Gianinna Brigneti

Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.

Post‐transcriptional gene silencing (PTGS) of a green fluorescent protein (GFP) transgene is suppressed in Nicotiana benthamiana plants infected with potato virus Y (PVY) or with cucumber mosaic virus (CMV), but not in plants infected with potato virus X (PVX). By expressing PVY and CMV‐encoded proteins in a PVX vector we have shown that the viral suppressors of gene silencing are the HCPro of PVY and the 2b protein of CMV. The HCPro acts by blocking the maintenance of PTGS in tissues where silencing had already been set, whereas the 2b protein prevents initiation of gene silencing at the growing points of the plants. Combined with previous findings that viruses are both activators and targets of PTGS, these data provide compelling evidence that PTGS represents a natural mechanism for plant protection against viruses.

Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase.

Three types of mutation were introduced into the sequence encoding the GDD motif of the putative replicase component of potato virus X (PVX). All three mutations rendered the viral genome completely noninfectious when inoculated into Nicotiana clevelandii or into protoplasts of Nicotiana tabacum (cv. Samsun NN). In order to test whether these negative mutations could inactivate the viral genome in trans, the mutant genes were expressed in transformed N.tabacum (cv. Samsun NN) under control of the 35S RNA promoter of cauliflower mosaic virus and the transformed lines were inoculated with PVX. In 10 lines tested in which the GDD motif was expressed as GAD or GED there was no effect on susceptibility to PVX. In two of four lines transformed to express the ADD form of the conserved motif, the F1 and F2 progeny plants were highly resistant to infection by PVX, although only to strains closely related to the source of the transgene. The resistance was associated with suppression of PVX accumulation in the inoculated and systemic leaves and in protoplasts of the transformed plants, although some low level viral RNA production was observed in the inoculated but not the systemic leaves when the inoculum was as high as 100 or 250 micrograms/ml PVX RNA. These results suggest for a plant virus, as reported previously for Q beta phage, that virus resistance may be engineered by expression of dominant negative mutant forms of viral genes in transformed cells.

Molecular mapping of the potato virus Y resistance gene Rysto in potato

Rysto is a dominant gene which confers resistance to potato virus Y (PVY) in potato. We have used bulked segregant analysis of an F1 tetraploid potato population to identify three AFLP markers linked to and on either side of Rysto. The tomato homologue of one of these AFLP markers was assigned to linkage group XI by analysis of an F2 mapping population of tomato, suggesting that Rysto is also on chromosome XI of the potato genome. This map position was confirmed by the demonstration that Rysto was linked to markers which had been previously mapped to chromosome XI of the potato genome. Four additional AFLP markers were identified that were closely linked to Rysto in a population of 360 segregating progeny of a potato cross between a resistant (Rysto) and a susceptible parent. Two of these markers were on either side of Rysto, separated by only a single recombination event. The other two markers co-segregated with Rysto.

Rpi-vnt1.1, a Tm-2(2) homolog from Solanum venturii, confers resistance to potato late blight.

Despite the efforts of breeders and the extensive use of fungicide control measures, late blight still remains a major threat to potato cultivation worldwide. The introduction of genetic resistance into cultivated potato is considered a valuable method to achieve durable resistance to late blight. Here, we report the identification and cloning of Rpi-vnt1.1, a previously uncharacterized late-blight resistance gene from Solanum venturii. The gene was identified by a classical genetic and physical mapping approach and encodes a coiled-coil nucleotide-binding leucine-rich repeat protein with high similarity to Tm-2(2) from S. lycopersicum which confers resistance against Tomato mosaic virus. Transgenic potato and tomato plants carrying Rpi-vnt1.1 were shown to be resistant to Phytophthora infestans. Of 11 P. infestans isolates tested, only isolate EC1 from Ecuador was able to overcome Rpi-vnt1.1 and cause disease on the inoculated plants. Alleles of Rpi-vnt1.1 (Rpi-vnt1.2 and Rpi-vnt1.3) that differed by only a few nucleotides were found in other late-blight-resistant accessions of S. venturii. The late blight resistance gene Rpi-phu1 from S. phureja is shown here to be identical to Rpi-vnt1.1, suggesting either that this strong resistance gene has been maintained since a common ancestor, due to selection pressure for blight resistance, or that genetic exchange between S. venturii and S. phureja has occurred at some time.

Solanum mochiquense chromosome IX carries a novel late blight resistance gene Rpi-moc1

Screening of a large number of different diploid Solanum accessions with endosperm balance number (EBN) 1 revealed segregation for strong resistance and sensitivity to Phytophthora infestans in accessions of Solanum mochiquense. Genetic analysis showed that resistance in S. mochiquense accession CGN18263 resides at the distal end of the long arm of chromosome IX, is linked to restriction fragment length polymorphism marker TG328 and is in the neighbourhood of the quantitative trait locus (QTL) Ph-3 conferring resistance to P. infestans in tomato. This is the first genetic study of S. mochiquense, a wild diploid species originating from fog oases in the Peruvian coastal desert.

Retraction: ‘Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana’

Post‐transcriptional gene silencing (PTGS) of a green fluorescent protein (GFP) transgene is suppressed in Nicotiana benthamiana plants infected with potato virus Y (PVY) or with cucumber mosaic virus (CMV), but not in plants infected with potato virus X (PVX). By expressing PVY and CMV‐encoded proteins in a PVX vector we have shown that the viral suppressors of gene silencing are the HCPro of PVY and the 2b protein of CMV. The HCPro acts by blocking the maintenance of PTGS in tissues where silencing had already been set, whereas the 2b protein prevents initiation of gene silencing at the growing points of the plants. Combined with previous findings that viruses are both activators and targets of PTGS, these data provide compelling evidence that PTGS represents a natural mechanism for plant protection against viruses.

Putting Plant Disease Resistance Genes to Work

Semi-dominant plant disease resistance (R) genes confer recognition of and response to specific races of pathogen that carry a corresponding Avirulence (Avr) gene. R proteins are presumed to recognise pathogen Avr gene-encoded products, or compatibility factors, that are likely to be involved in pathogenicity on the host. R genes against various important diseases have been used by plant breeders, but when deployed in monocultures, resistance frequently breaks down as races of the pathogen emerge that can overcome the R gene through recessive mutations in the corresponding Avr gene. Nevertheless, in nature, R genes have been maintained. In Arabidopsis, ~164 homologs of the largest class of R genes exist. These R genes encode proteins of the nucleotide binding-leucine rich repeat (NB-LRR) class (Dangl and Jones, 2001).