Source: PENNSYLVANIA STATE UNIVERSITY submitted to
PLANT IMMUNITY AND GENOME ENGINEERING FOR CROP IMPROVEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1016432
Grant No.
(N/A)
Project No.
PEN04659
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2018
Project End Date
Jun 30, 2023
Grant Year
(N/A)
Project Director
Yang, YI.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Plant Pathology & Environmental Microbiology
Non Technical Summary
Rice is a model plant for monocots and one of the most important food crops for over half of the world's population. Rice blast is the most devastating rice disease in the world. The proposed project will focus on understanding the mechanism of rice blast disease and developing novel genome editing strategies for crop improvement and disease management. The new knowledge about the rice-pathogen interactions and new technology for genome editing are applicable to other crop systems and genome engineering in general.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115301040100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1530 - Rice;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The overarching goal is to understand the mechanism of pathogen virulence and host immunity and to improve disease resistance and other important agronomic traits via plant genome engineering. The specific objectives are:1. To determine the roles of M. oryzae effectors (Nep1-like and zinc-finger protein effectors) and their host-interacting proteins in mediating pathogen virulence and host susceptibility.2. To elucidate the molecular and biochemical mechanisms of rice Ptr-mediated broad-spectrum disease resistance.3. To develop and improve CRISPR/Cas genome editing tools for precise and efficient plant genome editing.
Project Methods
Objective 1: A combination of molecular, biochemical and genetic approaches will be used to characterize M. oryzae Nep1-like and zinc-finger protein effectors. Three Nep1-like proteins and two zinc-finger protein effectors have been expressed and purified from E. coli. A number of rice interacting protein targets were also identified and produced from E. coli. These proteins are being characterized in vitro and in vivo for the protein-protein interaction, protein-DNA interaction and functional activities such as the ability for fungal effectors to induce plant cell death or suppress plant defense responses and the roles of host protein targets to mediate protein degradation and chromatin reconfiguration. Targeted mutagenesis of fungal effector genes (individually or in combination) in M. oryzae will be performed using the CRISPR/Cas genome editing approach. The resulting fungal mutants will be inoculated onto rice seedling leaves and evaluated for the potential reduction of pathogen virulence. In addition, targeted mutagenesis of host target genes in rice will be performed using the CRISPR/Cas genome editing. The resulting rice mutants will be characterized for altered biochemical functions (e.g., chromatin reconfiguration) and phenotypic traits such as disease susceptibility or resistance.Objective 2: The rice Ptr resistance gene encodes an atypical, non-NLR protein, however, its biochemical mechanism are unknown. Since we have already obtained CRISPR-edited ptr mutants, we propose to further characterize the ptr mutants using various molecular, biochemical and physiological approaches. Total RNAs will be isolated from the wildtype Katy rice cultivar and the ptr mutants with or without M. orzyae infection. RNA-seq will be performed based on Illumina sequencing. The resulting transcriptome data will be analyzed for altered gene expression pattern in wildtype vs. ptr mutant samples. This approach may lead to the identification of potential candidate genes which can provide some clues regarding the specific biochemical or physiological functions of Ptr. In addition, yeast two hybrid screening will be performed using Ptr protein as a bait to identify its interacting proteins. The resulting interacting proteins will be verified using in vitroprotein pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation. The identification of Ptr-interacting proteins should shed some lights on the potential biochemical mechanism of Ptr-mediated disease resistance and facilitate the genome engineering of broad-spectrum disease resistance in rice.Objective 3: Despite the high efficiency of CRISPR/Cas-mediated targeted mutagenesis, which depends on the nonhomologous end joining repair (NHEJ), it is still very difficult and inefficient to perform gene replacement in plants based on homology-dependent repair (HDR). By manipulating various editing components and endogenous DNA repair pathways, we hope to significantly improve the efficiency of HDR-mediated gene replacement. In addition, we will adapt and improve the single base editing systems for C to T (or G to A) and A to G (or T to C) conversion in rice genome. We have identified a number of rice SNPs responsible for important traits such as disease resistance and herbicide tolerance and will proceed with the multiplex editing of single base mutations. By improve the HDR and single base editing efficiency, we will be able to readily create allelic variation in rice and other crops and greatly facilitate precision breeding for various important agronomic traits.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audiences reached during the reporting period include graduate and undergraduate students as well as plant pathologists, molecular biologists and breeders in academia, industry and government agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training and professional development opportunities to three PhD students (Matthew Wheatley, Justin Shih and Vicki Hsieh-Feng) for studying host-pathogen interactions and/or genome editing technologies. Plant biology and CRISPR/Cas genome editing training were also provided to Debamalya Chatterjee, a PhD student I am co-advising, and to Jenna Reeger, for whom I serve as a member of her PhD Dissertation Committee. In addition, molecular biology and genome editing training were provided to two visiting scientists (Drs. Weixiao Yin and Zhaoyuan Zhang) and a research technician (Qin Wang). How have the results been disseminated to communities of interest?The data and findings from this project have been disseminated through journal articles, conference presentations, research seminars, and the CRISPR/Plant website. What do you plan to do during the next reporting period to accomplish the goals?I plan to further improve CRISPR/Cas tools for specific gRNA prediction and multiplex genome editing in fungal plant pathogens. Using both M. oryzae and rice knockout mutants, the roles of the fungal effectors such as MoNLPs and their interacting host targets will be elucidated. I would also like to improve CRISPR/Cas tools for precise genome editing in crop plants based on homology-dependent repair, base editing and prime editing. These improved genome editing tools will be used to study the Ptr-mediated disease resistance mechanisms and engineer a commercial rice cultivar for broad-spectrum disease resistance.

