Proteins synthesized in tobacco mosaic virus infected protoplasts

The study described here concerns the proteins, synthesized as a result of tobacco mosaic virus (TMV) multiplication in tobacco protoplasts and in cowpea protoplasts. The identification of proteins involved in the TMV infection, for instance in the virus RNA replication, helps to elucidate the infection process in the plant cell. Not only virus coded proteins, but possibly also host coded proteins may play a part in the TMV multiplication.Research on proteins encoded by the TMV RNA, carried out in cell-free protein synthesizing systems, has revealed that five polypeptides are synthesized under the direction of TMV (subgenomic) mRNAs (see table 1.2., chapter L). Whether the polypeptides, synthesized invitro with TMV RNA as messenger, are of functional significance for the TMV infection may only be determined by means of investigating TMV infected leaves and protoplasts.The TMV multiplication runs synchronously in all protoplasts that are infected. Therefore, proteins synthesized in small amounts upon infection, may be thus detected.The search for proteins sythesized in protoplasts as a result of TMV infection has long been hindered by the fact that various factors in the cultivation of the tobacco plants may adversely influence the quality of the protoplasts. The cultivation of the tobacco plants: Nicotiana Tabacum cv. L. Samsun, Samsun NN and Xanthi nc, could be standardized however, as described in chapter 2. When the tobacco plants were cultivated in this way, at least 50 % of the tobacco protoplasts could be infected with TMV and 70 % or more of the protoplasts survived the subsequent incubation period of 36 hours. This could be achieved every time the protoplasts were isolated. The intensity and quality of the light, the way of watering, the age of the tobacco plants and of the leaf, from which the protoplasts are isolated, among others, appeared to affect the quality of the protoplasts (chapter 3.).The proteins, synthesized upon TMV infection, have to be distinguished among a great variety of host proteins. For this reason it is important to determine the incorporation of radioactive amino acids into protein synthesized as a result of TMV multiplication, in comparison with the incorporation into host proteins that are formed independently from the virus infection. Therefore the specific activity of TMV coat protein (cpm/mg protein) and of the proteins of the 27,000 x g supernatant fraction, synthesized in infected tobacco protoplasts were compared. It appeared that the specific activity of TMV coat protein was at least four times higher than of the proteins in the 27,000 x g supernatant (chapter 4.).The proteins synthesized as a result of TMV multiplication were studied not only in tobacco protoplasts, but also in protoplasts from the primary leaves of cowpea ( Vigna unguiculata (L.) Walp. var. 'Blackeye Early Ramshorn'). The method used for the infection of tobacco protoplasts with TMV was not suitable for the infection of cowpea protoplasts with TMV. Best results were obtained when both protoplasts and virus were incubated in the presence of poly-D-lysine, for 7.5 min. before infection. The protoplasts were pre-incubated in 0.1 M potassium phosphate buffer (pH 5.4) at 0°C, at a concentration of 4 x 10 5 protoplasts/mI and 0.75 μg poly-D-lysine/ml. TMV was pre-incubated in the same buffer at room temperature at a concentration of 2 μg TMV/mI and 2 μg poly-D-lysine/ml. During infection the cowpea protoplasts were incubated together with TMV and poly-D-lysine in a concentration of 2 x 10 5 protoplasts/ml, 1 μg TMV/ml and 1 μg poly-D-lysine/ml, for 7.5 min, in the buffer mentioned above at 0°C. In this way 50 to 70 % of the cowpea protoplasts could be infected with TMV.The course of TMV synthesis in cowpea protoplasts was comparable with that in tobacco protoplasts. The TMV multiplication in cowpea protoplasts was preceeded, however, by a period of 16 hours, during which the increase of TMV is slight, while the TMV multiplication in tobacco protoplasts was preceeded by a lag period of 8 hours. A possible explanation is that a much smaller amount of TMV particles penetrates into cowpea protoplasts during inoculation and/or starts to multiply than is the case in tobacco protoplasts (chapter 5.).The proteins of TMV infected and mock-infected protoplasts were analysed therupon by means of SDS-polyacrylamide slabgel electrophoresis and the polypeptide patterns were visualized by autoradiography.Ten polypeptides were distinguished, which are synthesized as a result of TMV multiplication in polypeptide patterns of proteins from infected tobacco protoplasts. The molecular weights were estimated to be 260,000, 240,000, 170,000, 116,500, 96,000, 90,000, 82,000, 72,000, 30,000 and 17,500 (coat protein). Polypeptides of similar molecular weight were absent or were present to much less extent in polypeptide patterns of proteins from mock-infected tobacco protoplasts. Many polypeptides were observed for reason that the detection capacity was improved by means of subcellular fractionation of the protoplast homogenates.The polypeptides of molecular weight 170,000, 116,500, 72,000 and coat protein were present in the 31,000 x g supernatant fraction and the pellet fractions as well. The polypeptide of molecular weight of 30,000 was present exclusively in the pellet fractions. The other polypeptides were observed exclusively in polypeptide patterns of protein of the 31,000 x g supernatant fraction (see table 6. l., chapter 6.).Eight polypeptides were observed, which were synthesized as a result of TMV multiplication in cowpea protoplasts. The molecular weights of the