Progress 03/01/19 to 02/28/23
Outputs
Target Audience:The target audience for this information is all professional biologists, but particularly those interested in improvement of cereal crops. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Ms. Erin McGrann is a 2nd year undergraduate at Penn State. She conducted independent research on this project for BIOL296/496 course credit. She researched whether novel dwarf mutants of rice had defects in the RGA1/d1 gene sequence, and whether dwarf mutants were defective in their response to brassinosteroid plant hormones. This was Erin's first research experience and she learned techniques ranging from preparation of solutions to PCR to basic cloning to design of bioassays. Ms. Yue Han, a first-year graduate student in the Penn State Plant Biology Program did a rotation in our laboratory, learning how to isolate genomic DNA from rice and perform PCR and basic cloning and sequencing. The goal of her rotation project was to confirm the sequence of RGA1 in several rice cultivars. Yue had no previous molecular biology experience and significantly improved her wet bench skills during her rotation. Mr. Christian Cantos is a sixth-year Ph.D. student in Penn State's Plant Biology graduate program. Christian already had a background in CRISPR mutagenesis, but expanded his knowledge of CRISPR approaches through his work on Aim 2 of this project. In addition, Christian has learned how to identify and analyze natural variants using our HaploHammer software. Christian has had several opportunities for professional development. First, he presented a poster at the 2022 Penn State Biology student research symposium, and won first place. Second, he presented an in-person poster at the 2023 PAG conference in San Diego, CA. He also presented a seminar on his research in the Spring 2023 Plant Biology Seminar series at Penn State. In addition, he spent several weeks at IRRI in The Philippines, performing agronomic measurements on our second XLG variant field trial. Christian had not previously performed field work, so this significantly expanded his skill set. Ms. Jessica Walnut is a fourth-year Ph.D. student in Penn State's Plant Biology graduate program. She is currently working on Aim 1 of this project. New skills Jess has learned in this project to date include primer design, interpretation of sequencing chromatograms, sequence alignment using Clustal, motif searching using the motif search algorithms PROMO, PlantPromoDB, and PlantCare, construct design and cloning, and induction and propagation of rice callus tissue. How have the results been disseminated to communities of interest?We have authored an invited review on rice XLG phenotypes for Trends in Plant Science: Cantos, C.F. dePamphilis, C.W. Assmann S.M. Extra-large G proteins have extra-large effects on agronomic traits and stress tolerance in maize and rice, accepted. The results have been disseminated via an in-person poster presentation, "Natural variation of rice Galpha subunit affects plant architecture and drought response" by Christian Cantos at the 2023 Plant and Animal Genome (PAG) conference. The results have been disseminated via a poster by Christian Cantos in the 2022 Penn State Biology Student Research Symposium, which won first prize at the symposium. The results have been disseminated via a seminar, "Gene editing and allele mining reveal multifacted roles of rice Galpha proteins in plant architecture and drought stress response," by Christian Cantos in the Spring 2023 Penn State Plant Biology seminar series. Christian is currently analyzing data and will be writing several manuscripts on his research in 2023. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This research focuses on integrated approaches to manipulate the Gα genes (RGA1, four XLGs) of rice heterotrimeric G proteins toward the improvement of rice architecture and reproductive development under both well-watered and water-limited conditions, thereby improving yield, harvest index, and drought resistance. Progress on each Aim is described below. Aim 1. Improve Gα-related agronomic traits by genetic and biochemical manipulation of RGA1. 1a) Improve d1 phenotypes by panicle-specific expression of wild-type RGA1. Confining RGA1 expression to the panicle may help improve panicle exsertion while maintaining positive vegetative agronomic traits seen in d1/rga1 mutants. Panicle specific promoters, the Dense and Erect Panicle1 (DEP1) promoter and Homebox Gene 12 (HOX12) promoter, were selected to drive expression of RGA1 CDS. The final experimental constructs have been completed with the successful ligation of the HOX12 and DEP1 promoters into a rice specific vector, pHL1806, designed by Dr. Hong Li Chou of the Assmann Lab, to drive expression of RGA1. Nipponbare d1 calli have been induced from mature embryos and are in the process of being propagated for transformation. In the meantime, constructs have been transformed into Agrobacterium C58 in preparation for stable calli transformation. Calli will be transformed and regenerated in the near future. Changes in RGA1 gene expression may contribute to its regulation of development and drought tolerance. Therefore, we also designed a new experiment to address the goal of Aim 1a. The Nipponbare RGA1 promoter region was defined as 2958 bp upstream of the transcription start site because cis-regulatory elements (CRE), identified using CRE databases, became sparse beyond that region. Within the RGA1 promoter there is a ~1100bp region of unknown function that is fully conserved in various other chromosomes. Five sub-regions spanning the full length of the promoter were designed to serve two purposes: (1) assess the importance of the conserved region in RGA1 transcription, and (2) determine which cis-regulatory regions are required for development and drought stress responses. The RGA1 promoter segments and gene are being cloned into rice expression vector pHL1806, and preparation of constructs is almost complete. Constructs will be transformed into Nipponbare d1 calli to assess control of RGA1 expression and the extent of complementation of vegetative, panicle, and drought phenotypes. 