Progress 02/15/17 to 09/30/21
Outputs
Target Audience:The target audience for our work on the biology of C. elegans is the cell and developmental biology scientific community. The other target audiences of our work are researchers that are using C. elegans or other nematodes for applied biology and researchers working on plants and plant parasitic nematodes. During the term of this project, I and my collaborator interacted with scientists from industry by attending Seed Central events. Seed Central is a public-private partnership that hosts monthly meetings that typically feature a networking session and a seminar. These events bring together academics with local members of the seed industry and the broader agricultural biotechnology community. During the networking portion of these events, we are able to inform these community members of the work we are doing on gene expression in plants and nematodes. Through these interactions I established a relationship with Dr. F. Kaplan (CEO/CSO) and Mr. K. Schiller (COO) from the company Pheronym. Pheronym develops biologics for the control of nematodes that are agricultural pests. During the reporting period, I also interacted with Dr. A. Calderon-Urrea and his lab members (CSU Fresno). Dr. Calderon-Urrea studies plant parasitic nematodes and uses C. elegans to identify anti-nematode reagents and their mode of action; he in turn interacts directly with stakeholders such as biotechnology companies interested in his anti-nematode studies. Changes/Problems:Work on Objective 1, studying Meloidogyne development, was initiated in collaboration with the lab of Dr. A. Calderon-Urrea, CSU Fresno. While he had some undergraduates work on the project initially, the methods are technically challenging for students with limited time in the lab, and little progress was made. Dr. Calderon-Urrea did not have funding for a Masters student who could have devoted more time to the project. Similarly, although my Agricultural Experiment Station appointment generously funds part of my summer salary, it does not provide funds for other researchers or supplies, and my NIH funding is dedicated to the research in Objective 2. Thus, without personnel, we were unable to continue work on Objective 1, but made substantial progress on Objective 2. For Objective 3, progress has been relatively good because the work could be done by ourselves, undergraduates and Masters students (no personnel funds required) and supply funds from the A. Rose lab. We have additional experiments needed to publish this work and so they were proposed for the renewal project. What opportunities for training and professional development has the project provided?During the five-year project period, five Ph.D., two Masters students, and four research technicians worked on this project. Four of the students completed their Dissertations/Theses during the period and three technicians completed their two-year training as Jr. Specialists; these members pursued academic careers or now work in the biotechnology industry. All lab members received technical training from myself, Dr. A Rose and each other (with more senior students training new students). In addition, I aided in all personnel's professional development by mentoring them in the presentation of their results both in oral and written format. Graduate students and technicians were able to attend and present at various meetings (see the dissemination section below). The PhD level graduate students also attended UC Davis graduate program activities such as the annual retreat and the BMCDB Career and Professional Development Seminars. In addition, one Jr. Specialist was a post- baccalaureate student in the UCD PREP program, which helps disadvantaged students gain additional research experience and skills to help them pursue higher degrees. In addition to my mentoring, this student had access to additional training from the PREP program faculty for poster presentations, oral presentations and in applying for fellowships. The full-time lab members and I also used our expertise to train new undergraduates in the lab to help with the research projects. This increased the skill base of all the students and for the undergraduates, helped them obtain jobs upon graduation or pursue higher degrees. Finally, I also trained undergraduates from Dr. Calderon-Urrea's lab to carry out the antibody staining technique (Objective 1), and I provided genetic advice to his Masters student for her project using C. elegans to study anti-nematode compounds. How have the results been disseminated to communities of interest?I and my collaborator Dr. A. Rose, as well as Ph. D. students and laboratory technicians have presented our findings to the scientific community through several venues during the project period. Lab members presented posters at the following international meetings: the Annual Meeting for the Society for Cell Biology, the International C. elegans Meeting, the Society for Developmental Biology Annual Meeting, and the Santa Cruz Developmental Biology Meeting (~ one meeting per year per student). Dr. Alan Rose gave seminars at the NAIST Institute in Japan, and the UC Davis Plant Science Symposium, while I presented seminars at the NAIST Institute, Ohio State University, Washington State University and UC Davis. At the regional and campus level, lab members presented talks or posters at the Bay Area Worm Meeting, the UC Davis Cell Biology Research Meeting, the UC Davis MCB Training Grant Retreat, and at graduate program retreats (annual presentations for most lab members). Undergraduate students in the lab also presented their work at the UC Davis Undergraduate Research, Scholarship & Creative Activities Conference, whose audience included students outside of biology as well as family members. In addition during the first part of the project lab members presented at the UC Davis Worm Group Research Meeting. This meeting provided us with a venue to disseminate information about new techniques to researchers working in applied areas of nematode biology, and the forum provided those researchers with feedback on their experiments from myself and other C. elegans researchers. I also visited CSU Fresno to discuss our research on C. elegans with Dr. Calderon and explore how our expertise could benefit his projects. Since that meeting, I have provided feedback on C. elegans techniques for two of his grant proposals. We have also communicated our results via interactions with industry scientists at Seed Central Events. Events are scheduled monthly during the academic year and are attended by a variety of other academics, ag-business scientists, consultants and even local farmers. Dr. A. Rose is a standing member of Seed Central, and I attended many of these events from 2016-2020 (networking sessions were suspended due to Covid19 in 2020). During the networking portion of these events, we informed participants of the work we are doing on gene expression in plants and nematodes. My main discussions have been with research and business personnel from the company Pheronym, Dr. F. Kaplan (CEO/CSO) and Mr. K. Schiller (COO). Pheronym produces pheromone compounds that are being tested to enhance the efficacy of beneficial nematodes; other compounds could prevent infection by plant parasitic nematodes. By establishing this relationship, I was able to connect Dr. Kaplan with the regional C. elegans community and she presented a poster on their work at the April 2019 Bay Area Worm Meeting. (The 2020 and 2021 meetings were cancelled due to Covid19.) I have continued to have discussions with Dr. Kaplan since that time, to explore how Rose Lab knowledge and methods can aid Pheronym's projects. