Fig. 1

Construction and infectivity of a CsCMV-CM-based vector (pCsCMV-NC). a Schematic of infectious clone pCsCMV-CM and pCsCMV-NC vector. The full-length genomic cDNA of CsCMV-CM was cloned into between CaMV 35S promoter (35S P) and poly(A) signal of a T-DNA binary vector pGreenII-35S to generate pCsCMV-CM. A duplicated 90-bp putative CsCMV-CM CP subgenomic promote (SGP1) and a Nimble Cloning (NC) frame sequence (adapter 1–Sfi I–ccdB gene–Sfi I–adapter 2) were engineered into viral genome at upstream of the authentic CP promoter (SGP2), and the resultant vector was designated as pCsCMV-NC. The duplicated SGP includes 60 bp upstream of the CP start codon and ended 30 bp downstream (GenBank accession numbers MW175326, nt 5534–5623). The target gene fragment was flanked by adapter 1 and 2 of the NC frame and can be cloned into the pCsCMV-NC vector using Nimble Cloning. A total of 5 major open reading frames (ORFs) of the CsCMV genome are indicated by colored boxes: an RNA dependent RNA polymerase (RdRp), three triple gene block (TGB) proteins and a coat protein (CP). White rectangles and arrows indicate elements comprising the backbone of the pGreenII-35S vector. The nucleotide sequences of adapter 1 and 2 in the NC frame sequence are shaded in black. The Sfi I sites are underlined and the ccdB gene is marked in italics. Black arrows indicate primers used to construct agroinfectious clone pCsCMV-CM and pCsCMV-NC vector (Additional file 2: Table S2). b Systemic symptoms induced by the pCsCMV-CM and pCsCMV-NC on cassava leaves at 35 days postinoculation (dpi). c Detection of viral accumulation of the CsCMV-CM and CsCMV-NC in infected cassava (‘SC10’) leaves using RT-qPCR. Three independent experiments were performed and each included six plants per treatment group. Error bars indicate the SD