We used this technique to test hypotheses about how microbes living within whiteflies affect their ability to feed on different crop plants and which molecules in the whitefly are most important for feeding. Our results will help us to develop new biopesticides that target production of these molecules in the whitefly itself. Through these impacts, the project fulfills the need for AFRI-supported research to "lead to the development of innovative and environmentally sound strategies to manage agriculturally important plant pests." We also continued using this project as a platform for research experiences for undergraduate students from underrepresented minority (URM) groups, who may not be aware of career possibilities in agricultural sciences. UC Riverside is a leader in advancing involvement of URM students in STEM fields and student involvement in this project will synergize with a NIFA-funded educational grant to the primary investigator and other UCR Entomology Department faculty. 1. Major activities completed/experiments conducted Objective 1. Identify candidate effectors in saliva from B. tabaci with and without Rickettsia. TRANSCRIPTOMIC APPROACH: We previously validated a method for a hybrid single cell RNA sequencing approach that can be applied to individual whitefly salivary glands. This method was published in Gebiola et al. 2022, Insect Science, https://doi.org/10.1111/1744-7917.13008. Prior to the final project period we had dissected glands from individual insects to prepare for sequencing. However, during the final project period, we discovered an error in the library preparation of these samples. Following this quality check, new glands were dissected and sequencing libraries prepared. These libraries were validated to be high quality and suitable for sequencing, which is in progress. PROTEOMIC APPROACH: We continued to troubleshoot processing and recovery of proteins from saliva collections in consultation with a collaborator. This work will continue under alternative funding sources (Hatch funds). Objective 2. Identify candidate effectors that modify host responses to B. tabaci feeding. Work on this objective is ongoing pending the receipt of final RNA sequencing results from salivary glands. Effector identification will continue under alternative funding sources (Hatch funds, future competitive grants). Data collected (2) and Discussion of Results (3) We performed a factorial experiment exploring whitefly genotype (2 genotypes) x Rickettsia infection status (2 conditions). For each genotype x infection condition, recently emerged whiteflies were fed for 24 hours upon either a plant host (cowpea) which has defense responses, or a sachet of 10% sucrose, which lacks defense responses and represents the collection medium for harvesting saliva. After 24 hours, glands were dissected. Sufficient material was collected to have 10 replicates per genotype x bacteria x feeding treatment (80 samples total). An error occurred in library preparation of these samples, necessitating a repeat of the experiment. The experiment was repeated with new whiteflies from age standardized colonies as described above. Libraries were prepared again and staff were instructed in how to avoid any potential errors in the protocol. Libraries passed all quality control checks and are undergoing sequencing. Sequencing results will reveal the influence of Rickettsia infection status, genotype, and feeding substrate on gland gene expression, helping us identify effectors that are involved in different aspects of whitefly interactions with plants. This is the first time such an experiment has been possible, and it was made possible by our validation of single cell sequencing for gland gene expression studies. We attempted a few techniques for improving protein recovery from saliva samples. However, the results of these efforts were variable. We still have samples of collected saliva in long-term storage and plan to keep improving on options for protein recovery with our collaborator under other funding sources. As such, the resources collected during this grant period (saliva preserved in solution) can be leveraged for future funding opportunities. 4) Key outcomes/accomplishments - Presentation of project outputs at the 2022 Entomological Society of America Annual Meeting. - Successfully generated libraries for sequencing from an experiment examining the influence of whitefly genotype, Rickettsia infection status, and feeding substrate on gene expression in single salivary glands. - Refined methods for proteomic analysis of saliva from hemipteran insects
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Gebiola, M, Le, B, Mauck, KE. 2022. A transcriptomic look at the multifaceted interface between MEAM1 whiteflies, cucurbit yellow stunting disorder virus and melon plants. 2022 Annual Meeting of the Entomological Society of America, held jointly with the Entomological Society of Canada and Entomological Society of British Colombia, Vancouver, BC.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Gebiola, M, Le, B, Mauck, KE. 2022. A reproducible and sensitive method for generating high-quality transcriptomes from single salivary glands of whiteflies and psyllids. 2022 Annual Meeting of the Pacific Branch of the Entomological Society of America. Santa Rosa, CA.
