Source: COLD SPRING HARBOR LABORATORY ASSOCIATION, INC submitted to
HETEROTRIMERIC G PROTEIN SIGNALING IN MAIZE DEVELOPMENT AND PRODUCTIVITY.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1000953
Grant No.
2014-67013-21566
Project No.
NY.W-2013-02198
Proposal No.
2013-02198
Multistate No.
(N/A)
Program Code
A1101
Project Start Date
Dec 1, 2013
Project End Date
Nov 30, 2017
Grant Year
2014
Project Director
Jackson, D. P.
Recipient Organization
COLD SPRING HARBOR LABORATORY ASSOCIATION, INC
1 BUNGTOWN RD
COLD SPRING HARBOR,NY 11724-2209
Performing Department
OSMC
Non Technical Summary
Cereal crops provide the majority of our food and feed, and understanding the developmental mechanisms that generate the seed bearing inflorescences is thus critical to provide basic knowledge to enhance crop productivity. Maize is one of the most important crops worldwide, due to the high yielding ear. This project will study the mechanisms governing ear development. A maize mutant, compact plant2 (ct2), exhibits ear growth defects. The CT2 gene encodes the a subunit of a predicted heterotrimeric G protein, a well-known membrane-signaling complex in mammalian systems, whose function in plants is not well understood. The project enhances knowledge by challenging the standard dogma of G protein- receptor interactions, and could have wide ranging implications in other processes important to plant productivity, including defense. The project will investigate diverse phenotypes of ct2 mutants, will explore mechanistic aspects of how CT2 functions in ear growth, and will identify genetic variation that will have potential applications for control of crop plant growth and productivity. Crop improvements could be envisioned using the genes studied in this proposal in biotechnology or conventional breeding approaches.
Animal Health Component
(N/A)
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
The goals of this project are to investigate a potentially new signaling mechanism in maize inflorescence shoot development, and to ask if it has applications in traits of agricultural importance. We identified a maize mutant compact plant2 (ct2), which exhibits meristem proliferation defects similar to CLAVATA mutants, such as maize fasciated ear2 (fea2). CT2 encodes the a subunit of a predicted heterotrimeric G protein, a well-known membrane-signaling complex, and we have found that CT2 interacts genetically and biochemically with the FEA2 receptor. Heterotrimeric G proteins are well characterized in non-plant systems, where they transmit signals from G protein coupled receptors (GPCRs), which are typically 7-pass transmembrane proteins. Our results in plants challenge this dogma, because the CLAVATA receptors are predicted to be single pass transmembrane proteins. We will investigate these findings with the following major goals: (1) To characterize the role of the CT2 gene in maize growth, inflorescence development and seed productivity, (2) To use advanced imaging and biophysical methods to test the hypothesis that CT2/ G? interacts directly with a CLAVATA receptor in plants, and (3) to identify additional factors that contribute to CT2-mediated inflorescence development.
Project Methods
Several complementary methods will be used to achieve the project goals: 1. Genetic analysis of maize heterotrimeric G protein mutants. Here we will ask what other pathways and/or processes might be controlled by CT2/G? and other members of the heterotrimeric G protein complex in maize. Standard genetic methods, such as construction and analysis of double mutants will be used, and we will also collaborate with other scientists to assess root architecture changes and response to pathogens. Data analysis will use routing statistical tests conducted with our collaborators. 2. CT2/Gα-FEA2 interactions, and mechanism and isolation of novel interactors. Here we will investigate the mechanism of CT2/Gα interaction with the FEA2 receptor, using Fluorescence Resonance Energy Transfer (FRET) assays. These experiments will be performed in collaboration with the Scarlata lab at Stonybrook University, who are expert in biophysical analyses of Gα interactions in mammalian cells, and they will assist us with data analysis and evaluation. 3. CT2 natural variation and maize yield traits. Here we will test if variation in the CT2 locus can impact maize yield traits. For this, we will use TILLING to identify weak alleles of CT2. Such alleles could provide beneficial effects, such as an increase in kernel row number. We will use the maize TILLING facility at Purdue University, and molecular genetic methods to analyze sequences and to interpret their effects. Filed trials with random design and statistical testing will be used to test the effects of the new alleles on maize seed production and yield.