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Nematodes (roundworms) can be parasites of animals and plants and are a major source of agricultural damage. For example, in California, root-knot nematodes infect the roots of various plants including tomatoes and almonds, causing poor growth and reducing crop yield. Progress has been made in developing pesticides and plant varieties that cannot be infected. However, some pesticides are harmful to health and the environment, and nematodes can evolve the ability to infect resistant plants or survive treatments. Thus, improved methods for the control of parasitic nematodes are needed, which requires a better understanding of how nematodes grow and reproduce. Our research is aimed at expanding this knowledge and supports the basic biology mission of the Agricultural Experiment Station. One goal of our work is to understand a process called asymmetric division, which is essential for nematodes to develop from the egg into the functional reproducing organism. We also study how the amount of a gene product can be increased, which is useful for studying and modifying gene function in animals and plants. We use the model C. elegans, a free-living nematode which is easy to grow and is highly amenable to genetic and molecular analyses. The early development of C. elegans is similar to that of parasitic nematodes, and work in the C. elegans field has paved the way for technologies that are now being used on harmful species. In the past five years, our published findings on asymmetric cell division have resulted in changes in knowledge for the scientific community. This knowledge can be used by other researchers to design treatments that block growth in parasitic nematodes of agricultural importance. The specific objectives of the project and our accomplishments are outlined below. Objective 1: Determine if PAR polarity proteins and other asymmetric division proteins are required for development in Meloidogyne species. Objective 2: Further elucidate molecular mechanisms of spindle positioning proteins in C. elegans Objectives 1 and 2 were aimed at elucidating fundamental mechanisms by which nematodes develop from the fertilized egg/embryo into an adult. Critical to development is the process of asymmetric division, whereby one cell divides into two cells with different identities and functions. Genes required for asymmetric division have been identified and studied in C. elegans. These genes are present in most nematodes, and spindle alignment is a common feature of early nematode development. For an asymmetric division to occur, a cell becomes polarized such that molecules important for controlling cell identity are more abundant at one end. The cell must then divide in the proper plane so that the molecules are inherited unequally. In animals, the division plane is set by the position of a structure called the mitotic spindle. My lab identified the LET-99 protein as a key regulator of spindle positioning during the first division of the C. elegans embryo. LET-99 is localized in a polarized manner at the cell membrane in response to polarity-determining proteins called PAR proteins. LET-99 in turn regulates the localization of a force generating complex that moves the spindle into position. LET-99 is also needed redundantly for cytokinetic membrane furrowing, the final event in division that physically separates the cell into two. For Objective 1, we collaborated with Dr. Calderon-Urrea, CSU Fresno, to apply our knowledge to his work on embryo development in plant-parasitic nematodes. The sub-objectives were to (a) determine localization patterns of PAR proteins, and (b) to use RNA interference to inhibit the par genes and infer function. Based on sequence analysis, I advised Dr. Calderon-Urrea's lab on the best anti-PAR antibodies to use in Meloidogyne and trained his students. This project was discontinued but I have provided technical advice on other projects (see changes/problems and results dissemination sections) For Objective 2, sub-objective (a) was to further elucidate mechanisms for LET-99 localization and determine if LET-99 acts in different pathways for cytokinesis and spindle positioning. We also studied LET-99 in a later division, for which much less is known about spindle alignment. Our approach used a combination of genetics, mutant analysis, and quantitative examination of fluorescently tagged proteins. We found that LET-99 localization at cytokinesis is determined by the spindle, and not the PAR proteins, and that the force-generating complex for spindle positioning is not involved in the actual cytokinesis process (Price and Rose, 2017). In subsequent work, we have identified two new proteins that LET-99 works with to promote the normal organization of the actomyosin filament system needed for cytokinesis and overall cell shape (in preparation). We also determined that LET-99 and one component of the force generating machinery are required for spindle alignment in a specific cell at the four-cell stage, which was known to divide in response to cell signaling events (Liro and Rose, 2016). We also characterized a role for PAR proteins, which was significant because the PAR proteins were not thought to act in this division in concert with cell signaling (Liro et al., 2018). An open question in the asymmetric division field is how PAR domains reform during successive divisions when the initial cues and cellular contexts are changing. Thus, sub-objective (b) was to characterize genes required to reestablish PAR polarity for the second asymmetric division in C. elegans. Again, using a combination of genetics and microscopy, we characterized two candidate genes in this process. The results lead to a novel model in which a daughter cell inherits an asymmetric cue from the first division, a mechanism distinct from that used in the first division. We will test this model in the renewal project. Objective 3: Distinguish between different mechanisms by which introns boost expression. The use of engineered genes introduced back into organisms, called transgenes, is a powerful method to examine protein localization and activity that is starting to be used in nematodes of agricultural importance. Parasitic nematode genes can also be expressed in C. elegans to more easily and quickly study their functions, where methods are much better established. An increased understanding of gene regulation and identification of ways of boosting transgene expression will have many benefits. Our studies on gene expression in C. elegans are in collaboration with Dr. Alan Rose, an expert on intron mediated enhancement in plants. In plants, introns have been shown to boost gene expression substantially, but only when located near the start of the gene and only for a small subset of introns. These introns increase mRNA levels but do not contain traditional transcriptional enhancers; the effect is called intron-mediated-enhancement. Prior work revealed an enhancement for a few introns in C. elegans transgenes, and most transgenes include at least one small synthetic intron; however, systematic studies of the parameters required for enhancement are lacking. We found that a known C. elegans boosting intron had a positional effect similar to that seen in plants. During this project period, we tested additonal introns from genes with varying expression levels. Unlike the variable effect of different plant introns, we found that all 11 nematode introns boosted expression to the same degree, as did the commonly used synthetic intron. This suggests that the mechnism through which introns increase expression is different in plants and nematodes, even though intron location is important in both. The position effect is an important change in knowledge, which we have shared with other researchers from academia and biotechnology companies. We plan to write a manuscript after additional analyses are completed as part of the renewal project.
