Source: COLD SPRING HARBOR LABORATORY ASSOCIATION, INC submitted to
ROLE OF HETEROTRIMERIC G PROTEINS IN MAIZE DEVELOPMENT AND IMMUNITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1014839
Grant No.
2018-67013-27420
Project No.
NY.W-2017-06299
Proposal No.
2017-06299
Multistate No.
(N/A)
Program Code
A1152
Project Start Date
Jan 1, 2018
Project End Date
Dec 31, 2021
Grant Year
2018
Project Director
Jackson, D. P.
Recipient Organization
COLD SPRING HARBOR LABORATORY ASSOCIATION, INC
1 BUNGTOWN RD
COLD SPRING HARBOR,NY 11724-2209
Performing Department
Plant Biology
Non Technical Summary
Cereal crops provide the majority of our food and feed, and plant architecture is a key determinant of yield. Maize is one of the most important crops worldwide, due to the high yielding ear, and understanding the developmental mechanisms that generate this structure may provide basic knowledge to enhance productivity. However, crop yields are also severely impacted by immune responses to disease agents. A class of signaling molecules, heterotrimeric G proteins, have been found to function in maize inflorescence development, and in immunity. Prior work established that the maize COMPACT PLANT2 gene encodes the alpha subunit of a heterotrimeric G protein that interacts genetically and biochemically with the FASCIATED EAR2 clavata receptor. These results challenged the standard dogma of G protein- receptor interactions, and have wide ranging implications in other areas of plant biology, because LRR receptors are very common in plants and function in many other processes important to plant productivity, including defense. The proposal will investigate phenotypes of related G protein mutants, including in G beta and eXtra Large G proteins. These genes control yield traits as well as immune responses in maize, and could be applied to enhance crop productivity by understanding the tradeoff between growth and immunity. Crop improvements could be envisioned using the genes studied in this proposal in genome editing or conventional breeding approaches.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20615101050100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1510 - Corn;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
This proposal investigates the roles of heterotrimeric G proteins in maize inflorescence development and immune responses, to ask if it these genes can benefit traits of agricultural importance. Plant development and immune responses are major contributors to crop yields, and recent work has found evidence for tradeoff between immune signaling and growth and development, since plants defending themselves from pathogens limit the resources that they put into growth. However, the extent to which such tradeoffs occur in crop plants has received little attention, and here we will investigate a conserved set of signaling proteins that function in development and immunity in maize. We will investigate these findings with the following major goals:(1) To characterize the role of the CT2 gene and related ZmXLG genes in maize growth, inflorescence development and seed productivity,(2) To investigate the role of maize G beta in maize defense and development, and(3) to identify agronomic traits associated with variation in maize G protein genes.
Project Methods
Several complementary methods will be used to achieve the project goals:1. G alpha and ZmXLG phenotypes and signaling mechanisms. Here we will characterize mutations in a new receptor protein that we have found to interact with CT2/ G alpha. We will also characterize the roles of ZmXLGs, G alpha related genes, and potential G protein signaling associated genes, such as COLD1, in maize development. Mutations made using CRISPR/Cas9 genome editing will be characterized using standard genetic methods, such as construction and analysis of double mutants. Data analysis will use microscopy and quantification of phenotypes, and routine statistical tests.2. The role of maize G beta in defense and development.Here we will investigate the developmental roles of the maize G beta gene, using reporter genes and CLE peptide response assays. We will also characterize the immunity phenotypes of G beta, using immune markers and metabolic profiling, and by cloning a genetic suppressor that we identified in the maize CML103 inbred background. In addition, we will make an epitope tagged G beta transgene and use immunoprecipitation coupled with mass spectroscopy to find interacting proteins that could provide insights into how this protein provides crosstalk between development and defense pathways in maize.3. Agronomic traits of maize G protein alleles. Here we will test if variation in the maize Ga, XLG or G beta genes can impact maize yield traits. Various alleles that we have generated will be tested for their potential in increasing maize yields, for example by increasing kernel row number. Field trials with random design and statistical testing will be used to test the effects of the new alleles on maize seed production and yield. We will also collaborate with the Balint Kurti group (USDA-NCSU) to evaluate the role of G protein mutants in biotic stress responses.For all experiments, output(s) will beevaluated by data analysis, including appropriate statistical analyses, and by presentation to target audiences for discussion in seminars and in submitted manuscripts.