|
Progress 04/01/21 to 03/31/22
Outputs
Target Audience:The target audience consists of researchers in the plant-insect interactions community, including molecular plant biologists, entomologists, and virologists studying plant viruses. During research activities, undergraduate researchers are also a target audience. Long-term, a target audience will be growers of specialty crops affected by whiteflies. The target audience was reached through publication of work in a peer reviewed journal, public deposition of raw data, and presentations at scientific conferences. Changes/Problems:Our main impediment has been difficulties with reproducibility of replicate proteomics runs on collected saliva samples using the UCR core facility. Qualitative differences suggest that we may be missing detection of proteins that are present. We believe we are on the right path with this now thanks to involvement of a new collaborator who has provided guidelines for different sample processing approaches and data analysis. Testing these approaches using more readily available saliva material from psyllids began in the project period covered by this report and will be completed in the final project period. This is likely to yield additional insights into psyllid salivary proteins and the influence of symbionts on saliva composition, while allowing us to proceed with processing our whitefly saliva collections. Beyond this, we experienced some slowdowns due to staff/students being out of the lab for extended periods of time with COVID-19 infections during the December-January omicron surge. To address this, we applied for and received a second no-cost extension to wrap up work by the end of 2022. What opportunities for training and professional development has the project provided?The project scientist received training in bioinformatic analysis (RNAseq and associated tasks) from the UCR Bioinformatics Core Facility. Undergraduates assisted throughout with collection of saliva from whiteflies and psyllids. They were trained in handling and care of insects and techniques for saliva collection. How have the results been disseminated to communities of interest?The project scientist working on this project registered to present results at the 2022 Pacific Branch of the Entomological Society of America meeting. Registration and abstract submission occurred during the project period of this report. Presentation delivery occurred a few days into the final project period. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will finish the RNAseq experiment to evaluate symbiont influence on whitefly salivary components under different host environments. These data will be used to identify transcripts that are differentially expressed by symbiont, host, and symbiont x host treatments (Objective 2). We will also publish a manuscript on whitefly genome mega-assemblies generated during the prior two project periods from accumulated transcriptomic data that is publicly available. We will refine these into a meta-assembly resource using established protocols. This will be made publicly available to the whitefly genomics community. For the proteomic work, we will use the extracted and collected psyllid protein material to evaluate sample processing/clean-up options to determine if they improve protein hits following LC-MS/MS. We anticipate at least some improvements. However, if proteomics cannot be improved by these efforts, we will rely primarily on RNAseq to identify candidate effectors, and supplement with the proteomic data available from the 30,000 whitefly collections.
Impacts
What was accomplished under these goals?
IMPACT STATEMENT: The goal of this project is to identify and characterize the molecules that enable an invasive pest (sweet potato whitefly) to exploit crop plants for its own survival. In doing so, this project directly addresses the goals of the USDA AFRI-NIFA funding programs to "elucidate fundamental molecular processes affecting plant-associated pests, including vectors of diseases and microbes." During the current project period, we continued efforts to scale up two approaches to discover molecules that whiteflies use to feed and develop on plants. First, we collected spit from over 30,000 whiteflies of two different symbiont infection statuses and performed analysis of the proteins (chains of molecules that cause changes when injected into the plant). Next, we dissected the organs from the insect that produce these molecules (salivary glands) and measured the levels of RNA in these tissues. RNA is the instructions for proteins, and the levels of RNA give us information about what is being produced in the insect. From these efforts, we have validated and published a new technique for studying salivary gland RNA expression. We then used this technique to test hypotheses about how microbes living within whiteflies affect their ability to feed on different crop plants and which molecules in the whitefly are most important for feeding. Our results will help us to develop new biopesticides that target production of these molecules in the whitefly itself. Through these impacts, the project fulfills the need for AFRI-supported research to "lead to the development of innovative and environmentally sound strategies to manage agriculturally important plant pests." We also continued using this project as a platform for research experiences for undergraduate students from underrepresented minority (URM) groups, who may not be aware of career possibilities in agricultural sciences. UC Riverside is a leader in advancing involvement of URM students in STEM fields and student involvement in this project will synergize with a NIFA-funded educational grant to the primary investigator and other UCR Entomology Department faculty. 