VIGS-agroinfiltration assay

Virus-induced gene silencing (VIGS) is the most effective transient gene silencing system for plants (51). Combination of VIGS and agroinfiltration (VIGS-agroinfiltration) can be used to identify NSD trans factors. The VIGS-agroinfiltration system is described in detail in the Supplementary Data S1 file and Supplementary Figure S1.

To silence a gene (or a gene family), a segment from the target gene is incorporated into a Tobacco Rattle Virus (TRV) VIGS vector. If a gene family is targeted, a conserved segment is cloned, while specific regions are used for gene-specific inactivation. To silence a gene, N. benthamiana plants were infected with the recombinant TRV VIGS vectors (sequences of the VIGS fragments are shown at Supplementary Data S1). TRV infection induces antiviral RNA silencing response, thus the virus concentration will be very low in the upper leaves. Moreover, the silencing will specifically inactivate the host genes that are homologous to the incorporated sequence. To trigger VIGS, ∼21 days old N. benthamiana plants were co-agroinfiltrated with a mixture of three Agrobacterium cultures. One expressed P14, the second expressed TRV RNA1 and the third expressed TRV RNA2 containing segments from N. benthamiana PDS (phytoene desaturase) gene or from PDS and ∼600 nt long sequence from the coding region of the gene what we want to silence. PDS is used to monitor silencing. When the upper leaves started to bleach (indicating that PDS silencing was efficient and suggesting that the silencing of the gene of interest is also effective), leaves under the bleaching ones were agroinfiltrated (45). Quantitative RT-PCR (qRT-PCR) assays confirmed that the target mRNA levels were significantly reduced (Supplementary Figure S1B). Arabidopsis Pelota1 and Hbs1 genes were used for complementation assays. The domain structures of the predicted proteins and the alignments of their N. benthamiana homologs of Pelota, Hbs1 and SKI2 are shown at Supplementary Figures (Supplementary Figures S2–S5). For complementation assay (Supplementary Figure S6), the silenced leaves were co-infiltrated with three cultures, one expressing P14, the second expressing the NSD reporter and the third one expressing the tested factor.