Publications
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience for our work on basic cell biological mechanisms is the cell and developmental biology scientific community. The other target audiences of our work are researchers that are using C. elegans or other nematodes for applied biology and researchers working on plants and plant parasitic nematodes. During this reporting period, I continued to interact with scientists from industry by attending Seed Central events. Seed Central is a public-private partnership that hosts monthly meetings that typically feature a networking session and a seminar. These events bring together academics with local members of the seed industry and the broader ag-biotech community. Changes/Problems: The experiments in Objective 1 on Meloidogyne development were initiated in collaboration with the lab of Dr. A. Calderon-Urea, CSU Fresno, but his Masters students (after initial training by me) were not able to continue the project. Neither lab currently has sufficient funding for personnel to work on this Objective. Thus, we discontinued work starting in 2018-2019. In the coming year we will continue to focus on Objective 2 for which personnel are funded by my NIH award. We will also resume work on Objective 3, for which we have been able to make progress in the past with undergraduates and Masters students (no personnel funds required) and a small supply allowance obtained by the PIs. Our laboratory was shut down from March 2020- June 2020 due to Covid19; during this time only essential activities such as keeping worm strains growing were allowed. For June 2020- Sept 2020 we were allowed 33% of personnel. Even during the latter period, it was not feasible to recruit new Jr. Specialists or undergraduates because of social-distancing training restrictions. This severely impeded progress on all projects but especially Objective 3, which depends mainly on undergraduates and the PIs. What opportunities for training and professional development has the project provided? During the reporting period, two PhD, two Jr. Specialists (research technician) and three undergraduate students worked on the objectives of this project. All personnel receive some technical training from Dr. A. Rose or me, and the more senior lab members also used their expertise to train new undergraduates in the lab. This increases the skill base of all the students and for the undergraduates, helps them obtain jobs upon graduation. We also aid in professional development by mentoring the students in the presentation of their results both in oral and written format. One Ph. D. level student is a member of the MCB T32 Training Grant, which hosts multiple professional development workshops per year. In addition, one Jr. Specialist was a post- baccalaureate student in the UCD PREP program, which helps disadvantaged students gain additional research experience and skills to help them pursue higher degrees. In addition to my mentoring, this student had access to additional training from the PREP program faculty in poster presentations, oral presentations and in applying for fellowships. How have the results been disseminated to communities of interest? Dr. A. Rose and I presented a poster on the intron work, and one PhD gave an oral presentation on her asymmetric division research at the annual MCB Training Grant Retreat. This retreat is attended by numerous labs across campus. That student also presented her work orally at our Cell Biology Research Group Meeting, which is a cross campus weekly meeting. Both PhD students presented posters on their work at International conferences (The Allied Genetics Conference and the Society for Developmental Biology Meeting). The Jr. Specialist in the UCD PREP program presented her work in Poster form at ABRCMS, and annual research conference for minority and disadvantaged undergraduate and post-bacc students from across the US. We have also communicated our results via interactions with industry scientists at Seed Central Events. Events are scheduled monthly during the academic year and are attended by a variety of other academics and ag-business members and consultants at these events. My collaborator on Objective 3 of this project, Dr. Alan Rose, is a standing member of Seed Central, and attends these events regularly. I attended Seed Central in September and February (several spring events were cancelled due to Covid19). During the networking portion of these events, we have time to talk to others and inform them of the work we are doing on gene expression in plants and nematodes. My main discussions have been with research and business personnel from the company Pheronym. Pheronym produces pheromone compounds (chemical signals that nematodes naturally make but that can also be synthesized) to enhance the efficacy of beneficial nematodes as well as to prevent infection by plant parasitic nematodes. What do you plan to do during the next reporting period to accomplish the goals?Research on Objectives 2 and 3 will continue as outlined in the proposal. See below regarding Objective 1. I have been in discussions with Pheronym about potential collaborative projects that would utilize our expertise in either developmental biology or gene expression and would be an alternative to Objective 1. However, the execution of such work will depend on how soon Covid19 restrictions are relaxed.
Impacts
What was accomplished under these goals?
