Periodic Reporting for period 1 - RNASTIP (RNA Sprays as a Tool for Crop Improvement and Protection)
Reporting period: 2018-09-01 to 2020-08-31
With the increasing world population, reduced farmland and demand for heightened global food security, comes the need for new sustainable, effective and environmentally friendly agricultural solutions. The extensive use of toxic agrochemicals has been rendered obsolete, whereas the employment of transgenic plants and genetically modified organisms (GMOs) has raised considerable public and scientific concerns. Accordingly, this project aimed to develop novel RNA-based toxic-free and GMO-free tools to modify and protect crops. More specifically, in the framework of RNASTIP (i) single stranded RNAs (ssRNAs) that would serve as micro RNA decoys (Short Tandem Target Mimic, STTM) and would thus trigger the RNA activation (RNAa) pathway and (ii) double stranded RNAs (dsRNAs) that would trigger the RNA interference (RNAi) pathway were generated and exogenously applied in tomato plants. Concerning RNAa, our conclusions suggest that application of STTM RNAs in tomato plants is troubleshooting, since STTM RNAs are sensed by the plant cell as potentially dangerous aberrant transcripts and are thus channeled to RNA-directed RNA polymerase (RDR)-mediated degradation pathway. Concerning RNAi, our conclusions suggest that application of dsRNA in tomato plants confers limited resistance to selected viruses and viroids, due to the inefficient delivery of dsRNAs inside the plant cell and the high replication rate of these pathogens. In contrast, dsRNA delivery and RNAi induction in a soil-borne fungal endophyte was very efficient, since fungal cells lack the rigid cell walls that plant cells contain. In the future, we suggest that optimization such approaches should include the use of elements such as 3’ polyadenylation tails that inhibit RDR-mediated degradation of STTM RNAs (RNAa approaches) and nanoparticles such as carbon dots that increase the efficiency of dsRNA delivery inside the plant cell (RNAi approches).
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.
During this project, RNA molecules having the capacity to trigger RNA interference were exogenously applied into plants and fungi with state of the art methods such as high pressure spraying. According to the current legislative framework (EFSA assessment), exogenous application of RNA molecules is considered to be transgene-free. Given the public concern against the use of GMOs and the demand for more environmentally-friendly agricultural solutions, this project positively contributed towards the shaping of novel sustainable crop management tools, in line with EU policy objectives. Moreover, during this project the RNAi mechanistic details of a fungal endophyte were examined for the first time. Ongoing research aims to unravel the RNAi-based interactions between this endophyte and its plant host. Given the beneficial character of this endophyte such as stimulation of plant growth, protection against biotic and abiotic stress, these data will likely contribute towards the development of novel biostimulants, the production of which could be achieved by meager means and in huge quantities, even in third world countries.