Progress 01/01/19 to 12/31/19

Outputs
Target Audience:The target audience for this project will be academic scientists, who are interested in crop genetics, defense and yield traits, and industry scientists and breeders working to improve maize productivity. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher, Qingyu Wu, was trained in maize genetics, biochemistry, and imaging. He left last summer to take up a faculty position at the Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China. A new post doc, Thu Tran, joined the project in October, and has a background in maize genetics but is being trained in developmental studies and is now leading the project. How have the results been disseminated to communities of interest?Meetings / Seminars (PI): Jackson presented work from the project in Departmental seminars and conferences including at U Mass, Amherst, Feb 2019, Plant Stem Cell Symposium, Sendai, Japan May 11th- 13th, 2019, Huazhong Agricultural University Conference on Crop Genomics, Wuhan, China, June 2-5, 2019, Society for In Vitro Biology Meeting, Tampa, Florida, June 10- 12, 2019, FASEB Conference on Plant Development, New York, July 29- Aug 2, 2019, US- Mexico Plant Molecular Biology Meeting, Merida, Mexico, Oct 28- 31, 2019. He also organized the CSHL Cereal Genomics workshop in October 2019, funded by additional funds form USDA- NIFA, that trained 16 international students in state of the art genomics methods and theory. What do you plan to do during the next reporting period to accomplish the goals?The project goals are progressing according to the original plan, no major changes are anticipated.

Impacts
What was accomplished under these goals? 1. Galpha and ZmXLG phenotypes and signaling mechanisms. Progress: CT2/ Galpha interacts with FEA2, and ct2 mutants are insensitive to CLV3 peptide, indicating that CT2 acts in CLV signaling. However, FEA2 and CT2 proteins do not interact directly, but do interact in immunoprecipitation assays with an LRR receptor-like kinase, ZmIMK1 that is expressed in developing inflorescences. Knocking out ZmIMK1 and its paralog, ZmIMK2 did not show any obvious inflorescence phenotype. This could be due to functional redundancy.To solve this problem, we made a dominant negative ZmIMK1 construct by deleting its kinase domain, and transformed this construct into maize and are currently scoring inflorescence meristem phenotypes to understand the role of ZmIMK in maize inflorescence development. We also asked if GTPase activity of Galpha is important for its function. Studies in Arabidopsis suggest that Galpha is constitutively active, so we transformed a GTPase null allele into maize, and backcrossed to ct2 mutants, where we found that it worked like a weak allele. This told us that GTP hydrolysis is important for Galpha function in plants, and interestingly we found that this new allele could increase kernel row number, an important yield trait. This was published (Wu et al, PLOS Genetics 2018). Maize also has three non-canonical Galphas, extra-large GTP binding proteins (ZmXLGs). We knocked out all three by CRISPR-Cas9, and found that they enhance semi-dwarf and SAM size phenotypes in the ct2 mutant background. However, Zmxlg triple mutants were seedling lethal. We stained the triple mutants using trypan blue, and they had strong staining, suggesting cell death. We also measured the expression of two immune marker genes, PATHOGENESIS-RELATED PROTEIN 1 (PR1) and PR5, and found both were significantly up-regulated in the triple mutants, indicating that the lethality is due to over-activation of the immune system. These findings were also published, (Wu et al, PLOS Genetics 2018), and we recently initiated a new collaboration with Dr. Tiffany Jamann, Univ. Illinois, to test maize defense responses in these lines. The role of maize Gbeta in defense and development. Progress: Arabidopsis Gbeta (agb1) null mutants are fully viable, but have compromised immunity and are more susceptible to pathogens, presumably because Gbeta?acts in PAMP signaling in association with immune receptors. In contrast, we found that the corresponding maize mutants are lethal early in seedling development. Our working hypothesis is that lethality is caused by an autoimmune reaction, as mutants germinated under sterile conditions show the same lethal phenotype. We hypothesize that the reason for the lethal phenotype is that ZmGB1 protein is a "guardee". This occurs when a host protein is the target of a pathogen effector, and is "guarded" by a plant resistance (R) protein acting in effector triggered immunity (ETI). A prediction of this hypothesis