1c) Manipulate RGA1-based phenotypes by complementation of the d1 mutant with defined biochemical variants of RGA1 with altered GTPase activity/Gβγ interaction. The canonical Gα subunit has conserved G box domains, essential for GDP/GTP binding and GTPase activity. In Arabidopsis, mutation of the second Serine (S52) eliminates GTP binding, preventing the attainment of the activated state of the Gα and enhancing interaction with Gβγ. Interestingly, gpa1 mutants transformed with S52N/C non-phosphorylatable variants show restoration of some, but not all, phenotypes to wild-type. In rice, S53 is analogous to AtGPA1S52, and the RGA1S53C phosphonull protein similarly shows loss of GTP binding and increased interaction with rice Gβγ dimers (unpublished). Therefore, RGA1 S53 is an excellent candidate for this Aim. Constructs harboring the RGA1WT, RGA1S53C and mVenus vector control genes were designed and constructed, driven either by a constitutive promoter or by the native RGA1 promoter. Stable rice callus transformations in the d1/rga1 background have been performed. Rice calli are currently being manipulated for plantlet regeneration. Molecular screening to identify transgene insertion is scheduled for Spring 2023. Positive transgenic lines will be grown to the T0 generation in Summer 2023. Aim 2. Improve Gα-related agronomic traits by manipulation of XLG genes 2a) Manipulate Gα-based phenotypes by CRISPR knockout of genes encoding XLG Gα subunits. Evaluate these lines for known G protein-related phenotypes, and for drought resistance. CRISPR-induced mutations were successfully generated in each XLG in both the Nipponbare wild type and d1/rga1 backgrounds. To evaluate the performance of the resultant CRISPR-xlg lines, a greenhouse experiment was conducted in 2022 with twenty-five genotypes grown under two conditions: well-watered and drought-stress. Architectural and developmental phenotypes were measured. In the Nipponbare background, several CRISPR-xlg genotypes exhibited a shorter stature due to the reduction in flag leaf length and culm length. The combination of reduced flag leaf length and/or culm length further enhanced the shorter stature of plants in the d1/rga1 (semi-dwarf) background. Physiological parameters (e.g., photosynthetic rate and stomatal conductance) were measured daily until the flowering stage. Under well-watered conditions, our results indicated that the XLG CRISPR mutations had an additive effect with RGA1 null mutations on photosynthesis: all CRISPR-xlg lines demonstrated high photosynthesis rates in the Nipponbare background, with photosynthesis even higher in the d1/rga1 background. In drought-stressed conditions, xlg1 and xlg4 maintained a higher photosynthesis rate only in the Nipponbare background while xlg3b maintained a higher photosynthesis rate in both backgrounds. These results provide insight into the complex interactions between XLG and RGA1 in regulating photosynthesis in rice. These data, along with data on stomatal conductance, are currently being subjected to additional statistical analyses which will be followed by manuscript preparation and publication. 2b) Identify natural variants of XLGs associated with agronomic traits; for key variants, analyze G protein biochemistry, expression patterns and associated plant phenotypes. Because we experienced long-term delays with APHIS approval of our permit requesting import of the rice cultivars for Aim 2b, we instead performed a field trial of the XLG natural variants with our IRRI collaborator, Dr. Amelia Henry. We chose to grow cultivars in which computational analyses identified an RGA1 minor variant, as well as several XLG variants predicted to cause truncation of the corresponding XLG protein. Two field trials were conducted, with the first trial comprising 230 cultivars in 2021 and the second trial comprising 37 cultivars in 2022. Plants were grown under both well-watered and drought conditions, and architectural and developmental phenotypes were measured in both trials. The first trial revealed that cultivars with the RGA1 minor variant, which has six SNPs covarying together, exhibited d1-like phenotypes and performed better than the RGA1 major variant in both conditions. Under well-watered conditions, the RGA1 minor variant showed high grain yield and low biomass, resulting in a higher harvest index than the RGA1 major variant. Under drought-stress conditions, the RGA1 minor variant maintained a higher harvest index by having a higher grain yield than the RGA1 major variant. These are promising agronomic traits for breeders. The field data from the second trial are currently being processed to identify correlations between XLG variants and several phenotypes.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Cantos, Christian F. and Assmann, S.M. Natural variation of rice Galpha subunit affects plant architecture and drought response. Poster presentation, Plant and Animal Genome Conference 2023.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Cantos, C.F. dePamphilis, C.W. Assmann S.M. Extra-large G proteins have extra-large effects on agronomic traits and stress tolerance in maize and rice. Trends in Plant Science, accepted.
|
Progress 03/01/21 to 02/28/22
Outputs
Target Audience:The target audience for this information is all professional biologists, but particularly those interested in improvement of cereal crops. Changes/Problems:On Dec. 1, 2021, after over a year's delay, we finally received the APHIS permit to import rice seed from IRRI in The Philippines. However, the seed facility at IRRI, with minimal staffing due to COVID restrictions on in-person work, has a huge backlog in filling orders. Consequently, we still have not received the requested seeds as of today (5/25/22). To overcome this problem, our collaborator at IRRI, Dr. Amelia Henry, instead performed a field trial at IRRI with the selected lines, and a second field trial is currently underway there. What opportunities for training and professional development has the project provided?Mr. Eric Baron was a previous undergraduate on this project; he graduated in 2021 and is currently a full-time laboratory technician in the crystallography lab of Prof. Song Tan at Penn State. Ms. Erin McGrann is a 2nd year undergraduate at Penn State. She conducted independent research on this project for BIOL296/496 course credit. She researched whether novel dwarf mutants of rice had defects in the RGA1/d1 gene sequence, and whether dwarf mutants were defective in their response to brassinosteroid plant hormones. This was Erin's first research experience and she learned techniques ranging from preparation of solutions to PCR to basic cloning to design of bioassays. Mr. Christian Cantos is a