Progress 12/01/16 to 11/30/17

Outputs
Target Audience:The target audience for this project were academic scientists, who are interested in crop genetics, and yield traits. Also industry scientists and breeders working to improve maize productivity. Changes/Problems:No major problems have been encountered. Some unexpected results, for example lethality of G beta mutants, were exploited to expand the impact of our research 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?Results from this project have been presented to academic and industry scientists in seminars and posters, as follows: Conference abstracts: Wu QY, Jackson D, Role of heterotrimeric G proteins in maize development and immune responses, Plant Genomes & Biotechnology: From Genes to Networks, Cold Spring Harbor, NY, December 2017 Wu QY, Char SN, Yang B, Jackson D. Manipulation of heterotrimeric G proteins alters maize development, immune responses and agronomic traits. Maize Genetics Conference, St. Louis, MO, March 2017 In the past year, results from this project were presented in invited talks at: MU Pioneer Symposium, Indiana Univ., Japan Society for Plant Physiology, CRISPR conference, HZAU, Wuhan, China, VIB Conference 'At the forefront of Plant Biology', International Botanical Congress, Shenzhen, and at Inari Ag, Boston, MA. The project PI made a podcast in the CSHL "basepairs" series where he discussed the idea of improving corn yield through genetics, and framed in the idea that world population is increasing (the episode was called "The people problem"). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (1) To characterize the role of the CT2 gene in maize growth, inflorescence development and seed productivity, Having completed an in depth analysis of development of G alpha mutants, we moved on to explore additional G proteins. We previously completed an assessment of disease responses in maize G alpha mutants, in collaboration with Peter Balint Kurti, USDA, NCSU. We did not find any obvious effect on disease response. In Arabidopsis, a stronger disease phenotype is observed with G beta, so we collaborated with Bing Yang at Iowa State University to use CRISPR to knockout the gene. We found that the Zmgb1 null alleles are lethal at a very early stage of seedling development. The Zmgb1 mutants showed over-accumulation of H2O2and salicylic acid, constitutive activation of MAP-kinase, and up-regulation of PR1 (PATHOGENESIS- RELATED1) and PR5, two immune marker genes. These results suggest that ZmGB1 mutation causes autoimmune symptoms. Since the Zmgb1 null alleles die at a very early stage, we were unable to study shoot meristem and inflorescence phenotypes. Therefore, we introgressed Zmgb1 null mutants into different genetic backgrounds to see if the lethal phenotype can be rescued. Indeed, the lethal phenotype was partially suppressed in some genetic backgrounds, and the Zmgb1 null alleles could grow to the reproductive stage. In the suppressed background, the Zmgb1 mutants had larger SAMs and fasciated inflorescence meristems in both ear and tassel. These results suggest that ZmGB1 plays important roles in both meristem development and immune response. In addition to CT2, the canonical G protein alpha subunit, maize also has 3 non-canonical G alphas, named EXTRA LARGE GTP-BINDING PROTEINS (XLGs), which contain the G alpha domain at the C-terminus. We first demonstrated that the three ZmXLGs function in a heterotrimeric G protein complex by showing their interaction with a Gbeta gamma dimer in the a yeast 3 hybrid system. We next used CRISPR/Cas9 to knockout the three ZmXLGs and obtained different alleles for each. Single ZmXLG mutants did not alter development; whereas knocking out any two led to a modest but significant reduction in plant height, but did not affect SAM size, indicating that loss of any two ZmXLGs can be partially compensated by other XLGs or by CT2. Surprisingly, triple ZmXLG mutant plants showed a striking developmental arrest phenotype, and were lethal at the seedling stage. We also found that mutation of any two ZmXLGs in the ct2 background dramatically enhanced their dwarf and enlarged SAM phenotypes. In contrast, although both CT2 and ZmXLGs are expressed in the inflorescence, ZmXLG knockouts did not enhance ct2 inflorescence fasciation phenotype, suggesting that CT2 is the major G alpha functioning in inflorescence meristem development. 