This paper revealed that small RNAs in Boerichia cinerea could cross into Arabidopsis thaliana and tomato.
First, they sequenced infected plant leaves and pure cultured fungal samples. The criteria for screening fungal cross-kingdom sRNA were as follows:
- RPT100M;
- The expression level in infected leaves was higher than that of pure cultured fungal samples;
- The sequence should be completely matched with the fungal genome but not with the plant genome;
- The sequence should be at least two mismatches with the plant genome;
- There should be secondary structure of milRNA;
- Target genes are present in the host.
Then the authors conducted following experiments: quantify the cross-kingdom sRNAs and their target genes respectively, core-expression followed by western-blot in tobacco, view YFP fluorescence in tobacco, mutation sRNA target site, transgenic plant with cross-kingdom small RNA-OX, transgenic plant by knocking out target gene, AGO-IP in plant, plant AGO mutant lines, plant DCL mutants, fungal DCL gene knockout.
The above experiments more stably verified that three DCL-dependent cross-kingdom sRNAs of B. cinerea enhanced pathogenicity by hijack AGO1 in host plants.
