CLIMA ORGANIZACIONAL

The results described here show that the subgenomic RNA of PLRV is 2505 nucleotides in length, starting at position 3376 in the sequence of van der Wilk et al. (1989) with a 5’-untranslated leader sequence of 212

nucleotides. A direct repeat sequence of 8 nucleotides is present at the 5’- ends of the genomic and subgenomic RNAs which suggests that the extreme 5’-ends are functional, possibly as recognition sites for the replicase complex in the negative-strand RNA. They may also have a function in the translation of the ORFs.

Attempts to establish the presence or absence of a genome linked protein (VPg) on the subgenomic RNA were unsuccessful (not shown). A VPg is known to be located at the 5’-end of the genomic RNA (Mayo et ah,

1982b) and for some other viruses, it has been proposed to be also located at the 5’-end of the subgenomic RNA, e.g. southern bean mosaic virus (SBMV, Ghosh et al., 1981). The function of the VPg is unknown but it is thought that it may act as primer for RNA synthesis and there is a possibility that it may protect the RNA to some extent from degradation by intracellular

riboîlucleases. If these functions were fulfilled, it would be desirable for the virus to have a VPg at the 5’-end of the subgenomic RNA.

The proportion of genomic to subgenomic RNA seems to vaiy between the different samples examined. This probably reflects differences between protoplasts and plants in terms of virus multiplication, i.e.

differences in quantities of virus, timing of multiplication and preferred conditions of virus multiplication. The presence of the subgenomic RNA indicates that virus multiplication was taking, or had taken place in the cell. The results appear to have shown that PLRV spreads throughout the whole plant but does not necessarily multiply in every tissue. It is also evident that in plants such as the B3 transgenic line and the breeding clone G74451, which have been shown to have resistance to PLRV infection, virus spread is not restricted but multiplication is. These plants were grown up from infected tubers, so virus spread may be more thorough than if the infection was via aphid transmission. Protoplasts do not appear to show the same levels of resistance to virus multiplication that whole plants are capable of.

These results imply that resistance to PLRV, in secondary infections at least, inhibits virus multiplication in the cell rather than initial infection or cell to cell spread. Obviously, in the transgenic plants, r^istance is connected with the presence of the viral gene which may be expressing the virus coat protein. Whether this affects the uncoating of the virus or subsequent RNA transcription is unknown but it appears that something similar takes place in the cells of the resistant breeding clone, G74451.

This result outlines a problem in the detection of this type of

resistance. It suggests that if resistant plants can be infected but resist virus multiplication and show only slight symptoms, spread from plant to plant and therefore crop to crop is stül possible, although less likely than for a

susceptible crop. This may allow for the spread of the virus from one resistant potato crop to a susceptible one.

When RNA was extracted from different parts of a potato plant, differences in transcription of viral RNA were evident between the different tissues. Most appeared to take place in the stem of the plant where the major phloem vessels are and there appeared to be no PLRV-specific RNA present in extracts of root or tuber tissue. The apparent absence of PLRV-specific RNA in the tuber may be deceptive, since there may only be a very small quantity of virus present in this tissue. Otherwise the virus seems able to move into the phloem in most tissues of the plant.

These results seem to imply that the virus cannot or does not replicate in all plant tissues, further testing for this could be done by in situ

hybridisation or by sectioning followed by immunogold labelling or tissue printing. It is unknown whether this is due to some kind of resistance to uncoating or replication or simply due to the surrounding conditions being inappropriate for these processes. Virus multiplication is denoted on northern blots by the presence of the subgenomic RNA. This perhaps shows the importance of northern blots in the determination of virus infection. Serological tests can quantify accurately the amount of virus which is

present, but without performing several assays over a period of time, they cannot determine if it is actively multiplying in the plant.

Comparison of PLRV RNA extracted from plants and protoplasts of different species showed that only one subgenomic species was present and that both RNA species migrated to the same relative positions with each RNA extract. This result was the same when different PLRV isolates were used.

4. THE MÜLTffLICATION OF PLRV IN TOBACCO