1. Major activities completed/experiments conducted Objective 1.TRANSCRIPTOMIC APPROACH: In the prior project period, we tested a procedure for performing transcriptomics (RNAseq) on single salivary glands using specialized reagents for low input/single cell RNA samples. Based on the success of this pilot, we performed an experiment to compare RNAseq of single salivary glands and other body sections. The main goal was to test reproducibility of a "hybrid" single cell approach to get RNAseq data from minimal tissue and determinethe pitfalls and benefits of this approach relative to pooling of hundreds of glands per sample. During the project period for this report, we analyzed data from this experiment and published the results (see products: Gebiola et al. 2022, Insect Science, https://doi.org/10.1111/1744-7917.13008). Building on this approach, we have now dissected individual glands from age-standardized cohorts of whiteflies with and without Rickettsia infection. This includes two whitefly genotypes, each possessingRickettsia, or being Rickettsia-free. Each whitefly genotype x Rickettsia treatment is split between those fed upon a host (cowpea) or 10% sucrose (the medium for saliva collection to use in proteomic analysis). Within each whitefly x bacteria x feeding treatment, we have at least 10 replicates. PROTEOMIC APPROACH: We continued collecting saliva from whiteflies with and without Rickettsia infection. This activity involved research experiences for undergraduates. However, our core proteomics facility shut down during the project period. We established a collaboration with a proteomics expert (Dr. Michelle Heck, USDA-ARS) who will assist in the final steps of processing and running our samples. Prior to the UCR proteomics facility closure, we continued to experience large variability in outputs from sample runs that should have been quite similar. This is likely to introduce artefacts, so we will be proceeding with new sample processing suggestions provided by Dr. Heck. Objective 2. Identify candidate effectors that modify host responses to B. tabaci feeding. This objective will be completed during the final project period (2022) as we sequence and analyze whitefly genotype x infection x feeding treatment experiments. 2) Data Collected and 3) discussion of results Objective 1: TRANSCRIPTOMIC APPROACH: We worked with the publicly available, partially annotated whitefly genome, plus data from another paper using pooled salivary gland collections for transcriptomics. Cleaned libraries were mapped against the MEAM1 reference genome.Heatmaps and scatterplots were obtained to check for reproducibility and previously published libraries from a pool of 400 salivary glands and a pool of 100 whole whitefly bodies. Reproducibility was high across samples. Differential expression analysisrevealed significant differences by tissue type. By comparing our outputs to published bulk RNA-Seq datasets for whole whitefly bodies and pools of salivary glands, we demonstrate that this approach recovers similar numbers of transcripts relative to bulk RNA-Seq in a tissue-specific manner, and for some metrics, exceeds performance of bulk tissue RNA-Seq. Libraries generated from individual salivary glands also yielded additional novel transcripts not identified in pooled salivary gland datasets, and had hundreds of enriched transcripts when compared with whole head tissues. Overall, our study demonstrates that it is feasible to produce high quality, replicated transcriptomes of whitefly salivary glands and other low-input tissues. With the technique validated, we proceeded with a factorial experiment exploring whitefly genotype (2 genotypes) x Rickettsia infection status (2 conditions). For each genotype x infection condition, recently emerged whiteflies were fed for 24 hours upon either a plant host (cowpea) which has defense responses, or a sachet of 10% sucrose, which lacks defense responses and represents the collection medium for harvesting saliva. After 24 hours, glands were dissected. Sufficient material was collected to have 10 replicates per genotype x bacteria x feeding treatment (80 samples total). During the remaining project period, these samples will be prepared for RNAseq as described previously (Gebiola et al. 2022). PROTEOMIC APPROACH: With the assistance of Dr. Heck, we evaluated samples (saliva collections from 10k whiteflies) run in the UCR core facility for repeatability and quality. We detected large variability in peptide detection between samples that should have beensimilar, suggesting that sample processing prior to mass spec analysis is an issue. To troubleshoot this prior to processing our large-scale collections, we are using another hemipteran (psyllids) which produce much larger volumes of saliva per insect (fewer collections are needed to get sufficient saliva for testing methods). We have also extracted protein from whole bodies. During the final project period, we will use this protein to test recovery methods (by spiking into saliva collection fluid, then processing), then use psyllid saliva collections to validate the best approach. After doing so, we will process and run our 30k whitefly collections. 4) Key Outcomes/Accomplishments - Publication of a new method for performing transcriptomic analyses on low-volume whitefly tissues. - New genomic resources for the whitefly research community to improve future transcriptomic experiments. - Factorial experiment examining the influence of whitefly genotype, Rickettsia infection status, and feeding substrate on gene expression in salivary glands. - Troubleshooting proteomic analysis of saliva from Hemipteran insects.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Gebiola, M., Le, B. H., & Mauck, K. E. (2022). A reproducible and sensitive method for generating high?quality transcriptomes from single whitefly salivary glands and other low?input tissues. Insect Science. https://doi.org/10.1111/1744-7917.13008