polypeptides were approximately 150,000, 116,500, 86,000, 72,000, 17,500 (coat protein), 16,000,14,000 and 10,000. Polypeptides of similar molecular weight were absent or present on a far less extent in polypeptide patterns of proteins from mockinfected cowpea protoplasts.The polypeptides of molecular weight 116,500, 72,000 and coat protein were present in the 3 1,000 xg pellet and 3 1,000 xg supernatant. The other polyeptides were present exclusively in the 3 1,000 xg supernatant (table 7. l., chapter 7.).It was assumed that the TMV coded polypeptides are similar in different hosts and, on the other hand, that the host polypeptides, synthesized upon TMV infection differ from host to host. When the TMV specific polypeptides, synthesized in infected tobacco protoplasts were compared with the specific polypeptides synthesized in TMV infected cowpea protoplasts, it appeared that only the polypeptides of molecular weight 116,500, 72,000 and coat protein are of similar size in both hosts (table 7.2., chapter 7). This is an indication that not only the polypeptide of 116,500 daltons and coat protein are TMV coded polypeptides, but that also the polypeptide of 72,000 daltons is encoded in the TMV RNA. It has not been reported that a polypeptide of this size is observed when TMV RNAs are translated in cell-free protein synthesizing systems.A polypeptide of 170,000 daltons is synthesized in vitro under the direction of the TMV RNA. It appeared that the polypeptide synthesized in TMV infected tobacco leaves, has a slightly less electrophoretic mobility than the product of 170,000 daltons synthesized in vitro from TMV RNA as messenger. A polypeptide of similar electrophoretic mobility was present to a lesser extent in mockinfected tobacco protoplasts. Furthermore, a polypeptide of 170,000 daltons was not observed in TMV infected cowpea protoplasts. For these reasons it is likely, that the polypeptide of 170,000 daltons, synthesized in TMV infected tobacco protoplasts, is encoded in the genome of tobacco or is encoded in the TMV RNA, but then the polypeptide has no functional significance in the TMV multiplication process.Further the polypeptide of 30,000 was observed only in TMV infected tobacco protoplasts, whereas a polypeptide of similar molecular weight was shown to be synthesized in vitro from a TMV subgenomic mRNA. The polypeptide of 30,000 daltons was detected exclusively in the polypeptide patterns of protein from the pellet fractions of TMV infected tobacco protoplasts. Polypeptide patterns of protein from corresponding fractions of cowpea protoplasts had a predominant, grey background. Due to this the polypeptide of 30,000 daltons may not be distinguished in TMV infected cowpea protoplasts, whereas the polypeptide of 30,000 daltons synthesized in TMV infected tobacco protoplasts can in fact be a polypeptide coded by TMV RNA. The other polypeptides synthesized in infected tobacco protoplasts or cowpea protoplasts as a result of TMV multiplication are presumably synthesized under the genome of tobacco or cowpea respectively.Finally, it was attempted to examine in what way the polypeptides of 116,500 and 72,000 are involved in the TMV infection process. Both polypeptides were shown to be present in the 31,000 x g pellet of TMV infected tobacco and cowpea protoplasts. It was studied whether virus specific polypeptides of similar molecular weight can be observed in RNA-dependent RNA polymerase preparations isolated from the 31,000 x g pellet fraction of cowpea leaves infected with the cowpea strain of TMV (C-TMV). The RNA-dependent RNA polymerase preparations were isolated by extraction of the 31,000 x g pellet fraction and were further purified by means of subsequent DEAE-BioGel column chromatography and glycerol gradient centrifugation. The purification procedure used was the same procedure as described for the isolation of RNA-dependent RNA polymerase from cowpea leaves infected with cowpea mosiac virus (CPMV).Four specific polypeptides of molecular weight of 98,000, 90,000, 72,000 and 46,000 were distinguished in RNA-dependent RNA polymerase preparations from C-TMV infected cowpea leaves, after glycerol gradient purifications. A polypeptide of molecular weight 116,500 was not observed. Polypeptides of molecular weights 72,000 and 46,000 were not found and those of molecular weights 98,000 and 90,000 were distinguished to a less extent in polypeptide patterns of preparations isolated in exactly the same way from mock-inoculated cowpea leaves.RNA-dependent RNA polymerase activity was also observed in preparations isolated from mock-inoculated cowpea leaves. The specific activity (cpm/mg protein) of the preparation from mock-inoculated leaves was one sixth of the specific activity of the RNA-dependent RNA polymerase preparations from CTMV infected cowpea leaves. The RNA-dependent RNA polymerase activity in C-TMV infected cowpea leaves might therefore be attributed to the increase of one or several polypeptides, present already before inoculation. Since it was thought that the polypeptide of 72,000 daltons is a TMV coded polypeptide, it was examined which specific polypeptides are present in RNA-dependent RNA polymerase preparations isolated in a similar way from CPMV infected cowpea leaves. It appeared, that in addition to CPMV specific polypeptides, the polypeptides of molecular weight 98,000 and 90,000 were also observed in RNAdependent RNA polymerase preparations from CPMV infected leaves. The polypeptides of 72,000 and 46,000 daltons were distinguished only in preparations isolated from C-TMV infected cowpea leaves. These results suggest that the polypeptide of 72,000 daltons in involved is the synthesis of TMV RNA (chapter 8.).