The Role of a C‐Terminal Seven‐Amino Acid Motif in TbCSV C3 Protein and Its Interaction With NbPOLA2 in Enhancing Viral Replication

FIGURE 1. Deletion or replacement of the 3′ end region of the TbCSV C3 gene attenuates viral infection. (a) Schematic diagram of the TbCSV, TbCSVdC3 and TbCSVdC3‐iLOV genomes. (b) Symptoms of Nicotiana benthamiana plants inoculated with TbCSV, TbCSVdC3 or TbCSVdC3‐iLOV at 5, 10, 15 and 20 days post‐inoculation (dpi). The results were reproduced in three independent experiments using 12 plants per treatment. (c) Infection rate of TbCSV, TbCSVdC3 and TbCSVdC3‐iLOV in inoculated N. benthamiana plants, determined by PCR detection of the virus in upper new leaves at 5 and 15 dpi. Data are presented as the means from three independent experiments. Bars labelled with ‘n.s.’ indicate no significant difference (p > 0.05, t test). (d) Relative accumulation of TbCSV DNA in TbCSV‐, TbCSVdC3‐ and TbCSVdC3‐iLOV‐inoculated N. benthamiana plants at 5, 10, 15 and 20 dpi. Four plants were analysed for each treatment, and three technical replicates were used for each biological sample. Error bars represent means ± SEM. Significant differences were determined using Student's t test, **p < 0.01. Nicotiana benthamiana 25S ribosomal RNA was used as the internal reference. The experiment was repeated three times…

FIGURE 2. Trans expression of C3 rescues the functional deficiency of TbCSVdC3 mutant. (a–c) TbCXV C3 systemically overexpressed by virus vector PVX facilitates TbCSVdC3 mutant infection. (a) Symptoms of Nicotiana benthamiana plants co‐infected with TbCSV/PVX, TbCSVdC3/PVX or TbCSVdC3/PVX‐C3 at 7 days post‐inoculation (dpi). (b) Reverse transcription‐quantitative PCR (RT‐qPCR) analysis of the relative expression levels of C3. NbActin was used as the internal reference. (c) Relative accumulations of TbCSV DNA. (d, e) Complementation of TbCSVdC3 in C3‐overexpressing transgenic N. benthamiana plants. (d) Symptoms induced by TbCSVdC3 infection on wild‐type (Nb) and C3‐overexpressing transgenic N. benthamiana plants (L2) at 7 and 14 dpi. (e) Relative accumulations of TbCSV DNA. In (c) and (e), N. benthamiana 25S ribosomal RNA was used as the internal reference. Total RNA and DNA were extracted from the upper systemically infected leaves and subjected to RT‐qPCR (b) and qPCR (c, e). In (a) and (d), the results were reproduced in three independent experiments using 12 plants per treatment. In quantitative experiments, four plants were analysed for each treatment and three technical replicates…

FIGURE 3. The C‐terminal 123–129 amino acids of TbCSV C3 protein are critical for its replication enhancement ability. (a, b) Transient expression of C3 elevated the replication level of TbCSVdC3. (a) Reverse transcription quantitative PCR analysis of the relative expression levels of C3 using NbActin as the internal reference. (b) Relative accumulations of TbCSV DNA. (c) Relative accumulations of TbCSV DNA in Nicotiana benthamiana leaves transiently expressing C3 or truncated C3 mutants. (d) Symptoms of N. benthamiana plants co‐infected with TbCSVdC3 and PVX‐C3 or truncated C3 mutants. The results were reproduced in three independent experiments using 12 plants per treatment. (e) Relative accumulations of TbCSV DNA in infected N. benthamiana plants. N. benthamiana 25S ribosomal RNA was used as the internal reference in (b), (c) and (e). In quantitative experiments, four plants were analysed for each treatment, and three technical replicates were used for each biological sample. Error bars represent means ± SEM. The different letters above each bar in panels (a), (b), (c) and (e) indicate statistically significant differences as determined by one‐way analysis of variance followed by…

FIGURE 4. The 123–129 amino acid residues of TbCSV C3 are required for viral DNA accumulation and symptoms development. (a) Symptoms of Nicotiana benthamiana plants infected with wild‐type TbCSV and TbCSV(d123–129) mutant infectious clone at 7, 14 and 21 days post‐inoculation (dpi). Mock‐inoculated N. benthamiana plants served as controls. (b) Relative accumulation of TbCSV DNA. N. benthamiana 25S ribosomal RNA was used as the internal reference. The results were reproduced in three independent experiments using 12 plants per treatment. Error bars represent means ± SEM, and significant differences were indicated using Student's t test, **p < 0.01. These experiments were performed with three independent biological replicates with similar results.

