Progress 01/01/18 to 12/31/21
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:Yield trials that we had planned with collaborators in different institutions were delayed, due to the covid-19 pandemic. 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 in 2019 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 2019, 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 University of Georgia (Feb 2020), and virtual seminars at Univ. Missouri and UC Riverside in 2020, an in 2021 at Penn State (Feb 2021), and a Virtual International Single Cell Symposium, HZAU, Wuhan, China, April 2021 and University of Nebraska, Lincoln (Oct '21). He also presented an outreach talk to high school students about his career path and science, to the DNA Learning Center High School seminar series, in July 2021. 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. 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. Knocking out ZmIMK1 and its paralog, ZmIMK2 did not show any obvious inflorescence phenotype. Perhaps due to functional redundancy. We made a dominant negative ZmIMK1 construct by deleting its kinase domain, and transformed this construct into maize. However, these lines also did not produce any obvious phenotypes, so this line of investigation is paused. 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 ct2 phenotypes. However, the triple mutants were seedling lethal. They had strong trypan blue staining, suggesting cell death, and significantly up-regulated expression of two immune marker genes, PATHOGENESIS-RELATED PROTEIN 1 (PR1) and PR5, indicating that the lethality is due to over-activation of the immune system. These findings were published (Wu et al, PLOS Genetics 2018). We initiated a collaboration with Dr. Tiffany Jamann, Univ. Illinois, to test defense responses in these lines. The Arabidopsis proteins XLG2/3 and GPA1 regulate distinct aspects of immune responses. While GPA1 controls stomatal closure, XLG2/3 enhances PAMP-triggered ROS production, callose deposition, or MAPK activation. We performed disease trials withct2,aZmxlg1CRISPR mutant, and aZmxlg3a Zmxlg3bCRISPR double mutant, testing their response to Xanthomonas vasicola pv. Vasculorum (Xvv)andClavibacter nebraskensis (Cn). Xvvisa bacterial pathogen that causes bacterial leaf streak of maize gumming disease, and enters the leaf through stomata and wounds. This disease has been spreading rapidly through maize-growing areas of the USA.Cnis a bacterial pathogen that causes Goss's wilt and leaf blight in maize, and it requires a wound to enter host tissue. This disease is estimated to be in the top-ten most destructive diseases of corn in the northern U.S. Our preliminary results showed that compared to their wild-type siblings, ct2mutants were more resistant toXvvandCn.In Arabidopsis,agb1single mutants show enhanced bacterial growth and disease susceptibility. However, in our study, we found enhanced disease resistance in the ct2 mutant against both pathogens. This contrast in response is very interesting, and we are in the process of repeating these experiments to confirm the results. We will also ask if the differences are due to the different inoculation methods used; we used seedling infiltration whereas previous researchers used spray inoculation and flood-inoculation in Arabidopsis. We also found thatzmxlg1mutants were more sensitive toXvv,andzmxlg3a zmxlg3bdouble mutants were more susceptible to both Xvv andCn.These results agree with previous findings in Arabidopsis thatxlg123triple mutants were more disease susceptible. We are repeating these trials using additional CRISPR alleles ofZmxlg1, andZmxlg3a Zmxlg3b. 2. 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 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. We therefore crossed Zmgb1 (using viable heterozygotes) to the diverse maize NAM founder lines, and scored phenotypes in the F2s. We found that Zmgb1 lethality is suppressed in the tropical CML103 background. 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 using bulk segregant analysis to 200 - 205 Mb on chromosome 5, in a region with only one R gene. To ask if this gene was responsible for the lethality, we used CRISPR-Cas9 to knock it out. However, our results suggest that the R gene is not responsible for the autoimmune phenotype. We also crossed the heterozygous Zmgb1 mutants with ~100 CML103/B73 recombinant inbred lines (RILs), and performed QTL analysis in the F2 populations. We identified two linked QTLs ~ 30 cM apart on Chr5, and have made populations to fine-map them. As a backup plan, we also performed a screen for genes 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 400 M2 families. Indeed, we recovered some viable M2 plants, and confirmed that they are homogygous Gbeta mutant, suggesting they are real suppressors. Molecular analysis of these plants and mapping of the suppressor(s) is ongoing. We localized ZmGB1 to membranes, as expected, 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 also made a 3xFLAG-2xSTREP-ZmGB1 tagged line, and used tandem affinity purification (TAP) and mass spectrometry to identify interacting proteins. However, ZmGB1 is a signaling protein and interactions with other proteins may be transient, and no interesting interactors were identified. To overcome this problem, we made pZmGB1::TURBO ID-ZmGB1 transgenic plants, and in future will use a proximity labeling approach to identify transient interactors of ZmGB1. 