Virus-induced gene silencing and pathogen inoculation
Agrobacterium tumefaciens cells harboring TRV vectors were infiltrated into two fully expanded cotyledons of 10-day-old seedlings as described previously (Gao et al., 2013). The primers listed in Table S2 were used to evaluate the silencing effect based on the expression levels of the target genes in leaf and root tissues.
For pathogen inoculation, 3-week-old cotton seedlings of transgenic and VIGS plants (at least 30 plants for each lines) were infected withV. dahliae (V991) conidial suspension (1×10^5conidia·ml-1) using a root dip inoculation method, and the plants were transplanted into sterilized soil for observation of disease symptoms. A fungal recovery assay and a disease index analysis were performed as described previously (Xu et al., 2014a). For fungal colonization and biomass detection, longitudinal cross-sections of cotyledonary nodes were dissected at 16 days after inoculation and observed under a stereoscopic microscope (MZFLIII, Leica, Wetzlar, Germany). Infected stem sampleswere separately collected for DNA extraction and taken approximately 1 cm above the cotyledon node (CN) and first leaf node (FN). Each experiment was repeated three times.