|
Progress 04/01/20 to 03/31/21
Outputs
Target Audience:The target audience consists of researchers in the plant-insect interactions community, including molecular plant biologists, entomologists, and virologists studying plant viruses. During research activities, undergraduate researchers are also a target audience. Long-term, a target audience will be growers of specialty crops affected by whiteflies. Due to the ongoing pandemic of SARS-CoV-2, we were unable to connect with most target audiences to the extent planned. ? Changes/Problems:The major impediment has been the SARS-CoV-2 pandemic, which has resulted in university mandated laboratory shutdowns (March, April, May, June of 2020), followed by 25% occupancy restrictions (July 2020 to April 2021), which recently transitioned to 50% occupancy permitted (May) followed by full occupancy on June 24th, 2021. During the four months of shutdown, we halted ongoing experiments for this project, and were not able to start new experiments. We were able to maintain the whitefly colonies to continue experiments when the laboratory reopened for partial occupancy. Our work relies on core facility operations, which were also severely curtailed until about September 2020, so even though work in the lab could resume, we were not able to process samples in a timely manner. The overall impact of the pandemic restrictions has been inability to complete Objective 1 and move on to Objective 2, as well as reduced opportunities for education of undergraduates as a component of the project. However, refocusing efforts to more computational work (compatible with remote work requirements) has led to new products/outputs that we anticipate will be very useful for our continued work and the work of other researchers. Even though these are not anticipated products, they are closely aligned with the main goals of the project as a seed grant, will advance the long-term goals of the project, and are consistent with the same program area priorities. In addition to pandemic-related delays, we have as yet unresolved issues with proteomics of whitefly saliva, which mirror those we have encountered with saliva from potato psyllids and asian citrus psyllids (related organisms). Using these organisms as proxies, we are actively troubleshooting these issues, and will consult with additional colleagues about possible solutions. Based on these activities, we anticipate producing a methods paper to provide specific guidelines on best practices for saliva collection - a technique that has thus far been performed in haphazard manners across publications and study systems. What opportunities for training and professional development has the project provided?Opportunities for professional development were curtailed due to the SARS-CoV-2 pandemic. In-person meetings were not possible, laboratory interactions were reduced due to occupancy limits, and undergraduate researchers were not permitted in labs. However, the project scientist working on this project used the remote work requirement as an opportunity to drastically increase his expertise in bioinformatics, which will be of tremendous value in future project efforts and has led to two additional methods manuscripts currently in preparation. How have the results been disseminated to communities of interest?A talk on the project for the International Congress of Entomology (2020) was canceled due to the meeting being postponed. This will occur when the meeting is held (likely 2022). The project was discussed as a component of a talk on whiteflies delivered by the PI as part of a symposium on whitefly management organized for the Entomological Society of America annual meeting (November 2020, virtual) and as part of a talk delivered to stakeholder representatives at the CA Specialty Crops Technical Council August 2020 meeting (virtual). Undergraduate researchers were not permitted in laboratories from March 2020 through April 2021, therefore we were not able to directly engage with this target audience during the project period. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will publish a manuscript on the single cell salivary gland RNAseq approach and use this approach to perform an RNAseq experiment to evaluate symbiont influence on whitefly salivary components under different host environments. These data will be used to identify transcripts that are differentially expressed by symbiont, host, and symbiont x host treatments (for pursuit in Objective 2). We will also publish a manuscript on the mega-assemblies, which we will further refine into a meta-assembly resource using established protocols. This will be made publicly available to the whitefly genomics community. For the proteomic work, we will use factorial experiments to evaluate sample processing/clean-up options to determine if they improve protein hits following tryptic digest and data collection (LC-MS/MS) and evaluate strategies to inhibit protease activity in salivary collection devices. If proteomics cannot be improved by these efforts, we will rely primarily on RNAseq to identify candidate effectors, and supplement with the proteomic data available from the 30,000 whitefly collections. Objective 2. Candidate effectors identified from RNAseq or proteomic approaches will be used as the basis for construction of dsRNAs targeting relevant mRNA molecules, to be delivered along with dsRNAs inhibiting gut nucleases. We will evaluate the impact of dsRNA uptake on whitefly survival and ability to feed on hosts following dosing and quantify gene knockdown using quantitative RT-PCR.