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 in PNAS. We also collaborated with the Balint Kurti lab to assist in mapping of a different autoimmune mutant in maize, and published a paper together. 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 had planned to measure yield traits of different G protein alleles and combinations. However, these trials were delayed due to the covid-19 pandemic.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Tran, T., Demesa-Arevalo, E., Kitagawa, M., Garcia-Aguilar, M., Grimanelli, D, Jackson, D. (2021) An optimized whole-mount immunofluorescence method for shoot apices. Curr Prot Plant Bio Article DOI: 10.1002/cpz1.101.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Kim, S.B., Karre, S., Wu, Q., Park, M., Meyers, E., Claeys, H., Wisser, R., Jackson, D., Balint-Kurti, P. (2020). Multiple insertions of COIN, a novel maize Foldback transposable element, in the Conring gene cause a spontaneous progressive cell death phenotype. Plant J. Aug 4. doi: 10.1111/tpj.14945.
|
Progress 01/01/20 to 12/31/20
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 in 2019 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 2019, 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): Meetings and travel in 2020 were affected by the pandemic. Jackson presented work from the project in Departmental seminars and University of Georgia (Feb 2020), and virtual seminars at Univ. Missouri and UC Riverside. 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?
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. However these lines also did not produce any obvious phenotypes, and we are planning alternative approaches 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 Arabidopsis proteins XLG2/3 and GPA1 regulate distinct aspects of immune responses. While GPA1 controls stomatal closure, XLG2/3 enhances PAMP-triggered ROS production, callose deposition, or MAPK activation. In our collaboration with Tiffany Jamann's lab, we performed disease trials withct2,aZmxlg1CRISPR mutant, and aZmxlg3a Zmxlg3bCRISPR double mutant, testing their response to Xanthomonas vasicola pv. Vasculorum (Xvv)andClavibacter nebraskensis (Cn). Xvvisa bacterial pathogen that causes bacterial leaf streak of maize gumming disease, and enters the leaf through stomata and wounds. There is no evidence yet of yield or crop loss due toXvvin maize, but the disease has been spreading rapidly through maize-growing areas of the USA .Cnis a bacterial pathogen that causes Goss's wilt and leaf blight in maize, and it requires a wound to enter and infect host tissue. Goss's wilt and leaf blight has been estimated to be in the top-ten most destructive diseases of corn in the northern U.S. Our preliminary results showed that compared to their wild-type siblings, ct2mutants were more resistant toXvvandCn.In Arabidopsis,agb1single mutants show enhanced bacterial growth and disease susceptibility. However, in our study, we found enhanced disease resistance in the ct2 mutant against both pathogens. This contrast in response is very interesting, and we will repeat the experiments to confirm it. We will also ask if the differences are due to the different inoculation methods used; we used seedling infiltration whereas previous researchers used spray inoculation and flood-inoculation in Arabidopsis. We also found thatzmxlg1mutants were more sensitive toXvv,andzmxlg3a zmxlg3bdouble mutants were more susceptible to both Xvv andCn.These results agree with previous findings in Arabidopsis thatxlg123triple mutants were more disease susceptible. To further confirm these results, we will repeat these trials using additional CRISPR alleles ofZmxlg1, andZmxlg3a Zmxlg3bthis summer. 2. 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. So far our evidence suggests that the R gene is not responsible for the autoimmune phenotype, but these experiments are ongoing. As a backup plan, we also performed a screen for genes 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. Molecular analysis of these plants is ongoing. 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 in PNAS. 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 of different G protein alleles and combinations. We will set up some yield trials this summer to ask if the different alleles that affect kernel number are also able to enhance other yield related traits.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Tran, T., Demesa-Arevalo, E., Kitagawa, M., Garcia-Aguilar, M., Grimanelli, D, Jackson, D. (2021) An optimized whole-mount immunofluorescence method for shoot apices. Curr Prot Plant Bio Article DOI: 10.1002/cpz1.101. Accepted
|
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.
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