2. To use advanced imaging and biophysical methods to test the hypothesis that CT2/ G? interacts directly with a CLAVATA receptor in plants, We proposed to study the mechanism of CT2/Gaplha interaction with the FEA2 receptor, using Fluorescence Resonance Energy Transfer (FRET) assays. These experiments were performed in collaboration with the Scarlata lab at Stonybrook University, who are expert in biophysical analyses of Gα interactions in mammalian cells, and they assisted us with data analysis and evaluation. We previously completed the proposed FRET and BiFC assays, but did not find a direct interaction between CT2 and FEA2, suggesting that a third protein bridges their interaction in planta. We therefore used IP-mass spec to identify CT2 and FEA2 interacting proteins, and found one common protein, a novel LRR kinase, that interacts with both FEA2 and CT2. BiFC experiments suggested that this novel LRR kinase directly interacts with CT2 via its intracellular kinase domain. We also expressed FEA2, CT2, and the novel LRR kinase in Sf9 insect cell lines. Our results showed that the novel LRR kinase can "pull down" both FEA2 and CT2 in the heterologous expression system, suggesting that the three proteins form a complex. We are using the Sf9 system to ask if FEA2 and the LRR kinase work together to perceive CLE peptides and activate CT2. The novel LRR kinase gene is highly expressed in inflorescence meristems, and it has a close paralog in maize. We obtained knockouts in both paralogs, however the double mutants did not show any obvious developmental phenotypes, suggesting there might be other genes with redundant roles. Our phylogenetic analysis identified 19 other maize LRR kinases in the same clade. Interestingly, 6 of them interact with CT2 based on our IP-mass spec results. Since it is not feasible to knock out 19 LRR kinases, we generated dominant negative variants of the LRR kinases by introducing point mutations in the context of a native construct, and are crossing with the null alleles to see meristem phenotypes. In addition, we are introgressing the double mutants into other 'fasciation sensitive' genetic backgrounds, for instance NC350 and W23, to see if we can find more obvious phenotypes. 3. To identify additional factors that contribute to CT2-mediated inflorescence development. The maize TILLING facility was no longer active, so we adopted an alternative approach. We generated a constitutively-active (GTPase-dead) transgenic version of CT2 (CT2CA) in maize by introducing the Q223L mutation in the context of a native construct, and we backcrossed transformed plants to the ct2 mutant background. To ask if the mutation abolished GTPase activity, we performed in vitro GTP-binding and GTPase activity assays using fluorescent BODIPY-GTP, where an increase in fluorescence over time corresponds to GTP binding, and a subsequent decrease corresponds to GTP hydrolysis. The result showed that the CT2CA protein had similar GTP-binding activity as CT2, but lacked GTPase activity. We further tested if CT2CA interacted with Gbeta gamma in a yeast-3-hybrid (Y3H) system. In contrast to CT2, we found that CT2CA did not interact with the Gbeta gamma dimer, despite being expressed at a similar level as CT2 in the yeast cells. Both results confirmed that CT2CA is indeed constitutively active and could no longer form a heterotrimeric complex with Gbeta gamma. Expression of CT2CA in a ct2 mutant background resulted in interesting phenotypes, including higher spikelet density and kernel row number, larger ear inflorescence meristems and smaller leaf angles compared with normal sibs. We also found that the ct2 ear fasciation phenotype is enhanced in the NC350 background and attempted to map the natural modifier. Our bulk segregant analysis suggests that there are multiple loci responsible for the modification of ct2 phenotypes. We are backcrossing the modified plants with ct2 in B73 background to dissect the loci that are responsible for the modified phenotypes of ct2 for fine-mapping the natural modifiers. In addition, we identified several uncloned EMS fasciation mutants that synergistically enhanced the fasciation phenotype of ct2, and we are also mapping these modifiers. Mapping these loci could provide additional alleles for breeding enhanced seed traits. As part of these efforts, collaborations have been fostered in study of disease responses in the maize G protein mutants, with Alisa Huffaker and Eric Schmelz, UCSD, Peter Balint Kurti, at North Carolina State University and Nicole Clay, at Yale, with Suzanne Scarlatta, Stonybrook University, in G protein signaling and Hiro Furukawa, CSHL in membrane protein biochemistry.