Impacts
What was accomplished under these goals?
IMPACT STATEMENT: The goal of this project is to identify and characterize the molecules that enable an invasive pest (sweet potato whitefly) to exploit crop plants for its own survival. In doing so, this project directly addresses the goals of the USDA AFRI-NIFA funding programs to "elucidate fundamental molecular processes affecting plant-associated pests, including vectors of diseases and microbes." During the current project period, we continued efforts to scale up two approaches to discover molecules that whiteflies use to feed and develop on plants. First, we collected spit from thousands of whiteflies, and performed analysis of the proteins (chains of molecules that cause changes when injected into the plant). Next, we dissected the organs from the insect that produce these molecules, and measured the levels of RNA in these tissues. RNA is the instructions for proteins, and the levels of RNA give us information about what is being produced in the insect. From these efforts, we have validated a new technique for studying salivary gland RNA expression, created tools to improve the ability for other researchers to use whitefly genetic resources for many applications, and made progress on improving protein isolation and analysis from insect saliva. In the next project period, we will use this information to test hypotheses about how microbes living within whiteflies affect their ability to feed on different crop plants and which molecules in the whitefly are most important for feeding. Then, we can develop new biopesticides that target production of these molecules in the whitefly itself. Through these impacts, the project fulfills the need for AFRI-supported research to "lead to the development of innovative and environmentally sound strategies to manage agriculturally important plant pests." During the prior project period, pandemic restrictions excluded undergraduates from in-person research. However, now that undergraduates are again permitted in the laboratory, we will continue using this project as a platform for research experiences for undergraduate students from underrepresented minority (URM) groups, who may not be aware of career possibilities in agricultural sciences. UC Riverside is a leader in advancing involvement of URM students in STEM fields and student involvement in this project will synergize with a NIFA-funded educational grant to the primary investigator and other UCR Entomology Department faculty. 1. Major activities completed/experiments conducted Objective 1. Identify candidate effectors in saliva from B. tabaci with and without Rickettsia. TRANSCRIPTOMIC APPROACH: In the prior project period, we tested a procedure for performing transcriptomics (RNAseq) on single salivary glands using specialized reagents for low-input/single cell RNA samples. Based on the success of this pilot, we performed an experiment to compare RNAseq of single salivary glands and other body sections, and included samples from whiteflies feeding on multiple hosts. The goals were to 1. Test reproducibility of a "hybrid" single cell approach to get RNAseq data from minimal tissue (a single salivary gland, a single head, 10 heads), and 2. Test differential expression as related to feeding on different plant hosts (rearing host, susceptible melon variety, putatively resistant melon variety). The ages and feeding periods on different hosts were standardized for cohorts of whiteflies serving as a source of tissues. From each treatment, the following tissues were extracted from whiteflies: Salivary glands from a single specimen (N=3 per host), one whitefly head (N=3 per host), and a pool of 10 whitefly heads (N=3 pools per host). RNA was extracted from single glands, single heads and pools of 10 heads using the NEB low input RNA library prep kit. Libraries were prepared according to the manufacturer's protocol and checked on a BioAnalyzer before equimolar normalization and sequencing on 3 lanes of HiSeq2000 with PE 150 bp setting at UC Davis. In analyzing these samples (see data collection/results section), the poor annotation quality of the whitefly genomic resources became apparent (22% of the genome is uncharacterized and many of these unknowns are expressed in our data sets). To resolve this, we compared a reference genome-based approach with a de-novo assembly (from EST data) approach. We built two 'mega-assemblies': one based on paired end (PE) reads datasets and one based on both paired end and single end (SE) reads datasets. The PE+SE assembly is based on a separate in silico normalization procedure that effectively transforms PE reads into SE reads. Therefore using a PE+SE approach would be recommendable only if the PE+SE de-novo assembly was by far qualitatively better than the PE only assembly. PE and PE+SE assemblies were qualitatively checked for use as references in future mapping efforts. PROTEOMIC APPROACH: Based on pilot activities performing proteomic analysis on saliva collected from 5,000 whiteflies, we scaled up saliva collection to produce samples of symbiont + and symbiont - from at least 10,000 whiteflies per treatment, and following analysis of these data, to 30,000 whiteflies from each treatment. Objective 2. Identify candidate effectors that modify host responses to B. tabaci feeding. Activities in support of this objective have not yet begun. 