Plant Biotechnology Journal (2009) 7, pp. 49–58
doi: 10.1111/j.1467-7652.2008.00373.x
A temperature-controlled amplicon system derived from Plum pox potyvirus Gabriela Original Temperature-controlled Article Dujovny et al. Ltd potyvirus amplicon Blackwell Oxford, Plant PBI © 1467-7652 1467-7644 XXX 2008 Biotechnology UK Blackwell Publishing Publishing Journal Ltd
Gabriela Dujovny, Adrián Valli, María Calvo and Juan Antonio García* Centro Nacional de Biotecnología-CSIC, Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain
Received 7 May 2008; revised 31 July 2008; accepted 5 August 2008. *Correspondence (fax +34 915 854506; e-mail:
[email protected])
Summary The control of replication can facilitate a viral amplicon to reach high expression levels by enabling the virus to escape host defence mechanisms and reducing the deleterious effects of viral infection. We have developed a novel system to regulate amplicon expression by controlling the temperature of plant growth. Nicotiana benthamiana plants were transformed at two different temperatures with a cDNA copy of the Plum pox potyvirus genome harbouring the open reading frame 2 of Porcine circovirus 2 between the nuclear inclusion protein b and coat protein coding sequences. Although transformation at 27 °C mainly yielded nonexpressing amplicons, lines with a tight control of amplicon expression were obtained. Viral replication was not detected in these plants when germinated at 28 °C, but was observed when the plants were shifted to 20 °C. In lines transformed at 24 °C, although the amplicon was expressed at 28 °C, viral accumulation was low and caused only minor growing defects. Viral replication was enhanced in these plants by shifting the
Keywords: amplicon, controlled expression, expression vector, Plum pox virus, potyvirus, virus vector.
temperature to 20 °C; under such conditions, the amplicon reached higher and more persistent expression levels than in plants transformed at 27 °C. These results demonstrate the utility of temperature regulation to control viral amplicon expression.
Introduction Plants have the unique potential to serve as bio-factories because of their ability to produce large quantities of a protein of interest with facile manipulation, reduced cost and minimal risk of toxicity or contamination by animal pathogens (Horn et al., 2004; Streatfield, 2007). Foreign proteins are typically expressed from recombinant genes permanently inserted into the nuclear genome of transgenic plants; however, the establishment of such systems is a timeconsuming and costly process, and usually only yields low levels of expressed protein. Virus-based vectors offer an alternative approach for the transient expression of foreign proteins at high levels in plants (Scholthof et al., 1996; Cañizares et al., 2005; Gleba et al., 2007). Several plant viruses have been successfully engineered into vectors for the expression of foreign genes. The major difficulty associated with the use of these plant viral vectors is the genetic instability caused by the inserted foreign gene (Donson et al., 1991; Dolja et al., 1993; Guo et al., 1998; Barajas et al., 2006). One way to handle this problem is to use © 2008 Consejo Superior de Investigaciones Cientificas (CSIC) Journal compilation © 2008 Blackwell Publishing Ltd
replicating RNA viruses from plants transformed with fulllength viral genome cDNAs (Angell and Baulcombe, 1997). These expression systems, termed ‘amplicons’, combine the genetic stability of transgenic plants with the high gene copy number derived from viral replication. Various viruses have been expressed from amplicon lines, including Tobacco mosaic virus (Yamaya et al., 1988; Siddiqui et al., 2007), Brome mosaic virus (Kaido et al., 1995), Potato leafroll virus (Prüfer et al., 1997), Potato virus X (PVX) (Angell and Baulcombe, 1997; Mallory et al., 2002), Turnip yellow mosaic virus (Chen et al., 2004) and Cowpea mosaic virus (Liu et al., 2004). Unfortunately, these amplicons typically performed below expectations. They frequently yielded low viral accumulation levels, mainly because plants displaying disease symptoms could be counterselected during the transformation process and/or because the amplicon transgene induced an efficient antiviral RNA silencing response (Angell and Baulcombe, 1997; Liu et al., 2004). Therefore, special strategies, such as the co-expression of strong silencing suppressors (Mallory et al., 2002; Azhakanandam et al., 2007) or the use of chemically controlled promoters (Mori et al., 2001), have
