PLANT-Dx: A Molecular Diagnostic for Point of Use Detection of Plant Pathogens

M. Verosloff; J. Chappell; K. L. Perry; J. R. Thompson; J. B. Lucks

To develop PLANT-Dx, we first sought to create pathogen detecting molecular sensors based upon the Small Transcription Activating RNA (STAR) regulatory system (11) . This transcription activation system is based upon conditional formation of a terminator hairpin located within a target RNA upstream of a gene to be regulated: alone, the terminator hairpin forms and halts transcription of the downstream gene, while in the presence of a specific transacting STAR the hairpin cannot form and transcription proceeds (SI Figure 2) . Previous work showed that the STAR linear binding region can be changed to produce highly functional and orthogonal variants (15) . Here we sought to utilize this by replacing the linear binding region with sequences derived from genomic pathogen DNA to create new viral sensors. To do this, we utilized the secondary structure prediction algorithm NUPACK to identify regions within the genomes of cucumber mosaic virus (CMV) and potato virus (PVY), that are predicted computationally to be unstructured for target RNA design (Supplementary Note 1) (16) . Once viral STARs were designed, reporter DNA constructs were then created in which these target RNA sequences were placed downstream of a constitutive T7 promoter and upstream of the CDO reporter gene. We next designed RPA primer sets to amplify and transform a pathogen's genomic material into a DNA construct capable of synthesizing a functional STAR. Specifically, a T7 promoter and anti-terminator STAR sequence were added to the 5' end of a reverse RPA primer, which when combined with a forward primer, amplified a 40 nt viral sequence to produce a double-stranded DNA encoding the designed STAR which contained the target viral . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/498998 doi: bioRxiv preprint sequence. In this way, we anticipated that combining the CDO-encoding reporter construct and RPA amplified DNA into a cell-free gene expression reaction (12 , 17) would lead to the production of a detectable colorimetric output signal.