2) Data Collected and 3) discussion of results Objective 1: TRANSCRIPTOMIC APPROACH: We worked with the publicly available, partially annotated whitefly genome, plus data from another paper using pooled salivary gland collections for transcriptomics. Cleaned libraries were mapped against the MEAM1 reference genome using STAR. Mapping rates were very high across samples. Heatmaps and scatterplots were obtained to check for reproducibility and previously published libraries from a pool of 400 salivary glands and a pool of 100 adults, not replicated, were included for comparison. Reproducibility was high across samples. Differential expression analysis with the EdgeR program revealed significant differences by tissue type across all hosts. These results suggest that our single cell approach can be used to analyze RNA expression in salivary glands. Our de-novo reference assembly efforts also suggest that the new resources will result in greater mapping rates, ensuring that relevant transcripts are not missed due to poor reference genome resources for whiteflies. PROTEOMIC APPROACH: We did not obtain much improvement in protein matches due to our scale up. Based on this, we have now collected saliva from at least 30,000 whiteflies from each symbiont status and stored future analysis. In the interim, to improve the proteomic side, we used psyllids (a related insect in the Hemiptera) as a proxy for whiteflies because they produce more saliva and collections are quicker (just a few thousand to produce enough protein for multiple runs). We detected persistent issues with obtaining high quality proteomic analyses despite having sufficient material input and high quality LC-MS/MS runs producing many fragments. We are currently testing sample cleanup approaches and exploring whether protease activity during saliva collection is interfering with controlled protein fragmentation during preparation for LC-MS/MS runs. These activities will provide useful information for improving saliva collection based proteomics for whiteflies and psyllids, both of which are serious crop pests. ? 4) Key Outcomes/Accomplishments - New method for analyzing gene expression in salivary glands that requires less tissue and is amenable to more complex factorial designs due to reduced limitations on tissue amounts (manuscript in preparation). - New genomic resources for the whitefly research community to improve future transcriptomic experiments (manuscript in preparation). - Identification and troubleshooting progress on key issues impeding analysis of saliva from Hemipteran insects.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Mauck, K., Kenney, J.R., Sun, P., Chesnais, Q., Gebiola, M. November 2020. Managing whitefly-transmitted viruses when inoculation is guaranteed: lessons from southwestern agroecosystems. Presented in the member symposium "Ecology, Epidemiology, and Management of Whiteflies and Whitefly-transmitted Viruses in the United States" during the virtual meeting of the Entomological Society of America.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Kerry Mauck. August 2020. Ecology of plant pathogens and their vectors in managed ecosystems. Presented to the CA Specialty Crops Technical Council.
|
Progress 04/01/19 to 03/31/20
Outputs
Target Audience:In the near term, researchers in the plant-insect interactions community, including molecular plant biologists, entomologists, and virologists studying plant viruses. Longer-term, research will benefit growers of specialty crops affected by whiteflies if results are useful for breeding more resistant crops or developing RNA interference-based biopesticides. Throughout the project, undergraduate researchers will also be a target audience as we provide laboratory research experiences on whitefly biology. Changes/Problems:We spent some time improving saliva collection methods and testing out some new techniques in the beginning. Some of this took longer than expected (e.g., waiting on sequencing facilities, proteomics runs, and testing new saliva chambers involved working with undergraduate mentees, so this involved education components that added time). However, the time invested yielded useful improvements and we will likely be able to publish some methods papers describing these approaches. The major impediment has been the SARS-CoV-2 pandemic, which has resulted in university mandated laboratory shutdowns since March 16, 2020. We halted ongoing experiments for this project, and were not able to start new experiments. We were able to maintain the whitefly colonies to continue experiments when the laboratory reopens. We are now operating in Phase II as of this report, which allows some research to resume as lab personnel work in individual shifts (one person to the lab at a time). Given the ongoing public health challenges in the area (Riverside County, CA) we expect this may be temporary, and another shutdown is likely. Progress on Objective 2 may be substantially hindered by the nearly 4 months we have lost thus far and limited research activities for the foreseeable future this year. If this continues, we will focus on ensuring that all anticipated outputs of Objective 1 are produced, as this objective is the most amenable to the limited work schedules and remote work options. For example, saliva collections can be set up in short periods of time and then left unattended for the duration of collection, then harvested and stored at ultralow temps for later processing. And data analysis work can be completed remotely using access to cloud-based computing. Given the challenges of limited in-person work, we will identify opportunities to adapt the project