fifth-year Ph.D. student in Penn State's Plant Biology graduate program. Christian already had a background in CRISPR mutagenesis, but expanded his knowledge of CRISPR approaches through his work on Aim 2 of this project. In addition, Christian has learned how to identify and analyze natural variants using our HaploHammer software. Christian has had several opportunities for professional development. First, he presented his research on this project via a virtual poster at the Plant and Animal Genome (PAG) conference, which went from in-person to virtual at the last minute. Second, he spent several weeks at IRRI in The Philippines, performing agronomic measurements on our second XLG variant field trial. Christian had not previously performed field work, so this significantly expanded his skill set. Ms. Jessica Walnut is a third-year Ph.D. student in Penn State's Plant Biology graduate program. She is currently working on Aim 1 of this project. New skills Jess has learned in this project to date include primer design, interpretation of sequencing chromatograms, sequence alignment using Clustal, motif searching using the motif search algorithms PROMO, PlantPromoDB, and PlantCare, construct design and cloning, and induction and propagation of rice callus tissue. Dr. Ángel Ferrero-Serrano is a Research Assistant Professor in the Assmann lab. He assisted in the RGA1 variant analysis, thereby extending his training in population genetics. Dr. Ferrero-Serrano also contributed data to the New Phytologist publication described below. Dr. Yotam Zait is a postdoctorate in the Assmann lab, currently supported on his own USDA post-doctoral fellowship. Yotam performed research on gas exchange characteristics of the d1 (rga1 mutant) and demonstrated that the d1 mutant has greater mesophyll conductance under drought, which results in improved water use efficiency. A manuscript on these findings was published in New Phytologist. How have the results been disseminated to communities of interest?The results have been disseminated via a virtual poster presentation by Christian Cantos at the 2022 Plant and Animal Genome (PAG) conference, "Generation of novel alleles of rice extra-large G proteins (XLGs) via CRISPR/Cpf1 gene editing" Results have also been disseminated through a peer-reviewed scientific publication: Zait Y, Ferrero-Serrano Á, Assmann SM. 2021. The α subunit of the heterotrimeric G protein regulates mesophyll CO2 conductance and drought tolerance in rice. New Phytol., doi: 10.1111/nph.17730. Moreover, the above publication was featured in Faculty Opinions (previously F1000), and thereby brought to the attention of a large circle of scientists. See https://facultyopinions.com/article/740803603 What do you plan to do during the next reporting period to accomplish the goals?Aim 1. 1a and 1b) In the next year of funding, d1/rga1 calli will be transformed with the DEP1 and HOX12 promoter constructs. Molecular screening will be performed in the T0 generation to identify successful transgene insertion. Positive transgenic lines will be regenerated and seeds will be harvested for the T1 generation. A copy number analysis will be performed to identify T1 lines with one copy of the transgene. Selected lines will be grown to maturity for seed collection. In the T2 generation, vegetative and reproductive phenotypes, particularly panicle exsertion and yield, will be quantified in comparison to control lines. 1c) In the next year of the project, T0 lines of RGA1WT and RGA1S53C constructs as well as empty vector control constructs will be regenerated. Molecular screening will be performed in the T0 generation to identify transgene insertion. Positive transgenic lines will be grown to the T1 generation. Vegetative and reproductive phenotypes and drought tolerance will be quantified and compared between the RGA1WT and RGA1S53C transgenic lines vs. vector control lines. Aim 2. 2a) In the next year of the project, transgene-free, homozygous T2 lines of the current XLG-CRISPR lines will be grown. Phenotypic analysis of agronomic traits and physiological analysis drought tolerance will be performed. The resultant information on XLG mutants will also inform on which XLG natural variants are most likely to be of agronomic interest. 2b) and Aim 3) In the next year of the project, haplotypes with significant correlation with several advantageous agronomic phenotypes from our field trials will be selected to perform a small controlled greenhouse experiment to confirm the phenotypes observed.
Impacts
What was accomplished under these goals?
This research focuses on integrated approaches to manipulate the Gα genes (RGA1, four XLGs) of rice heterotrimeric G proteins toward the improvement of rice architecture and reproductive development under both well-watered and water-limited conditions, thereby improving yield, harvest index, and drought resistance. Progress on each Aim is described below. Aim 1. Improve Gα-related agronomic traits by genetic and biochemical manipulation of RGA1. 1a) Improve d1 phenotypes by panicle-specific expression of wild-type RGA1. 1a) Confining RGA1 expression to the panicle may help improve panicle exsertion while maintaining positive vegetative agronomic traits seen in d1/rga1 mutants. Panicle specific promoters, the Dense and Erect Panicle1 (DEP1) promoter and Homebox Gene 12 (HOX12) promoter, were selected to drive expression of RGA1 cDNA. The final experimental constructs have been completed with the successful ligation of the HOX12 and DEP1 promoters into a rice specific vector, pHL1806, designed by Dr. Hong Li Chou of the Assmann Lab, to drive expression of RGA1. 1b) The Assmann lab identified a 4 SNP haplotype in RGA1 that is correlated with increased panicle exsertion in rice cultivars. All 4 SNPs are in introns. Further assessment raised questions about the reliability of the sequencing information and thus the correlation, so this subaim is not being pursued further. 