Impacts
What was accomplished under these goals? Goal 1. To facilitate the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated(Cas9)genome editing and functional discovery of fungal or oomycete genes, we performed genome-wide prediction of guide ribonucleic acid(gRNA) spacers in over twenty fungal and oomycete genomes and identified highly specific gRNA spacers that are useful for CRISPR/Cas9 genome editing in these species. To avoid double strand breaks and associated cell death, we also tested base editing and bacterial retron approaches for multiplex genome editing in the rice blast fungus (Magnaporthe oryzae). Goal 2. Based on the previous yeast two-hybrid screening results, we further verified that at least two candidate proteins were strongly interacting with Ptr, a key resistance protein for broad-spectrum resistance against multiple races of M. oryaze. In addition, different allele editing methods have been tested to create the resistant Ptr allele from the susceptible ptr allele in Jupiter, an elite commercial rice cultivar widely grown in the southern US. Goal 3. After the generation and initial evaluation of IPA1 and Bsrd1 promoter-edited T0 and T1 lines of Jupiter rice cultivar in the previous years, transgene-free and promoter-edited T1 and T2 progeny were selected and further evaluated in the greenhouse. Significant variations in heading date, total grain weight and panicle number were observed in theIPA1promoter-edited lines. Using the adenine base editor, single nucleotide editing was performed at Wsl5 and Zebra3 loci in Kitaake cultivar, which yielded the corresponding phenotypic changes in leaf morphology. Furthermore, the susceptible pid3 allele in Jupiter cultivar was successfully converted to the blast resistant Pid3 allele using the adenine base editor. In addition, we have developed a CRISPR/Cas12a-based method for highly sensitive and rapid detection of the Group IV phytoplasma pathogens.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zeng, Z., Han, N., Liu, C., Buerte, B., Zhou, C., Chen, J., Wang, M., Zhang, Y., Tang, Y., Zhu, M., Wang, J., Yang, Y., and Bian, H. 2020. Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing. Annals of Botany (doi.org/10.1093/aob/mcaa115)
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Molla, K.A. and Yang, Y. 2020. CRISPR-Cas-mediated single base editing at more than one locus in rice genome. In: Islam M., Bhowmik P., Molla K. (eds) CRISPR-Cas Methods. Springer Protocols Handbooks. Humana, New York, NY. (doi.org/10.1007/978-1-0716-0616-2_4)
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hsieh-Feng, V. and Yang, Y. 2020. Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing. aBIOTECH 1:123-134. (doi.org/10.1007/s42994-019-00014-w)
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Molla, K.A., Shih, J., and Yang, Y. 2020. Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors. aBIOTECH 1:106-118. (doi.org/10.1007/s42994-020-00018-x)
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Osdaghi, E., Martins, S.J., Ramos-Sepulveda, L., Vieira, F.R., Pecchia, J.A., Beyer, D.M., Bell, T.H., Yang, Y., Hockett, K.L., and Bull, C.T. 2019. 100 years since Tolaas: Bacterial blotch of mushrooms in the 21st century. Plant Disease 103:2714-2732.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Target audience includes plant pathologists, molecular biologists, breeders, and general public who are interested in understanding the mechanisms of host-pathogen interactions and genome editing technology. Changes/Problems:The goal of this project has been expanded to include the development of highly sensitive and rapid assays for early detection and diagnosis of plant pathogens. This change was made in response to the evolving capabilities of CRISPR/Cas technology and an urgent need for highly sensitive and field-deployable methods to detect citrus greening pathogen. What opportunities for training and professional development has the project provided?This project provided excellent opportunities for three PhD students (Matthew Wheatley, Justin Shih and Vicki Hsieh-Feng) to learn the knowledge and techniques to study plant-pathogen interactions and be trained to perform CRISPR/Cas9 editing in plant and fungal systems. Plant molecular biology and genome editing training were also provided to Debamalya Chatterjeewere, a PhD student I am co-advising, and to Jenna Reeger, for whom I serve as a member of her PhD Disseration Committee. In addition, molecular biology and genome editing training were provided to a Fulbright postdoctoral fellow (Dr. Kutubuddin Molla) and two visiting scientists (Drs. Weixiao Yin and Zhaoyuan Zhang). Presentations at research conferences and other opportunities for professional development were also provided to graduate students, postdocs and visiting scholars. How have the results been disseminated to communities of interest?The results from this project have been disseminated through research articles, conference presentations, seminars and workshops. What do you plan to do during the next reporting period to accomplish the goals?I plan to optimize the Cas12a-based assay for early detection and accurate diagnosis of citrus greening and other important plant pathogens. I plan to characterize the Ptr-interacting proteins and elucidate the underlying molecular and biochemical mechanism of the Ptr-mediated broad-spectrum resistance in rice. I will improve CRISPR/Cas-based methods and tool for precise genome editing in plants and fungi. In collaboration with rice breeders, I plan to conduct field trials of gene-edited elite rice lines for yield improvement.

