Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector

Abstract

The stability of the inserted genes in the viral expression vector varied depending on the sequence introduced and the position of insertion. Infectious cDNA to Clover yellow vein virus (pClYVV) was modified to insert a foreign gene at two independent sites: one, along with a polylinker, between the NIb and CP genes (pClYVV/CP/ W) and the other between P1 and HC-Pro (pClYVV-Pst/ CP). The green fluorescent protein (GFP) gene was inserted into either pClYVV/CP/W or pClYVV-Pst/CP. GFP gene was stably maintained and expressed in both vectors following serial passages in plants. Progeny viruses from both constructs accumulated in similar amounts and at rates of 70%-80% of that of the wild-type virus. On the other hand, progeny viruses carrying the human interferon-α (hIFN) gene cloned in pClYVV-Pst/CP were genetically unstable owing to frequent deletions of the cloned gene during passage through plants. In contrast, the hIFN sequence cloned in pClYVV/CP/W was stably maintained in viruses after several passages in broad bean plants, and the progeny virus accumulated at the rate of about 50%-100% of that of the wild-type virus. The nucleotide sequence analyses indicated that the genetic instability of the inserted sequence results from homologous recombination of viral vector and inserted DNA sequences; it is not due to the inserted sequence alone.

Activation of the Salicylic Acid Signaling Pathway Enhances Clover yellow vein virus Virulence in Susceptible Pea Cultivars

“…Constructs were described previously (Wang et al 2003;Yambao et al 2008). B, Viruses were inoculated mechanically.…”

“…In pea, ClYVV systemically induces cell death, a process that is controlled by a single incompletely dominant gene, Cyn1, and ultimately results in plant death (Ravelo et al 2007). ClYVV infectious cDNA was synthesized and developed as a vector in legumes (Masuta et al 2000;Takahashi et al 1997;Wang et al 2003). Point mutations in P1/HCPro of ClYVV attenuate symptom development in broad bean and compromise the RSS activity of P1/HCPro mutants (Yambao et al 2008).…”

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas

“…(20,21). These chimeric viruses were based on Cl-No.30 infectious cDNA that we previously constructed and developed for use as a gene expression vector (7,23,37,38). The following chimeric viruses tagged with GFP that covered almost all regions of the ClYVV genome were created: Cl-P1HC/GFP, Cl-BB/GFP, Cl-NS/GFP, and Cl-SB/GFP (Fig.…”

Recent Advances in In Planta Transient Expression and Silencing Systems for Soybean Using Viral Vectors

“…In addition, a second position between nuclear inclusion b (NIb) and coat protein (CP) has also been used ( Fig. 15.1; Wang et al 2003), with greater stability of foreign inserts compared to the P1/HC-Pro site, though there are no reports that this version has been used in soybean. Independent attempts to use this NIb/CP site in SMV were not successful (A.L.E.…”

“…The use of other positions in the viral genome as cloning sites was investigated, and the junction of NIb/CP could also be used to express foreign sequences. For proteins that cannot be stably expressed at the P1/HC-Pro junction, it may be possible to express them from the junction of NIb/CP and vice versa (Wang et al 2003). Thus, context within the viral genome can affect the expression of foreign proteins.…”