1c) Manipulate RGA1-based phenotypes by complementation of the d1 mutant with defined biochemical variants of RGA1 with altered GTPase activity/Gβγ interaction. The canonical Gα subunit has conserved G box domains, essential for GDP/GTP binding and GTPase activity. In Arabidopsis, mutation of the second Serine (S52) eliminates GTP binding, preventing the attainment of the activated state of the Gα and enhancing interaction with Gβγ. Interestingly, gpa1 mutants transformed with S52N/C non-phosphorylatable variants show restoration of some, but not all, phenotypes to wild-type. In rice, S53 is analogous to AtGPA1S52, and the RGA1S53C phosphonull protein similarly shows loss of GTP binding and increased interaction with rice Gβγ dimers (unpublished). Therefore, RGA1 S53 is an excellent candidate for this Aim. Constructs harboring the RGA1WT, RGA1S53C and mVenus vector control genes were designed and constructed, driven either by a constitutive promoter or by the native RGA1 promoter. Stable rice callus transformations in the d1/rga1 background have been performed. Aim 2. Improve Gα-related agronomic traits by manipulation of XLG genes 2a) Manipulate Gα-based phenotypes by CRISPR knockout of genes encoding XLG Gα subunits. Evaluate these lines for known G protein-related phenotypes, and for drought resistance. CRISPR-induced mutations of each XLG have been successfully produced in the Nipponbare and d1/rga1 backgrounds. The Nipponbare background has a mutation efficiency of 45-100% for all XLGs while for d1/rga1 background, mutation efficiency is 36-100%. Two different types of CRISPR-xlg alleles were selected for T1 analysis: those encoding truncated proteins or proteins with 1-2 amino acid deletions. For T1 analysis, T-DNA PCR was performed to select lines that are CRISPR-free. Homozygosity test was performed by sequencing the target sites for each XLG to select biallelic, homozygous mutant lines. CRISPR-free, homozygous T2 lines have been collected. To generate different mutation combinations of rice XLGs, a CRISPR-OsXLGquad construct, targeting the four XLGs, was transformed into Nipponbare and d1/rga1 backgrounds. For Nipponbare background, six transgenic lines were generated and analyzed. Based on mutation analysis, osxlg3a, osxlg3b, osxlg1 are all targeted resulting in triple-xlg CRISPR lines. Fifty-six T1 lines were generated and analyzed. Sequence analyses resulted in different mutant combinations. Three plants have xlg3a/xlg3b/xlg3c/xlg1 quadruple combination, 41 plants have xlg3a/xlg3b/xlg1 triple combination and 12 plants have xlg3b/xlg1 double mutant combination. Currently, T1 lines are being grown to produce the T2 generation. For d1/rga1 background, thirty independent rice calli were selected and analyzed. Sequence analysis showed that 20 rice calli events have all the xlgs (xlg3a/xlg3b/xlg3c/xlg1) mutated while ten rice calli events have triple-xlgs (xlg3a/xlg3b/xlg1) mutated. 2b) Identify natural variants of XLGs associated with agronomic traits; for key variants, analyze G protein biochemistry, expression patterns and associated plant phenotypes. Because we have experienced long-term delays with APHIS approval of our permit requesting import of the rice cultivars for Aim 2b, and to this day are still waiting for those seeds, we instead performed a field trial of the XLG natural variants at IRRI, with our IRRI collaborator, Dr. Amelia Henry. In this study, two computational analyses were performed initially to identify the different haplotypes present in the 3K panel. The first approach was to computationally evaluate whether the natural variants of RGA1 have a significant correlation with several different phenotypes. In this approach, the RGA1 genotypic data and 3K panel phenotypic data were extracted from the rice SNP-seek database (https://snp-seek.irri.org/_snp.zul). A correlation matrix between the RGA1 genetic variants and specific phenotypes was tested using Student's t-test (continuous phenotypes) and chi-square test (categorical phenotypes). RGA1 genetic variants with significant correlation with several phenotypes were selected as haplotypes of interest. For the second approach, the Haplohammer package was used (https://github.com/StructureFold2/HaploHammer). Haplohammer is a suite of Python scripts designed to analyze and align many <.vcf> sequence files back to the reference genome, identifying nucleotide and peptide variants. The <.vcf> files of the 3,024 rice accessions were downloaded, filtered, and aligned to Nipponbare reference genome to produce nucleotide-specific haplotypes. The resulting haplotypes were translated into peptide-specific haplotypes. A total of 230 rice accessions was selected that comprised approximately equal number of lines for each major and minor haplotype. For the IRRI field experiment, two treatments were performed: well-watered (WW) and drought stress (DS). WW was transplanted in a paddy rice field while DS was transplanted in a rain-out shelter. After 60 days, drought was introduced in the DS treatment. Continuous (plant height, flag leaf length, and width, tiller and panicle numbers, grain size) and categorical (flag leaf angle, panicle type, panicle exsertion) phenotypes were measured. Yield, biomass and harvest index were also determined. Initial analyses showed a correlation between DS and biomass and harvest index for OsXLG1 variants, suggesting a role in drought stress. Accordingly, a second field trial is being conducted at IRRI focusing on OsXLG1 natural variants. Aim 3. Perform field trials of d1 drought resistance and planting density x drought resistance at the International Rice Research Institute (IRRI). Our collaborator at IRRI, Dr. Amelia Henry, performed one field trial of d1 drought resistance under moderate drought conditions. In this field trial, d1 showed higher photosynthesis, higher stomatal conductance, and greater root length than the corresponding wild type, as well as smaller shoot biomass (as expected for this dwarfing mutation) which led to increased harvest index relative to wild-type. However, d1 did not yield better than wild-type on a seed biomass basis. Because we have experienced long-term delays with APHIS approval of our permit requesting import of the rice cultivars for Aim 2b, we are instead performing field trials of the XLG natural variants at IRRI, with our IRRI collaborator, Dr. Amelia Henry. One field trial has been completed and the second one is currently ongoing (see Aim 2b above).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zait Y, Ferrero-Serrano �, Assmann SM. 2021. The Galpha subunit of the heterotrimeric G protein regulates mesophyll CO2 conductance and drought tolerance in rice. New Phytol., doi: 10.1111/nph.17730.
- Type:
Other
Status:
Other
Year Published:
2021
Citation:
Poster presentation at the 2022 Plant and Animal Genome (PAG) conference, Generation of novel alleles of rice extra-large G proteins (XLGs) via CRISPR/Cpf1 gene editing .
