The transgenic soybean QQS interactor overexpressing mutants (Williams 82) will be tested for seed composition (protein and oil content) in the field; the transgenic QQS interactor overexpressing mutants with increased seed protein will be identified. (Timeline: by Year 1)
Soybean disease and insect pest tests on transgenic soybean QQS interactor overexpressing mutants (Williams 82) (Timeline: by Year 1)
The plasmid construct for non-transgenic approach via CRISPR/Cas9 will be generated and submitted for transformation. (Timeline: by Year 1)
The soybean Williams 82 plants containing CRISPR/Cas9-edited soybean QQS interactor will be generated. Homozygous, overexpressed and ideally transgene-free QQS interactor Williams 82 will be identified. (Timeline: by Years 1 and 2)
The composition of transgene-free QQS interactor overexpressing Williams 82 plants will be tested (protein and oil content) in the field and those with increased seed protein will be identified. (Timeline: by Year 2)
Soybean disease and insect pest tests on non-transgenic QQS interactor overexpressing plants (Williams 82) (Timeline: by Year 2 and Year 3)
At least two lines of non-transgenic QQS interactor overexpressing-Williams 82 and control will be planted in Ames and two other different locations, yield and composition of mature seeds (protein and oil content) will be evaluated. (Timeline: by Year 3)
If funding available, conduct 1) soybean disease, nematode, insect pest and abiotic stress tests and 2) RNA-Seq and metabolomics analysis to transgenic soybean QQS interactor overexpressing mutants (Williams 82) and transgene-free soybean QQS interactor overexpressing plants. Expression levels of individual genes by RNA-Seq will be identified; candidate molecular markers of protein content, and genes involved in defense of soybean disease, nematode, insect pest and abiotic stress will be identified using the RNA-seq data. (Timeline: by Year 2 and Year 3)
Transcripts and metabolites that are differentially accumulated from overexpressing of transcription factor (QQS interactor) will be identified; our understanding of the carbon partitioning metabolic and regulatory network will be deepened by a combination of transcriptomics and metabolomics analyses. (Timeline: by Year 2 and Year 3)

Update:
1. Analyses of the composition of the mature soybean seeds overexpressing Target Gene 2 harvested from the field in 2015 indicated they have increased seed protein plus oil content; decreased seed fiber, seed yield per plant is not affected.
2. Developed transgenic soybeans expressing Target Gene 1 or overexpressing Target Gene 2 with increased resistance to soybean pathogens and pests.
a. Both foci number and focus size of viral infection are decreased in transgenic Arabidopsis overexpressing Target Gene 1 or 2; Bacterial growth is decreased in transgenic Arabidopsis overexpressing Target Gene 1 or 2 inoculated with bacterial Pst DC3000.
b. Bacterial growth (bacterial infection: PsgR4) was similarly decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
c. Soybean Cyst Nematode female count numbers were decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 2.
d. Aphid numbers were decreased on transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
e. Preliminary experiment indicated that viral foci size is decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 2. Will repeat the experiment when more seeds are available.
f. Experiment to test SDS screening in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2 indicated these transgenic soybeans had decreased scores of foliar SDS symptoms.
g. Seeds have been planted in the field in spring 2016 to bulk more seeds for transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2 for disease experiments, and to confirm their seed composition. The bulked seeds have been harvested, cleaned, and being organized. The seed composition will be analyzed by NIRS soon. Seeds will be prepared for disease experiments, including disease experiments in the field in 2017.
h. RNA-Seq experiment is undergoing for molecular mechanism of the increased disease resistance for for transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
3. Non-transgenic approach.
a. Promoter regions have been identified for Target Gene 2 that deletion of these promoter regions can overexpress Target Gene 2, make route for targeted mutagenesis via CRISPER/Cas9 technologies to increase the seed protein content of agronomic species, to reduce crop susceptibility to pathogens and pests.
b. The construct is under construction, four constructs are generated and confirmed, will be submitted to ISU PTF for soybean transformation to generate soybean plants.
4. Developing molecular tools of both Transgenic and Non-transgenic approaches, for soybeans with normal growth and development, increased soybean seed protein, reduced soybean susceptibility to pathogens and pests, and to increase sustainability.
5. Manuscript for disease experiment results is under review for publication.

1. Analyses of the composition of the mature soybean seeds overexpressing Target Gene 2 harvested from the field in 2015 indicated they have increased seed protein plus oil content; decreased seed fiber, seed yield per plant is not affected.
2. Developed transgenic soybeans expressing Target Gene 1 or overexpressing Target Gene 2 with increased resistance to soybean pathogens and pests.
a. Both foci number and focus size of viral infection are decreased in transgenic Arabidopsis overexpressing Target Gene 1 or 2; Bacterial growth is decreased in transgenic Arabidopsis overexpressing Target Gene 1 or 2 inoculated with bacterial Pst DC3000.
b. Bacterial growth (bacterial infection: PsgR4) was similarly decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
c. Soybean Cyst Nematode female count numbers were decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 2.
d. Aphid numbers were decreased on transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
e. Preliminary experiment indicated that viral foci size is decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 2. Will repeat the experiment when more seeds are available.
f. Experiment to test SDS screening in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2 indicated these transgenic soybeans had decreased scores of foliar SDS symptoms.
g. Seeds have been planted in the field in spring 2016 to bulk more seeds for transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2 for disease experiments, and to confirm their seed composition. The bulked seeds have been harvested, cleaned, and being organized. The seed composition will be analyzed by NIRS soon. Seeds will be prepared for disease experiments, including disease experiments in the field in 2017.
h. RNA-Seq experiment is undergoing for molecular mechanism of the increased disease resistance for for transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
3. Non-transgenic approach.
a. Promoter regions have been identified for Target Gene 2 that deletion of these promoter regions can overexpress Target Gene 2, make route for targeted mutagenesis via CRISPER/Cas9 technologies to increase the seed protein content of agronomic species, to reduce crop susceptibility to pathogens and pests.
b. The construct is under construction, four constructs are generated and confirmed, will be submitted to ISU PTF for soybean transformation to generate soybean plants.
4. Developing molecular tools of both Transgenic and Non-transgenic approaches, for soybeans with normal growth and development, increased soybean seed protein, reduced soybean susceptibility to pathogens and pests, and to increase sustainability.
5. Manuscript for disease experiment results is under review for publication.