Progress 05/01/18 to 04/30/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:As previously stated, a decision was made to focus on a single rice cultivar rather than a broad panel of cultivars. This was in part due to the emergence of nanopore sequencing technologies and my desire to learn to use this technology to generate an annotatedreference genome for our resistant rice cultivar. I believe that our work can be used as a template for others that wish to generate these data and identify disease resistance genes inother cultivars. The use of long-read sequencing allowed us to resolve the complex disease resistance locus in Carolina Gold Select and, eventually, clone the resistance gene. What opportunities for training and professional development has the project provided?This project allowed me to have academic freedom over the last few years of my PhD. With the support from the fellowship I was able to attend and present at several domestic and international conferences where I was able to introduce my work (and myself) to a broader academic community. The manucripts that resulted from my research provided me opportunities to hone my scientific writing and communication skills, which are very important in crafting future fellowship and grant applications. It is unfortunate that I was never able to attend the national predoc fellow meeting in Washington DC due to extenuating circumstances, butI am very thankful for the opportunities that I was afforded as a USDA-NIFA predoc fellow. How have the results been disseminated to communities of interest?Results have been disseminated through publications as well as posters and presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The project evolved over the course of the fellowship and the focus shifted from a broad survey of rice cultivars to a focused examination of a specific resistant cultivar, Carolina Gold Select. The genome of this cultivar was published and the disease resistance gene repertoire was identified. A candidate resistance gene was identified and later cloned and confirmed to be sufficient for the resistance. A unique protein interaction assay was developed and used to identify plant proteins that interact directly with the bacterial defense suppressing truncTALE Tal2h. These are important steps to understand the mechanism of triggering and suppressing host defenses and will inform future studies aimed at the identification, development, and deployment of resistant cultivars.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Spelling changes and fluorescent tagging with prime editing vectors for plants. Li Wang, Hilal Betul Kaya, Ning Zhang, Rhitu Rai, Matthew R Willmann, Sara CD Carpenter, Andrew C Read, Federico Martin, Zhangjun Fei, Jan E Leach, Gregory B Martin, Adam J Bogdanove. Frontiers in Genome Editing
|
Progress 05/01/19 to 04/30/20
Outputs
Target Audience:Findings from this project were shared with the scientific community at the International Society of Molecular Plant-Microbe Interactions Meeting in Glasgow Scotland July 2019 as well as the Plant and Animal Genome meeting in San Diego, CA in January 2020. Changes/Problems:Due to the COVID situation, I postponed my thesis defense from May 2020 until August 2020. I applied for a no cost extension to help support my research during this time. What opportunities for training and professional development has the project provided?The project has provided travel funds to attend important scientific conferences including IS-MPMI in Glasgow, Scotland and PAG in San Diego, CA. At IS-MPMI I reconnected with many colleagues in the plant-microbe interaction community (many of whom I met during my extended visit to Norwich, UK paid for by this fellowship). At PAG I was exposed to broader range of scientific ideas which laid the groundwork for my pursuit of a post-doctoral position. How have the results been disseminated to communities of interest?The results of the project were presented at the 2019 IS-MPMI meeting as a talk at the satellite rice-pathogen interactions meeting as well as a poster at the main meeting. A poster was presented at PAG 2020. The project resulted in two manuscripts in the last year. The first focused on the genome assembly of Carolina Gold Select rice and was published in Plos Genetics, and the second is currently posted to BioRxiv and is in a second round of review at MPMI. What do you plan to do during the next reporting period to accomplish the goals?Due to the COVID situation, I postponed my thesis defense from May 2020 until August 2020. I applied for a no cost extension to help support my research during this time.
Impacts
What was accomplished under these goals?
The TALE recognition spectra of Xanthomonas resistant rice cultivars is ongoing - germplasm has been collected and preliminary screens have been carried out. The status of Tal2h to suppress these resistances is also in progress. Importantly, we recently cloned the Xo1 gene from Carolina Gold Select rice. This allowed us to begin functional characterization of the suppression of Xo1 by Tal2h. We have done comparative transcriptomics, sub-cellular localization, and co-immunoprecipitation to begin to understand the potential interactions between these two proteins. These results are part of our BioRxiv paper that is currently under review at MPMI. The decision was made last year to focus on a full genome assembly for Carolina Gold Select rather than a sequence enrichment for a broad variety of rice cultivars. We used nanopore sequencing which provided long reads and allowed assembly of a high quality genome. This work was published in our Plos Genetics manuscript.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Read, A. C., et al. (2020). "Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing." PLoS Genetics 16(1): e1008571.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Cloning of the rice Xo1 resistance gene and interaction of the Xo1 protein with the defense-suppressing Xanthomonas effector Tal2h
Andrew C. Read, Mathilde Hutin, Matthew J. Moscou, Fabio C. Rinaldi, Adam J. Bogdanove
bioRxiv 2020.05.26.116731; doi: https://doi.org/10.1101/2020.05.26.116731
|
Progress 05/01/18 to 04/30/19
Outputs
