Small RNA sequencing of different microRNA-induced gene silencing constructs
To test whether a single MIGS consturct can inhibit different viruses, a MIGS construct was generated consisting of 4 modules targetting PVX and TMV (pMIGS_TMV/PVX) and agro-infiltrated with or without pmiR173. To test the maximum number of modules in a single MIGS molecule that can successfully lead to sRNA production, MIGS constructs were generated consisting of either six (MIGS_6X), nine (MIGS_9X) or fifteen modules (MIGS_15X). Each module was made using a unique random sequence, allowing for the back-mapping of sRNAs to the fragment of origin. These constructs were agroinfiltrated in N. benthamiana leaves with or without the miR173, and the sRNA population was assessed by sRNA northern blot and deep sequencing. To test the effect of the first nucleotide at the 5 end of migsiRNA on MIGS efficiency, MIGS constructs were generated, either with the wild-type target sequence (MIGS_TMV_A) or with a modified sequence where the first nucleotide at the 5 end of all predicted phased migsiRNAs originating from the minus strand was replaced with a U (MIGS_TMV_A (U)) or a G (MIGS_TMV_A (G)).