Protocol
One-step site-directed mutagenesis of viroid dimeric cDNA

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Abstract

The study of viroids (plant pathogens constituted by small non-coding RNA) has greatly benefited from the use of site-directed mutagenesis tools. However, compared to viral systems, this technique is complicated by the fact that, usually, infectious cDNAs carry two copies of the viroid genome. A simple method for a one-step site-directed mutagenesis of viroids is described and tested by estimating the rate of mutation incorporation of three random nucleotide substitutions in each Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd). The protocol is essentially based on the original QuickChange™ Stratagene methodology; dimeric cDNA templates are amplified directly using Pfu DNA polymerase and self-complementary mutagenic primers. The reaction typically yields dimeric, but also monomeric clones which can be easily distinguished by electrophoretic analysis. The data show that approximately 50% of the dimeric clones carry the desired mutation in both viroid copies. Since the proposed protocol is simple technically and rapid compared to previous methods, it could be applied routinely for site-directed mutagenesis studies in viroids.

Introduction

Viroids are plant pathogens constituted by a small (246–401 nt), non-coding, circular, single-stranded RNA. Since they do not encode any protein, viroids rely entirely on the host to complete their infectious cycle. Despite their extreme simplicity, viroids can sustain plant infections varying from latent to very severe, both in herbaceous and woody species, agronomic as well as ornamental. The 29 viroid species known currently are grouped into the families Pospiviroidae and Avsunviroidae, based on the presence of particular sequence domains, sequence homology, and properties of their infectious cycle (Elena et al., 2001, Flores et al., 2005). In both groups, replication takes place through a rolling-circle mechanism. Host RNA polymerases transcribe longer-than-unit replication intermediates that are cleaved and ligated to form circular monomers of (+) polarity (Branch and Robertson, 1984, Flores et al., 2005). In pospiviroids, only the (+) RNA intermediate is processed, whereas in avsunviroids both (+) and (−) intermediates are cleaved autocatalitically by hammerhead ribozymes and ligated subsequently (Daròs et al., 1994).

The investigation of viroid replication and pathogenesis has been strengthened by the creation of infectious cDNAs clones, which can be used directly for in vitro runoff transcription and plant inoculation. Since viroids have circular genomes, the only way to ensure that cloning does not interrupt any physiologically relevant domains is to use cDNAs with head-to-tail dimers of the entire viroid sequence. Such dimeric transcripts mimic the longer-than-unit replication intermediates and thus can be processed into unitary, circular RNAs within the cell (Cress et al., 1983, Tabler and Sanger, 1985). In contrast to transcripts of dimeric cDNAs, the infectivity of those derived from monomeric cDNAs depends on the cloning site (Cress et al., 1983, Visvader et al., 1985) and, in all cases, is lower than that of dimeric cDNAs.

Site-directed mutagenesis of viroids is carried out typically by PCR amplification of a monomeric cDNA using mutagenic primers, followed by cloning into Escherichia coli and sequencing (de la Peña et al., 1999). However, prior to transcription/inoculation, an additional excision-cloning step is needed to create infectious head-to-tail dimeric constructs. A restriction fragment analysis is also required to discard head-to-head clones and head-to-tail clones inserted in the non-desired orientation.

One of the simplest and most rapid methods for site-directed mutagenesis is the QuickChange™ non-PCR protocol originally developed by Stratagene (La Jolla, CA, USA). This strategy is based on the amplification of the entire plasmid using self-complementary mutagenic primers and a DNA polymerase lacking 5′exonuclease activity (Papworth et al., 1996). Two general advantages of this technique are that the in vitro ligation step is obviated and that, since DNA amplification is linear rather than exponential, the incorporation of undesired mutations is minimized. This strategy could in principle be applied to monomeric viroid cDNAs. However, this would not eliminate the most time-consuming step, which is the construction of head-to-tail dimeric clones from the mutagenized monomeric ones. This problem was solved here by using directly dimeric constructs as templates of the mutagenesis reaction. This protocol was tested by estimating the efficiency of incorporation of each of three random point mutations into Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd), two species representative of the families Pospiviroidae and Avsunviroidae, respectively.

Section snippets

Dimeric cDNA construction

A dimeric CSVd clone was constructed from a CSVd-infected plant RNA preparation. Briefly, RT-PCR was done with phosphorylated primers and the PCR products were ethanol precipitated, and autoligated. The ligation fragments were separated on an agarose gel to isolate dimers, which were cloned into the EcoRV site of a pUC18 derivative containing a T7 promoter. The ligation reaction was precipitated with ethanol, used for electroporation of E. coli DH5α, and plasmid DNA was isolated from

Results

Fig. 1 shows a scheme of the protocol followed for site-directed mutagenesis of dimeric cDNA templates. The basic steps are: (i) mutagenesis reaction according to the Stratagene QuickChange™ guidelines; (ii) E. coli transformation; (iii) screening of clones by rapid phenol DNA extraction and electrophoresis; (iv) plasmid purification and sequencing. To evaluate the efficiency of this protocol to produce plasmids with dimeric cDNA and the desired mutation in both copies, mutagenic primers were

Discussion

Site-directed mutagenesis studies in viroids usually require the construction of dimeric clones carrying the mutation in each of the two copies of the genome. This is a time-consuming process when done sequentially because it involves intermittent transformation and screening steps. Most of these steps can be avoided using the methodology suggested in the current study, making site-directed mutagenesis easier and more rapid (the entire protocol can be completed in approximately 3 days). Dimeric

Acknowledgements

We thank Dr. S. Gago for kindly gifting us the pCChMVd++ clone, Prof. S.F. Elena for financial support, and A. Cuadrado for technical support. This work was financed by grant BFU2005-23720 from the Spanish Ministerio de Educación y Ciencia to S.F.E. and grant GV06/031 from the Spanish Generalitat Valenciana to R.S.

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