Publications

  • 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: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wu QY, Jackson D, Role of heterotrimeric G proteins in maize development and immune responses, Plant Genomes & Biotechnology: From Genes to Networks, Cold Spring Harbor, NY, December 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wu QY, Char SN, Yang B, Jackson D. Manipulation of heterotrimeric G proteins alters maize development, immune responses and agronomic traits. Maize Genetics Conference, St. Louis, MO, March 2017


Progress 12/01/15 to 11/30/16

Outputs
Target Audience:The target audience for this project will be academic scientists, who are interested in crop genetics, and yield traits. Also industry scientists and breeders is 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. In addition, a visiting MSC student from France, Kevin Kercmar, was trained in maize genetics and genomics, and a high school student, Shaina Zafar, is being trained in plant developmental biology. How have the results been disseminated to communities of interest?Results from this project have been presented in seminars and posters, as follows: Oral presentation (post doc): Wu QY, Bommert P, Jackson D. A maize leucine-rich repeat receptor-like kinase interacts with Ga and a CLVATA LRR receptor-like protein: a new play in shoot meristem regulation? Keystone Symposia Plant Receptor Kinase Meeting, Taos, NM, Feburary, 2015. Meeting abstract/poster presentations (post doc): Wu QY, Jackson D, Understanding the functions of maize heterotrimeric G proteins in meristem regulation. Maize Genetics conference. Jacksonville, FL, March, 2016. Meetings / Seminars (PI): In the past year, results from this project were presented in invited talks at: European Maize Conference, Hamburg, Germany (Plenary Speaker), Arabidopsis Conference, Korea, ComBio, Brisbane, Australia (Plenary Speaker), CSH Asia Conference, Awaji, Japan, Pioneer Conference, Univ. Missouri (Plenary Speaker), Univ. Indiana, Bloomington. 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, although I expect an acceleration of some of our genetic analyses by the application of CRISPR technology, which we recently showed to be highly effective in maize.

Impacts
What was accomplished under these goals? To characterize the role of the CT2 gene in maize growth, inflorescence development and seed productivity. Progress: We previously completed an assessment of disease responses in maize G alpha mutants, in collaboration with Peter Balint Kurti, NCSU. We did not find any obvious effect on disease response. In Arabidopsis, a stronger disease phenotype is observed with G beta, so we collaborated with Bing Yang at Iowa State University to use CRISPR to knockout the gene. We found that the Zmgb1 null alleles are lethal at a very early stage of seedling development. The Zmgb1 mutants showed over-accumulation of H2O2, constitutive activation of MAP-kinase, and up-regulation of PR1 (PATHOGENESIS-RELATED1) and PR5, two immune marker genes. These results suggest that ZmGB1 mutation causes autoimmune symptoms. In addition, the Zmgb1 mutants showed abnormal cell divisions, but meristems were normal. Although the null alleles of Zmgb1 were lethal, we were fortunate to isolate a 3-bp deletion allele using CRISPR, resulting in mutation of two conserved amino acids between the second and third WD domain (HK55Q). These mutants did not show any obvious phenotype, however, after we crossed the 3-bp deletion allele with the Zmgb1 null allele, our preliminary results suggest that the 3-bp deletion/ null allele plants develop higher kernel row numbers. We are also introgressing Zmgb1 null mutants into different genetic backgrounds to see if the lethal phenotype can be rescued. We also used CRISPR to knockout the CT2- related eXtra Large G proteins (XLGs), which have a G alpha domain at their C-terminus. There are 3 XLGs in maize, and we got different alleles for all three. Knocking out XLGs in a ct2 mutant background enhanced its dwarf phenotype. Surprisingly, the xlg123 triple mutants are also lethal in the seedling stage, although appear to be distinct to Zmgb1 null alleles. Our preliminary observation suggested that the triple XLG mutants have defects in primary root development. 2.) To use advanced imaging and biophysical methods to test the hypothesis that CT2/ G? interacts directly with a CLAVATA receptor in plants, Here we proposed to study the mechanism of CT2/Gα interaction with the FEA2 receptor, using Fluorescence Resonance Energy Transfer (FRET) assays. These experiments were performed in collaboration with the Scarlata lab at Stonybrook University, who are expert in biophysical analyses of Gα interactions in mammalian cells, and they assisted us with data analysis and evaluation. Progress: We previously completed the proposed FRET and BiFC assays, and do not find a direct interaction between CT2 and FEA2, suggesting that a third protein bridges their interaction in planta. We therefore used IP-mass spec to identify CT2 and FEA2 interacting proteins, and found one common protein, a novel LRR kinase, that interacts with both FEA2 and CT2. The BiFC experiments suggested that the novel LRR kinase directly interact with CT2 via its intracellular kinase domain. We also expressed FEA2, CT2, and the novel LRR kinase in Sf9 insect cell lines. Our results showed that the novel LRR kinase can "pull down" both FEA2 and CT2 in