Infectivity of turnip mosaic potyvirus cDNA clones and transcripts on the systemic host Arabidopsis thaliana and local lesion hosts
Introduction
Turnip mosaic virus (TuMV) is a member of the genus potyvirus, the largest genus among plant viruses. Potyvirus virions are long, flexuous particles, containing 2000 copies of the coat protein and a single positive RNA molecule, approximately 10 kb long, covalently linked to a VPg in its 5′ end (Shukla et al., 1994). The genomic RNA is translated into a single polyprotein of approximately 3000 amino acids. The polyprotein is processed into at least eight mature proteins by at least three viral encoded proteinases (Riechmann et al., 1992; Verchot et al., 1992). The complete nucleotide sequence of one Canadian and one Japanese isolate have been reported (Nicolas and Laliberté, 1992; Ohshima et al., 1996). Several partial nucleotide sequences of different TuMV isolates are also available (Kong et al., 1990; Nakashima et al., 1991; Sano et al., 1992; Choi and Choi, 1993; Nakashima et al., 1993; Kim et al., 1995; Petrzik and Lehmann, 1996; Lehman et al., 1997). The genome is 9830–9833 nt long, excluding the poly (A) tail. The single open reading frame consists of 3163–3164 aa.
TuMV infects many economically important cruciferous crops, i.e. oilseed rape, swede, Brussels sprout, white cabbage or cauliflower among others, causing significant losses (Shattuck, 1992). TuMV isolates have been classified by different authors into groups according to host range and symptomatology (Pound and Walker, 1945; Yoshii, 1963; McDonald and Hiebert, 1975; Choi et al., 1980) or into strains based on Chinese cabbage or Brassica napus differentials (Provvidenti, 1980; Green and Deng, 1985; Stobbs and Shattuck, 1989; Walsh, 1989; Liu et al., 1990). A recent classification, including 124 isolates, differentiates 12 pathotypes according to the response of oilseed rape (Brassica napus var. oleifera) and swede (Brassica napus var. napobrassica) resistant or susceptible lines to TuMV infection (Jenner and Walsh, 1996).
TuMV also infects Arabidopsis thaliana, a cruciferous weed currently considered as the model system for genetic and host-pathogen interaction studies (Dangl, 1993), inducing yellow mosaic and stunting (Martı́nez-Herrera et al., 1994). Genetic studies of virus/host interactions require a good amount of variability in the response of the plant to the virus infection. The availability of hundreds of different ecotypes and chemical, physical and T-DNA induced mutants of A. thaliana assures variability on the plant side of the interaction. To characterize viral determinants relevant to specific interactions, an essential tool is an infectious clone.
Infectious transcripts or cDNA clones of plant RNA viruses have been described for members of most plant RNA virus groups (Boyer and Haenni, 1994). Among potyviruses, infectious transcripts have been described for papaya ringspot virus (PRSV) (Chiang and Yeh, 1995), pea seedborne virus (PSbMV) (Johansen et al., 1996), peanut stripe virus (PStV) (Flasinski et al., 1996), plum pox virus (PPV) (Riechmann et al., 1990), potato virus A (PVA) (Puurand et al., 1996), tobacco etch virus (TEV) (Dolja et al., 1992), tobacco vein mottling virus (TVMV) (Domier et al., 1989; Nicolas et al., 1996) and zucchini yellow mosaic virus (ZYMV) (Gal-On et al., 1991). Infectious cDNA clones have been described for PPV (Maiss et al., 1992), PSbMV (Johansen, 1996) and ZYMV (Gal-On et al., 1995). The availability of a cell-free clone of potato virus Y has also been reported (Fakhfakh et al., 1996). These tools have contributed enormously to deepen our knowledge of potyvirus genomic organization and to clarify the involvement of each viral genomic protein in potyvirus basic biology (replication, polyprotein processing, cell-to-cell movement, long-distance movement, aphid transmission, seed transmission, etc.) (Dolja et al., 1995; Johansen et al., 1996; Kasschau et al., 1997; Maia and Bernardi, 1996; Schaad and Carrington, 1996; Blanc et al., 1997).
The construction of two types of infectious cDNA clones of TuMV UK1 isolate is reported in this paper: (i) cDNA clones under the control of the 35S promoter of cauliflower mosaic virus, whose plasmid DNA is directly infectious upon mechanical inoculation, and (ii) a cDNA clone under the control of the bacteriophage T7 promoter, to be used as a template for the synthesis of infectious in vitro transcripts. These tools should facilitate the mapping of avirulence determinants among TuMV pathotypes in the Brassica napus or Chinese cabbage pathosystems and the general study of potyvirus-host interactions in Arabidopsis.
Section snippets
Virus
Turnip mosaic virus, isolate UK1 (Tomlinson and Ward, 1978), a generous gift from Dr John Walsh (HRI, Wellesbourne, UK), was propagated in plants of Indian mustard (Brassica juncea) cv. Tendergreen. TuMV virions were purified as reported (Spence, 1992). Purified virus particles were stored at 4°C in the CsCl gradient buffer, or at −20°C after centrifugation, resuspension in 10 mM Tris–HCl pH 7.5, 10 mM EDTA, and addition of glycerol to a final concentration of 40% (v/v).
Viral genomic RNA was
Full-length cDNA synthesis, restriction map and cloning under T7 or 35S promoters
Overlapping cDNA clones of TuMV UK1 covering the whole genome except the most 28 5′ nucleotides were obtained by priming first strand synthesis with oligo d(T)14 or with an internal primer. Fragments covering the 5′ end of the virus were generated by applying the amplifinder RACE kit (Clontech). The sequence of these clones and direct sequencing of RNA confirmed the described heterogeneity of the 5′ end (5 or 6 As) (Nicolas and Laliberté, 1992).
cDNA clones obtained priming first strand
Discussion
The construction of full-length TuMV cDNA clones under the control of T7 or 35S promoters is reported in this work. These and some of the partial clones obtained in the construction process, presented problems of low plasmid yields and plasmid instability in bacteria grown at 37°C. Instability of full-length cDNA clones in bacteria have been reported for several animal and plant viruses, possibly due to potential toxicity and/or instability of viral sequences in bacteria (Boyer and Haenni, 1994
Acknowledgements
We thank Margarita Calvo for the managing of Arabidopsis plants. The suggestions of Dr Montserrat Agüero, and the help of Dr Begoña Blanco-Urgoiti and Dr Alicia Romero with the immunocapture and transcription techniques are also acknowledged. We also thank Dr John Walsh and Dr Carol Jenner for Brassica juncea seeds and the virus purification protocol. This work was supported by the European Commission research Contract number ERBCIPA-CT92-3007 and by grant BIO 95-0766-C02-01 from the Spanish
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