as needed to achieve some or all of the stated goals. What opportunities for training and professional development has the project provided?Saliva collection optimization was performed by an undergraduate student in collaboration with a project scientist and the PI. The student assisted with design and testing of novel collection chambers that allowed aspiration of whiteflies directly into the chamber to which collection membranes are already affixed. The student received training in the methods associated with this project and the downstream applications of the work and we were able to improve whitefly survival during saliva collections. A second student received training in whitefly biology, rearing, and saliva collection methods later in the project, but her involvement was cut short by the SARS-CoV-2 pandemic and associated laboratory shutdown. ? How have the results been disseminated to communities of interest?A presentation for the International Congress of Entomology was planned for July 2020, but this was postponed to July 2021 due to the SARS-CoV-2 pandemic. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will complete saliva collections from Rickettsia +/- whiteflies and perform qualitative and quantitative proteomics on these samples using TMT mass tagging. Glands will be dissected from standardized-age individuals (female and male) and mRNA will be sequenced using the single cell library prep method described in prior sections. Both data sets will be subjected to bioinformatic analyses. We will work with the director of the UCR Proteomics Core facility for protein identification. RNAseq outputs will be quality checked and analyzed using a Galaxy-based workflow (described in prior sections). Effector discovery will be performed using annotated genome resources for Bemisia tabaci and its symbionts. We will also use both RNAseq and proteomics data to measure the relative expression of putative effectors identified in recent publications on B. tabaci MED salivary proteins and compare this between Rickettsia positive and Rickettsia negative whiteflies. Expression/quantitative proteomics data will be combined with qualitative information (e.g., presence/absence) to select putative effectors for subsequent characterization. ?Objective 2. A subset of putative effectors will be knocked down in whiteflies using artificial membrane feeding and delivery of complementary dsRNAs that will trigger silencing of the target gene. To improve RNAi we will incorporate dsRNAs that also induce RNAi of gut nucleases. Whitefly feeding and mortality will then be monitored following an acquisition access period, first by honeydew deposition (for rapid phenotyping) and then by electrical penetration graphing analysis for a subset of candidates showing robust phenotypes. Gene knockdown will be quantified using RT-qPCR. These data will be used to generate 1-2 publications and serve as the basis for a standard AFRI foundational grant. Undergraduate researchers will be involved in performing dsRNA screening with whiteflies.
Impacts
What was accomplished under these goals?
Impact statement The goal of this project is to identify and characterize the molecules that enable an invasive pest (sweet potato whitefly) to exploit crop plants for its own survival. In doing so, this project directly addresses the goals of the USDA AFRI-NIFA funding programs to "elucidate fundamental molecular processes affecting plant-associated pests, including vectors of diseases and microbes." During the current project period, we used two approaches to discover molecules that whiteflies use to feed and develop on plants. First, we collected spit from thousands of whiteflies, and performed analysis of the proteins (chains of molecules that cause changes when injected into the plant). Next, we dissected the organs from the insect that produce these molecules, and measured the levels of RNA in these tissues. RNA is the instructions for proteins, and the levels of RNA give us information about what is being produced in the insect. From these efforts, we have generated a list of molecules that are important for whitefly feeding and compared the levels of some of these molecules when the insects are feeding on whitefly-resistant or whitefly-susceptible crop plants. In the next project period, we will use this information to more carefully quantify molecule production in the whiteflies and also determine whether the molecules are produced by the whitefly or the microbes that live within it. Once we are sure which molecules are most important for whitefly feeding, we can develop new biopesticides that target production of these molecules in the whitefly itself. Through these impacts, the project fulfills the need for AFRI-supported research to "lead to the development of innovative and environmentally sound strategies to manage agriculturally important plant pests." The project is also being used as a platform for research experiences for undergraduate students from underrepresented minority (URM) groups, who may not be aware of career possibilities in agricultural sciences. UC Riverside is a leader in advancing involvement of URM students in STEM fields. As the project continues it will support student researchers participating in UCR's support programs targeted at recruiting and retaining URM students in STEM, including through synergism with a NIFA-funded educational grant to the primary investigator and other UCR Entomology Department faculty. ?1) Major activities completed / experiments