the heterologous expression system, suggesting that the three proteins might form a complex. We are using the Sf9 system to ask if FEA2 and the LRR kinase work together to perceive CLE peptides and activate CT2. The novel LRR kinase gene is highly expressed in inflorescence meristems, and it has a close paralog in maize. We obtained knockouts in both paralogs, however the double mutants did not show any obvious developmental phenotypes, suggesting there might be other genes with redundant roles. Our phylogenetic analysis identified 19 other maize LRR kinases in the same clade. Interestingly, 6 of them interact with CT2 based on our IP-mass spec results. Since it is not feasible to knock out 19 LRR kinases, we generated dominant negative variants of the LRR kinases by introducing point mutations in the context of a native construct, and expect to see meristem phenotypes for the dominant negative mutants. In addition, we are introgressing the double mutants into other 'fasciation sensitive' genetic backgrounds, for instance NC350 and W23, to see if we can get more obvious phenotypes. We also used phospho-proteomics to ask if CT2 is phosphorylated, and more interestingly, if CT2 phosphorylation occurs in response to CLV3 peptide treatment (collaboration with Steve Briggs, UCSD). We are studying if our newly identified LRR kinase affects the phosphorylation state of CT2, and how this could affect CT2 signaling. (3) To identify additional factors that contribute to CT2-mediated inflorescence development. Progress: The maize TILLING facility was no longer active, so we adopted an alternative approach. We generated a constitutively-active (GTPase-dead) transgenic version of CT2 (CA-CT2) in maize by introducing the Q223L mutation in the context of a native construct, and we backcrossed transformed plants to the ct2 mutant background. Using yeast-3-hybrid we showed that the Q223L mutation abolished the interaction between CT2 and the Gbg dimer, confirming that it is indeed constitutively active. Expression of CA-CT2 in a ct2 mutant background resulted in interesting phenotypes, including higher spikelet density and kernel row number, larger ear inflorescence meristems and smaller leaf angles compared with normal sibs. Furthermore, we are collaborating with Doreen Ware at Cold Spring Harbor Laboratory and Zhanguo Xin at USDA to get weak alleles of a G alpha ortholog in Sorghum, using their TILLING populations. We also screened for genetic modifiers of ct2 in the NAM parental inbreds, and have identified three enhancers and one fasciation suppressor. The fasciation suppressor does not affect other phenotypes of ct2, such as plant height, leaf width and erectness, indicating it might be an inflorescence specific gene. We observed the development of the modified ct2 ears at early stage by using scanning electron microscopy. Our bulk segregant analysis suggests that there are multiple loci responsible for the modification of ct2 phenotypes. We are backcrossing the modified plants with ct2 in B73 background to dissect the loci that are responsible for the modified phenotypes of ct2. Mapping these modifier loci could provide additional alleles for breeding enhanced seed traits.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Urano, D., Maruta, N., Trusov, Y., Stoian, R., Wu, Q., Liang, Y., Jaiswal, DK., Thung, L., Jackson, D., Botella, JR., Jones, AM. (2016). Saltational evolution of the heterotrimeric G protein signaling mechanisms in the plant kingdom. Science Signaling. 9(446): ra93
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Urano, D. and Miura, K. and Wu, Q. and Iwasaki, Y. and Jackson, D. and Jones, A. M. (2016) Plant Morphology of Heterotrimeric G protein Mutants. Plant Cell Physiology. 57(3):437-45


Progress 12/01/14 to 11/30/15

Outputs
Target Audience:The target audience for this project will be academic scientists, who are interested in crop genetics, and yield traits. Also 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. In addition, a visiting MSC student from France, Kevin Kercmar, was trained in maize genetics and genomics. How have the results been disseminated to communities of interest?Results from this project have been presented in seminars and posters, as follows: Oral presentation (post doc): Wu QY, Bommert P, Jackson D. A maize leucine-rich repeat receptor-like kinase interacts with Ga and a CLVATA LRR receptor-like protein: a new play in shoot meristem regulation? Keystone Symposia Plant Receptor Kinase Meeting, Taos, NM, Feburary, 2015. Meeting abstract/poster presentations (post doc): Wu QY, Bommert P, Jackson D. Understanding maize G? signaling in shoot meristems, are additional receptor-like proteins involved? Maize Genetics Conference. St. Charles, IL, March, 2015. Wu QY, Jackson D. Identification of natural modifiers of the maize heterotrimeric G protein ?-subunit mutant ct2. Plant Genomes & Biotechnology: From Genes to Networks. Cold Spring Harbor, NY, December, 2015 Meetings / Seminars (PI): First Annual Maize Conference, Wuhan, China (plenary speaker), Crops Conference, Alabama, FASEB Plant Development Conference, Vermont, invited seminars at Nagoya Univ, and Tokyo Univ, Japan, China Agricultural University, Yale. 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, although I expect an acceleration of some of our genetic analyses by the application of CRISPR technology, which we recently showed to be highly effective in maize.

