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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

cRNA

probes

and

antibodies for the viroid (e.g. PSTVd)

www.ugent.be www.ilvo.vlaanderen.be www.insects.ugent.be

gold-labelled

anti-biotin