is that the autoimmune phenotypes may be suppressed in some genetic backgrounds, because R genes are highly polymorphic between accessions. We therefore crossed Zmgb1 (using viable heterozygotes) to the diverse maize NAM founder lines, and then scored phenotypes in the F2s. We found that Zmgb1 lethality is suppressed in the tropical CML103 background. We also found that in this suppressed background, the mutants had significantly larger SAMs and fasciated ear and tassel inflorescence meristems, suggesting ZmGB1 plays an important role in meristem development. To gain insights into the mechanism of Zmgb1 lethality, we mapped the CML103 suppressor of Zmgb1 lethality. Using bulk segregant analysis, we mapped the suppressor between 200 to 205 Mb on chromosome 5, in a region with only one R gene. To ask if this gene is responsible for the lethality, we used CRISPR-Cas9 to knock it out. We obtained CRISPR edited alleles, and backcrossed with Zmgb1. We are now making F2 populations, to ask if the double mutants can survive when the R gene is mutated. As a backup plan, we also tried to knock out the gene responsible for Zmgb1 lethality in B73 using EMS mutagenesis. We treated B73 pollen with EMS, and crossed onto viable heterozygous Zmgb1 ears. The M1 plants were selfed, and we screened M2 families, to ask if Zmgb1 lethality can be suppressed. Indeed, we recovered some viable M2 plants, and confirmed that they are homogygous Gbeta mutant, suggesting they are real suppressors. We localized ZmGB1, using a translation fusion to YFP- streptavidin-binding peptide (SBP) tag under the control of its native promoter and terminator. The transgene was able to complement the lethal and developmental phenotype of Zmgb1 mutants, suggesting that tagging did not affect its function. We are bulking up these lines and plan to use them to identify protein interactors using IP mass spectroscopy. Independently of these studies, we mapped another fasciated mutant, fea*GN183, from an EMS population generated by Gerry Neuffer. Remarkably, fea*GN183 contains an amino acid change in a conserved residue of the same gene, ZmGB1. This weak EMS allele is a great tool to further explore the dual role of Gbeta in development and immunity. Our results on the crosstalk between developmental and immune signaling conditioned by Gbeta was published this past year in PNAS. Agronomic traits of maize G protein alleles. Progress: Several studies have found enhanced yield associated with heterotrimeric G protein alleles. We have collected an array of weak and strong G protein mutants in maize, and are planning to measure yield traits and biotic stress responses of different G protein alleles and combinations. In the past summer, we tested ct2 and weak Gbeta mutants, in collaboration with Peter Balint Kurti. As expected from results in Arabidopsis, we didn't see any obvious effect for CT2 mutants, and the Gbeta mutants had too many spontaneous lesions to assess their disease symptoms, so we will repeat these tests using Gbeta mutants introgressed into the CML103 suppressive background, as well as with our new XLG CRISPR mutants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wu, Q., Xu, F., Liu, L., Char, S., Ding, Y, Je, B., Schmelz, E., Yang, B., Jackson, D. (2019) The maize heterotrimeric G protein beta subunit controls shoot meristem development and immune responses. Proc. Natl. Acad. Sci. USA doi/10.1073/pnas.1917577116.


Progress 01/01/18 to 12/31/18

Outputs
Target Audience:The target audience for this project will be academic scientists, who are interested in crop genetics, and yield traits, and industry scientists and breeders working to improve maize productivity. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher, Qingyu Wu, has been trained in maize genetics, biochemistry, and imaging. How have the results been disseminated to communities of interest?Meetings / Seminars (PI): Jackson presented work from the project in Departmental seminars and conferences including Univ Toronto,Canada, Univ Lausanne, Switzerland, Gatersleben Institute, Germany, The Agriculture Genetics Institute, Ha Noi, Viet Nam, Molecular Plant Conference, Xian, China, The 29th International Conference on Arabidopsis Research (ICAR) 2018, Turku, Finland, The Max Plank Institute, Golm, Germany, IV European Workshop on Plant Peptides & Receptors, Antequera (Málaga), Spain, The John Innes Institute, Norwich, UK, Univ. North Texas, and U Mass, Amherst. What do you plan to do during the next reporting period to accomplish the goals?The project goals are progressing according to the original plan, no major changes are anticipated.

