Rapidly Reprogramming Plants with RNA Vectors

Transcriptional programs sculpt plant morphology and metabolism to meet environmental challenges. These same programs have been manipulated through selective breeding over generations to increase agricultural productivity and robustness. Studying the genetic basis of these improved traits has elucidated the specific alterations in the expression levels of key master regulator genes that lead to phenotypic improvement. The use of synthetic transcription factors has enabled precise alterations to the transcriptional landscape, but their application in crop plants has been challenging due to difficulties posed by plant transformation and long life cycles of crops. We are developing a system called VipariNama (ViN, from the Sanskrit: to change) to allow rapid reprogramming of transcription in adult plants. It uses systemically mobile and stably maintained RNA scaffolds in conjunction with genomically integrated transcription factor components to assemble transcription factors at loci of interest. To experimentally validate the ViN system, we have built lines of the model plants Arabidopsis thaliana, Nicotiana benthamiana and the crop Oryza sativa with genomically integrated copies of functionally validated Cas9-based activators and repressors. We demonstrate the capacity to alter gene expression via guide RNAs delivered on mobile RNA viruses. We also explore how these transcription factors themselves can be delivered via viral ViN vectors. Finally, we are optimizing the systemic mobility and stability of our RNA vectors by using next generation sequencing enabled screening of variant libraries. By putting these optimized ViN vectors and deconstructed transcription factors together we hope to facilitate rapid reprogramming of transcriptional landscapes in adult plants and thereby accelerate the phenotypic improvement of crops as well as enable generation of necessary traits in the field to ensure food security in rapidly changing agricultural environments.