In order to trigger RNAi against (i) a virus (Cucumber Mosaic Virus, CMV), (ii) a viroid (Potato Spindle Tuber viroid, PSTVd), (iii) a fungus (Fusarium solani strain Κ, FsK) and (iv) a plant gene (acetolactate synthase, ALS), T7 promoter-containing plasmid constructs designed to produce the corresponding dsRNAs were generated. Subsequently, dsRNA molecules were produced either by in vitro or in vivo transcription and exogenously applied by high pressure spraying in tomato leaves against CMV, PSTVd and ALS, or by in vitro application in FsK conidia. The data we obtained revealed that the onset of RNAi against these targets exhibited variable degrees of efficiency. Thus, while CMV, PSTVd and ALS were poorly targeted by RNAi, FsK exhibited very strong RNAi response to dsRNA application. These data underpin that the efficiency of RNAi sprays is influenced by the morphological and anatomical properties of the tissue upon which the RNAi spray is applied. Thus, fungal cells seem to be more receptive to exogenous dsRNA delivery than plant cells, most likely due to the presence of the rigid cell wall in the latter. In order to trigger RNAa in tomato by sequestering specific micro RNAs (miRNAs), Short Tandem Target Mimics (STTM) RNA molecules designed to bind tomato miR156 and miR165 were generated and sprayed in tomato plants. Yet, these exogenously applied single stranded STTM RNA molecules were recognized by the host machinery (and particularly by the RNA-dependent RNA polymerase 6) as aberrant transcripts and were rapidly degraded before managing to exhibit any miRNA decoy action. These findings suggest that in contrast to endogenously expressed STTM (e.g. STTM-expressing transgenic plants), exogenously applied STTM RNAs are channeled to host degradation pathways, underpinning the complexity of the RNAi surveillance pathways in plants. Importantly, during RNASTIP, the core RNAi machinery of the beneficial endophyte Fusarium solani strain K (FsK) was for the first time examined. It is well established that not all fungi encode RNAi components and not all fungi are able to take up dsRNAs from their environment. By performing next generation genome and transcriptome sequencing we could reveal the presence of 2 Dicer-like endonucleases (DCLs), 2 Argonaute effectors (AGOs) and 4 RNA-dependent RNA polymerases (RDRs) in FsK, suggesting that this fungus encodes all the core RNAi components. By exogenously applying in vitro transcribed dsRNA in FsK conidia we could record efficient onset of RNAi of a FsK reporter gene, underlining that FsK is not only able to take up dsRNA from its environment but also process it into small RNAs (siRNAs) that trigger the RNAi mechanism. Finally, by generating a FsK strain that expresses dsRNA against a reporter plant gene, we aim to investigate the possibility of RNA-based cross-talk between this fungus and its host plant. Given the beneficial effect this endophyte fungus exhibits on plant growth, these data may not only shed light to the molecular mechanisms involved but also lead towards novel biostimulants.
