Effect of mutations in the 2b protein of tomato aspermy virus on RNA silencing suppressor activity, virulence, and virus-induced gene silencing

Virus-Induced Gene Silencing in Chrysanthemum seticuspe Using the Tomato Aspermy Virus Vector

“…The TAV ChJ strain, isolated from Chrysanthemum in Japan, was used (GenBank, accession numbers: LC634031, LC634032, and LC634033) [ 13 ]. pTOPOT1, pTOPOT2, and pTOPOT3, in which TAV RNA1, 2, and 3 are cloned downstream of a T7 RNA polymerase promoter, respectively, were used for the mechanical inoculation of TAV RNA transcripts [ 13 ].…”

“…The TAV ChJ strain, isolated from Chrysanthemum in Japan, was used (GenBank, accession numbers: LC634031, LC634032, and LC634033) [ 13 ]. pTOPOT1, pTOPOT2, and pTOPOT3, in which TAV RNA1, 2, and 3 are cloned downstream of a T7 RNA polymerase promoter, respectively, were used for the mechanical inoculation of TAV RNA transcripts [ 13 ]. pJL89T1, pJL89T2, and pJL89T3, in which TAV RNA1, 2, and 3 are cloned downstream of a cauliflower mosaic virus 35S promoter, respectively, were used for agroinoculation [ 13 ].…”

“…pTOPOT1, pTOPOT2, and pTOPOT3, in which TAV RNA1, 2, and 3 are cloned downstream of a T7 RNA polymerase promoter, respectively, were used for the mechanical inoculation of TAV RNA transcripts [ 13 ]. pJL89T1, pJL89T2, and pJL89T3, in which TAV RNA1, 2, and 3 are cloned downstream of a cauliflower mosaic virus 35S promoter, respectively, were used for agroinoculation [ 13 ]. pJL89T2 2b ΔC61 , pJL89T2 2b ΔC23 , pJL89T2 2bR46C , and pJL89T2 2bS4042A containing deletions in C-terminus of 2b gene (ΔC61 and ΔC23) or amino acid substitutions in 2b gene (R46C and S4042A) in pJL89T2 were used as TAV 2b mutants with decreased VSR activity for agroinoculation [ 13 ].…”

“…Approximately 20 viruses, such as tomato aspermy virus (TAV), cucumber mosaic virus (CMV), chrysanthemum virus B, tomato spotted wilt virus, and chrysanthemum stem necrosis virus, infect chrysanthemums [ 10 , 11 , 12 ]. Among these viruses, we previously developed a TAV VIGS vector based on the TAV ChJ strain isolated from chrysanthemums, which induced silencing in N. benthamiana [ 13 ]. TAV belongs to the Cucumovirus genus of the Bromoviridae family and has positive-sense ssRNA genomes [ 14 ].…”

“…Vacuum infiltration into sprouts was successful for the inoculation of the TAV VIGS vector in C. seticuspe , whereas the mechanical inoculation of viral RNA transcripts and agroinoculation into leaves did not work. Although efficient TAV VIGS in N. benthamiana was achieved by the attenuation of the viral suppressor of RNA silencing (VSR) activity of the 2b protein [ 13 ], we demonstrated that the TAV vector with the wild-type 2b protein harboring the partial C. seticuspe phytoene desaturase ( CsPDS ) sequence caused photobleaching phenotypes in C. seticuspe . The TAV vector will be a useful rapid functional genomics tool in chrysanthemums.…”