FIGURE 5. Interactions between TbCSV C3 and NbPOLA2 in yeast and in planta. (a) Yeast two‐hybrid (Y2H) analysis of intra‐ and intermolecular interactions of TbCSV C3. Recombinant plasmid pairs as that indicated were co‐transformed into yeast strain Y2H‐Gold. Serial dilutions (10‐fold) of transformants were spotted on SD/−Trp−Leu and SD/‐Trp−Leu−His−Ade selective media. Co‐transformation with AD‐T+BK‐53 served as a positive control. Yeast co‐transformed with AD‐Lam+BK‐53, AD‐NbPCNA+BK, AD‐NbpRBR+BK, AD‐NbPOLA2+BK and AD‐NbPOLD2+BK were negative controls. Images were taken after 72 h. (b) Bimolecular fluorescence complementation (BiFC) analysis of the intra‐ and intermolecular interactions of TbCSV C3 in planta. Leaves co‐infiltrated with cYFP‐C3 and one of the candidates including nYFP‐C3, ‐C1, ‐NbPCNA, ‐NbpRBR, ‐NbPOLA2 and ‐NbPOLD2 were examined under confocal microscope. Co‐infiltration with cYFP‐C3+nYFP, cYFP+nYFP‐NbPOLA2 and cYFP+nYFP‐NbpRBR served as negative controls. Histone 2B‐RFP (H2B‐RFP) was used as a nuclear marker. (c) Luciferase complementation imaging (LCI) analysis of the interaction between C3 and candidate proteins in Nicotiana benthamiana leaves. Leaves were…

FIGURE 6. Interaction analysis between NbPOLA2 and C3 mutants. (a) Yeast two‐hybrid (Y2H) analysis of the amino acid region in TbCSV C3 required for interaction with NbPOLA2. The schematic diagram indicates the truncated mutants of C3. Transformants were serially diluted 10‐fold and spotted on SD/−Trp−Leu−His−Ade selective medium. Images were captured after 72 h. (b) Bimolecular fluorescence complementation (BiFC) analysis of the interaction between NbPOLA2 and the C3(123–129A) mutant. Fluorescence in Nicotiana benthamiana leaves co‐infiltrated with nYFP‐NbPOLA2 and cYFP‐C3(123–129A), in which the amino acids from residues 123 to 129 were substituted with seven alanines, was examined using a confocal microscope. Co‐infiltration with nYFP‐NbPOLA2+cYFP and nYFP+cYFP‐C3(123–129A) served as negative controls, while co‐infiltration of nYFP‐NbPOLA2 with cYFP‐C3 was used as a positive control. (c) Luciferase complementation imaging (LCI) analysis of the interaction between NbPOLA2 and the C3(123–129A) mutant in N. benthamiana. Agrobacterium strains harbouring C3(123–129A)‐nLUC and cLUC‐NbPOLA2 were co‐infiltrated into N. benthamiana leaves, with luciferase detection performed 3 days…

FIGURE 7. NbPOLA2 positively regulates TbCSV infection. (a, b) Overexpression of NbPOLA2 facilitates TbCSV infection. (a) Symptoms of Nicotiana benthamiana plants infected with PVX‐NbPOLA2 or the PVX‐GUS control at 7 days post‐inoculation (dpi). (b) Quantitative PCR (qPCR) analysis of TbCSV DNA accumulation at 7 and 14 dpi. (c, d) Silencing of NbPOLA2 inhibits TbCSV infection. (c) Symptoms of N. benthamiana plants infected with PVX‐Nbpola2 or PVX‐GUS at 7 and 14 dpi. (d) qPCR analysis of TbCSV DNA accumulation at 7 and 14 dpi. In panels (b) and (d), N. benthamiana 25S ribosomal RNA was used as the internal reference. Each experiment was conducted independently three times with four plants for each treatment each time. Error bars represent means ± SEM, and significant differences were determined using Student's t test, *p < 0.05, **p < 0.01. These experiments were performed with three independent biological replicates with similar results.

FIGURE 8. NbPOLA2 enhances TbCSV replication. In local infection assays, pGD‐NbPOLA2 or pGD empty vector was infiltrated into Nicotiana benthamiana leaves pre‐inoculated with TbCSV for 2 days, and the leaf samples were collected at 3 days post‐agroinfiltration. (a) Reverse transcription quantitative PCR (RT‐qPCR) analysis of NbPOLA2 expression levels in pGD‐NbPOLA2 and pGD‐infiltrated leaves. NbActin was used as the internal reference. (b) qPCR analysis of TbCSV DNA accumulations in pGD‐NbPOLA2 and pGD‐infiltrated leaves. (c, d) Quantifications of the viral complementary strand (CS) (c) and viral strand (VS) (d) in N. benthamiana plants overexpressing NbPOLA2 (pGD‐NbPOLA2) or the control (pGD). In panels (b) to (d), N. benthamiana 25S ribosomal RNA was used as the internal reference. Data represent the mean of five independent biological replicates. Error bars represent means ± SEM. Significant differences were determined using Student's t test, **p < 0.01.

FIGURE 9. Proposed working model for the seven‐amino acid motif‐dependent viral replication enhancement activity of TbCSV C3. A C‐terminal seven‐amino acid motif spanning from residues 123 to 129 of the C3 protein is responsible for recruiting the host factor NbPOLA2 to facilitate viral replication. Mutation of the 123–129 motif to seven alanines abolishes the interaction between C3 and NbPOLA2, hence impairing the replication enhancement activity of C3.