Objective 1 and 3 of the proposed research are aimed at elucidating the fundamental mechanisms by which nematodes (roundworms) develop from the fertilized egg into an adult organism. Specifically, our work using the model nematode C. elegans seeks to understand the molecular basis of asymmetric division, an essential developmental process that results in cells with different identities that have different functions. C. elegans is easily manipulated in the lab and many genes required for the proper execution of asymmetric division have been identified and studied in this model. In our current project, we are continuing our work to characterize the molecular mode of action of a subset of these genes in C. elegans, which are conserved. The results of our studies should aid in understanding the basic development of other nematodes, including those of agriculture importance. For example, Meloidogyne (root knot nematodes) are plant parasites that cause significant agricultural damage in California and worldwide. Although progress has been made in studying how these nematodes infect plants, and some methods can inhibit nematode infection, there is still very little known about the early development of these nematodes, which is of course necessary for them to grow and spread. Our work on C. elegans mechanisms has the potential to identify developmentally important genes that could be targeted in order to inhibit Meloidogyne embryo development. Objective 2 of the project includes collaborative work to understand fundamental aspects of gene expression in C. elegans. The main goal is to understand how the amount of product from a gene is influenced by the presence of specific intron sequences. These studies should help shed light on similar processes in plant or animal parasitic nematodes of agricultural importance. In addition, as part of this work we are testing how introns and other parameters can be used to optimize expression of genes reintroduced into organisms, called transgenes. Transgenes are powerful tools for studying molecular mechanisms of many processes in nematodes and in plants, and can also be used to introduce extra genes in to organisms, for example to make plants with added nutritive value or that are resistant to nematodes. The results of our basic studies should have an impact on the design of transgenes for use in studying the biology of parasitic or beneficial nematodes, as well as for designing anti-nematode strategies in plants. Our progress on these projects is outlined below. Objective 1: Determine if PAR polarity proteins and other asymmetric division proteins are required for development in Meloidogyne species. No progress; see Changes/Problems section. Objective 2: Further elucidate molecular mechanisms of spindle positioning proteins in C. elegans. For an asymmetric division to occur and produce daughters with distinct fates, several key events must occur. First polarity of the cell must be established, such that molecules important for cell fate are asymmetrically localized. Second, the spindle must be positioned in parallel with the polarity axis. In animal cells, the spindle determines the placement of the cytokinetic furrow that divides the cell, and thus this parallel position ensures that fate molecules are differentially distributed to daughter cells. For many years, my lab has studied the key spindle positioning protein LET-99 during the first asymmetric division in C. elegans. We recently started studying LET-99 in other divisions. In the past year, one PhD student has made progress in determining how LET-99 is localized by polarity cues in the embryo, and is now generating transgenic reagents to test the hypothesis that LET-99 is a direct target of phosphorylation by the conserved PAR polarity kinases. She has also begun experiments to determine how PAR and LET-99 protein localization asymmetry is re-established during the second asymmetric division. Another PhD student has carried out experiments aimed at elucidating how LET-99 and the RAC protein (an actin-myosin regulator) regulates spindle positioning in a later asymmetric cell division in response to cell signaling events. Finally, we continued experiments on the role of LET-99 during the process of membrane furrowing during cell division, which we had found before is distinct from its role in spindle positioning. Interestingly in this process we showed that LET-99 antagonizes the RAC protein. We carried out additional genetic and molecular analyses of LET-99's interactions during cytokinesis in the past year, and the data are consistent with the model that LET-99 inhibits the function of Rac. We are currently completing additional experiments to resubmit a manuscript on this work. Objective 3: Distinguish between different mechanisms by which introns boost expression. Work of my collaborator Alan Rose in plants has shown that sequences called introns can have a large effect on the expression of transgenes if the introns are located near the start of the gene; however only a subset of introns have this ability in plants. This affect causes increased mRNA levels, but is distinct from known mechanisms affecting splicing or nuclear export, and thus has been termed intron-mediated-enhancement (IME). Some reports in the literature were consistent with the presence of IME in C. elegans, and in past project periods we began to test several C. elegans introns using transgenic constructs. We were surprised to find that all, even a synthetic intron, had a position effect similar to that seen for IME introns. However, the synthetic intron was previously shown to act at the level of nuclear export, which suggested the effect was due to splicing and deposit of the exon- junction complex, a process distinct from IME. If confirmed, this would be a novel finding because the EJC is not known to have position-dependent effects. Additional independent transgenic lines are required to support the findings. In the current reporting period, we generated several new transgenic lines but a few more are needed. The generation of these, and the quantitative analysis of mRNA levels in these lines has been delayed due to Covid19 (see Changes/Problems section).