|
Progress 03/01/20 to 02/28/21
Outputs
Target Audience:We have published a topical review article on crop phenotypes related to G protein signaling pathways, with a particular focus on the rice literature: Ferrero-Serrano Á., Cantos C., and Assmann SM. 2019. The Role of Dwarfing Traits in Historical and Modern Agriculture with a Focus on Rice. Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect. a034645. The target audience for the above article is all professional biologists, particularly those interested in crop improvement. Ph.D. student Christian Cantos presented on his analysis of G protein natural variants in rice and on CRISPR/Cas-based manipulation of XLG signaling to a broad biology audience in the Penn State Biology Department 2020 weekly seminar series. Changes/Problems:Aim 2a) We had outsourced some of our transgenic rice production to the Cornell Plant Transformation facility and when they shut down in March, 2020 due to the pandemic, we received the heartbreaking news that they were forced to throw out our nascent transgenics, which delayed that part of the project by many months. Here is an excerpt from an email from the director of that facility, dated 3/19/20: "Yesterday was a very rough day because we had to scale back all of our work, letting all projects at early stages go until the university is back up and running at full capacity. This included having to toss the callus we had been growing so that we could do your transformations next week (it takes a month for us to grow callus for transformation). As of today, I am the only person working in my lab and am focused on getting projects that are at the regeneration stage to completion. I am sorry to deliver the bad news." The Cornell Plant Transformation facility became operational again in summer 2020 but underwent a change in leadership in January 2021. They are now slowly providing us with some of the promised transgenic lines, but they have reneged on a prior agreement to produce transgenics of the redesigned CRISPR-OsXLGquad construct due to staffing limitations at their facility. Therefore, as personnel and resources allow, we will produce those transgenics at Penn State. Aim 2b) We have been waiting many months for APHIS to approve our (routine) permit for import of the seeds for our XLG natural variant analysis from IRRI, but have seen no action on our application, despite inquiries. The process seems to be stalled at the phase of local inspection of our facilities. This is quite frustrating, especially since we can only conduct these experiments in Pennsylvania in the summer months. Accordingly, we are now instead planning to evaluate these lines through a field trial at IRRI. What opportunities for training and professional development has the project provided?Mr. Eric Baron is a senior undergraduate assisting with rice growth and genotyping of the CRISPR rice lines. Eric has learned fundamental research skills, including basic principles of experimental design, how to grow research plants, how to extract DNA and perform PCR and gel electrophoresis, and how to analyze sequencing data. As a result of this experience, Eric was offered a job as a full-time laboratory technician in the crystallography lab of Prof. Song Tan at Penn State upon his graduation. Mr. Christian Cantos is a fourth-year Ph.D. student in Penn State's Plant Biology graduate program. Christian already had a background in CRISPR mutagenesis, but expanded his knowledge of CRISPR approaches through his work on Aim 2 of this project. In addition, Christian has learned how to identify and analyze natural variants using our HaploHammer software. Christian has had several opportunities for professional development. First, he applied for and was awarded a slot in the 2019 Cold Spring Harbor Laboratory Workshop on Cereal Genomics. His attendance at this course provided excellent training in all aspects of grass genomics. Second, Christian received training in oral scientific presentation through an invited seminar he gave to Penn State's Biology Department in February 2020. Third, Christian served as a teaching assistant for the undergraduate course BIOL240, "Function and Development of Organisms," thereby receiving training and practice in scientific pedagogy. Fourth, Christian has submitted a (virtual) poster on his research for this project to the 2021 Penn State Graduate Research Exhibition. Ms. Jessica Walnut is a second-year Ph.D. student in Penn State's Plant Biology graduate program. She is currently working on Aim 1 of this project. New skills Jess has learned in this project to date include primer design, interpretation of sequencing chromatograms, sequence alignment using Clustal, motif searching using the motif search algorithms PROMO, PlantPromoDB, and PlantCare, construct design and cloning, and induction and propagation of rice callus tissue. Jess also served as a teaching assistant for the undergraduate course BIOL110, "Basic Concepts and Biodiversity," thereby receiving training and practice in scientific pedagogy. Dr. Ángel Ferrero-Serrano is a Research Assistant Professor in the Assmann lab. He was partially supported on this grant, and performed the RGA1 variant analysis, thereby extending his training in population genetics. Dr. Yotam Zait is a postdoctorate in the Assmann lab. He was partially supported on this grant and partially supported by his own U.S.-Israel BARD (Binational Agriculture Research and Development) postdoctoral fellowship. Yotam has been performing research on gas exchange characteristics of the d1 (rga1 mutant) and has demonstrated that the d1 mutant has greater mesophyll conductance under drought, which results in improved water use efficiency. A manuscript on these findings has been reviewed by New Phytologist and is currently under revision. How have the results been disseminated to communities of interest?We have published a topical review article on crop phenotypes related to G protein signaling pathways, with a particular focus on the rice literature: Ferrero-Serrano Á., Cantos C., and Assmann SM. 2019. The Role of Dwarfing Traits in Historical and Modern Agriculture with a Focus on Rice. Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect. a034645. The target audience for the above article is all professional biologists, particularly those interested in crop improvement. Ph.D. student Christian Cantos presented on his analysis of G protein natural variants in rice and on CRISPR/Cas-based manipulation of XLG signaling to a broad biology audience in the Penn State Biology Department 2020 weekly seminar series. What do you plan to do during the next reporting period to accomplish the goals?Aim 1a and 1b) In the next year of funding, d1/rga1 calli will be transformed with the DEP1 promoter constructs. Molecular screening will be performed in the T0 generation to identify successful transgene insertion. Positive transgenic lines will be grown to the T2 generation. Vegetative and reproductive phenotypes, particularly panicle exsertion and yield, will be quantified in comparison to control lines. Also in the next year of funding, the "4 SNP" construct will be completed and transformed into d1/rga1 calli, the T0 generation of these transgenics will be produced and screened, and T1 generation seeds will be collected. Aim 1c) In the next year of the project, RGA1WT and RGA1S53C constructs as well as empty vector control constructs will be transformed into d1/rga1 rice calli. Molecular screening will be performed in the T0 generation to confirm transgene insertion. Positive transgenic lines will be grown to the T2 generation. Vegetative and reproductive phenotypes and drought tolerance will be quantified and compared between the RGA1WT and RGA1S53C transgenic lines vs. vector control lines as soon as the T2 generation is available. Aim 2a) In the next year of the project, transgene-free, homozygous T2 lines of the current lines will be identified and grown. Phenotypic analysis of agronomic traits and drought tolerance will be performed. The redesigned CRISPR-OsXLGquad construct will be used for transformation of WT and d1 calli and the T0 generation will be produced and screened. This phenotypic information on XLG null mutants will also inform on which XLG natural variants are most likely to be of agronomic interest. Aim 2b) and Aim 3) In the next year of the project, we will perform field trials on the natural variant lines, with our collaborator at IRRI, Dr. Amelia Henry.
