Engineering resistance against potato virus Y

Potato virus Y is the type species of the potyvirus genus, the largest genus of the plant virus family Potyviridae. The virus causes serious problems in the cultivation of several Solanaceous crops and although certain poly- and monogenic resistances are available, these can not always be employed, e.g. R y genes in potato cv. 'Bintje'. The aim of the research described in this thesis was to establish new forms of resistance against PVY by genetic modification of host plants. One such form of genetic engineered resistance is 'coat protein-mediated resistance', whereby expression of a viral coat protein (CP) in a transgenic plant may confer resistance against infection with the homologous virus, and some closely related viruses.At the start of this investigation no sequence data on the RNA genome of PVY were available, therefore cDNA synthesis and subsequent sequence determination was performed to obtain the necessary PVY CP gene sequence as well as additional sequences from the 3'-terminal region of the viral genome (Chapter 2 and Van der VIugt et al., 1989). This enabled the determination of the exact taxonomic position of the PVY N('tobacco veinal necrosis strain') isolate used in these experiments, among other PVY isolates from at least two different strains. Detailed comparisons of the PVY NCP and 3'-non translated (3'-NTR) sequences with those from a large number of geographically distinct PVY isolates that became available during the course of this investigation, showed that these sequences, in addition to distinguish between different potyvirus species (Ward and Shukla, 1991; Frenkel et al., 1989), can also be used for the distinction between strains of one potyvirus (Chapter 3, Van der VIugt et al., 1992a). Several strain specific amino acid sequences in the CPs and nucleotide sequences in the 3'-NTRs could be discerned, that are possibly involved in virulence and/or symptom expression. Further experiments are required to elucidate the precise biological significance of these sequence motifs. Interestingly the sequence comparisons as complied in Chapter 3 also confirmed the high levels of CP and 3'-NTR sequence identity between the PVY isolates at one hand and one putative isolate of pepper mottle virus (PepMoV, Dougherty et al., 1985) at the other, as described previously (Van der VIugt et al., 1989; Van der Vlugt, 1992). Initially described as an atypical strain of PVY (PVY-S, Zitter, 1972) PepMoV was later found to be serologically and biologically distinct from PVY (Purcifull et al., 1973, 1975; Zitter and Cook, 1973). Recent determination of the complete genomic RNA sequence of a Californian isolate of pepper mottle virus (PepMoV-C; Bowman-Vance et al, 1992a,b) and comparisons between a Florida isolate of PepMoV and PVY (Hiebert and Purcifull, 1992) however, suggest that PepMoV represents a distinct potyvirus though more closely related to PVY than to any other potyvirus. Additional sequence information of other, biologically well characterized, isolates of PepMoV, like a virus isolate apparently intermediate between PepMoV and PVY (Nelson and Wheeler, 1978), will hopefully aid in establishing the exact taxonomic position of this pepper infecting virus in the genus Potyvirus. Generally it is to be recommended that of all virus isolates whose (partial) sequences are under investigation, precise origin and other relevant biological characteristics are also accurately documented.In contrast to all other viruses for which 'CP-mediated resistance' has been described sofar, potyviruses do not express their CPs from a distinct, separate gene but through proteolytic cleavage of a polyprotein precursor. This necessitated theaddition of translational start signals, directly upstream of the CP encoding sequence, in order to enable expression of the PVY NCP in transgenic potato and tobacco plants. Potato tuber disc and tobacco leaf disc transformations with these constructs resulted in large numbers of transgenic plants (Chapters 4 and 5). Despite the fact that a large number of transgenic plants was tested for CP expression, using a highly sensitive enzyme-amplification based ELISA format, in none of the plants significant amounts of viral CP could be detected. Whether this is caused by the extra N-terminal methionine residue, or improper folding of the CP, resulting in decreased stability of the protein, or by inefficient protein extractions, possibly resulting from protein insolubility, is not known. It remains to be tested whether transformation of plants with a construct in which a functional protease domain is coupled to a potyviral CP with an intact protein processing sequence, will result in high levels of expression of the CP. For more practical purposes however, PVY CP expression levels appear not to be of significant importance since the