Progress 06/15/21 to 06/14/23
Target Audience:Plant biology researchers, graduate and undergraduate students, and scientific journal editors/reviewers. Changes/Problems:Two major changes/problems were encountered during the term of this project; they are as follows: 1) Transition to a DAP-seq dataset for determining regulatory targets of ZmARF27 As previously mentioned, the challenge of generating enough experimental material for anin-vivoChIP-seq experiment was hampering progress in discovering ZmARF27 binding targets. Instead, PD McReynolds and group decided best to use an alternative dataset of DAP-seq binding targets that had been published by a collaborator of the lab. This dataset included target information for a group of maize ARF transcription factors including ZmARF27. We were able to mine the dataset and obtain the targets for ARF27 with confidence. This change in research allowed us to validate and format our putative genetic regulatory network of ZmARF27. 2) Abandonment of the Turbo-ID approach to identify protein interactors of ZmARF27 Much like the ChIP-seq experiment mentioned above, there were also issues in obtaining sufficient amounts of successfully transformed maize root protoplast containing the ZmARF27 and Turbo-ID casette. The use of stable transgenic maize lines with the ZmARF27 and TurboID casettesexist as an alternative source of biological material for the Turbo-ID approach but the sheer amount of time to generate such lines was not feasible during the funding period or remaining time PD McReynolds had during his graduate program.Anin-vitroapproach such as yeast two hybrid is another viable approach but was not implemented during the funding period. Future efforts can explore this area of ZmARF27's protein interactionfunctionality. Challenges aside, PD McReynolds was able to accomplish a majority of the body of work exploring ZmARF27 in auxin signaling and maize root development. What opportunities for training and professional development has the project provided?PD McReynolds has benefited both intellecually and professionallythrough USDA NIFA project funding to studyZmARF27. This funding opportunity allowed PD McReynolds to devote full-time efforts to his research project while also providing adequate material resources for experimental activities.He used this time to increase the breadth of his expertise in the subject of plant molecular biology as well as learn new skills in the ever-growing field of computational and genetic network biology. This has opened up future professional opportunities for PD McReynolds to explore outside of the wet lab bench.He also expanded his professionalnetwork throughattendance ofthe Annual Maize Genetics Research Conferenceand collaborative efforts that were necessary for completion of the project. Finally, this project allowed PD McReynolds to successfully complete his PhD program and move into post-graduate school employment. How have the results been disseminated to communities of interest?Publication pre-print will be available on servers available for the general public to view. What do you plan to do during the next reporting period to accomplish the goals?
What was accomplished under these goals?
The major accomplishment during the project period was the generation of biological data filling a major knowledge gap in maize root development and hormone signaling. More specifically, during this final reporting period the following activities were undertaken toward completion of the defined goals previously outlined by PD Maxwell McReynolds: 1) Identify auxin responsive target genes that are bound and regulated by ZmARF27 Multiple attempts were made during this final reporting period to generate sufficient amounts of transformed ProCsVMV:ZmARF27-GFP maize root protoplast material for the ChIP-seq experiment. Unfortunately, low yield of root protplast as well as thesmall percentage of successful transformations resulted in the ChIP-seq experiment being abandoned. Alternatively, a comprehensive transcription factor binding targetDAP-seq (DNA affinity purification sequencing) dataset was obtained from our collaborators. This dataset including transcription factor binding data for various ARFs including maize ARF27 and served as a suitable substitute for ChIP-seq data. ARF27 binding targets were mined from this dataset and later utilized in ourgenerated gene regulatory networks to identify the direct targets of ARF27. Although their number were not sufficient for ChIP-seq analysis, transformed root protoplast were used for fluorescent microscopy studies to determine localization of intracellularZmARF27-GFP following auxin treatment.Following RNA expression level quantification (see goal 2 below), theDE genes were then input into the creation of gene regulatory networks to predict putative regulatory targets of the ZmARF27 transcription factor. A directed gene regulatory network wasgenerated with ZmARF27 serving as the central hub node. First neighbor nodes were included in the resulting network and represented genes that were predicted to be targets of ZmARF27. In total, 605 target genes were identified through our computational approach. ZmARF27 targets mined from the DAP-seq dataset were overlayed into the network and overlapping targets were identified. In total, 46% of the network predicted ZmARF27 targets were also bound by ZmARF27 suggesting they are direct binding targets.Some of these overlapping targets represented well-characterized genetic regulators of maize root development, auxin-related genes, and cell wall modifying proteins which highlights the accuracy of our generated network. Our network represents an enroute into candidate genes to study for further elucidating the complex relationship between maize root development and auxin. This will prove to be a valuable tool to the community as we envision researchers will be able to mine the network and data therein contained to better understand maize root and develop their own follow-up scientific questions, hypotheses, and findings. 2) Quantify gene expression changes caused by ZmARF27 in response to auxin Transcriptome analysis had previously revealed that hundreds of genes weredifferentially expressed (DE) between both genotype comparison(wildtype versuszmarf27 mutant)and auxin treatment (+ or - indole-3-acetic acid) illustrating ZmARF27's role in controlling auxin basedroot development. DE genes in these comparisons were compared and contrasted to reveal unique and overlapping sets of transcripts to elucidate effects of genotype versus auxin treatment or genotype + auxin treatment. The zmarf27 mutant was revealed to have a large number of unique genes expressed when treated with auxin compared to the wildtype treated with auxin. This result highlights the importance of ZmARF27 in auxin signaling within maize root development. Additionally, gene ontology (GO) analysis was performed to further classify the sets of DE genes by their biological functions. This assignment of biological classification to genes of unknown function will allow the community to parse genes within their areas of interest (i.e., hormone signaling, pathogenesis, etc.) and promote future gene discovery. 3) Elucidate the protein interactors of ZmARF27 that fine tune its transcriptional activity We had hypothesized that ZmARF27 interacts with sets of different proteins depending on its spatial positioning within the cell (nucleus versus cytoplasm). While theTurboID system was going to be our method of choice for investigating such interactions, time constraints with generating stable transgenic mutants coupled with difficulties in in-vitro protoplast transformation efficiency prevented completion of this goal. Alternative approaches for probing protein-protein interactions have been looked into including a yeast two hybrid approach. These activities may be pursued at a future date by a different PD.
Progress 06/15/21 to 06/14/22
Target Audience:Plant biology researchers, graduate and undergraduate students, and scientific journal editors/reviewers. Changes/Problems:
What opportunities for training and professional development has the project provided?Training activities: During this period PD Maxwell McReynolds worked with several post-doctoral scientists as well as a visiting faculty member. Training for PD McReynolds involved expanding molecular biology techniques, improving protoplast isolation, and advancing his skillset with the confocal microscope. Professional development: PD McReynolds traveled to St Louis Missouri to the 64th Annual Maize Genetics Meeting. This conference is heavily attended by evolutionary, developmental, computational, and molecular biologists as well as plant breeders in the field of maize research. Not only is this meeting frequented by members of the academic community several government and private industries are sponsors and featured at this event. PD McReynolds attended lecture talks, networked with other attendees across disciplines, and shared the preliminary research findings from this project. How have the results been disseminated to communities of interest?PD McReynolds traveled to the 64th Annual Maize Genetics Meeting. He presented his preliminary findings to the conference on a research poster. PD McReynolds communicated his results to conference attendees during the scheduled poster session times. What do you plan to do during the next reporting period to accomplish the goals?The following activities will be conducted on PD McReynold's defined goals during the extended funding period: 1)Identify auxin responsive target genes that are bound and regulated by ZmARF27 Improvements will be made to the transient protoplast system to generate material for the ChIP experiment. Once material is obtained the ChIP-seq experiment will be carried out and ARF27 targets will be revealed. 2) Quantify gene expression changes caused by ZmARF27 in response to auxin Continued analysis of the differentially expressed gene targets will be explored. Gene regulatory networks will be generated to indentify targets of ZmARF27. 3)Elucidate the protein interactors of ZmARF27 that fine tune its transcriptional activity The Pro35S:ZmARF27-TurboID plasmid will be transformed into isolated maize protoplasts. The protplasts will then be treated with IAA or a mock treatment, lysed, and proteins interacting with the ZmARF27 protein will be pulled down using streptavidin beads and analyzed via liquid-chromatography coupled mass spectrometry.
What was accomplished under these goals?
During this project period, the following activities were undertaken toward completion of the defined goals previously outlined by PD Maxwell McReynolds: 1)Identify auxin responsive target genes that are bound and regulated by ZmARF27 To acomplishthis goal a ChIP-seq experiment will be conducted to to pull-down and subsequently sequence the nucleotides bound by ARF27 to identify its target genes. The input material for this experiment will be generated using transient expression system in which maize root protoplasts are to be isolated and transformed with a ProCsVMV:ZmARF27-GFPvector. The majority of the work during this progress period was dedicated to improving the isolation total, viability and transformation efficiency of maize root protoplast. Typical protoplast transformations require ~106protoplast per replicate. Not only did maize root yield far less protoplast compared to leaf tissue, the protoplast exhibited reduced viability following isolation as well as a much reduced transformation frequency. Efforts are being made to modify isolation medium as well as cell incubation medium for post-transformation recovery. The ChIP-seq experiment will be delayed until suitable amounts of biological material is available. 2) Quantify gene expression changes caused by ZmARF27 in response to auxin Wild-type and zmarf27mutant plants were grown and exposed to either the auxin indole-3acetic acid (IAA) or a mock treatment. The roots were then collected, RNA quantified/extracted, and Illumina HiSeq reads from QuantSeq libraries were prepared. During this period the reads were mapped to the W22 genome counts obtained, and differential expression analysis performed. Thousands of genes were shown to be differentially expressed between genotypes and the auxin treatment suggesting the extent of ZmARF27's role in controlling auxin related root processes. These genes will be input into the creation of gene regulatory networks to predict putative regulatory targets of the ZmARF27 transcription factor. 3)Elucidate the protein interactors of ZmARF27 that fine tune its transcriptional activity We hypothesize that ZmARF27 interacts with sets of different proteins depending on its spatial positioning within the cell. The TurboID system is our method of choice for investigating such interactions. During this time period the ZmARF27 gene cassette was cloned into the empty Pro35S:TurboID plasmid. No other activities involving this goal was accomplished during this reporting period.