Publications
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience for our work on basic cell biological mechanisms is the cell and developmental biology scientific community. The other target audiences of our work are researchers that are using C. elegans or other nematodes for applied biology and researchers working on plants and plant parasitic nematodes. During this reporting period, I continued to interact with scientists from industry by attending Seed Central events. Seed Central is a public-private partnership that hosts monthly meetings that typically feature a networking session and a seminar. These events bring together academics with local members of the Agbiotech community (ie it is not limited to seed biology). My collaborator on Objective 3 of this project, Dr. Alan Rose, is a standing member of Seed Central, and attends these events regularly. I also attended 5 events during the reporting period. During the networking portion of these events, we have time to talk to others and inform them of the work we are doing on gene expression in plants and nematodes. Changes/Problems:My current NIH award on spindle positioning focuses on the work described in Objective 2 of this project, and thus far is only able to fund personnel for that Objective. Work on Objective 1, studying Meloidogyne development, was initiated in collaboration with the lab of Dr. A. Calderon-Urea, CSU Fresno, but neither lab currently has funding for personnel to work on this Objective. Thus, we have not pursued this Objective and in the coming year we will continue to focus on Objective 2. We will also continue work on Objective 3, for which we have been able to make progress with undergraduates and Masters students (no personnel funds required) using supply funds from the A. Rose lab. What opportunities for training and professional development has the project provided?During the reporting period, one PhD and one Masters student, a research technician and three undergraduates worked on this project. All students receive technical training from Dr. A. Rose or me. We aid in professional development by mentoring the students in the presentation of their results both in oral and written format (see below). The more senior lab members also used their expertise to train new undergraduates in the lab. This increases the skill base of all the students and for the undergraduates, helps them obtain jobs upon graduation. The Masters student completed his thesis in September 2019 and moved on to a job in a Biotechnology company. One Ph. D. level student is a member of the MCB T32 Training Grant, which hosts multiple professional development workshops per year. How have the results been disseminated to communities of interest?Dr. A Rose and I both gave seminars at the NAIST Institute in Japan in December 2018, and I presented a seminar at Ohio State University. The Research Technician presented a poster at the international American Society for Cell Biology Meeting and at the Bay Area Worm Meeting. The Technician and one PhD student also presented their work orally at our local Cell Biology Research Group Meeting, which is attended by a number of labs from across campus. The Ph. D. level student on the T32 Training Grant, gave an oral presentation on her research at the annual retreat. As mentioned earlier, we have also communicated our results more informally via interactions with industry scientists at Seed Central Events. I attended 5 of the events in the past year. In addition to meeting new members of the community, I continued my interactions with research and business personnel from the company Pheronym, which generates nematode pheromones as a biocontrol agent to protect plants from agricultural pests. In addition to informing them of our work on plant and nematode gene expression, I was able to connect them with the C. elegans community and they presented a poster on their work at the May 2018 Bay Area Worm Meeting. What do you plan to do during the next reporting period to accomplish the goals?Research on Objectives 2 and 3 will continue as outlined in the proposal. See below regarding Objective 1.
Impacts
What was accomplished under these goals?
The main goal of the proposed research is to elucidate the fundamental mechanisms by which nematodes (roundworms) develop from the fertilized egg into an adult organism. Our work is carried out using the model organism C. elegans, a nematode that is easily manipulated in the lab and has a long history of research. Specifically, our work on C. elegans embryo development is aimed at understanding the molecular basis of asymmetric division, a process that results in cells with different identities that have different functions. Asymmetric divisions are essential for normal development in all animals. The very first divisions of C. elegans and many nematodes are asymmetric, and in C. elegans normal execution of the divisions is essential for the embryo to survive and develop properly. Many genes required for the proper execution of asymmetric division have been identified and studied in the C. elegans model. In our current project, we are continuing our work to characterize the molecular mode of action of a subset of these genes in C. elegans. The results of our studies should aid in understanding the basic development of other nematodes, including those of agriculture importance. For example, Meloidogyne (root knot nematodes) are plant parasites that cause significant agricultural damage in California and worldwide. Although progress has been made in studying how these nematodes infect plants, and some methods can inhibit nematode infection, there is still very little known about the early development of these nematodes, which is of course necessary for them to grow and spread. Our work on C. elegans mechanisms could potentially identify genes that could be targeted in order to inhibit Meloidogyne embryo development. Our basic research on C. elegans also includes collaborative work to understand fundamental aspects of gene expression- how the amount of product from a gene is regulated. These studies should help shed light on similar processes in plant or animal parasitic nematodes of agricultural importance. In addition, as part of this work we are testing parameters needed for optimal expression of genes reintroduced into organisms, called transgenes. Transgenes are powerful tools for studying molecular mechanisms of many processes in nematodes and in plants, and can also be used to introduce extra genes in to organisms, for example to make plants with added nutritive value or that are resistant to nematodes. The results of our basic studies should have an impact on the design of transgenes for use in studying the biology of parasitic nematodes, as well as for designing anti-nematode strategies in plants. Our progress on these projects is outlined below. Objective 1: Determine if PAR polarity proteins and other asymmetric division proteins are required for development in Meloidogyne species. No progress. Objective 2: Further elucidate molecular mechanisms of spindle positioning proteins in C. elegans. For an asymmetric division to occur and produce daughters with distinct fates, several key events must occur. First polarity of the cell must be established, such that molecules important for cell fate are asymmetrically localized. Second, the spindle must be positioned in parallel with the polarity axis. In animal cells, the spindle determines the placement of the cytokinetic furrow that divides the cell, and thus this parallel position ensures that fate molecules are differentially distributed to daughter cells. For many years, my lab has studied the