Impacts
What was accomplished under these goals? Ga and ZmXLG phenotypes and signaling mechanisms. Progress: We previously found that CT2/Ga interacts with FEA2 using in vivo co-IPs, and ct2 mutants are partially insensitive to CLV3 peptide treatment, indicating that CT2 is involved in maize CLV signaling. However, FEA2 and CT2 proteins do not interact directly, but do interact in immunoprecipitation assays with an LRR receptor-like kinase, ZmIMK1 that is expressed in developing inflorescences. Knocking out ZmIMK1 and its paralog, ZmIMK2 did not show any obvious inflorescence phenotype. This could be due to functional redundancy, since phylogenetic analysis revealed that there are 19 additional LRR-RLKs closely related to ZmIMK1 and 2. To solve this problem, we made a dominant negative ZmIMK1 construct by deleting its kinase domain driven by a constitutive promoter, pEF1A. We transformed this construct into maize and will score the inflorescence meristem phenotypes to understand the role of ZmIMK in maize inflorescence development. We also asked if GTPase activity of Ga is important for its function. Studies in Arabidopsis suggest that Ga is constitutively active, so we made a constitutively active allele with a single amino acid change predicted to block GTP hydrolysis. We transformed this GTPase null allele into maize, and backcrossed to ct2 mutants, where we found that it worked like a weak allele. This told us that GTP hydrolysis is important for Ga function in plants, and interestingly we found that this new allele could increase kernel row number, an important yield trait. This work is now published (Wu et al, PLOS Genetics 2018). In addition to the single canonical Ga, CT2, maize also has three non-canonical Gas, extra-large GTP binding proteins (ZmXLGs). We knocked out all three by CRISPR-Cas9, and found that they enhance semi-dwarf and SAM size phenotypes in the ct2 mutant background. However, Zmxlg triple mutants were seedling lethal. We stained the triple mutants using trypan blue, and they had strong staining, suggesting cell death. We also measured the expression of two immune marker genes, PATHOGENESIS-RELATED PROTEIN 1 (PR1) and PR5, and found both were significantly up-regulated in the triple mutants, indicating that the lethality is due to over-activation of the immune system. Since maize Gb mutants also show immune symptoms (see next section), we are also crossing the Zmxlg triple mutants into different inbreds to see if lethality is suppressed. Heterotrimeric G protein signaling in Arabidopsis relies on the REGULATOR OF G PROTEIN SIGNALING (RGS) gene, which encodes a seven-pass transmembrane protein, however most grasses lack RGS orthologs. In rice, the COLD1 gene is not related by sequence, but functions as an RGS. COLD1 functions in temperature responses in rice, but the mutants were not analyzed for developmental phenotypes. We generated knockouts of the maize COLD1 ortholog using CRISPR-Cas9; however did not find obvious meristem phenotypes, nor did they affect ct2 mutant phenotypes. We plan to assess cold stress phenotypes using this mutant. 2. The role of maize Gb in defense and development. Progress: Arabidopsis Gb (agb1) null mutants are fully viable, but have compromised immunity and are more susceptible to pathogens, presumably because Gb acts in PAMP signaling in association with immune receptors. In contrast, we found that the corresponding maize mutants are lethal early in seedling development. Our working hypothesis is that lethality is caused by an autoimmune reaction, as mutants germinated under sterile conditions show the same lethal phenotype. We hypothesize that the reason for the lethal phenotype is that ZmGB1 protein is a "guardee". This occurs when a host protein is the target of a pathogen effector, and is "guarded" by a plant resistance (R) protein acting in effector triggered immunity (ETI). A prediction of this hypothesis is that the autoimmune phenotypes may be suppressed in some genetic backgrounds, because R genes are highly polymorphic between accessions. We therefore crossed Zmgb1 (using viable heterozygotes) to the diverse maize NAM founder lines, and then scored phenotypes in the F2s. We found that Zmgb1 lethality is suppressed in the tropical