DEVELOPMENT OF A WIDE HOST-RANGE VIRUS VECTOR FOR EXPRESSION OF PROTEINS AND/OR MULTIPLE SIRNAS TO TARGET PATHOGENS AND PROVIDE VALUE ADDED BENEFITS TO TREES AND VINES

The world is facing an unprecedented pandemic of tree diseases, leading to billions of tree losses each year, exasperating climate change and decimating entire industries. Currently, there are no treatments or cures for any pathogen-associated tree diseases. What is missing is the ability to deliver efficacious agents inside the tree. Viruses can be exceptional delivery vehicles, but their limited host ranges and instability with inserted sequences have greatly limited their use in trees. We have discovered a new type of subviral RNA called umbra-virus-like (ula)RNAs that have the potential to be game changers as vectors due to their extensive host range, lack of silencing suppressors, symptom-free infections (in nearly all hosts), lack of plant-to-plant transmission, and vascular location (where most of the worst pathogens reside). In addition, a successful delivery vector for trees would be transformative for many researchers studying plants that are recalcitrant to direct genetic modification. CYVaV is a ulaRNA that can deliver small RNAs (that can target pathogens and change traits in the trees) but is so far incapable of delivering peptides and proteins. However, the strict structural requirements of highly structured CYVaV should not be a factor in three other potential ulaRNA vectors, BabVQ and CYVaV dimers and defective (D)RNAs. BabVQ, found naturally in all babaco (mountain papaya) trees, is a Class 1 ulaRNA with a mainly unstructured and lengthy 3' UTR, which suggests tolerance for weakly structured inserted sequences. We will generate a full-length infectious construct of BabVQ, determine its structure and infectivity, and examine its ability to be stable with inserted sequences of sufficient size to specify proteins that can be used to target bacterial pathogens. CYVaV also produces very interesting defective versions of itself naturally in plants. CYVaV dimers are replicating molecules in the presence of CYVaV and a significant portion of the dimer genome is likely not contributing to infectivity of the molecule, and thus at least a portion of this sequence should be either replaceable or tolerant to inserts. CYVaV (D)-RNAs are replicons that should be dependent on co-infecting CYVaV for their RdRp, but have also been found in infected plants in the absence of CYVaV, suggesting they may be capable of being replicated by host enzymes. We will determine if these two additional RNAs have potential as delivery vectors for small peptides and proteins, either alone or when being accompanied by CYVaV, by completing the following objectives: Objective 1- Examine the structure and biology of BabVQ and investigate its potential as a vector for siRNAs, peptides and proteins; Objective 2- Determine the structure of CYVaV dimers and investigate their potential as vectors for siRNAs, peptides and proteins; Objective 3- Determine the structure of a CYVaV D-RNA and investigate its potential as a VIGS vector. The simultaneous examination of three additional potential ulaRNA vectors is important since the window of opportunity is shrinking for developing a solution to the critical biotic (e.g., Citrus Greening) and abiotic stresses (e.g., climate change) facing trees and vines.