key spindle positioning protein LET-99 during the first asymmetric division in C. elegans. Earlier during this project, we found that LET-99 regulates the acto-myosin cytoskeleton during cytokinesis of the one-cell, to promote robust furrowing for an accurate asymmetric division. This role is genetically distinct from LET-99's involvement in spindle positioning. In particular, while LET-99 regulates trimeric G proteins (which ultimately regulate microtubule motors) during spindle positioning, we identified a genetic interaction at cytokinesis with the small G protein Rac (an actin regulator). A continuing PhD. Student and a Research Technician have carried out additional genetic and molecular analyses of LET-99's interactions during cytokinesis in the past year, and the data are consistent with the model that LET-99 inhibits the function of Rac. We are currently completing additional experiments to resubmit a manuscript on this work. The Ph.D. student also carried out experiments aimed at elucidating how LET-99 regulates spindle positioning in a later asymmetric cell division in response to cell signaling events. Interestingly, this pathway also includes Rac and genetic and localization experiments are under way to determine if LET-99 inhibits Rac and actin in this context. All together, our results suggest that LET-99 is a novel G protein regulator that can act downstream of several different polarity cues. A new Ph.D. student has started investigating polarity reestablishment, which is needed in order to execute successive asymmetric divisions during development. She has generated many strains for the project and is now testing genes for a role in polarity reestablishment. Objective 3: Distinguish between different mechanisms by which introns boost expression. Work of my collaborator Alan Rose in plants has shown that sequences called introns can have a large effect on the expression of transgenes if the introns are located near the start of the gene; however only a subset of introns have this ability in plants. This affect has been termed intron-mediated-enhancement (IME); it results in increased mRNA levels and is distinct from known mechanisms affecting splicing or nuclear export, and Dr. Rose is attempting to determine the mechanism behind IME in plants. Similar phenomena have been reported in several animal systems and work published in C. elegans many years ago reported an "intron effect" using one natural intron from the unc-54 gene and a very small synthetic intron. However, no systematic studies of these introns had been carried out. In our prior AES project, we began characterizing the unc-54 intron and found that its ability to boost expression had similar position requirements to those in plants - that is, the intron must be close to the start of transcription. This result was interpreted as consistent with an IME mechanism in C. elegans. For Aim 3 of the current project, our goal was analyze the synthetic intron and compare the requirements for its activity to that of the unc-54 intron, using transgenic constructs that were identical except for the intron. We were surprised to find that the synthetic intron boosted expression to the same degree as unc-54 intron, if it was placed in the same position. As with unc-54, the synthetic intron boosted less when far from the start of the gene. We also tested other introns, from genes with varying levels of expression. We found that seven different introns gave the same moderate but significant boost as the unc-54 intron when placed near the start of the gene. This effect is weaker quantitatively than that seen in plants. Further, the synthetic intron was previously shown to act at the level of nuclear export, which suggested the effect was due to splicing and deposit of the exon- junction complex, a process distinct from IME. We plan to test whether all of the introns affect nuclear export (versus mRNA levels) in the coming year. If these introns, including unc-54, affect export, it would indicate that we have not yet identified a "true" IME intron in C. elegans, and more introns would be tested for larger enhancing effects. Nonetheless, the finding that unc-54 and the synthetic intron have a position-dependent enhancement effect would be a novel finding for the mechanism of exon-junction complex mediated RNA transport (which is conserved in many organisms). These results have important implications for the design of transgenes.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience for our work on basic cell biological mechanisms is the cell and developmental biology scientific community. The other target audiences of our work are researchers that are using C. elegans for applied biology and researchers working on plants and plant parasitic nematodes. During this reporting period, I interacted with several scientists from industry by attending two Seed Central events (May and September 2018). Seed Central is a public-private partnership that hosts monthly meetings that bring together academics with local members of not only the seed industry but the broader agbiotech community. These events include a networking session, where we have time to talk to others and inform them of the work we are doing on plants and nematodes. In particular, I interacted with members of Pheronym, a company that is developing the use of nematode pheromones as a biocontrol agent for agricultural pests. My collaborator on Objective 3 of this project, Dr. Alan Rose, is a standing member of Seed Central, and attends these events regularly where he interacts with numerous members of the agbiotech industry. Changes/Problems:We are studying Meloidogyne development in collaboration with Dr. A. Calderon-Urea from CSU Fresno. Work on this objective has been delayed due to lack of funding and personnel in both labs. What opportunities for training and professional development has the project provided?During the reporting period, two PhD and one Masters student, a research technician and several undergraduates worked on this project. In addition to the technical training they receive from me, Dr. A Rose and each other (with more senior students training new students), I aid in all personnel's professional development by mentoring them in the presentation of their results both in oral and written format. The PhD level graduate students also attended several of their graduate program (BMCDB) activities such as the annual retreat and the MCB Training Grant retreat and professional developmental seminars. The more senior lab members and myself also used our expertise to train new undergraduates in the lab to help with the research projects. This increases the skill base of all the students and for the undergraduates, helps them obtain jobs upon graduation. How have the results been disseminated to communities of interest?Two PhD students and myself presented posters at the regional Bay Area Worm Meeting as well as the international Annual Meeting for the Society for Cell Biology and the Santa Cruz Developmental Biology Meeting. In addition, several lab members spoke about their research at our UC Davis Cell Biology Research Meeting, which provides a venue for students and postdoctoral fellows to present their work in oral format. As described earlier, we have also communicated our results more informally via interactions with industry scientists at Seed Central Events, so that these community members are more aware of the research being carried out at UC Davis. What do you plan to do during the next reporting period to accomplish the goals?Research on objectives will continue as outlined in the proposal.
Impacts
What was accomplished under these goals?