Impacts
What was accomplished under these goals? A comparison of actual accomplishments with the goals established for the reporting period (where the output of the project can be expressed readily in numbers, a computation of the cost per unit of output should be submitted if the information is considered useful); This project investigates a new signaling mechanism in maize inflorescence shoot development, and its applications in traits of agricultural importance. A maize mutant compact plant2 (ct2), exhibits meristem proliferation defects similar to CLAVATA mutants, such as maize fasciated ear2 (fea2). CT2 encodes the a subunit of a predicted heterotrimeric G protein, a well-known membrane-signaling complex, and interacts genetically and biochemically with the FEA2 receptor. Heterotrimeric G proteins are well characterized in non-plant systems, where they transmit signals from G protein coupled receptors (GPCRs), which are typically 7-pass transmembrane proteins. Our results in plants challenge this dogma, because the CLAVATA receptors are predicted to be single pass transmembrane proteins. We proposed to investigate these findings with the following major goals: To characterize the role of the CT2 gene in maize growth, inflorescence development and seed productivity. Progress: We have completed an assessment of disease responses in the maize G alpha mutants, in collaboration with Peter Balint Kurti, at North Carolina State University. We did not find any obvious effect on disease response. In Arabidopsis, a stronger disease phenotype is observed with G beta, so we have invested considerable effort in identifying a maize G beta mutation. Initially we identified 4 transposon insertions, however they are in non-coding regions and none have a phenotype, suggesting they are not causing any significant expression change. We therefore collaborated with Bing Yang at Iowa State University to use CRISPR to knockout the gene. We found that the Zmgb1 null alleles are lethal at a very early stage of seedling development. We are trying to figure out the reasons for lethality, by checking the embryo development and immune responses of the Zmgb1 alleles. In addition, we luckily obtained a 3bp deletion allele using CRISPR, resulting in a conservation of reading frame but mutation of two conserved amino acids between the second and third WD domain (HK55Q). This could be a potential weak allele; we have not observed any obvious phenotype yet but will continue to study it. We are crossing the Zmgb1 null alleles with the Uniform-Mu and the 3-bp deletion alleles, to see if we can get hypomorphic phenotypes. We are also using CRISPR to knockout the eXtra Large G proteins (XLGs), which have a domain with homology to G alpha at their C-terminus. There are 3 XLGs in maize, and we got different alleles for the three proteins by using CRISPR. We are crossing the xlgs with ct2 mutants to get the quadruple mutants to study the genetic relationship between ct2 and xlgs. We are also investigating interactions with brassinosteroid (BR) signaling. We obtained RNAi lines from Phil Becraft, ISU, and have made double mutants with ct2. In addition, we backcrossed BES1-YFP, a BR signaling reporter, into ct2 mutants. BES1 is dephosphorylated upon BR treatment in Arabidopsis. We found similar responses in maize as well; however, our preliminary results suggested that the BES1-YFP phosphorylation state responded to BR treatment similarly between ct2 heterozygous and homozygous mutants. Further BR responses will be characterized using the bri1 RNAi/ct2, bin2 RNAi/ct2 double mutants. To use advanced imaging and biophysical methods to test the hypothesis that CT2/ G? interacts directly with a CLAVATA receptor in plants, Here we proposed to study the mechanism of CT2/Gα interaction with the FEA2 receptor, using Fluorescence Resonance Energy Transfer (FRET) assays. These experiments are performed in collaboration with the Scarlata lab at Stonybrook University, who are expert in biophysical analyses of Gα interactions in mammalian cells, and they assist us with data analysis and evaluation. Progress: We have completed the proposed FRET and BiFC assays, and do not find a direct interaction between CT2 and FEA2, suggesting that a third protein bridges their interaction in planta. We therefore used IP-mass spec to identify CT2 and FEA2 interacting proteins, and found one common protein, a novel LRR kinase, that interacts with both FEA2 and CT2. The BiFC experiments suggested that the novel LRR kinase directly interact with CT2 via its intracellular kinase domain. This gene is highly expressed in inflorescence meristems, however it has a close paralog in maize. We have obtained knockouts in both