Table 1

Primers used in this study"

Fig. 1

Expression response of wheat Paf1 subunit genes to BSMV infection A: schematic diagram showing the genome structures of the BSMV clones constructed and used in this study, red arrows indicate the positions of the RT-PCR primers used for viral RNA detection. CP: coat protein; TGB1, 2, and 3: triple gene block 1, 2, and 3, respectively. B: RT-qPCR analysis of the expression responses of wheat Paf1 subunit genes to BSMV infection. For each treatment (mock and BSMV:GFP infection), three independent seedlings were used as biological replicates for analysis of all target genes, and each biological replicate was analyzed in technical triplicate for each gene. * indicates significant differences (P < 0.05)."

Fig. 2

Efficiency of silencing wheat Paf1 subunit genes using BSMV-VIGS The silencing efficiency of the target genes was assessed by RT-qPCR. Three independently infected seedlings were used as biological replicates for each BSMV clone, with BSMV:GFP as the control. Each biological replicate was analyzed in technical triplicate. * indicates significant differences (P < 0.05)."

Fig. 3

Paf1 subunit genes negatively regulate BSMV-induced hypersensitive response-associated cell death in wheat A-C: comparison of cell death in the systemic leaves of wheat seedlings inoculated with infectious RNA of BSMV or BSMV:CDC73. Cell death in systemic leaves was assessed based on symptom observation (A), trypan blue staining (B), and electrolyte leakage analysis (C). For the electrolyte leakage analysis, three independent seedlings for each treatment were used as biological replicates. ns indicates no significant difference (P > 0.05). D: comparison of cell death in the systemic leaves of wheat seedlings inoculated with virion preparations of BSMV or BSMV:CDC73. The left panel shows cell death symptoms in systemic leaves, and the right panel shows the comparable virion concentrations of the two virion preparations used for inoculation, as indicated by coat protein abundance. CP: coat protein; RbcL: Rubisco large subunit. E: BSMV clones capable of silencing different Paf1 subunit genes consistently enhanced cell death when infecting wheat seedlings."

Fig. 4

Silencing of TaCDC73 does not alter BSMV-induced ROS levels in wheat ROS accumulation in the systemic leaves of seedlings inoculated with infectious RNA (A) or virions (B) was examined by DAB staining for H2O2 and NBT staining for O2?."

Fig. 5

TaCDC73 silencing enhances wheat resistance to BSMV A-B: immunoblot detection of viral proteins in immature, unexpanded (A) and fully expanded (B) fourth leaves, corresponding to the second systemic leaves, of BSMV-infected wheat seedlings. Numbers 1-3 represent three individual plants. TGB1: triple gene block 1. C: immunoblot detection of viral proteins in the leaves of BSMV-infected adult wheat plants. CP: coat protein; RbcL: Rubisco large subunit. D: RT-PCR detection of viral RNA in the leaves of BSMV-infected adult wheat plants, with Tubulin used as an internal control. For RNAγ detection, amplification from the plasmid vector was used as a size reference for the amplicon containing the full-length insert. P: plasmid; M: molecular marker."

Fig.6

TaCDC73 silencing inhibits wheat growth during the response to BSMV infection A: growth phenotypes of wheat plants infected with BSMV:GFP or BSMV:CDC73. B: measurement and statistical analysis of plant height and the length and width of the uppermost fully expanded leaves of the two groups of plants shown in panel A. Six plants per group were used as biological replicates for all measurements. * indicates significant differences (P < 0.05)."

Fig. 7

TaCDC73 silencing reduces the efficiency of BSMV- mediated VIGS VIGS: virus-induced gene silencing. The extent of TaPDS silencing, reflected by the degree of leaf bleaching, was used to evaluate VIGS efficiency."