The main goal of the proposed research is to learn about fundamental mechanisms of how nematodes develop from the fertilized egg into an adult organism. Most of our work is done in the model organism C. elegans, a nematode that is easily manipulated in the lab and has a long history of research. Our basic research on C. elegans also includes collaborative work to understand fundamental aspects of gene expression- how the amount of product from a gene is regulated. These studies should help shed light on similar processes in plant parasitic nematodes of agricultural importance. For example, Meloidogyne (root knot nematodes) are plant parasites that cause significant agricultural damage in California and worldwide. Although progress has been made in studying how these nematodes infect plants, and some methods can inhibit nematode infection, there is still very little known about the early development of these nematodes, which allows them to grow and spread. Our work on C. elegans embryo development is aimed at understanding the molecular basis of asymmetric division, a process that results in cells with different identities and thus have different functions. Asymmetric division are essential for normal development in all animals. The very first divisions of C. elegans and many nematodes are asymmetric, and in C. elegans normal execution of the divisions is essential for the embryo to survive and develop properly. Many genes required for the proper execution of asymmetric division have been identified and studied in the C. elegans model. In our current project, we are continuing our work to characterize the molecular mode of action of a subset of these genes in C. elegans, and we also plan to determine which genes are utilized in Meloidogyne species. The results of our studies could potentially identify genes that could be targeted in order to inhibit Meloidogyne embryo development. In addition, as part of this work we are testing parameters needed for optimal expression of genes reintroduced into organisms, called transgenes. Transgenes are powerful tools for studying molecular mechanisms of many processes in nematodes and in plants, and can also be used to introduce extra genes in to organisms, for example to make plants with added nutritive value or that are resistant to nematodes. The results of our basic studies should have an impact on the design of transgenes for use in studying the biology of parasitic nematodes, as well as for designing anti-nematode strategies in plants. In the past year we have made progress on the the project as outlined below. Objective 1: Determine if PAR polarity proteins and other asymmetric division proteins are required for development in Meloidogyne species. No progress. Objective 2: Further elucidate molecular mechanisms of spindle positioning proteins in C. elegans. Our work in the prior reporting period showed that LET-99 and some of its G protein pathway targets also play a key role in another asymmetrically dividing cell, which divides in response to cell signaling (rather than in response to intrinsic PAR cues characterized before). During the current reporting period, we identified a new component of the pathway, a PAR-1 related kinase. The results were published in December 2017. We also made progress in the analysis of LET-99 in another pathway, cytokinesis, which again involves distinct partners. Our results suggest that LET-99 regulates the actin-myosin cytoskeleton via effects on the small G protein Rac; a publication on this project was submitted and is now in revision. Objective 3: Distinguish between different mechanisms by which introns boost expression. Work of my collaborator Alan Rose in plants has shown that sequences called introns can have a large effect on the expression of transgenes if the introns are located near the start of the gene. Our prior work characterizing an intron known to boost expression in C. elegans (the first normal intron from the unc-54 gene) revealed that it has similar position requirements. During the current period, we extended our work to characterize short "synthetic" introns that are thought to act by a different mechanism. Our preliminary results suggest they also work better near the start of a gene, but provide a much smaller boost to expression than the normal unc-54 intron. In addition, we have generated several new constructs to test other introns that are predicted to be high or low boosters (using a computational approach designed by our collaborators Alan Rose and Ian Korf). Additional quantitative analyses are in progress and we hope to combine the results for publication in the coming year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Malgorzata J. Liro, Diane G. Morton and Lesilee S. Rose. The kinases PIG-1 and PAR-1 act in redundant pathways to regulate asymmetric division in the EMS blastomere of C. elegans. Dev Biol. 444(1):9-19. doi: 10.1016/j.ydbio.2018.08.016.
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Progress 02/15/17 to 09/30/17
Outputs
Target Audience:The target audience for our work on basic cell biological mechanisms is the cell and developmental biology scientific community. The other target audiences of our work are researchers that are using C. elegans for applied biology and researchers working on plant parasitic nematodes. During the reporting period, I interacted with Dr. A. Calderon-Urrea from CSU Fresno, who studies plant parasitic nematodes and uses C. elegans to identify anti-nematode reagents and their mode of action; he interacts directly with stakeholders such as biotech companies interested in his anti-nematode studies. We have begun a collaboration with Dr. Calerderon-Urea to study the basic development of plant parasitic nematodes (Objective 1), with the long term goal of inhibiting development as a nematode control strategy. I also gave him advice on a grant proposal for using C. elegans to study pathways for chalcone killing of nematodes, and helped his Master student design genetic mapping experiments for that project. I also collaborate with Dr. A. Rose for our work on gene expression in Objective 3. Dr. Rose has consulted for plant biotechnology companies and is a member of Seed Central, a public-private partnership that hosts monthly meetings that bring together academics with local members of the seed and agbiotech community. He attends these events regularly, and I also attended several during the reporting period, where I interacted with scientists from biotechnology companies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting period, two PhD and one Masters student, a research technician and several undergraduates worked on this project. In addition to the technical training they receive from me, Dr. A Rose and each other (with more senior students training new students), I aid in all personnel's professional development by mentoring them in the presentation of their results both in oral and written format. The PhD level graduate students also attended several of their graduate program (BMCDB) activities such as the annual retreat and the BMCDB Career and Professional Development Seminars. These lab members and myself also used our expertise to train new undergraduates in the lab to help with the research projects. This increased the skill base of all the students and for the undergraduates, helped them obtain jobs upon graduation. In addition to training the undergraduates from Dr. Calderon-Urrea's lab on the antibody staining technique, I also provided genetic advice to his Master's student for a project aimed at using C. elegans to study anti-nematode compounds identified in his lab. How have the results been disseminated to communities of interest?During the reporting period, my PhD students presented posters at the Annual Meeting for the Society for Cell Biology, and at the regional Bay Area Worm Meeting. Undergraduate members presented posters at the UC Davis Undergraduate Research, Scholarship & Creative Activities Conference. In addition, several lab members spoke about their research at our UC Davis Cell Biology Research Meeting and Worm Group Research Meeting. These campus meetings provide opportunities for students and postdoctoral fellows to present their work in oral format. Importantly, the UC Davis Worm Group Research Meeting also gives C. elegans researchers like myself a venue to disseminate information about new techniques to researchers working in applied areas of nematode biology, and the forum provides those researchers feedback on their experiments. I also visited CSU Fresno to discuss our research on C. elegans with Dr. Calderon. What do you plan to do during the next reporting period to accomplish the goals?Research on all objectives will continue as outlined in the proposal, time and personnel permitting. This NIFA award provides salary from the PI (which is much appreciated) but does not provide any salary or supply funds for other personnel to carry out the Objectives. My NIH R01 funding covers Objective 2, the most basic aim. The other areas that are more directly applicable to agriculture are currently unfunded.
