RNAi based approaches like Host-Induced Gene Silencing (HIGS) and Spray-Induced Gene Silencing (SIGS) have shown great potential in inhibiting pathogen infection in plants. In earlier studies it was demonstrated that inhibiting the sterol 14 alpha-demethylase through silencing of the three FgCYP51 genes, after delivery through transgene expression or external application of the double-stranded CYP3RNA, effectively controls Fusarium graminearum (Fg) infection in planta (Koch et al., 2013; Koch et al., 2016). In the attempt to silence only one or two of the FgCYP51 genes, co-silencing effects in the respective non-target FgCYP51 genes largely influenced the silencing efficiency in HIGS as well as SIGS approaches and masked the influence of individual constructs on Fg growth. Therefore, single and double constructs showed efficiencies in inhibiting Fg growth similar to the original CYP3RNA. By increasing the length of the single dsRNA constructs, silencing efficiency and co-silencing effects, proven by off-target analysis and qRT-PCR, were enhanced. The results suggest that up to 1500 bp the longest construct analysed here - there is no length limitation for a dsRNA to function in HIGS. However, increasing the dsRNA length did not constitute a boost for Fg resistance, suggesting that target gene selection is more important than the length of dsRNA for effective fungal growth inhibition by HIGS. This was further supported by HIGS studies with constructs in which the design of the dsRNA was largely changed in comparison to the original single, double or the CYP3RNA construct. The changes involved switching the position of the dsRNA in the target gene, triplication of single constructs and transposition of the individual CYP51 fragments in the CYP3RNA.With the objective to clarify how siRNAs are transferred between plant and fungus during HIGS and SIGS, two different methods were successfully used for the isolation of exosome-like nanoparticles or extracellular vesicles (EVs) from plants. The isolated vesicles shared characteristic features from known exosome preparations of mammalian cells and plant EVs that have been reported recently (Rutter and Innes, 2017). My work further confirmed that EVs from CYP3RNA-expressing plants contained siRNAs that originated from the dsRNA precursor, as proven by small RNA sequencing. The same was shown for EVs from CYP3RNA sprayed leaves. This indicates that the siRNAs, that are transferred during HIGS and SIGS, are indeed transported via vesicles, what was shown for the first time within this study. Therefore, the study gives new insight into the mechanism of RNA-based cross-kingdom communication. Altogether, this PhD project could clarify some important mechanistic details about HIGS and SIGS what is indispensable for a successful future application of these technologies as plant protection measure.
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