Enzyme-assisted Purification of two Phloem-limited Plant Viruses: Tobacco Necrotic Dwarf and Potato Leafroll

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Abstract
Tobacco necrotic dwarf (TNDV) and potato leafroll (PLRV), viruses which are normally phloem-limited and are transmitted by aphids in the persistent manner, were successfully purified using Driselase, an enzyme that macerates plant tissue. Frozen tissue of Physalis floridana infected with TNDV or PLRV was homogenized in buffer containing Driselase and incubated with shaking. The virus particles were then purified by clarifying extracts with chloroform and n-butanol, precipitation of virus with polyethylene glycol, differential centrifugation and density-gradient sedimentation. The average yield of TNDV purified without the enzyme was 0.5 mg/kg of fresh tissue and of that purified with the enzyme was 4.7 mg. Yield of PLRV purified using the enzyme was 1.3 mg/kg. The sedimentation coefficients of both TNDV and PLRV were s20, w = 115S. Enzyme treatment had no influence on the infectivity of the viruses, assessed either using tobacco mesophyll protoplasts or using aphids fed through membranes. The enzyme method described here may be useful for purification of other phloem-limited viruses.