Biochemical aspects of plant virus infection of cucumbers: a studyof the viral-induced protein and RNA species, and the RNA-dependant RNA polymerase

Abstract

An investigation has been carried out on the biochemical aspects of the infection of cucumbers by two plant viruses Cucumber Mosaic Virus (CMV) and Tobacco Ringspot Virus (TRSV). 1. Attempts were made to characterise the TRSV-induced RNA species (replicative form, replicative intermediate and viral RNA genome); RNA was extracted from cucumber cotyledons using the phenol method and analysed on 2.4e" polyacrylamide gels. No differences in gel patterns between healthy and infected plants could be found in either single labelling studies (plants labelled through the roots with 32p-orthophosphate) or with double-labelling studies (plants labelled through the roots with either 32p-orthophosphate or 3H-uridine); actinomycin D, although completely abolishing ribosomal RNA synthesis, did not show up any differences between healthy and TRSV-infected cucumber RNA extracts. 2. Attempts were also made to characterise the TRSV-induced protein species. As radioactive amino acids could not be incorporated into cucumber proteins by absorption through the roots, 35s-=sulphate was used as the label. No differences could be found between healthy and infected protein extracts analysed. by polyacrylamide gel electrophoresis. Further attempts to incorporate radioactive amino acids using tissue slices and other methods were also unsuccessful. An alternative approach of differentially labelling healthy and infected plant proteins after extraction by reduction and carboxymethylation with either 3H or I4c-io.loacetic acid produced variable and unreliable radioactive protein profiles on polyacrylamide gels. 3. A comparison of the properties was made between the CMV-induced and. the TRSV-induced RNA polymerases, both soluble and particulate enzymes; these enzymes were undetectable in healthy plants. Both the soluble and particulate RNA polymerases from TRSV-infected cucumbers were detectable two days after infection and declined rapidly after four days, whereas the CMV-induced enzymes appear after about five days, reach a plateau level at 10 days and remained at this 1evel for several more days. The mol. wt. of the soluble CMV-induced RNA polymerase was calculated by sucrose gradient centrifugation to be 123,000 daltons while the soluble TRSV-induced enzyme sedimented over a wide range of 120-180,000 daltons. Solubilising the particulate CMV-induced RNA polymerase could be accomplished by incubating with 50-100mM MgSO4 or by freezing and thawing, but these methods did not release the particulate TRSV-induced enzyme. All other properties tested v/ere similar in both cases. This provided circumstantial evidence for the differences being due to viral-coded functions in the RN polymerase molecules. 4. Several- purification methods have been tried in an attempt to purify the CMV-induced RNA polymerase. Those methods that gave reasonable recoveries were protamine sulphate precipitation, phosphocellulose chromatography (stepwise elution with either KCI or (NH4) 2SO4) and poly c-sepharose chromatography (stepwise elution with M9SO4). A possible method of purification is outlined.