Target Audience:In the first year of my fellowship I presented my research at several scientific conferences to an audience of academic, government, and industry scientists. Specifically, I presented at the Xanthomonas Genomics Conference in Halle Germany and the International Congress on Plant Pathology in Boston, MA. Both conferences are attended by members of the international scientific community. Additionally, I contributed to the design and implementation of a two day genome editing module attended by members of theCornell graduate student community. Changes/Problems:As previously noted, wemade the decision to sequence a single resistant genotype, Carolina Gold Select, rather than using an enrichment sequencing approach on a panel of cultivars. This decision was made because it allowed because the resistance in Carolina Gold Select had been mapped to a one megabase locus and we wanted to determine the sequence of the locus and know exactly which resistance genes were encoded in this specific locus. Our approach also allowed us to look at the global resistance gene composition of Carolina Gold Select and we determined that it also encodes a recently cloned rice blast resistance gene. We have identified a candidate resistance gene we believe is responsible for resistance in Carolina Gold Select. Importantly, we also have the up and downstream sequences flanking this gene. This is not available for Xa1 and will be useful in comparative analyses of functional and non-functional alleles. We are still interested in diversity of resistance genes in our rice panel and will likely use a PCR-based approach to address this. What opportunities for training and professional development has the project provided?As part of the fellowship I proposed visiting thegroup of Dr. Jonathan Jones at The Sainsbury Laboratory (TSL) in Norwich, Englandto learn the enrichment sequencing techniques developed in his group. With the decision to sequence the entire genome, I shifted my plans and decided to do a one month visit with Dr. Matthew Moscou at TSL. I spent the month of October working as a member of Dr. Moscou's group and integrating with peers at TSL and the John Innes Centre - two premier plant science research institutions. During this visit I was trained in genomic and phylogenetic analysis and visualizations using the Carolina Gold Select genome. I also had the opportunity to make connections with my European peers in the graduate programs at TSL and the John Innes Centre. I was also able to help design and carry out a two day genome editing module for ~20 members of the Cornell graduate student community. I designed a 30 minute lecture providing background on genome editing technologies and developed a lab exercise in which studentsused PCR to genotype potentiallyedited rice plants. In addition to the lecture and lab components, I also facilitated a paper discussion on a recent genome editing paper from the scientific literature. I received written feedback from the students and reviewed this with my academic supervisor. How have the results been disseminated to communities of interest?Results from the genome sequencing project have been summarized in a submitted manuscript that is also posted to BioRxiv. As of July 23, 2019 the BioRxiv manuscript has been viewed over 1,000 times and tweeted by over 20 members of the research community. A progress report was given in the form of a poster at the 2018 International Congress on Plant Pathology meeting, and most recently the results were shared at the 2019 International Congress on Molecular Plant-Microbe Interactions as both an oral presentation at the New Insights into Rice-Pathogen Interactions satellite meeting as well as a poster. What do you plan to do during the next reporting period to accomplish the goals?The genome sequencing project was very important, however I still have a lot of data to collect in order to characterize the recognition and suppression spectra in my proposed rice panel. I have collected most of the proposed rice cultivars, and sent out requests for the remainder. Over the course of the next year I will continue cloning a set of TALEs from diverse Xanthomonas species and test these against my rice panel to determine recognition spectra. Using much of the same bacterial and plant material, I will then determine the ability of a truncTALE to suppress each of the resistances. The Carolina Gold Select genome is a valuable resource as I work to amplify coding and regulatory sequences for resistance genes of interest in the panel in order to determine the genetic variability and start elucidating the mechanisms of resistance and suppression.
Impacts
What was accomplished under these goals?
Data collection for the first two goals (defining TALE recognition spectra and truncTALE suppression in a rice panel) is ongoing, however the primary focus of my work in the past year wassequencingthe Xanthomonas oryzae pv. oryzae (Xoo) and pv oryzicola (Xoc) resistant cultivar of rice Carolina Gold Select. We initially proposed doing sequence capture enrichment, however as Oxford Nanopore Technologies (ONT) long-read sequencing platform matured, it became an attractive alternative. We elected to do whole genome sequencing of Carolina Gold Select using ONT long-reads combined with existing short-read data for this cultivar. Together these read types were used to generate a high quality assembly, the first on NCBI of a tropical japonica rice cultivar. The genome and all raw read data are publically available and have been publicized on twitter, at meetings, and in our pre-print on BioRxiv. This approach revealed complexity at our disease resistance locus of interest and allowed comparative genomics to look at diversity of Xa1-like resistance genes across publically available Oryzeae genomes. We are still very interested in generating additional sequence data in order to look at diversity of this gene available in rice germplasm and will likely use a PCR-based approach to amplify and sequence specific genes of interest. I believe that the genomic resource generated for Carolina Gold Select will allow for more rapid and accurate primer design and analysis of the diversity of this gene family.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Read, Andrew, Hutin, M, Triplett, L, Zimin, A, Salzberg, S, and Bogdanove, A. Characterization of a bacterial leaf streak of rice resistance locus aided by nanopore sequencing. Poster presented at the International Congress on Plant Pathology, Boston, MA. 2018
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing
Andrew C. Read, Matthew J. Moscou, Aleksey V. Zimin, Geo Pertea, Rachel S. Meyer, Michael D. Purugganan, Jan E. Leach, Lindsay R. Triplett, Steven L. Salzberg, Adam J. Bogdanove
bioRxiv 675678; doi: https://doi.org/10.1101/675678
|
|