Earlier, identifying viruses found in plants took some effort. First, attempts were made to deduce the identity of the virus on the basis of visible symptoms, after which the existence of the virus was proven by identifying an antibody associated with that particular pathogen. Viruses can also be found by using an electron microscope.
However, screening for viruses in plants that appear symptomless on visual inspection is difficult. Many viruses harmful to plant health can cause no symptoms in certain plants, yet cause great devastation after spreading to crops.
A state-of-the-art method of analysis based on the formation of small interfering RNA (siRNA) sequences makes it possible to identify all viruses contained in a sample, even when the plant in question is free of symptoms.
“The technique is based on the fact that all viruses and viroids, pathogens smaller than viruses, form double-stranded RNA structures at some point of their multiplication. Specialised enzymes functioning in cells are able to identify double-stranded RNA, attach to it and split it into bits of 21, 22 or 24 bases,” as Professor Jari Valkonen from the Faculty of Agriculture and Forestry, University of Helsinki, describes the initial stages of the process.
The bits of RNA are not free to float within the cell, as another enzyme binds with them and simultaneously breaks down one of the strands. What remains is a single-stranded RNA sequence with an enzyme attached.
Subsequently, the strand carrying the enzyme serves as a detective. When the strand locates its pair in the single-stranded genome of a virus, it latches on to it. The enzyme riding along bonds with the virus genome, incapacitating it by splitting.
“Then you still need a third enzyme to change the RNA from a single-stranded to a double-stranded structure, after which the entire splitting process starts over with accelerating speed. More and more viral RNA molecules are broken down, increasing the number of small RNA sequences,” Valkonen explains.
Viral identification specifically employs these short bits of RNA cut to size. They are extracted from the plant material for sequencing, or determining the sequence of their bases. An entire viral genome, or at least large parts of one, can be constructed by overlapping millions of short sequences of RNA. Based on the sequence of bases, potential matches can be looked for in virus libraries, gaining indications on the genus and species of the virus. At the same time, the library content is constantly growing.
The most significant benefit of the method is that it identifies all viruses and viroids in a sample at once. From time to time, data on the same sequence of bases can be reanalysed, enabling the identification of new viruses and viroids as information on their genomes is accrued in databases.
“Determining the genome of crops also boosts testing for viruses using this technique, since plant-based small interfering RNA sequences can be excluded from the sequence dataset before looking for viruses. Subsequently, even viruses with a sequence of bases notably different from known viruses are easier to detect.”
Great financial impact
Potentially great crop losses make identifying viruses in crops important. Most viruses are also able to migrate between plant species, which makes the prevention of the spread of viruses and the use of uncontaminated propagating materials essential.
Due to the increasingly active import of plants, the importance of virus identification has grown. Finland wishes to avoid such harmful organisms, which could, for example, halve horticultural crops and which have even forced the discontinuation of farming elsewhere.
“This is why it’s important to first identify plant enemies in order to prevent them from finding their way to Finland or moving from farm to farm,” says Johanna Santala, senior scientist from the Finnish Food Authority.
For a couple of years already, the Finnish Food Authority, located on Viikki Campus, has been using deep sequencing equipment with which to determine the sequence of bases in small RNA sequences. Last year the equipment was deployed in earnest in a research project investigating viral infections in potatoes. In the future, the method will be utilised, among other things, to ensure the purity of vegetable and berry plant propagation material.
Similar deep sequencing methods can be used to test for viruses also in insects, eelworms and warm-blooded animals, in addition to which the technology enables the identification of bacteria and fungi. At the Finnish Food Authority, deep sequencing is in fact used, in addition to identifying plant viruses, for typing bacterial strains in connection with food poisoning epidemics.
It all started with sweetpotatoes
Ten years ago Jan Kreuze, PhD, who studied at the University of Helsinki and the Swedish University of Agricultural Sciences, and who is currently working at the International Potato Centre in Peru as a Crop and Systems Science Division Leader, got the idea to rely on small interfering RNA analysis when looking for viruses. A sweetpotato strain cultivated in cell culture was supposed to be free from viruses, yet three previously unknown viruses were identified in it. More viruses were found, for example, in plant cell cultures and core plant materials.
So far, the technique has been successfully used to identify viruses in dozens of plant species, including potato, tomato, pepino, vine, raspberry and cocksfoot, as well as many wild plants. In Finland, the method has uncovered the black raspberry necrosis virus (BRNV) in wild raspberries, the raspberry leaf blotch virus (RLBV) in raspberries grown in plastic tunnels and outdoors, as well as the Lily virus X and the woolly burdock yellow vein virus (WBYVV) in woolly burdocks, previously not known to occur in Finland.