Impacts
What was accomplished under these goals?
The main goal of the proposed research is to learn about fundamental mechanisms of how nematodes develop from the fertilized egg into an adult organism. Most of our work is done in the model organism C. elegans, a nematode that is easily manipulated in the lab and has a long history of research. Information learned in the system is applicable to to many organisms including parasitic nematodes. We also plan to directly apply our basic knowledge of C. elegans to the understanding of development in the parasitic nematode Meloidogyne. Meloidogyne (root knot nematodes) are plant parasites that cause significant agricultural damage in California and worldwide. Although progress has been made in studying in how these nematodes infect plants, and some methods can inhibit nematode infection, there is still very little known about the development of these nematodes, which allows them to grow and spread. Our basic research on C. elegans development, as well as our collaborative work to understand fundamental aspects of gene expression, should help shed light on similar processes in plant parasitic nematodes. Our work on C. elegans embryo development is aimed at understanding the molecular basis of asymmetric division, a process that results in cells with different identities that is critical for normal development in all animals. The very first divisions of C. elegans and many nematodes are asymmetric, and in C. elegans normal execution of the divisions is essential for viability. Many genes required for the proper execution of asymmetric division have been identified and studied in the C. elegans model. For the current project, we are continuing our work to characterize the molecular mode of action of a subset of these genes in C. elegans. In collaboration with Dr. Calderon-Urrea (CSU-Fresno) we are also trying to determine whether these genes are utilized in Meloidogyne species.. Although several of the proteins under study are conserved, and thus identifiable in Meloidogyne at the protein level, the DNA sequences are often highly divergent; this raises the possibility that the results of our studies could identify genes that could be specifically targeted in order to inhibit Meloidogyne development, without detrimental effects to other animals including beneficial nematodes. In addition, as part of this work we are examining the parameters needed for optimal expression of genes reintroduced into organisms as transgenes, with particular focus on sequences called introns. Transgenes are powerful tools for studying molecular mechanisms of many processes in nematodes and in plants. The results of our work will be relevant to designing transgenes for use in parasitic nematodes, as well as for designing transgenes for plants to make them resistant to nematode activities. We will interact with and disseminate knowledge to several investigators working on plant parasitic nematodes during this work. In the past year we have made progress on all three Objectives of the project as outlined below. Objective 1: Determine if PAR polarity proteins and other asymmetric division proteins are required for development in Meloidogyne species. We are studying Meloidogyne development in collaboration with Dr. A. Calderon-Urrea from CSU Fresno. Based on sequence analysis, I advised Dr. Calderon-Urrea's lab on the best antibodies to test in Meloidogyne and I trained his students in the immunolocalization techniques needed to test them. Objective 2: Further elucidate molecular mechanisms of spindle positioning proteins in C. elegans. We have determined that LET-99 and some of its G protein pathway targets also play a key role in another asymmetrically dividing cell, which divides in response to cell signaling (rather than in response to intrinsic PAR cues characterized before). This work shows that LET-99 can act in two different types of pathways to connect polarity to spindle positioning. This work was published in Dec. 2016. We also determined that LET-99's role in cytokinesis is separable from its role in spindle positioning and we defined its place relative to other known regulators of cytokinesis. This work, published in 2017, suggests LET-99 is acting with different partners in this process. Objective 3: Distinguish between different mechanisms by which introns boost expression. For this objective, we completed a quantitative analysis of gene expression for a set of transgenic constructs that allowed us to confirm that the unc-54 intron increases gene expression in a manner similar to that shown for IME (Intron mediated enhancement) in plants. We also began to test other types of introns to determine their mechanism and compare their ability to boost expression. This work forms the basis for a manuscript in preparation, and a version was submitted as a Master's Thesis by a student in the lab.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Malgorzata J. Liro and Lesilee S. Rose. Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA. Genetics 204: 1177-1189
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Starr Daniel A, and Lesilee S. Rose. TorsinA regulates the LINC to moving nuclei. J Cell Biol. 216(3):543-545
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Price, Kari L. and Lesilee S. Rose. LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring. Mol Biol Cell. 28:2360-2373
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