Impacts
What was accomplished under these goals?
This research focuses on integrated approaches to manipulate the Gα genes (RGA1, four XLGs) of rice heterotrimeric G proteins toward the improvement of rice architecture and reproductive development under both well-watered and water-limited conditions, thereby improving yield, harvest index, and drought resistance. Progress on each Aim is described below. Aim 1a) To achieve panicle specific expression of RGA1, the Dense and Erect Panicle1 (DEP1) promoter was selected to drive expression of RGA1 cDNA. The DEP1 promoter was amplified from wild type Nipponbare leaf tissue and RGA1 cDNA was amplified from a previous pDEST-GADT7 construct. Primers included restriction enzyme sites to allow for ligation into the final rice specific vector, pHL1806, designed by Dr. Hong Li Chou of the Assmann Lab. The construct has been completed, verified, and transformed into Agrobacterium for rice callus transformation. Aim 1b) The Assmann lab identified a 4 SNP haplotype in RGA1 that is correlated with increased panicle exsertion in rice cultivars. Toward complementation of the d1 mutant with this haplotype, the RGA1 gene and promoter have been amplified from wild type tissue, and SNPs will be introduced by GeneBlock technology. Aim 1c) The canonical Gα subunit has conserved G box domains, essential for GDP/GTP binding and GTPase activity. In Arabidopsis, mutation of the second Serine (S52) eliminates GTP binding, preventing attainment of the activated state of the Gα and enhancing interaction with Gβγ. Interestingly, gpa1 mutants transformed with S52N/C non-phosphorylatable variants show restoration of some, but not all, phenotypes to wild-type. In rice, S53 is analogous to AtGPA1S52, and the RGA1S53C phosphonull protein similarly shows loss of GTP binding and increased interaction with rice Gβγ dimers (unpublished). Therefore, RGA1 S53 is an excellent candidate for this Aim. In the past year, constructs harboring the RGA1WT and RGA1S53C genes have been designed and constructed, driven either by a constitutive promoter or by the native RGA1 promoter. Callus transformation will be performed in the near future. Aim 2a) A major focus in this year has been on producing the transgenic plants with CRISPR-based knockout of the four rice XLG genes. To produce targeted knockout lines of rice XLGs for subsequent phenotypic analysis, we used the CRISPR/Cpf1 genome editing system. We identified CRISPR RNA (crRNA) sequences for all four rice XLGs using the Benchling CRISPR design tool (https://benchling.com/crispr). The target sites with the highest specificity were chosen and were analyzed using BLAST to confirm the absence of off-target matches. CRISPR constructs with sequence confirmed clones were used for Agrobacterium-mediated rice transformation of both Nipponbare wild type and d1/rga1 mutant background calli induced from mature embryos. Regenerated transgenic rice were analyzed for the presence of the mutation in the target sites by Sanger sequencing of the target regions and analyzing the sequence files using DSCODEM online software (http://skl.scau.edu.cn/dsdecode/) which automatically decodes sequencing chromatograms with biallelic, heterozygous, and homozygous mutations. Mutation of each XLG has been achieved in the Nipponbare wild type background. OsXLG3B and OsXLG1 have a mutation efficiency of 100% with no wild-type sequence identified in the T1 lines. OsXLG3C has a 92.86% mutation efficiency with some lines still inheriting the wild-type sequence. T1 analysis for OsXLG3A is ongoing. Currently, the homozygous lines are being grown to produce the T2 generation. To identify the effect of knocking out all the XLGs, a CRISPR-OsXLGquad construct, targeting the four XLGs, was transformed into rice. Thirty independent events were regenerated and analyzed for mutation efficiency. With the CRISPR-OsXLGquad construct, OsXLG3B and OsXLG1 have the highest mutation rate (100%) followed by OsXLG3A (93.33%) and OsXLG3C (40%). Several XLG knockout (KO) combinations were also identified: xlg3a/xlg3b/xlg3c/xlg1 (12 events), xlg3a/xlg3b/xlg1 (16 events) and xlg3b/xlg1 (2 events). The crRNA for XLG3C, with lowest CRISPR efficiency, has been redesigned and the redesigned CRISPR-OsXLGquad construct is being generated. To determine the dependent and independent roles of XLGs with RGA1, the CRISPR constructs were also transformed into the d1/rga1 background. Knockout lines of xlg3b and xlg1 have been identified while analysis for OsXLG3A and OsXLG3C is in progress. Currently, T0 lines are being grown for production of the T1 generation. The CRISPR-OsXLGquad construct was also transformed into the d1/rga1 background with the goal to knock out all the rice G proteins. Aim 2b) We have identified natural variants of XLGs that are associated with agronomic traits. In particular, we have used our custom HaploHammer software (https://github.com/StructureFold2/HaploHammer) to identify XLG haplotypes within the 3K rice genomes. We have determined that within the 3K genomes there are 29 haplotypes of OsXLG3a, 29 haplotypes of OsXLG3b, 11 haplotypes of OsXLG3c, and 35 haplotypes of OsXLG1 with altered protein sequence. Variants of OsXLG3C and OsXLG1 that result in deletions of almost the entire Gα region are of particular interest. Aim 3) Our collaborator at IRRI, Dr. Amelia Henry, performed one field trial of d1 drought resistance under moderate drought conditions. In this field trial, d1 showed higher photosynthesis, higher stomatal conductance, and greater root length than the corresponding wild type, as well as smaller shoot biomass (as expected for this dwarfing mutation) which led to increased harvest index relative to wild-type. However, d1 did not yield better than wild-type on a seed biomass basis. Because we have experienced long-term delays with APHIS approval of our permit requesting import of the rice cultivars for Aim 2b, we will instead perform a field trial of the XLG natural variants at IRRI, with our IRRI collaborator, Dr. Amelia Henry. Cultivars have been selected and the trial will be performed in the next field season.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Ferrero-Serrano �., Cantos C., and Assmann SM. 2019. The Role of Dwarfing Traits in Historical and Modern Agriculture with a Focus on Rice. Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect. a034645.