protection against PVY, observed in the transgenic tobacco plants (Chapter 5 and 6), is apparently RNA-mediated, i.e. prima rily based on the presence of the CP encoding RNA rather than on the coat protein itself. Transgenic tobacco lines expressing PVY CP transcripts devoid of a translational start signal (CP -ATG), possess equal levels of protection against both mechanically inoculated virus and virus transmitted by the natural aphid vector Myzuspersicae (Chapter 5 and 6). It seems highly unlikely that the protection in these CP -ATGplants is based on minute amounts (i.e. less then 0.0001 % of the total soluble protein) of a truncated viral polypeptide since the presence of six translational stopcodons preceding the first in-frame AUG startcodon, 162 nucleotides down stream the 5'-end of the CP encoding sequence, will prevent expression of such a polypeptide.Analysis of the transgenic potato lines (Chapter 4) showed that most lines, as the transgenic tobacco lines, expressed CP specific RNA transcripts. Under the given greenhouse conditions, however, in none of the transgenic plants protection to PVY could be determined. In view of the results obtained with the transgenic tobacco lines, it may be anticipated that virus challenging of additional transgenic potato lines, under more optimal greenhouse conditions, will reveal similar levels of RNA-mediated virus resistance as observed in tobacco. For all practical purposes genetically engineered resistance based on the presence of RNA molecules is to be preferred over forms of resistance that are based on the expression of a (foreign) protein. Apart from being energetically more favourable for the plant, it is likely to aid in the acceptance of genetically modified crop plants by both politicians and the public, something which might, in the next few years, turn out to be the major obstacle in the successful application of plant transformation techniques.At this stage one can only speculate on the mechanism(s) on which this RNAmediated resistance is based. Transformation of plants with partial CP or other PVY Ngenomic sequences will help in identifying the protection mechanism(s) involved and show whether regions other than the CP-encoding domain can be equally effective in conferring virus resistance. If the resistance is based on a 'sense-RNA' effect, i.e. hybridization of the positive sense transgenic RNA to negative-sense viral RNA replication intermediates, thereby blocking further virus replication, the ribozyme technology might prove an efficient expansion of this genetically engineered type of resistance. Ribozymes, RNA sequences capable of specific and catalytic cleavage of other RNA-sequences, are able to cleave target RNAs efficiently and catalytically in vitro . The antiviral application of ribozymes in transgenic plants however has sofar demonstrated not to be very successful and reported protection levels are not yet exceeding those obtained with antisense RNAs (Edington and Nelson, 1992). Chapter 7 describes the design and synthesis of hammerhead ribozymes capable to cleave a highly conserved region from the PVY RNA dependent RNA-polymerase cistron. It was shown that the correct formation of the hammerhead cleavage complex, determined at least in part by the lengths of the antisense arms of the ribozyme, forms an important factor in the efficiency of cleavage. Cellular and full-length viral RNA molecules generally posses extended, unknown secondary structures which are likely to hamper precise formation of hammerhead structures, which requires bimolecular basepairing. Correct hammerhead formation and efficient cleavage of these RNAs will therefore require ribozymes with rather long basepairing arms. These long antisense arms however will make catalytic cleavage rather unlikely since complex dissociation will probably become the rate limiting factor. For this reason one can assume that ribozymes will only be successful when introduced into specific antisense RNA molecules, directed against the less abundant viral complementary strands, rather than as highly efficient RNA cleaving "enzymes".

Bibliographic Details

Main Author:	van der Vlugt, R.A.A.
Other Authors:	Goldbach, R.W.
Format:	Doctoral thesis biblioteca
Language:	English
Published:	Landbouwuniversiteit Wageningen
Subjects:	evolution, genetic engineering, genetic variation, genetics, immunization, immunogenetics, induced resistance, plant diseases, plant viruses, plants, potatoes, potyvirus, recombinant dna, solanum tuberosum, speciation, aardappelen, evolutie, genetica, genetische modificatie, genetische variatie, geïnduceerde resistentie, immunisatie, immunogenetica, planten, plantenvirussen, plantenziekten, soortvorming,
Online Access:	https://research.wur.nl/en/publications/engineering-resistance-against-potato-virus-y
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