Auxin-Response Factors (ARFs) together with Aux/IAA proteins mediate auxin responses including, floral development, fertilization, fruit set and development, and ripening process1. Among them, from our past research, the auxin response factor 4, VviARF4 a likely “key genetic regulator” during the onset of ripening2. The research project is proposed 1) to characterize the function of VviARF4 through genetic engineering, and to identify potential regulatory protein partners of ARF4* (see comments at the end of the document), 2) to determine the ripening-related genes targeted by VviARF4 during the onset of ripening, and 3) to evaluate the impact of altered expression of VviARF4 on the final fruit composition.
Since the commencement of the project in June 2016, significant efforts were made towards the objective 1. We first finalized the agreement for shipping the microvine lines with the USDA (See supplemental data). The signature of the Material Transfer Agreement is on its way between OSU and the CSIRO. Meanwhile, we concluded the logistics of the complex cloning strategies and finalized several vector constructs to transform the microvines designed to either turn on or turn off the activity of VviARF4 specifically during the fruit maturation stage. In the preliminary experiments, we are testing the gene silencing strategy in strawberry (Fragaria ananasa), which shows similar developmental pattern of ARF4 expression during the ripening initiation stage. The plasmid vectors designed to silence the endogenous FaARF4 and to over express the ARF4 from grapevine will into an aggressive Agrobacterium bacterium strain (EHA105). We plan the transient transformation experiments in strawberry within three weeks from now. This will help to establish the role of ARF4 in another non-climacteric fruit model and will enable us to optimize our cloning strategy for the microvines.
The second part of the objective 1 is to find the regulatory protein partners of VviARF4 for which, we initiated the Yeast Two Hybrid Screens (Y2H). Through this approach, we expect to identify potential protein partners of VviARF4, which could have major regulatory role in VviARF4 gene function during the ripening initiation stage. We concluded the experiments to estimate the efficiency of library transformation in yeast and obtained satisfactory results. We are now preparing the final library in order to find the protein candidates that interact with ARF4.
Finally, as part of the objective 3, we are currently building our library of primary and secondary metabolites that will be analyzed when the microvines are transformed during the second year. The shipping of the microvines is expected by mid-February. Meanwhile, we are preparing the various media necessary to maintain the microvine calluses, to induce embryogenesis, to promote the regeneration, and to propagate the transformed materials. We anticipate being ready with the gene constructs cloned in to the Plant Gene Switch Vector to over-and under-express ARF4 before the microvine materials from CISRO, Australia arrive. The post-doctoral researcher is scheduled to fly to Australia in late March to get trained for the critical steps of the microvine transformation in collaboration with Dr. Thomas.