RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of inverted repeat (IR) constructs during cloning and plant transformation. Although such constructs have been shown to be stabilized with introns and efficiently induce RNA silencing, we found that the Pdk intron did not stabilize South African cassava mosaic virus (SACMV) silencing constructs. Therefore, we developed a method for producing long SACMV IR constructs through bisulfite-induced base pair mismatches on the sense arm prior to IR assembly. Expression of SACMV BC1 mismatched IR constructs in the model test plant Nicotiana benthamiana resulted in a reduction in viral BC1 transcript levels, hence viral replication, upon SACMV infection. Mismatched SACMV AC1 IR constructs induced PTGS more efficiently in a N. benthamiana callus system than nonmismatched IR constructs. Our novel method for IR construct generation should be applicable to many sequences where the generation of these constructs has proven difficult in the past.

Currently, posttranscriptional gene silencing (PTGS) techniques are being applied for engineering resistance against viral plant pathogens. Many of these strategies involve the use of either sense or antisense constructs (1-7). Inverted repeat (IR) transgenes, whereby sense and antisense gene fragments are expressed from a single promoter in a head-to-head orientation, have been developed, and these IR constructs are reportedly more efficient inducers of PTGS than any of the previously used constructs (8). IR transgenes now form the basis of experimental RNA interference (RNAi) in plant systems, and the process has been termed IR-PTGS (9).

Despite potential applications of IR-PTGS, the inverted repeats generally result in genomic instability, particularly in prokaryotes and lower eukaryotes (10); with Escherichia coli, inverted repeats were shown to cause cell death in some cases, and deletions or rearrangements in others (10, 11). DNA sequences in an IR conformation have the tendency to form a four-way helical junction, known as a cruciform structure or Holliday junction (12). Several nucleases that specifically cleave Holliday junctions and other large secondary structures of DNA have been identified (13-15). In addition, these junctions are key intermediates for homologous recombination, and are therefore substrates of recombination enzymes (16, 17). IR transgenes are therefore difficult to manipulate during cloning and plant transformation procedures, where the process depends on the replication and maintenance of the plasmid containing the construct.

To prevent formation of cruciform structures and stabilize DNA conformation during cloning, constructs containing a sufficiently large spacer sequence between the IR sense and antisense arms were tested. However, these spacer sequences significantly reduced PTGS induction efficiency (18). Subsequently, vectors were designed to stabilize the IR constructs by placing a spliceable intron from the Pdk gene of Flaveria between the sense and antisense arms (19). However, our studies with South African cassava mosaic virus (SACMV) found the Pdk intron did not prevent SACMV IR construct instability during cloning and plant transformation (Taylor and Rey, unpublished). We believe that the difficulty in cloning the IR constructs may be due to the formation of Holliday junctions that can be created between two cruciform arms (12), altering the electrophoretic mobility of the DNA and rendering the DNA insensitive to restriction endonucleases (20). Instability of inverted repeats has been suggested as a likely cause of cloning failure (21, 22). In addition, the large intron (∼700 bp) increases the overall size of the T-DNA, and studies have shown that large-insert T-DNAs are prone to rearrangements and deletions in both Agrobacterium and plant cells (23).

Here we describe a method to produce long mismatched IR constructs, using SACMV BC1 and AC1 open reading frame (ORF)-derived sequences, wherein the sense arm is bisulfite-treated to randomly convert cytosine residues to thymine. When DNA is treated with sodium bisulfite, cytosines are converted into uracil. Subsequent amplification of the DNA fragment by PCR results in the uracils being replaced by thymine residues in the sense strand, resulting in a mismatched IR construct that should prevent cruciform formation of the DNA. The mismatched SACMV BC1 IR DNA could easily be cloned and sequenced and was expressed in the host plant Nicotiana benthamiana. When compared with a nonmismatched AC1 IR construct, the mismatched SACMV AC1 IR construct showed improved efficiency in reducing SACMV replication.

Materials and methods

Isolation of gene fragments

SACMV is a geminivirus consisting of two circular ssDNA components [DNA-A and DNA-B, that have six ORFs (AC1, AC2, AC3, AC, AV1, and AV2) and two ORFs (BC1 and BV1), respectively] (24). A 222-bp fragment of the SACMV BC1 ORF (involved in movement of virus) was PCR-amplified using unmodified primers [BC1 (unmod) F (5'-AAACATTCCACGGACATACG-3') and BC1 (unmod) R (5'-TGGTAG-CCCAATCTGAGACCTT-3')] from 15 ng template DNA (full-length genomic SACMV DNA-B clone) (Figure 1) using 0.4 µM each forward and reverse primer at an annealing temperature of 52°C. Amplification products were purified using a High Pure PCR Product Purification kit (Roche Applied Science, Mannheim, Germany).

Figure 1. Method used to generate a SACMV BC1 mismatched inverted repeat expression cassette in the plant transformation vector pCambia 1303 (pC). (Click to enlarge)