Note: Virology

In planta agroinfection by Canadian and GermanPotato leafroll virus full-length cDNAs1

Lawrence Kawchuk, Hannah Miriam Jaag, Karen Toohey, Robert Martin,Wolfgang Rohde, and Dirk Prüfer

Abstract: Full-length infectious cDNA clones of Canadian (PLRVflc) and German (PLRVflg) Potato leafroll virusisolates were constructed and their biological activities examined in planta. For both constructs, agroinfection by PLRVcDNA resulted in virus multiplication in the primary leaf followed by systemic movement and the production oficosohedral PLRV particles. Differences between the two constructs occurred with respect to infection, movement, andtitres of infectious virus produced from the two full-length cDNAs. This study reports the first PLRV agroinfection andcomparison of biological characteristics between two independent infectious Luteoviridae cDNA clones.

Key words: luteovirus, agroinfection, full-length infectious cDNA, clone comparison.

Résumé : Des clones infectieux d’ADNc pleine longueur d’un isolat canadien (PLRVflc) et d’un isolat allemand(PLRVflg) du Virus de l’enroulement de la pomme de terre ont été construits et leur activité biologique a été observéein planta. Pour les deux constructions, l’agroinfection par l’ADNc du PLRV a causé une multiplication virale dans lesfeuilles inoculées, suivie d’un transport systémique et de la production de particules icosaédriques du PLRV. Desdifférences sont apparues entre les deux constructions quant à l’infection, le transport viral et le titre des virusinfectieux produits à partir des deux ADNc pleine longueur. Cette étude est la première sur l’agroinfection avec lePLRV et la comparaison des caractéristiques biologiques de deux clones indépendants et infectieux d’ADNc deLuteoviridae.

Mots clés : luteovirus, agroinfection, ADNc infectieux pleine longueur, comparaison de clones.

243Kawchuk et

al.: Potato leafroll virus / agroinfectionIntroduction

Potato leafroll virus (PLRV) is the type member of thePoleroviruses. It is transmitted in a circulative manner byaphids and confined to the phloem tissue of the host plant(Harrison 1984). PLRV particles encapsidate a single-stranded positive-sense RNA of 5.9 kb with a 5′ covalentlylinked VPg (Mayo et al. 1982). Eight major open readingframes (ORFs) are expressed from the genomic RNA(ORF0, ORF1, and ORF2), subgenomic RNA1 (ORF 3,ORF4, and ORF5), or subgenomic RNA2 (ORF6 and

ORF7) (Mayo et al. 1989; van der Wilk et al. 1989; Keeseet al. 1990; Ashoub et al. 1998). Translation of these vari-ous ORFs involves several noncanonical strategies includ-ing ribosomal frameshifting and suppression of an amberstop codon (for review see Rohde et al. 1994).

The biological activity of several plant viruses has beenexamined in protoplasts or transformed plants by introduc-ing full-length viral constructs. Infectious full-length cDNAclones of the Luteoviridae Beet western yellows (BWYV)(Leiser et al. 1992; Veidt et al. 1992), Barley yellow dwarf(BYDV) (Young et al. 1991), Cucurbit aphid-borne yellows(CABYV) (Prüfer et al. 1995), and the German (Prüfer etal. 1997) and Canadian (Franco-Lara et al. 1999) isolates ofPLRV have also been developed and characterized in proto-plasts or transformed plants. In this study, we demonstrateby agroinfection, the biological characteristics of two inde-pendently developed full-length PLRV cDNAs representingisolates from Germany and Canada. Furthermore, their im-portance for the study of virus infection, multiplication, andmovement is described.

Materials and methods

The German and Canadian isolates of PLRV selected toprepare cDNA were chosen because they produced high in-

Can. J. Plant Pathol. 24: 239–243 (2002)

239

Accepted 25 January 2002.

L. Kawchuk2 and K. Toohey. Agriculture and Agri-FoodCanada, Lethbridge Research Centre, PO Box 3000,Lethbridge, AB T1J 4B1, Canada.H.M. Jaag and W. Rohde. Max-Planck-Institut fürZüchtungsforschung, Carl-von-Linné Weg 10, 50829 Köln,Germany.R. Martin. USDA-ARS, 3420 NorthWest Orchard Avenue,Corvallis, OR 97330, U.S.A.D. Prüfer. Fraunhofer-Institut IUCT, Abteilung fürangewandte Genomforschung, Auf dem Aberg 1, 57392Schmallenberg, Germany.