Impacts
What was accomplished under these goals? 1. We significantly improved the efficiency of CRISPR/Cas9-mediated multiplex genome editing in the rice blast fungus (Magnaporthe oryzae). The improved method allows targeted mutagenesis of multiple fungal genes such as those encoding virulence protein effectors and is expected to facilitate the molecular elucidation of fungal pathogenesis. We also identified the cell death problems associated with fungal genome editing and developed alternative tools for improved genome editing in rice blast and other filamentous fungi. (Goal 1) 2. We conducted yeast two-hybrid screenings and identified a number of candidate proteins that interact with Ptr, a key resistance protein for broad-spectrum rice blast resistance. These putative interacting proteins are being validated using in vitro protein pull-down and in vivo co-immunoprecipitation and bimolecular fluorescence complementation. Further molecular and genetic characterization of these interacting proteins is expected to help elucidate the molecular and biochemical basis of the Ptr-mediated blast resistance and facilitate the precision breeding of new rice cultivars with durable and broad-spectrum blast resistance. (Goal 2) 3. We performed CRISPR/Cas9-mediated promoter editing of agronomically important genes in a commercial rice cultivar widely grown in the US. Over 250 independent transgenic lines were generated and genotyped for genomic modifications and phenotyped for quantitative trait variations related to yield or rice blast resistance. Transgene-free and gene-edited progeny were selected and further evaluated in the greenhouse with the goal of potential field trials in the future. In addition, we have successfully developed a CRISPR/Cas12a-based assay for highly sensitive and rapid detection of citrus greening pathogen (Candidatus Liberibacter asiaticus). This assay is expected to facilitate the early detection and field-deployable diagnosis of the devastating citrus greening disease. (Goal 3).

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Molla, K.A. and Yang, Y. 2019. Predicting CRISPR/Cas9-induced mutation for precise genome editing. Trends in Biotechnology (doi.org/10.1016/j.tibtech.2019.08.002).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Xie, S., Wang, Y., Wei, W., Li, C., Liu, Y., Qu, J., Meng, Q., Lin, Y., Yin, W., Yang, Y., and Luo, C. 2019. The Bax inhibitor UvBI-1, a negative regulator of mycelial growth and conidiation, mediates stress response and is critical for pathogenicity of the rice false smut fungus Ustilaginoidea virens. Current Genetics 65:1185-1197.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Molla, K.A. and Yang, Y. 2019. CRISPR/Cas-mediated base editing: Technical considerations and practical applications. Trends in Biotechnology 37:1121-1142.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Yang, Y. and Wheatley, M. 2019. CRISPR/Cas9 genome editing tools for functional genomics and biocontrol of fungal plant pathogens. Annual Meeting of American Phytopathological Society, Cleveland, OH, August 3-7, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Yin, W., Jia, Y. and Yang, Y. 2019. Elucidating the molecular mechanism of the Ptr-mediated rice blast resistance. Annual Meeting of American Phytopathological Society, Cleveland, OH, August 3-7, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wheatley, M. and Yang, Y. 2019. CRISPR/Cas9-enabled multiplex genome editing and locus-dependent DNA repair in Magnaporthe oryzae. Annual Meeting of American Phytopathological Society, Cleveland, OH, August 3-7, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Fountain, J.C., Clevenger, J., Vaughn, J., Chu, Y., Starr, D., Youngblood, R.C., Korani, W., Pandey, M., Chen, Z.-Y., Wang, K., Yang, Y., Kemerait, R., Varshney, R., Scheffler, B., Ozias-Akins, P. and Guo, B. 2019. Dissecting the role of oxidative stress in host-Aspergillus flavus interactions using genomics and genetic engineering. Annual Meeting of American Phytopathological Society, Cleveland, OH, August 3-7, 2019.