paralogs, and are currently screening for phenotypes. In addition, we are introgressing the double mutants into other 'fasciation sensitive' genetic backgrounds, for instance, NC350 and W23, to see if we can get more obvious phenotypes. We also used phosphoproteomics to identify three serines of CT2 protein that can be phosphorylated, and more interestingly, CT2 phosphorylation responses to CLV3 peptide treatment (collaboration with Steve Briggs, UCSD). We are studying if the newly identified LRR kinase affects the phosphorylation state of CT2, and how this could affect CT2 signaling. We are also trying to ascertain whether CLAVATA signaling affects G alpha GTP binding status, however high background signal in the experiments has prevented us from obtaining useful data so far. We are therefore expressing the proteins in insect cells to create an in vitro system. (3) to identify additional factors that contribute to CT2-mediated inflorescence development. Field trials will be used to test the effects of the new alleles on maize seed production and yield. Progress: Unfortunately the maize TILLING facility is no longer active, due to lack of grant support. Therefore we adopted an alternative approach, to make a weak transgene allele based on a published report in rice. The construct has been transformed into maize and is being backcrossed to the mutant to make weak allele plants for agronomic analysis. In addition, we generated a constitutive active (GTPase dead) transgenic version of CT2 in maize, and we are backcrossing to the ct2 mutant background. Rice plants harboring a constitutively active G alpha have 20% greater seed weight compared with wild type. Furthermore, we are collaborating with Doreen Ware at Cold Spring Harbor Laboratory and Zhanguo Xin at USDA to get the weak alleles of a G alpha ortholog in Sorghum, using their TILLING populations. We also screened for genetic modifiers of ct2 in the NAM parental inbreds, and have identified three enhancers and one fasciation suppressor. The fasciation suppressor does not affect other phenotypes of ct2, such as plant height, leaf width and erectness, indicating it might be an inflorescence specific gene. We observed the development of the modified ct2 ears at early stage by using scanning electron microscopy. We are also crossing ct2 mutants with the RIL population of the strongest enhancer background. We are mapping these modifier loci that could provide additional alleles for breeding. The reasons for slippage if established goals were not met; As described above, alternative approaches have been designed if the original plan was not feasible. Additional pertinent information including, when appropriate, analysis and explanation of cost overruns or unexpectedly high unit costs. The project is progressing extremely well and there are no issues in meeting the goals in the funding period. The post doc on the project, Qingyu Wu, presented some of his work in a selected talk at the Keystone Meeting on Plant Receptor Kinases, and was awarded a fellowship to cover his meeting costs.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Urano, D. and Miura, K. and Wu, Q. and Iwasaki, Y. and Jackson, D. and Jones, A. M. (2016) Plant Morphology of Heterotrimeric G protein Mutants. Plant Cell Physiol, doi: 10.1093/pcp/pcw002
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Urano, D. and Jackson, D. and Jones, A. M. (2015) A G protein alpha null mutation confers prolificacy potential in maize. Journal of Experimental Botany, 66(15) pp. 4511-4515.


Progress 12/01/13 to 11/30/14

Outputs
Target Audience: The target audience for this project will be academic scientists, who are interested in crop genetics, and yield traits. Also industry scientists and breeders is 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. In addition, a visiting MSC student from Germany, Michael Fuchs,was trained in maize cloning and genomics. How have the results been disseminated to communities of interest? Results from this project have been presented in seminars to the North Carolina plant molecular biology consortium, at a plant genomics Congress in Malaysia, the Japanese society for plant physiology conference in Japan, a Gordon research conference in plant molecular biology, in New Hampshire, USA, and seminars at the University of Missouri, the Danforth plant science center, and Monsanto Company. In addition, the postdoc gave a selected oral presentation at the recent Keystone conference on receptor kinase signaling. 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, although I expect an acceleration of some of our genetic analyses by the application of CRISPR technology, which we recently showed to be highly effective in maize.