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:We have published a topical review article on crop phenotypes related to G protein signaling pathways, with a particular focus on the rice literature: Ferrero-Serrano Á., Cantos C., and Assmann SM. 2019. The Role of Dwarfing Traits in Historical and Modern Agriculture with a Focus on Rice. Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect. a034645. The target audience for this article is all professional biologists, particularly those interested in crop improvement. Graduate student Christian Cantos presented on his analysis of G protein natural variants in rice and on CRISPR/Cas-based manipulation of XLG signaling to a broad audience in the Penn State Biology Department 2020 weekly seminar series. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Mr. Christian Cantos is a third-year Ph.D. student in Penn State's Plant Biology graduate program. Christian already had a background in CRISPR mutagenesis, but expanded his knowledge of CRISPR approaches through his work on Aim 2 of this project. In addition, Christian has learned how to identify and analyze natural variants using our HaploHammer software. Christian has had several opportunities for professional development. First, he applied for and was awarded a slot in the 2019 Cold Spring Harbor Laboratory Workshop on Cereal Genomics. His attendance at this course provided excellent training in all aspects of rice genome research. Second, Christian received training in oral scientific presentation through an invited seminar he gave to Penn State's Biology Department in February 2020. Third, Christian served as a teaching assistant for the undergraduate course BIOL240, "Function and Development of Organisms," thereby receiving training and practice in scientific pedagogy. Ms. Jessica Walnut is a first-year Ph.D. student in Penn State's Plant Biology graduate program who joined the Assmann lab in December 2019. As a first-year student, she is supported by program funds, but she is currently working on Aim 1 of this project, and has just finished resequencing the RGA1 promoter in Nipponbare and IR64. New skills Jess has learned in this project to date include primer design, interpretation of sequencing chromatograms, sequence alignment using Clustal, and motif searching using the motif search algorithms PROMO, PlantPromoDB, and PlantCare. Dr. Ángel Ferrero-Serrano is a Research Assistant Professor in the Assmann lab. He was partially supported on this grant, and performed the RGA1 variant analysis, thereby extending his training in population genetics. How have the results been disseminated to communities of interest?We have published a topical review article on crop phenotypes related to G protein signaling pathways, with a particular focus on the rice literature: Ferrero-Serrano Á., Cantos C., and Assmann SM. 2019. The Role of Dwarfing Traits in Historical and Modern Agriculture with a Focus on Rice. Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect. a034645. The target audience for this article is all professional biologists, particularly those interested in crop improvement. Graduate student Christian Cantos presented on his analysis of G protein natural variants in rice and on CRISPR/Cas-based manipulation of XLG signaling to a broad audience in the Penn State Biology Department 2020 weekly seminar series. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. In the next year, we will make the CRISPR constructs for promoter bashing of RGA1 and will (in collaboration with the Qi lab) produce the T0 generation of the CRISPR transformants. We will also perform the aforementioned common garden experiment with the RGA1 natural variants. Aim 2. In the next year, our major focus will be on analyzing the XLG CRISPR edited lines. Our initial analyses will focus on the identification of successful editing events. Regenerated transgenic rice will be analyzed for the presence of mutations in the target sites by Sanger sequencing of the target regions and analysis of the sequence files using DSCODEM online software (http://skl.scau.edu.cn/dsdecode/), which automatically decodes sequencing chromatograms with biallelic, heterozygous, and homozygous mutations into allelic sequences. Candidate transgenic lines with a stable mutation will be grown for T2 to T3 generations to conduct phenotyping of agronomic traits. This phenotypic information on XLG null mutants will then inform on which XLG natural variants are most likely to be of agronomic interest, which we will then target for subsequent phenotypic analyses in common garden experiments. Aim 3. Dr. Henry, our IRRI collaborator, will be visiting Penn State University in March 2020, and we will make plans for the next field trial then.
Impacts
What was accomplished under these goals?
This research focuses on integrated approaches to manipulate the Gα genes (RGA1, four XLGs) of rice heterotrimeric G proteins toward the improvement of rice architecture and reproductive development under both well-watered and water-limited conditions, thereby improving yield, harvest index, and drought resistance. Progress on each Aim is described below. Aim 1. The RGA1 knockout exhibits advantageous vegetative traits such as semi-dwarf stature and drought survival but some disadvantageous reproductive traits, including smaller seeds and reduced panicle exsertion. We are aiming to drive panicle-specific expression of wild-type RGA1 in the d1 (harboring a null mutation of the sole rice canonical Gα subunit gene, RGA1) mutant background. In addition, we have initiated a new approach. In collaboration with Prof. Yiping Qi at the University of Maryland, we plan to use CRISPR to edit the RGA1 promoter in wild-type plants, which may confer organ-specific expression. In order to design these CRISPR constructs, we have performed Sanger sequencing of the RGA1 promoter region in Nipponbare (the reference genome) and IR64 (an agronomically relevant cultivar), and we will target sequences that are identical in both cultivars. In another approach, we have determined that within the 3K sequenced rice genomes there are seven RGA1 haplotypes that alter RGA1 protein sequence, as well as six intronic RGA1 SNPs. We have correlated this natural variation with phenotypic assessment of the 3K population as described at the IRRI SNP-Seek database (https://snp-seek.irri.org/). We are about to order from IRRI seeds of cultivars showing significant correlation of