1LRC Contribution 387-01063.2Corresponding author (e-mail: kawchuk@em.agr.ca).

fection levels, severity of disease symptoms, and virustitres. A full-length cDNA clone designated p35SPL-WT(PLRVflg) of the German PLRV isolate was constructedfrom cDNA clones modified by site-directed mutagenesisas described by Prüfer et al. (1997). cDNA clones of a Ca-nadian PLRV isolate (Kawchuk et al. 1989) were modifiedby in vitro mutagenesis and used to prepare the full-lengthcDNA clone in plasmid p35SUP110 (PLRVflc) as describedby Franco-Lara et al. (1999). Site-directed mutagenesis wasused to add missing 5′ cDNA and remove nonviral se-quences. To facilitate transcription, the full-length PLRVconstructs are flanked by the CaMV 35S promoter (Guilleyet al. 1982) that confers constitutive and high expression offoreign genes in plants (Odell et al. 1985) and the Agro-bacterium tumefaciens nos terminator. The Escherichia colistrain JM109 was used to develop and maintain PLRVflclones since cell toxicity and cDNA deletions were prob-lematic in other E. coli strains (Prüfer, Richards, andKawchuk, unpublished results).

The A. tumefaciens strain LB4404 (BRL) was trans-formed with PLRVflg or PLRVflc DNAs and subsequentlyintroduced in three independent experiments by injectioninto three leaf midribs (Leiser et al. 1992) of eight, two-week-old Nicotiana clevelandii and Solanum tuberosumplants. Plants were maintained in a level two containmentfacility to exclude aphids and prevent PLRV movement be-tween plants. Uninoculated control plants were includedwith each experiment to detect external contamination.

Accumulation and movement of PLRV was determinedand quantified by indirect ELISA as described by Kawchuket al. (1990). Samples were taken 3 and 5 weeks after inoc-ulation from distal and proximal portions of inoculated anduninoculated leaves. Positive ELISA extracts were exam-ined by immunosorbent electron microscopy with PLRVpolyclonal antisera as described by Gill and Chong (1975).Various versions of the BLAST algorithm (Altschul et al.1997) and PCGENE software version 6.85 were used tosearch DNA and protein databases for homology amongstPLRV isolate sequences.

Injection of A. tumefaciens LBA4404 carrying full-lengthcDNA copies indicated that German and Canadian full-length PLRV cDNA were infectious and could be intro-duced by agroinfection into N. clevelandii (Table 1). Injec-tion into the smaller vascular tissues of potato was moredifficult and no infection occurred. After 3 and 5 weeks, vi-rus was detected by indirect ELISA in agroinoculatedN. clevelandii leaves and in the newly formed upper leaves.Both PLRVflc and PLRVflg cDNAs behaved in a similarmanner in that inoculated primary leaves became infected

and PLRV was detected by ELISA 3 weeks postinoculation.In most inoculated N. clevelandii plants, systemic move-ment was observed and PLRV could be detected by ELISAin newly formed leaves after 5 weeks. However, in a fewcases, the virus was localized within the primarily infectedleaf and then was detected in only certain sections of this leaf.Confirmation that the Canadian and German full-length cDNAclones formed virions was obtained by immunosorbent TEMof extracts from agroinfected N. clevelandii plants.

Results and discussion

Interestingly, strain-specific infectivity was observed withPLRVflc and PLRVflg cDNAs. Infection of the primaryagroinoculated and uninoculated leaves occurred at a higherfrequency with the PLRVflg cDNA compared to the PLRVflccDNA (Table 1). Furthermore, viral titres after agro-infection with PLRVflc cDNA were substantially lower inall PLRV ELISA-positive leaves when compared to identi-cal experiments using the PLRVflg cDNA.

Sequence comparisons, performed with GenBank acces-sions of the Canadian (D13954) and German (submitted)PLRV isolates, detected an overall homology of 97.48%with some unique nucleotide differences among the isolates.Interestingly, the most obvious sequence alterations werefound in the overlap region between ORF 1 and ORF 2(Fig. 1; nts 1620 to 2000) (van der Wilk et al. 1989). Thisregion shows a considerable reduction in strain similarity(92.9%) with an exchange of 27 nucleotides leading to analtered amino acid composition of the corresponding pro-teins.

The molecular analysis of Luteoviridae infection is diffi-cult because of the relatively low titres of these phloem-confined RNA viruses. The most effective strategy to datefor characterizing the biological function of different genesinvolves “reverse genetics” and full-length cDNA clonesinto which specific mutations were introduced. For exam-ple, the impact of site-directed mutations within Luteo-viridae RNA on replication or disease symptoms can beexamined in protoplasts and transgenic plants (Prüfer et al.1997; Franco-Lara et al. 1999). However, the strategy ofagroinfection facilitates characterization of these andadditional characteristics such as movement in a system thatmore closely resembles the natural infection. Since only afew cells receive T-DNA by agroinoculation, the in plantasynthesized virus RNA transcripts need to express all pro-teins necessary for infection, replication, and movement,thereby completing the entire virus replication cycle.