Impacts
What was accomplished under these goals? A comparison of actual accomplishments with the goals established for the reporting period (where the output of the project can be expressed readily in numbers, a computation of the cost per unit of output should be submitted if the information is considered useful); The goals of this project are to investigate a potentially new signaling mechanism in maize inflorescence shoot development, and to ask if it has applications in traits of agricultural importance. We identified a maize mutant compact plant2 (ct2), which exhibits meristem proliferation defects similar to CLAVATA mutants, such as maize fasciated ear2 (fea2). CT2 encodes the a subunit of a predicted heterotrimeric G protein, a well-known membrane-signaling complex, and we have found that CT2 interacts genetically and biochemically with the FEA2 receptor. Heterotrimeric G proteins are well characterized in non-plant systems, where they transmit signals from G protein coupled receptors (GPCRs), which are typically 7-pass transmembrane proteins. Our results in plants challenge this dogma, because the CLAVATA receptors are predicted to be single pass transmembrane proteins. We proposed to investigate these findings with the following major goals: To characterize the role of the CT2 gene in maize growth, inflorescence development and seed productivity. Here we proposed to ask what other pathways and/or processes might be controlled by CT2/G? and other members of the heterotrimeric G protein complex in maize. Standard genetic methods, such as construction and analysis of double mutants will be used, and we will also collaborate with other scientists to assess root architecture changes and response to pathogens. Data analysis will use routing statistical tests conducted with our collaborators. Progress: We have completed an assessment of disease responses in the maize G alpha mutants, in collaboration with Peter Balint Kurti, at North Carolina State University. We did not find any obvious effect on disease response. In Arabidopsis, a stronger disease phenotype is observed with G beta, so we have invested considerable effort in identifying a maize G beta mutation. Initially we identified 4 transposon insertions, however they are in non-coding regions and none have a phenotype. We are therefore collaborating with Bing Yang at Iowa State University to use CRISPR to knockout the gene, and just identified several heterozygous knockouts that are being propagated. We are also investigating interactions with brassinosteroid signaling, we obtained RNAi lines from Phil Becraft, ISU, and have made double mutants with ct2/ G alpha. These will be characterized in the coming months. To use advanced imaging and biophysical methods to test the hypothesis that CT2/ G? interacts directly with a CLAVATA receptor in plants, Here we proposed to study the mechanism of CT2/Gα interaction with the FEA2 receptor, using Fluorescence Resonance Energy Transfer (FRET) assays. These experiments are performed in collaboration with the Scarlata lab at Stonybrook University, who are expert in biophysical analyses of Gα interactions in mammalian cells, and they assist us with data analysis and evaluation. Progress: we have completed the proposed FRET and BiFC assays, and do not find a direct interaction between CT2 and FEA2, suggesting that a third protein bridges their interaction in planta. We therefore used IP-mass spec to identify CT2 and FEA2 interacting proteins, and found one common protein, a novel LRR kinase, that interacts with both FEA2 and CT2. This gene is highly expressed in inflorescence meristems, however it has a close paralog in maize. We have obtained knockouts in both paralogs, and are currently screening for phenotypes. We also used phosphoproteomics to identify three serines of CT2 protein that can be phosphorylated, and we are studying if the newly identified LRR kinase affects the phosphorylation state of CT2, and how this could affect CT2 signaling. We are also trying to ascertain whether CLAVATA signaling affects G alpha GTP binding status, however high background signal in the experiments has prevented us from obtaining useful data so far. We are attempting to express the proteins in insect cells to create an in vitro system. (3) to identify additional factors that contribute to CT2-mediated inflorescence development. Here we will test if variation in the CT2 locus can impact maize yield traits. For this, we will use TILLING to identify weak alleles of CT2. Such alleles could provide beneficial effects, such as an increase in kernel row number. We will use the maize TILLING facility at Purdue University, and molecular genetic methods to analyze sequences and to interpret their effects. 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. Progress: Unfortunately the maize TILLING facility is no longer active, due to lack of grant support. Therefore we adopted an alternative approach, to make a weak allele transgene based on a published report in rice. This is being transformed into maize and will be backcrossed to the mutant to make weak allele plants for agronomic analysis. In addition, we generated a constitutive active (GTPase dead) version of CT2 in transgenic maize, and we are backcrossing to the ct2 mutant background. Rice plants harboring a constitutively active G alpha have 20% greater seed weight compared with wild type. Furthermore, we also screened for genetic modifiers of ct2 in the NAM parental inbreds, and have identified ~ 3 potential enhancers and a suppressor, we will proceed with mapping of these modifier loci that could provide additional alleles for breeding. The reasons for slippage if established goals were not met; As described above, alternative approaches have been designed if the original plan was not feasible. Additional pertinent information including, when appropriate, analysis and explanation of cost overruns or unexpectedly high unit costs. I believe that this project is progressing extremely well and there are no issues in meeting the goals in the funding period. The post doc on the project, Qingyu Wu, presented some of his work in a selected talk at the recent Keystone Meeting on Plant Receptor Kinases, and was awarded a fellowship to cover his meeting costs.

Publications