Progress 07/01/18 to 09/30/18

Outputs
Target Audience:Target audience includes plant pathologists, molecular biologists, breeders, other researchers and public who are interested in understanding themechanisms of host-pathogen interactions and genome editing technology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three PhDstudents, Matthew Wheatley, Justin Shih andVicki Hsieh-Feng, have learned knowledge and techniques for studying molecular plant-pathogen interactions and trained to perform CRISPR/Cas9 editing for fungal or plant genome engineering. Molecular biology and genome editing trainingwere also provided to Debamalya Chatterjee and Jenna Reeger, for whom I serve asa member of theirPhD DisserationCommittees. In addition, CRISPR/Cas9 and base editing technology training were provided to Dr. Kutubuddin Molla, who is a Fulbright postdoctoral fellow. How have the results been disseminated to communities of interest?Our results have been disseminated through research articles, conference presentations, seminars and workshops. What do you plan to do during the next reporting period to accomplish the goals?I plan to characterize M. oryzaeeffector mutants and rice protein target mutants using a combination of molecular, biochemical, physiological and pathological approaches to elucidate the mechanism of fungal virulence and plant immunity. I will identify and characterizethe Ptr-interacting proteins and understand the molecular and biochemical basis of the Ptr-mediated broad-spectrum resistance in rice. In addition, I will develop and improve CRISPR tools and strategies for more efficient homology-dependent repair and base editing in plants and fungi.

Impacts
What was accomplished under these goals? 1. We have successfully developed and improved the CRISPR/Cas9 tools and methods for genome editing in the rice blast fungus (Magnaporthe oryzae).Targeted mutagenesis of the fungal genes encoding Nep1-like proteins and zinc-finger effectors were achieved using CRISPR/Cas9. The resulting fungal mutants are being evaluated for the potential reduction of pathogen virulence in rice plants. In addition, targeted mutagenesis of rice genes encoding three host target proteins were also achieved using the CRISPR/Cas9 genome editing. Therice mutant lines are being characterized for altered biochemical functions and phenotypic traits such as disease susceptibility or resistance. 2. Rice ptr mutant lines have been generated using the CRISPR/Cas9 genome editing and evaluated for the loss of disease resistance. The second and third generations of the mutant lines were further characterized for preparation of total RNA isolation andIllumina sequencing. Putative Ptr-interacting proteins have been identified and will be verified using in vitro and in vivo protein interaction assays. 3.Phylogenetic analysis of DNA repair gene families has been performed to help understand the evolution of DNA repair mechanism in plants. Novel strategies are being tested to improve the precise genome editing in plants via homology-dependent repair. Single base editing system was successfully established for C to T (or G to A) and A to G (or T to C) conversion in rice plants. Using an elite commercial rice cultivar, we performed promoter editing of two rice genesimportant for yield and/or rice blast resistance. The promoter-edited rice lines are being characterized for altered gene expression and evaluated for high yield and broad-spectrum disease resistance.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Minkenberg, B., Zhang, J., Xie, K., and Yang, Y. 2018. CRISPR-PLANT v2: An online resource for highly specific guide RNA spacers based on improved off-target analysis. Plant Biotechnology J. (doi.org/10.1111/pbi.13025)
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2018 Citation: Xie, K. and Yang, Y. 2018. A multiplexed CRISPR-Cas9 editing system based on the endogenous tRNA processing. Methods in Molecular Biology: Plant Genome Editing with CRISPR systems (in press)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Yang, Y. 2018. Improving CRISPR/Cas9 tools for precise genome editing of host plants and fungal pathogens. International Congress of Plant Pathology 2018, Boston, MA, July 29-August 3, 2018. Phytopathology 108:10S1.288
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Shih,J.W., Hsieh-Feng,V. and Yang, Y. 2018. Phylogenetic and evolutionary analysis of DNA repair gene families in plants. Genome Engineering: The CRISPR-Cas Revolution, Cold Spring Harbor, NY, August 22-25, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wheatley, M. and Yang, Y. 2018. CRISPR/Cas9-enabled multiplex genome editing in Magnaporthe oryzae. Genome Engineering: The CRISPR-Cas Revolution, Cold Spring Harbor, NY, August 22-25, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kutubuddin, A.M., Shih, J.W., and Yang, Y. 2018. Multiplex CRISPR-mediated base editing in rice. Genome Engineering: The CRISPR-Cas Revolution, Cold Spring Harbor, NY, August 22-25, 2018.