RGA1 variants with variation in agronomic traits. In particular, we have seen statistically significant correlations with panicle exsertion, leaf width, and seed sphericity, which are of interest given that d1, the complete null mutant of RGA1, has broader leaves, reduced panicle exsertion, and smaller, rounder seeds than wild-type. We will grow cultivars with the key RGA1 haplotypes together in a "common garden" experiment in the Penn State greenhouse and phenotype them for the agronomic traits known to be affected by the d1 null mutation of RGA1. Aim 2. A major focus in this first year of funding has been on producing the constructs for CRISPR-based knockout of the four rice XLG genes: CRISPR design and vector construction: To produce targeted knockout lines of rice XLGs for subsequent phenotypic analysis, we used the CRISPR/Cpf1 genome editing system. We chose the CRISPR/Cpf1 system due to its reported higher specificity, high frequency of biallelic conversion and potential for multiplex targeting of a single gene or multiple genes using a relative smaller construct (Tang et al., 2017, DOI: 10.1038/nplants.2017.18; Wang et al., 2017, DOI: 10.1016/j.molp.2017.03.001). We identified CRISPR RNA (crRNA) sequences for all four rice XLGs using the Benchling CRISPR design tool (https://benchling.com/crispr), which scans the genomic sequence of interest for CRISPR target sites that are 24bp long and upstream of the PAM sequence (TTTN) required for the Cpf1 nuclease recognition. The target sites with the highest specificity were chosen and were analyzed using BLAST to confirm the absence of off-target matches. We are performing single and multiple targeting of the rice XLGs to produce short indels, large deletions, and truncations. Two types of CRISPR/CpF1 systems are being used: the pCRISPR_ribozyme system based on Tang (2017, DOI: 10.1038/nplants.2017.18) and the pCRISPR_SSTU system based on Wang (2017, DOI: 10.1016/j.molp.2017.03.001 ). For the pCRISPR_ribozyme, each XLG concatenated with direct repeat (DR) sequences was introduced into the pYPQ141 vector, encoding the crRNA expression array and the ribozyme system. Sequence confirmed clones of pYPQ141_crRNA were used for the final Gateway cloning assembly with pYPQ230, encoding the LbCpf1 nuclease, and pYPQ203, containing the T-DNA vector backbone. For the pCRISPR_SSTU system, each XLG concatenated with DR repeats and with BamHI overhangs was introduced into pSSTU_LbCpF1 and pSSTU_FnCpF1 CRISPR constructs via the T4 DNA ligation system (Wang et al., 2017). The pCRISPR_SSTU is driven by the Zea mays ubiquitin promoter to produce a single transcript, which makes it easier to transform in Agrobacterium. In summary, four CRISPR constructs were built with a single target per XLG, five CRISPR constructs were built with multiple targets per XLG, and another CRISPR construct was built that contains a quadruple target for all XLGs. Rice transformation: For both CRISPR systems, sequence confirmed clones were used for Agrobacterium-mediated rice transformation of Nipponbare and Nipponbare-d1 calli (harboring a null mutation of the sole rice canonical Gα subunit gene, RGA1) induced from mature embryos, following the rice transformation protocol of Nishimura (2006; DOI: 10.1038/nprot.2006.469). Table 1 summarizes the status of each rice transformation. Currently, pSSTU_FnCpF1_OsXLG1_MT Nipponbare rice transformation regenerated 11 independent transgenic events. The pCRISPR_OsXLG lines with single targets are in the regeneration stage, while the pSSTU_FnCpF1_OsXLG constructs with multiple targets are in the process of rice transformation. Table 1. Summary of CRISPR-XLGs rice transformation. Construct name: pCRISPR_LbCpF1_OsXLG3A_ST Targeted gene: OsXLG3A Construct type: One target site Status: Regeneration stage Construct name: pCRISPR_LbCpF1_OsXLG3B_ST Targeted gene: OsXLG3B Construct type: One target site Status: Regeneration stage Construct name: pCRISPR_LbCpF1_OsXLG3C_ST Targeted gene: OsXLG3C Construct type: One target site Status: Regeneration stage Construct name: pCRISPR_LbCpF1_OsXLG1_ST Targeted gene: OsXLG1 Construct type: One target site Status: Regeneration stage Construct name: pCRISPR_LbCpF1_control Targeted gene: empty Construct type: control Status: Regeneration stage Construct name: pSSTU_FnCpF1_OsXLG3A_MT Targeted gene: OsXLG3A Construct type: Multiple target sites Status: Rice transformation awaiting Construct name: pSSTU_FnCpF1_OsXLG3B_MT Targeted gene: OsXLG3B Construct type: Multiple target sites Status: Rice transformation awaiting Construct name: pSSTU_FnCpF1_OsXLG3C_MT Targeted gene: OsXLG3C Construct type: Multiple target sites Status: Rice transformation awaiting Construct name: pSSTU_FnCpF1_OsXLG1_MT Targeted gene: OsXLG1 Construct type: Multiple target sites Status: T0 molecular characterization Construct name: pSSTU_FnCpF1_OsXLGQUAD Targeted gene: All OsXLGs Construct type: One target site per XLG Status: Rice transformation awaiting Construct name: pSSTU_FnCpF1_control Targeted gene: control Construct type: control Status: Rice transformation awaiting XLG natural variants: We have also identified natural variants of XLGs that are associated with agronomic traits. In particular, we have used our custom HaploHammer software (https://github.com/StructureFold2/HaploHammer) to identify XLG haplotypes within the 3K rice genomes. We have determined that within the 3K genomes there are 29 haplotypes of OsXLG3a, 29 haplotypes of OsXLG3b, 11 haplotypes of OsXLG3c, and 35 haplotypes of OsXLG1 with altered protein sequence. Variants of OsXLG3C and OsXLG1 that encode deletions of almost the entire Gα region are of particular interest. Aim 3. Our collaborator at IRRI, Dr. Amelia Henry, has performed one field trial of d1 drought resistance under moderate drought conditions. In this field trial, d1 showed higher photosynthesis, higher stomatal conductance, and greater root length than the corresponding wild type, as well as smaller shoot biomass (as expected for this dwarfing mutation) which led to increased harvest index relative to wild-type. However, d1 did not yield better than wild-type on a seed biomass basis.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Ferrero-Serrano �., Cantos C., and Assmann SM. 2019. The Role of Dwarfing Traits in Historical and Modern Agriculture with a Focus on Rice. Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect. a034645.
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