CML103 background. We also found that in this suppressed background, the mutants had significantly larger SAMs and fasciated ear and tassel inflorescence meristems, suggesting ZmGB1 plays an important role in meristem development. To gain insights into the mechanism of Zmgb1 lethality, we mapped the CML103 suppressor of Zmgb1 lethality. Using bulk segregant analysis, we mapped the suppressor between 200 to 205 Mb on chromosome 5, in a region with only one R gene. To ask if this gene is responsible for the lethality, we used CRISPR-Cas9 to knock it out. We obtained CRISPR edited alleles, and backcrossed with Zmgb1. We are now making F2 populations, to ask if the double mutants can survive when the R gene is mutated. As a backup plan, we also tried to knock out the gene responsible for Zmgb1 lethality in B73 using EMS mutagenesis. We treated B73 pollen with EMS, and crossed onto viable heterozygous Zmgb1 ears. The M1 plants will be selfed to produce M2 families, to ask if Zmgb1 lethality can be suppressed by mutations in some families. To examine the localization of ZmGB1, we made a translation fusion of ZmGB1 genomic sequence and YFP- streptavidin-binding peptide (SBP) tag under the control of its native promoter and terminator. The construct was transformed into maize and backcrossed twice to Zmgb1 heterozygotes in B73. The transgene was able to complement the lethal and developmental phenotype of Zmgb1 mutants, suggesting that tagging with YFP-SBP did not affect its function. YFP-SBP-ZmGB1 protein was detected in both cytosol and on the plasma membrane, confirmed by co-localization with FM4-64 after plasmolysis, throughout the meristem. We also fused a 3XFLAG-2XStrep tag at the N-terminus of ZmGB1 and this construct also fully complemented Zmgb1 mutants. We will use these transgenic lines to identify protein interactors using IP mass spectroscopy. Independently of these studies, we mapped another fasciated mutant, fea*GN183, from an EMS population generated by Gerry Neuffer. Remarkably, fea*GN183 contains an amino acid change in a conserved residue of the same gene, ZmGB1. This allele is viable, though makes some necrotic lesions. We hypothesize that this allele can make a Gb protein that allows it to mostly evade the R gene autoimmunity, yet is non-functional in its developmental role. The amino acid change in this allele is predicted to be in a region that makes an interface with Ga. We tested this hypothesis using yeast-three-hybrid experiments, and indeed found that the Gb-fea*183 protein failed to form a complex with the alpha subunit, CT2, and the gamma subunit, RGG1. Using double mutant analysis, we found that FEA2 is epistatic to ZmGB1, and that ZmGB1 functions in the same pathway as CT2, suggesting that ZmGB1 and CT2 function together downstream of FEA2. This weak EMS allele is a great tool to further explore the dual role of Gb in development and immunity. 3. Agronomic traits of maize G protein alleles. Progress: Several studies have found enhanced yield associated with heterotrimeric G protein alleles. We have collected an array of weak and strong G protein mutants in maize, and are planning to measure yield traits and biotic stress responses of different G protein alleles and combinations. In the past summer, we backcrossed the ct2;Zmxlg mutants to the B73 background, and made homozygous populations. In addition, we are further introgressing Zmgb1 mutants to the CML103 background. These populations will be used for yield and biotic stress field tests, in collaboration with Peter Balint Kurti and Nick Lauter.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wu, Q., Regan, M., Furukawa, H., Jackson D. (2018). Role of heterotrimeric Ga proteins in maize development and enhancement of agronomic traits. PLoS Genetics 14(4): e1007374.
  • Type: Book Chapters Status: Published Year Published: 2018 Citation: Wu, Q. and Jackson, D. (2018) Detection of MAPK3/6 Phosphorylation During Hypersensitive Response (HR)-Associated Programmed Cell Death in Plants. Methods in Molecular Biology. 1743:153-161.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wu, Q., Xu, F., Jackson, D. (2018). All together now, a magical mystery tour of the maize shoot meristem. Current Opinion in Plant Biology 45: 26-35.