Pepino mosaic virus

Pepino mosaic virus (PepMV) is a relatively new plant virus that has become a signifi cant agronomical problem in a relatively short period of time. It is a member of the genus Potexvirus within the family Flexiviridae and is readily mechanically transmissible. It is capable of infecting tomato (Solanum lycopersicum) and other Solaneceous host plants. Since its description in 1980 from pepino plants (Solanum muricatum) collected in 1974 in Peru, the virus remained unknown for a long time until it manifested itself in commercial tomato crops in Europe in 1999. Since then the virus has been reported worldwide and the disease it causes has become important in commercial tomato production. Since 1999, new strains of the virus have been described which diff er from the original pepino isolate. The fast spread of the virus and the appearance of mixed infections with the new strains may play an important role in the increase of the agricultural importance of this viral diseas

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"

Kalanchoë blossfeldiana, a new host for Sonchus yellow net virus

The agent causing chlorotic spots in Kalanchoë blossfeldiana `Isabella¿ was investigated. A virus isolated from this naturally infected kalanchoë was mechanically transmissible to several indicator plants. Observation of suspension preparations in the electron microscope revealed rhabdovirus-like particles. On the basis of symptoms on indicator plants, serology, electron microscopy, molecular characterisation and back inoculation to K. blossfeldiana 'Isabella', the causal agent was identified as an isolate of Sonchus yellow net virus (SYNV). This is the first report of an ornamental plant species naturally infected by SYNV

The control of PVY in Dutch seed potato culture

Over the recent years Potato virus Y presents a growing problem in Dutch seed potato culture. In recent years a significant % of seed potato lots was de-classified due to PVY infections. This apparent increase in PVY infections was unexpected since no increase in field symptoms were observed and the numbers of aphids caught in the yellow water traps and high suction traps showed a clear decline over the last 10 years http://www.aab.org.uk/images/VIRO_CONF_PROG.pd

The use of attenuated isolates of Pepino mosaic virus for cross-protection

Pepino mosaic virus (PepMV) has recently emerged as a highly infectious viral pathogen in tomato crops. Greenhouse trials were conducted under conditions similar to commercial tomato production. These trials examined whether tomato plants can be protected against PepMV by a preceding infection with an attenuated isolate of this virus. Two potential attenuated isolates that displayed mild leaf symptoms were selected from field isolates. Two PepMV isolates that displayed severe leaf symptoms were also selected from field isolates to challenge the attenuated isolates. The isolates with aggressive symptoms were found to reduce bulk yields by 8 and 24% in single infections, respectively. Yield losses were reduced to a 0–3% loss in plants that were treated with either one of the attenuated isolates, while no effects were observed on the quality of the fruits. After the challenge infection, virus accumulation levels and symptom severity of the isolates with aggressive symptoms were also reduced by cross-protection. Infection with the attenuated isolates alone did neither affect bulk yield, nor quality of the harvested tomato fruits

Tomato marchitez virus, a new plant picorna-like virus from tomato related to tomato torrado virus

A new virus was isolated from a tomato plant from the state of Sinaloa in Mexico. This plant showed symptoms locally known as `marchitez disease¿: severe leaf necrosis, beginning at the base of the leaflets, and necrotic rings on the fruits. A virus was isolated from the infected plant consisting of isometric particles with a diameter of approximately 28¿nm. The viral genome consists of two (+)ssRNA molecules of 7221 (RNA1) and 4898¿nts (RNA2). The viral capsid contains three coat proteins of 35, 26 and 24¿kDa, respectively. The abovementioned characteristics: symptoms, morphology, number and size of coat proteins, and number of RNAs are similar to those of the previously described tomato torrado virus (ToTV). Sequence analysis of the entire viral genome shows that this new virus is related to, but distinct from, ToTV and that these members of two obviously new virus species belong to the recently proposed plant virus genus Torradovirus. For this new virus, the name tomato marchitez virus (ToMarV) is proposed

Lang leve het virus!

“Dat virussen ziekten kunnen veroorzaken, in allerlei organismen, weet iedereen. Maar dat virussen ook gunstige effecten kunnen hebben is veel minder bekend.” Dit artikel geeft een gesprek weer met Marilyn Roossinck van Pennsylvania State University, die in Wageningen was voor het geven van de tweede Rob Goldbach Virology Lecture. “Naast pathogene virussen is er een enorm scala aan virussen met juist een gunstig effect op de plant. Voorbeelden daarvan zijn een verbeterde droogtetolerantie, hitte- of koudetolerantie of zouttolerantie. Ook bepaalde stammen van pathogene soorten kunnen deze effecten veroorzaken. Van verreweg de meeste soorten weten we simpelweg niet wat ze doen.

Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus

The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae