FROM VIRAL DYNAMICS TO EPIGENETIC INTERVENTION: INSIGHTS INTO PLUM POX VIRUS DYNAMICS IN A PERENNIAL HOST AND CRISPRA-MEDIATED ACTIVATION OF ANTIVIRAL RNAI DEFENSE GENES

Abstract

The emergence of novel plant virus strains poses an increasing threat to global agriculture. Viruses evolve rapidly, accumulating advantageous mutations that drive disease emergence. Plum pox virus (PPV), a major pathogen of Prunus species, has caused millions of dollars in economic losses and threatens cultural icons within the species. PPV persists in perennial tree hosts, creating an ideal environment for viral variation. At least 10 PPV strains have been identified, underscoring its evolutionary potential. This study used ribosome-bound RNA sequencing, termed TRAP (Translating ribosome affinity purification), to examine how PPV populations evolve over time and in the vascular tissues of infected plum trees. RNA-seq analysis over two consecutive growth periods revealed that PPV populations contain distinct variant categories—some stable, others fluctuating—during leaf development. Notably, some high fluctuating variants were phloem-specific. Further, leaf buds exhibited lower infection levels but retained a greater proportion of viral variants than mature leaves. Plants use RNA interference (RNAi) to defend against viruses, with RNA-dependent polymerases (RDRs) amplifying viral dsRNA that dicer-like proteins (DCLs) process into siRNAs which guide viral RNA degradation. Previous analysis of this RNA-seq dataset found that antiviral RNAi-associated gene expression was activated in leaves during the growth season but not in bud tissues, where PPV overwinters. To target the diverse viral population in bud tissues in a low-replication phase, efforts in this project sought to increase RNAi expression, as a broad-spectrum defense pathway. RDR6 and DCL2 are key contributors in the production and amplification antiviral RNAi signal molecules termed vsiRNA. Thus, RDR6 and DCL2 were simultaneously targeted for transcriptional activation in Arabidopsis using a novel CRISPR activation system, CRISPR-Act3.0. Transcript analysis showed a moderate increase of DCL2 (2.28-fold) with little change in RDR6 expression. Following Turnip mosaic potyvirus (TuMV) infection, transgenic lines with increased DCL2 expression showed a 29% reduction in systemic infection 6 days post-inoculation compared to WT plants. This research advances our understanding of viral adaptation within plant hosts and presents a novel application of CRISPR activation for engineering host plant defense.