240 Can. J. Plant Pathol. Vol. 24, 2002

Three weeks Five weeks

Inoculated (N = 24) Inoculated (N = 24) Uninoculated (N = 24)

Incidence Titre Incidence Titre Incidence Titre

PLRVflg 18 0.65 ± 0.32 18 0.54 ± 0.31 18 0.52 ± 0.25

PLRVflc 6 0.27 ± 0.07 2 0.29 ± 0.19 2 0.20 ± 0.03

Note: Virus levels were determined in agroinoculated and uninoculated leaves by ELISA (A405). Uninoculated controls (N = 20),ELISA (A405) = 0.07 ± 0.02.

Table 1. Incidence of infection and average PLRV titres and standard error in infected plants.

Kawchuk et al.: Potato leafroll virus / agroinfection 241

Fig. 1. Sequence comparison of the PLRVflc (upper sequence) and PLRVflg (lower sequence) clones in the ORF 1/ORF 2 overlapregion. Location of the ribosomal “slippery sequence” is indicated by underlined nucleotides. Putative amino acid sequences of thePLRVflc are shown below the nucleotide sequences. Sequence differences are shaded.

Agroinfection was first described as an alternate route ofvirus infection of plants by Grimsley et al. (1986, 1987) andsubsequently applied to BWYV and CABYV by placing thecDNA under the control of the CaMV 35S promoter (Leiseret al. 1992; Prüfer et al. 1995). Our study describesagroinfection with full-length cDNA clones of PLRV anddelineates similarities and differences between infectiouscDNA clones for two different PLRV isolates. By analogyto BWYV and CABYV, agroinfection with PLRV clones re-sulted in replication, movement, and production of virions.However, the efficiency of infection was much lower withPLRVflc and the titres were consistently lower than that ob-served with PLRVflg.

Differences in the severity of disease symptoms producedby various isolates of luteoviruses have been observed(Wright and MacCarthy 1963) and the PLRV clones weremade to isolates that caused severe disease symptoms. Se-quence comparisons of PLRVflc and PLRVflg showed a highlevel of homology (97.48%) but identified remarkable dif-ferences in the ORF 1/ORF 2 overlap region (Fig. 1) thatcontains the ribosomal frameshift site for the synthesis ofthe transframe protein (Prüfer et al. 1992). The proteins en-coded by ORF 1 and ORF 2 are responsible for viral multi-plication. Thus, one possible explanation for the observedstrain differences may be that a more “active” replicase isproduced by PLRVflg resulting in a higher virus titer andsubsequent level of infection. Indeed, initial experiments re-placing this region in the Canadian clone with the corre-sponding sequence of the German isolate resulted in asubstantial increase in replication efficiency (Jaag et al., inpreparation). These results demonstrate that minor sequencedifferences can influence the infectivity of full-length infec-tious virus clones and need to be considered when choosinga clone for genetic analysis and biological assays in reversegenetic approaches.

Isolates of PLRV from Australia, Canada, Germany, theNetherlands, Poland, and Scotland have been sequenced,but full-length infectious clones were successfully devel-oped only for the two isolates described in this study. Se-quence comparisons identified many unique differences inthese other PLRV isolates that may affect infectivity. Thesedifferences could represent natural virus genomic variationsthat occur in an infected host or may have resulted from er-rors in reverse transcription during preparation of cDNA.

In planta infection by full-length PLRV cDNA viaagroinfection will overcome several intrinsic difficulties as-sociated with other methods of PLRV expression. Leafprotoplasts, which are used in transient expression experi-ments, are mainly derived from mesophyll cells, whilePLRV is almost entirely confined to cells of the phloem tis-sue. Similarly, expression of full-length PLRV cDNA intransgenic plants occurs in most cells and does not resemblethe conditions of natural infections. Agroinfection repre-sents a strategy that closely resembles natural PLRV infec-tion and is well suited to the in planta study of proteins andregulatory virus RNA sequences. Agroinfection of infec-tious full-length PLRV cDNAs as described in this study al-lows site-directed mutagenesis and the exchange of genesbetween various viruses to advance our understanding ofgene function and host–pathogen interactions.

Acknowledgements

This study was in part supported by DFG grant 330/9-2to W.R. The authors would like to thank F. Kulcsar for tech-nical assistance and Dr. N. Emans for critical reading of themanuscript.

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