49
50 Gabriela Dujovny et al.
been necessary in order to obtain amplicon lines with high expression levels. Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae, the largest group of plant viruses (García and Cambra, 2007). Potyviruses have a single-stranded messenger polarity RNA genome of approximately 10 kb in length. This genomic RNA is translated into a large polyprotein and a shorter frameshift product, which are further processed by three self-encoded proteinases (Revers et al., 1999; UrcuquiInchima et al., 2001; Chung et al., 2008). PPV has a wide host range that extends from its natural Prunus hosts (James and Thompson, 2006; Llácer and Cambra, 2006) to herbaceous plants, such as Nicotiana benthamiana and Arabidopsis thaliana (Decroocq et al., 2006; Llácer and Cambra, 2006). Expression vectors based on PPV have been used for the efficient expression
Results Construction of PPVO2PCV transgenic plants RNA silencing appears to be a major limitation to the achievement of high plant viral amplicon expression levels (Angell and Baulcombe, 1997). As RNA silencing is temperature dependent (Szittya et al., 2003), we assessed the relevance for amplicon expression of the temperature conditions of plant transformation and subsequent plant culture. N. benthamiana plants were transformed at 24 °C (series D24) or 27 °C (series G27) with a full-length cDNA copy of the PPV genome, including the open reading frame 2 (ORF2) of Porcine circovirus 2 (PCV2) inserted between the nuclear inclusion protein b (NIb) and CP coding sequences (PPVO2PCV, Figure 1). Twenty-seven
of whole independent proteins (Fernández-Fernández et al., 2001) and of small peptides fused to the viral coat protein (CP) (Fernández-Fernández et al., 1998, 2002). Recently, we have produced PPV amplicons that displayed either severe developmental constraints because of the harmful effects of early viral infection or stochastic and delayed expression of the replicating virus (M. Calvo, J. A. García and G. Dujovny, unpubl. data). Plant–virus interactions are strongly conditioned by environmental factors, particularly by temperature (Hull, 2002). High temperature is frequently associated with weakened infection, which may be the result of a stronger RNA silencingmediated antiviral defence (Szittya et al., 2003). We took advantage of this temperature sensitivity to design a PPV amplicon system controlled by temperature switching. The goal of this system was to allow plants to germinate and grow at high temperatures without severe disease symptoms before inducing strong viral expression by culture at low
regenerated shoots were shown to be transformed with PPVO2PCV sequences, as ascertained by polymerase chain reaction (PCR) (Tables 1 and 2). Eight of the 12 D24 primary transformants displayed disease symptoms, whereas none of the 15 G27 primary transformants appeared to be symptomatic (Tables 1 and 2). Segregation ratios for the neomycin phosphotransferase II (nptII) reporter gene of the progeny of self-fertilized primary transformants (F1 plants) suggested single transgene inserts for most lines (segregation ratios of approximately 3 : 1) in both the D24 and G27 series (Tables 1 and 2).
temperature.
(plants D24x and G27x) were germinated in vitro at either 20
Effect of seed germination temperature on amplicon expression in PPVO2PCV transgenic seedlings As a first assessment of the effect of temperature on the expression of the PPV amplicon, seeds derived from the selfpollination of initially regenerated PPVO2PCV transgenic plants
Figure 1 Schematic representation of the Plum pox virus (PPV) cDNA clones used in this study. The PPV open reading frame (ORF) is represented by a rectangular box and vertical lines indicate the cleavage sites giving rise to the different PPV mature proteins. The insertion sites in the pICPPV-NK and pBin19 vectors are indicated. The ORF2 of Porcine circovirus 2 (PCV2) (ORF2PCV) cloned in PPV is shown as a black box. Lac Z′, α fragment of β-galactosidase gene; Nos, terminator of the nopaline synthase gene; NPTII, neomycin phosphotransferase II gene; p35S, 35S promoter of cauliflower mosaic virus; RB and LB, right and left T-DNA borders. © 2008 Consejo Superior de Investigaciones Cientificas (CSIC) Journal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 49–58
Temperature-controlled potyvirus amplicon 51
Table 1 Characterization of PPVO2PCV amplicon plants: series D24 Line 2 Symptoms*
F0 F1
F1 WB†
Line 3
Line 4
Line 5
Line 6
Line 8
Line 9
Line 10
Line 11
Line 14
Line 17
Line 19
+
+
–
–
–
+
+
+
–
+
+
+
20 °C
–
+
+
+
–
+
+
+
–
NP
+
NP
28 °C
–
–
–
–
–
–
–
–
–
NP
–
NP
20 °C
–
+++
+++
+++
–
+++
+++
+++
–
NP
+++
NP
28 °C F1 Seg‡
–
+
++
+
–
++
++
+++
–
NP
+++
NP
~3 : 1
~3 : 1
~15 : 1
~3 : 1
~3 : 1
~15 : 1
~3 : 1
~3 : 1
~3 : 1
NP
~15 : 1
NP
*Plants with (+) or without (–) disease symptoms. †Virus accumulation as assessed by Western blot analysis. The number of + signs reflects the estimated accumulation levels. ‡Segregation ratios of kanamycin resistance. NP, no progeny obtained.
Table 2 Characterization of PPVO2PCV amplicon plants: series G27 Line 1 Line 4 Line 5 Line 6 Line 7 Line 8 Symptoms*
F1 WB†
Line 10 Line 13 Line 14 Line 15 Line 16 Line 18 Line 19 Line 20 Line 24
F0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
F1
20 °C –
–
–
NP
–
–
–
–
–
–
–
–
–
–
– –
28 °C –
–
–
NP
–
–
–
–
–
–
–
–
–
–
20 °C –
+++
–
NP
–
–
–
+++
–
–
–
–
+
–
+++
28 °C –
–
–
NP
–
–
–
–
–
–
–
–
–
–
+++
~3 : 1
