Phytophthora cinnamomi is a globally important oomycete pathogen responsible for root rot and dieback diseases in diverse agricultural and natural ecosystems. Although RNA interference (RNAi)-based technologies represent a promising alternative to conventional disease management, the barriers limiting exogenous RNA-mediated gene silencing in P. cinnamomi remain poorly understood. Thus, this study investigated exogenous RNA uptake across developmental stages of P. cinnamomi and evaluated factors that may influence RNA-induced gene silencing.
Uptake of Cy3-labelled double-stranded RNA (dsRNA) was examined by fluorescence microscopy, while functional silencing assays targeting the translation elongation factor (TEF) gene were conducted using both long dsRNA and 30-nt RNA molecules. Because reliable aseptic production of sporangia was not achieved under laboratory conditions, mycelial cultures were used for gene-expression analyses. To improve RNA internalisation, a lysozyme-assisted uptake approach was also evaluated following determination of a sublethal lysozyme concentration.
Microscopy revealed consistent uptake of Cy3-dsRNA in sporangia and internal zoospores, whereas mycelia displayed heterogeneous, patchy uptake. No detectable uptake was observed in chlamydospores or encysted germinated zoospores. Despite evidence of RNA uptake, neither lysozyme-assisted dsRNA delivery nor treatment with 30-nt RNA molecules resulted in statistically significant suppression of TEF expression.
These findings suggest that developmental-stage-specific RNA accessibility represents a major barrier to exogenous RNA-mediated gene silencing in P. cinnamomi. The results further indicate that limitations in RNA delivery, rather than the absence of endogenous RNAi machinery, may constrain the effectiveness of RNA-based disease control strategies against this pathogen.