In this study, we developed a VIGS vector derived from CsCMV-CM for use in cassava plants. The CsCMV-based vector has several advantages over previous ACMV, EACMV, and TRV VIGS vectors in cassava [17–19, 23]. First, the vector caused milder mosaic symptoms than ACMV and EACMV in cassava leaves, making it suitable for functional genomics in cassava because severe viral symptoms may be confused with the effects of the VIGS vector and its resulting phenotypes in test plants. Second, unlike ACMV and EACMV and TRV, which consist of bipartite DNA and RNA genomes, respectively, CsCMV has a single-stranded RNA genome. A virus vector-based single viral genome is usually easier to manipulate than those of multipartite genomes . For example, compared with vectors based on multipartite viruses, agroinfiltration with CsCMV vectors does not require the preparation of a mixture of agrobacterium suspensions, each one carrying discrete portions of the genome. Third, CsCMV spreads by mechanical transmission, which makes infection of plants easier through leaf agroinfiltration, resulting in effective VIGS. Conversely, agroinoculation of cassava plants with geminivirus-based vectors requires injection of agrobacteria suspensions near the axillary buds and through superficial cuts in the stem. This inoculation method could damage the meristems and affect plant growth . Biolistic delivery has also been used for geminivirus inoculation of cassava plants, but it is a high-cost method . In this study, 100% infection efficiency by agroinfiltration of each CsCMV-based construct was achieved. Fourth, ACMV and EACMV are transmitted by whitefly. Their viral vectors have a potentially higher risk of escape into the environment than the CsCMV vector by mechanical transmission. Fifth, we inserted the NC frame of Nimble Cloning into the CsCMV genome to facilitate the rapid cloning of desired target genes. Accordingly, each PCR-generated target fragment with NC adapters can be cloned into a circular pCsCMV-NC vector via a simple mixture of the rare-cutting restriction enzyme Sfi I and T5 exonucleases, which simultaneously accomplish linearization of the vector and the ligation reaction . In previous studies, Gateway-based and ligation-independent VIGS vectors have been used for rapidly cloning a target fragment without multiple digestion and ligation steps [22, 38]. In contrast to these approaches, Nimble Cloning does not require an additional step to linearize the VIGS vector; therefore, it is simpler and more cost-effective than Gateway-based and ligation-independent cloning methods . Furthermore, the NC frame includes the ccdB gene, which is a positive selection marker to facilitate more rapid and accurate screening of putative recombinant colonies. A similar strategy is widely applied in Gateway methods [22, 38]. In this study, greater than 95% of clones were positive in each transformation using Nimble Cloning with mixtures of pCsCMV-NC vector and PCR amplicons of individual target gene fragments. Thus, the CsCMV-based VIGS system could also be applied in the construction of a cassava VIGS library for high-throughput forward genetics screening in the future.
Application of CsCMV as a VIGS vector requires the insertion of foreign sequences into the viral genome at positions that do not affect viral infectivity. CsCMV is the type member of the genus Potexvirus. There are two strategies for construction of potexvirus-based vectors according to their viral genome organization. Introduction of an additional SGP upstream of the CP gene for expression of a gene of interest is a commonly used approach and has successfully been used in PVX, PepMV, and FoMV [31–33]. The other strategy is development of a FoMV-based VIGS vector by insertion of the cloning site after the CP stop codon . In our study, we constructed the CsCMV vector by duplicating the 90-bp putative CsCMV CP SGP, including the potexvirus-specific octanucleotide motif (GUUAAGUU) . Our result showed that engineering the duplicated copy of the putative CsCMV CP SGP and NC cloning frame into the viral genome did not affect infectivity of the CsCMV-NC vector in cassava and N. benthamiana. Moreover, the anti-sense fragments of PDS and ChlI cloned into the pCsCMV-NC vector caused obvious silencing phenotypes in both host plants.
In this study, the 487-bp fragment of PDS and the 345-bp fragment of ChlI with best target region score were selected by genome-wide off-target gene silencing assessment. The predicted result showed that ChlI345-derived siRNAs can target two ChlI homologous genes located on the 16th and 17th chromosomes of cassava while the predicted siRNAs only can match PDS gene in the 5th chromosome of cassava. More target sites might result in more accumulation of the target fragment-derived siRNAs and induced more severe silencing phenotype. Indeed, we observed that the ChlI345-silenced cassava plants exhibited more severe silencing phenotype with large areas of yellow–white leaf than PDS487-silenced cassava plants. Certainly, the gene silencing efficiency is related to various factors including sequence space, target availability, the position of nucleotides, secondary structures of mRNA and intrinsic characteristics of siRNA and target mRNA . In addition, we assessed whether the size of the host-derived sequence insert affects CsCMV based VIGS efficiency. Our results showed that the CsCMV-vectors carrying partial ChlI genes of different sizes (133, 236, 345, and 439 bp) in antisense orientation could effectively induce silencing in cassava, and the more severe silencing phenotype was observed when the insert length was more than 300 bp. Similarly, infection with the PVX VIGS vector harboring PDS sequences of 412-bp in antisense orientation resulted in strong photobleaching phenotypes in both diploid and cultivated tetraploid Solanum species . However, FoMV vector with a duplicated FoMV CP SGP was used to induce effective silencing of endogenous genes in barley when target sequence insert was a short inverted-repeat fragment but not an antisense one . Therefore, the effect of length of target genes on silencing depends on the different potexviruses-derived vectors and hosts.
As VIGS approaches induce transient gene knockdowns, increasing the duration of endogenous gene silencing will widen the application of VIGS in functional genomics. In this work, strong yellow-white silencing phenotype in systemic leaves infected with CsCMV-ChlI345 can persist for more than two months. The longer silencing period will facilitate characterization of the gene functions involved in developmental and biosynthetic pathways and stress tolerance in cassava. However, the phenotype gradually became less severe in the upper leaves and almost disappeared in the top leaves, which was related to partial or complete loss of inserted ChlI345 fragment because of the sequence redundancy of the duplicated SGP in potexvirus-based vectors [34, 37]. To address the problem, an PVX-based expression vector was improved to stabilize the foreign inserts by replacing the duplicated SGP with a heterologous SGP combined with an N-terminal CP deletion . In addition, change of the position of insertion was used to increase stability of the insert. The cloning site in FoMV was placed after the stop codon following the CP coding sequence instead a duplicated subgenomic promote . However, the loss of PDS inserts still occurred when this FoMV vector was used to silence PDS in maize . Therefore, insert stability of viruses is regard as a surprisingly complex problem involved in the genome characteristics, the host environment and the demography of a virus population .
Over the years, numerous cassava varieties with different traits have been released in the world . Theoretically, CsCMV-based VIGS system are applicable to cassava lines susceptible to CsCMV. Here, we induced ChlI gene silencing in 6 popular lines (TMS60444, ZM9781, SC5, SC8, SC9 and SC10) in China using pCsCMV-ChlI345, which will contribute to use this vector to analysis some functional genes involved in important biological and agronomical traits among these cultivars. In addition, CsCMV were detectable in fbrous and storage roots of CsCMV-NC-infected cassava plants (Additional file 1: Fig. S4), thereby we will further broaden the use of CsCMV vector in gene silencing from leaves to root tissues like ACMV-based vector.