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.