2.10 Establishment of virus-induced gene silencing (VIGS) to wheat bunt pathogens
The cDNA fragment of TaEC with ApaI restriction site were obtained by reverse transcription PCR and inserted into the original Barley strip mosaic virus (BSMV) vector for gene silencing. Fragment had no similarity to any other wheat gene in BLAST analysis (Http:// blast.ncbi.nlm.nih.gov/Blast/). In addition, to confirm the specificity of fragments used in the VIGS assays, the expression level ofTaEC were measured in TaEC- knockdown plants, respectively. The fragment of wheat phytoene desaturase (TaPDS) was inserted into the BSMV vector as the positive control.
The BSMV viruses (BSMV:γ, BSMV:TaPDS; BSMV: TaEC) were inoculated in wheat tassel at (0, 10, 15, 22, 35 and 42d) and then inoculated T. controversa and T. laevis at 5 dpi following modification of previous method. Virus infection on Nicotiana benthamiana were achieved by Agrobacterium -mediated transient gene expression of infectious constructs from the T-DNA of a binary plasmid (GV3101). The bacterial solution was centrifuged at 5000rmp for 10 min, resuspended in infiltration buffer (10 mM MgCl2, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES) and 0.1 mM acetosyringone (AS) and adjusted to 0.7 (OD600)and then incubated at room temperature for 2 h. The culture was then infiltrated to the underside of a leaf using a 1 ml syringe without a needle. The N. benthamiana leaves were grounded in the pestle and mortar and were inoculated on the wheat leaves at the booting stage and repeated three times. At the same time, the wheat inoculated with BSMVγ was used as the control. The disease index was analyzed between BSMVγ and BSMV: TaEC. RNA was extracted from the ears of the infected plants and the control group using the plant total RNA extraction kit (Tiangen, Beijing). The integrity, concentration and purity of RNA were detected by electrophoresis of agarose gel and Spectrophotometer (DeNovix DS-11+Spectrophotometer,US). According to TaEC sequences using the SnapGene design primers (AGGGACAACCATTTCCAAGGATGCC/CGCGTTCTGGCAA
GTACTCTCG), wheat Actin gene was used as the internal reference gene for qRT-PCR detection of transcripts. SuperReal PreMix Plus(SYBR Green) kit (Tiangen, Beijing) was used to perform qRT-PCR reaction, the 20 µl reaction mixtures contain 1 µl template, 0.4 of each primer (10 μM), 10 µl SuperMix (TransGen Biotech, Beijing, China) and 8.2 µl ddH2O. The amplification protocol was 95 ℃ for 15 min, followed by 40 cycles of 95 ℃ for 10 s, 60 ℃ for 32s. All cDNA and qRT-PCR reactions were carried out in triplicate. The 2−∆∆CT method was used to calculate the relative expression of each gene (Ma et al., 2012).