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Detection and visualization of pospiviroids in potato,. Potato leafroll virus (PLRV) and aphids in support of transmission studies. Background. Viroids are highly ...
Detection and visualization of pospiviroids in potato, Potato leafroll virus (PLRV) and aphids in support of transmission studies Noémi Van Bogaert1,2, Kris De Jonghe2, Martine Maes2, and Guy Smagghe1 1Department
of Crop Protection, Faculty Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium 2Plant Sciences Unit - Crop Protection, Institute for Agricultural and Fisheries Research (ILVO), Burgemeester Van Gansberghelaan 96, 9820 Merelbeke, Belgium
Background During virus replication in a plant cell, the virus Viroids are
highly stable, single-stranded RNA
coat opens and viroids can get encapsulated
plant pathogens that lack a protein coat. In potatoes (S. tuberosum, L.), Potato spindle tuber
Virion
viroid (PSTVd) and other pospiviroids cause severe malformations of the tubers and foliage, leading to important agricultural losses. Mixed
Viroid
infections of plant viruses and viroids might lead to “trans-encapsidation” of the viroid within the protein coat of the virus (e.g. Potato leafroll virus, PLRV). This system can potentially be vectored by aphids and ultimately (co-)infect new plants.
Aphids could transmit the virus with
Objectives
encapsidated viroid to new host plants
- How can we investigate transencapsidation (indirectly/directly)? - How common is transencapsidation (for different viruses/viroids)? - How can further transport to host plants (e.g. by aphids) occur?
Viruses and viroids in doubly inoculated potato leaves can be visualized microscopically by using specific antibodies
Indirect detection
Gold-labelled antibody
Start with potato material that is doubly infected (e.g. PLRV + PSTVd)
Specific antibody
Virus purification by ultracentrifugation
Target Detection of both pathogens by (q)RT-PCR
Treatment with endo-exonuclease
Direct detection
Detection of both pathogens by q(RT)-PCR
Via transmission electron microscopy (TEM) If both pathogens are still detected after nuclease treatment it can be assumed that the viroid was protected from degratation by being encapsulated within the protein coat of the virus
- Specific antibodies and Protein-A-Gold for the virus (e.g. PLRV) - Biotinylated