conducted; Objective 1. Identify candidate effectors in saliva from B. tabaci with and without Rickettsia. Activities: First, we optimized methods for collecting saliva from whiteflies to make this more high throughput. Second, we tested two salivation-enhancing compounds (pilocarpine and resorcinol) used in prior studies to enhance salivation in other Hemipteran insects. Third, we performed a proteomic study of saliva collected from different numbers of whiteflies and pilot-tested a labeling method that would allow multiplexing of samples in the same run. This is important for distinguishing quantitative vs. qualitative effects of symbionts on whitefly saliva composition. Fourth, we performed a preliminary analysis of whitefly saliva gland transcriptomes using a novel method for isolation and RNA sequencing from single whitefly salivary glands. Most studies with hemipterans pool whole heads to try to obtain salivary transcriptomes and one has pooled actual salivary glands. The dangers of introducing artefacts during sequential dissection and pooling of glands presents challenges. We took a different approach and used a library preparation kit intended for single-cell and low-RNA-input samples to characterize the transcriptomes of whitefly salivary glands. As a test of this approach to ensure it would be robust for detecting treatment differences due to Rickettsia presence/absence, we fed age-standardized whiteflies on resistant and susceptible host plants for 24 hours prior to dissection of glands and preparation of libraries. Libraries were sequenced as 100bp single end reads using Illumina HiSeq. Objective 2. Identify candidate effectors that modify host responses to B. tabaci feeding. Activities in support of this objective have not yet begun. 2) Data Collected and 3) discussion of results Objective 1. Effects of salivation inducers on whiteflies were mixed. Resorcinol proved to have negative effects on whiteflies, so further work with this compound was not pursued. Proteomic surveys of saliva collected from whiteflies with and without pilocarpine did not indicate a positive effect on saliva quantity. We have decided to simply focus on collecting sequentially from more whiteflies to obtain greater quantities of saliva for each proteomics study involving whitefly lines with and without Rickettsia symbionts. Proteomic studies with different quantities of whiteflies (2000 vs. 4000) suggest that at least 4000 whiteflies are required for robust proteomics. Based on this, we have revised our total estimate of the number of whiteflies needed to 10,000 per treatment to be safe, and we will attempt to generate several biological replicates for proteomics using this target number. Even with lower number of whiteflies, however, we detected over 100 proteins with hits to the Bemisia tabaci protein database. Saliva collection from whiteflies with and without the Rickettsia symbiont was ongoing until the laboratory was shut down due to the SARS-CoV-2 pandemic in early March. The single-cell/low-input RNA library preparations passed all quality checks and libraries were successfully sequenced. Data underwent standard quality control checks for an RNAseq workflow. Some checks indicate that mapping to the reference genome was not ideal, however, based on other studies with whiteflies, this appears to be an issue with the annotation of the genome. Still, we will explore ways to optimize library preparation and sequencing. Although salivary glands are about 8 cells, (16 cell input per individual whitefly), we think this should be equivalent enough RNA input to be treated similarly to single cell approaches. We compared outputs of our experiment with those of a prior study using pooled whitefly salivary glands (about 160 glands) and found good overlap in gene ontology (GO) IDs within pathway categories (26/38 IDs in "molecular function" category, 40/60 IDs in the "Biological Process" category). We detected comparable numbers of unique transcripts relative to this study as well (15,633 vs. 13,615 in the prior study) with the advantage that our study mapped to an annotated genome, and the other study mapped to a whole-body transcriptome (EST database). We also detected about one third of "housekeeping genes" commonly expressed by hemipterans in saliva (similar to other papers using pooling of glands). In terms of treatment effects, we found 90 differentially expressed genes (DEGs) between whiteflies feeding on cowpea (rearing host) and whiteflies feeding on susceptible melon, 22 DEGs between whiteflies feeding on susceptible melon and resistant wild melon, and 25 DEGs between whiteflies feeding on cowpea and resistant wild melon. Treatment effects suggest that the method of using a single cell sequencing library prep kit is suitable for detecting subtle differences in gene expression in whitefly salivary glands. 4) Key Outcomes/Accomplishments - Higher throughput saliva collection methods and equipment to streamline future work. - Undergraduate training in hands-on research with insects for two students. - Effects of variation in whitefly numbers and salivation stimulants on saliva output and proteomic composition. Preliminary proteomic analysis and collection of saliva from Rickettsia +/- whiteflies for future proteomic profiling. - Improved method for salivary gland transcriptome sequencing using single insects, partially validated against previous data using pooled salivary glands. Objective 2. These activities will take place during the next project period.
Publications
|
|