Progress 01/15/24 to 01/14/25
Outputs
Target Audience:Academics at various universities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project director: Reuben J. Peters, oversight of project, including setting long and intermediate term goals, along with appropriate direction of research. Technician: Meimei Xu, construction of transgenic vectors and genotyping. PhD students: Yiling Feng, investigating effect of DPC inhibition; Ahmed Raslan, mutagenesis of OsCPS1 to increase affinity for DPC. UG student: Griffin Humphreys, assisting with protein purification and assays. How have the results been disseminated to communities of interest?Project has been presented in a talk at the 2024 USDA-NIFA Physiology of Agricultural Plants PD meeting in Hawaii as well as a virtual talk presented at the International Conference for Molecular Plant Sciences. What do you plan to do during the next reporting period to accomplish the goals?We recently sent the OsCPS1:K417Y knock-in lines to our collaborators, who will test larger numbers of plants (i.e., in the field) to determine if increasing the sample size will reveal any statistical significance in effect of DPC on growth. If this is accomplished, we will test relative effects on chemical defense, both diterpenoid production and resistance to both Xanthomonas oryzae and Magnaporthe oryzae. Finally, we are testing the effect of our current set of (additional) mutants on susceptibility of OsCPS1 to inhibition by DPC, if time permits we will then add the most successful of these to our OsCPS1:K417Y variant to determine if such increased affinity enables more significant growth inhibition.
Impacts
What was accomplished under these goals?
We have generated the targeted K417Y site-directed mutant within the endogenous OsCPS1 in the semi-dwarf cv Kitaake and have homozygous plants. Unfortunately, as I indicated in my talk at last summer's PD meeting, while our results are suggestive, the difference between vegetative growth for this variant in the presence and absence of DPC is not significant. We also attempted to introduce this mutation into the 'tall' cv Koshihikari but have been unsuccessful to date (through two separate attempts). Building on our determination of a cryo-EM structure for susceptible GhCPS (from cotton) complexed with DPC and crystal structure of OsCPS1:K417Y, which revealed binding of dual molecules in the active site, we have designed additional mutations to improve the susceptibility of OsCPS1:K417Y.
Publications
|
Progress 01/15/23 to 01/14/24
Outputs
Target Audience:Academics from various universities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project director: Reuben J. Peters, oversight of project, including setting long and intermediate term goals, along with appropriate direction of research. Technician: Meimei Xu, construction of transgenic vectors, genotyping and X. oryzae infection assays. PhD student: Yiling Feng, investigating effect of DPC inhibition. UG student: Caroline Nichols, protein purification and enzyme analysis. How have the results been disseminated to communities of interest?Project has been presented in talks to the Crop Bioengineering Center here at Iowa State Univ. and at other institutions (UC Davis, UC San Francisco & SUNY Buffalo) What do you plan to do during the next reporting period to accomplish the goals?With the Kittake cultivar (cv) knock-in now in-hand we will test these lines for relative effects on yield and chemical defense, both diterpenoid production and resistance to various microbial pathogens and parasitic nematodes. Having proven our CRISPR Prime Editing approach works, we further will apply this to the 'tall' cv Koshihikari, where this is expected to have a more dramatic effect and, critically, enable investigation of the hypothesized ability to retain more efficient nitrogen uptake relative to the more commonly grown semi-dwarf varieties such as cv Kitaake. Finally, we will test the effect of our current set of (additional) mutants on susceptibility of OsCPS1 to inhibition by DPC.
Impacts
What was accomplished under these goals?
Building on our prime editing approach we have now generated the targeted K741Y site-directed mutant within the endogenous OsCPS1 and now have homozygous plants, which are susceptible to inhibition of vegetative growth by DPC. While this validates one of the central tenants of the project, indicating an effect on gibberellin biosynthesis, any effect on more specialized diterpenoid metabolism and, hence, plant defense (against microbes and herbivores), as well as yield, remains to be determined. We also continue to build on our determination of a cryo-EM structure for susceptible GhCPS (from cotton) complexed with DPC and crystal structure of OsCPS1:K741Y, which revealed binding of dual molecules in the active site, designing additional mutations to improve the susceptibility of OsCPS1:K741Y. Fortunately, this focuses our attention on the more easily defined active site, rather than the previously proposed allosteric inhibition mechanism. Accordingly, we are focusing on accommodation of the amine in the second DPC bound in the active site (as the first interacts with the catalytic/necessary aspartic acid).
Publications
|
Progress 01/15/22 to 01/14/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project director: Reuben J. Peters, oversight of project, including setting long and intermediate term goals, along with appropriate direction of research. Technician: Meimei Xu, construction of transgenic vectors, genotyping and X. oryzae infection assays. PhD student: Yiling Feng, investigating effect of DPC inhibition. UG student: Preston Pellatz, protein purification and enzyme analysis. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Continue the planed work.
Impacts
What was accomplished under these goals?
We have continued our efforts to generate suitable transgenic plant lines using the proposed parallel approaches. Fortunately, a prime editing approach has now been successful in generating the targeted K741Y site-directed mutant within the endogenous OsCPS1, albeit the T0 plants are heterogenous. We are now growing T1 plants with the expectation of finding homozygous plants, which will be used for the proposed studies. With regards to further investigation of the structural basis for the observed biphasic nature of DPC inhibition of OsCPS1:K741Y, we have successfully used cryo-EM to determine structures in complex with DPC, revealing binding of dual molecules within the active site. We are now designing additional mutations to probe this surprising finding and improve affinity.
Publications
|
Progress 01/15/21 to 01/14/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project director: Reuben J. Peters, oversight of project, including setting long and intermediate term goals, along with appropriate direction of research. Technicians: Meimei Xu, construction of transgenic vectors and X. oryzae infection assays. PhD student: Kristin Roach, investigating DPC inhibition. UG student: Preston Pellatz, assisting with protein purification. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Continue the planed work
Impacts
What was accomplished under these goals?
We have continued our efforts to generate suitable transgenic plant lines using the proposed parallel approaches. This has primarily focused on the originally proposed gene-replacement approach to create the targeted K741Y mutant in the endogenous OsCPS1. Unfortunately, this method has failed to yield this point mutant, although OsCPS1 knock-out lines were generated. These cps1 mutant plants are difficult to grow and transform in homozygous form, but we expect that our next set of transformations, using heterozygous cps1 plants, will be successful. The resulting transformants can be selfed to generate the targeted homozygous cps1+OsCPS1:K417Y lines. In addition, we have now shifted our focus on the more desirable K741Y site-directed mutant within the endogenous OsCPS1 to use a prime editing approach, which we are hopeful will yield the targeted mutant. With regards to further investigation of the structural basis for the observed biphasic nature of DPC inhibition of OsCPS1:K741Y, we have engaged in further crystallographic studies as well as initiated cryo-EM work targeting its interaction with DPC, either alone or in tandem with bound substrate and magnesium co-factor.
Publications
|
|