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Upon infection with tobacco mosaic virus (TMV) sensitive tobacco varieties develop systemic mosaic symptoms in the developing leaves. These symptoms are the visible result of the interaction of the virus with its host and the nature and the severity of the symptoms are determined by the genetic constitutions of both partners. Their interaction involves several stages, including virus entry, replication and spread, the latter of which are dependent on viruscoded proteins. How the virus induces symptoms remains unclear.TMV codes for proteins of 183, 126, 30 and 17.5 kDa, that are necessary for replication (183 kDa and presumably 126 kDa), cell- to-cell transport (30 kDa) and long-distance transport, as well as for coating the viral RNA (17.5 kDa). Virus multiplication leads to cellular disturbances in the chloroplasts, cytoplasm and nucleus, as reviewed in chapter 1. Since viral multiplication most likely occurs in the cytoplasm, changes in the nucleus seem to be secondary and might be related to the developmentally- controlled appearance of mosaic symptoms. As shown by Van Telgen (Changes in chromatinassociated proteins of virus-infected tobacco leaves, thesis Agricultural University Wageningen, 1985), during the development of systemic mosaic symptoms the viral non-structural 126-kDa protein was present among the chromatin- associated proteins in fractionated leaf homogenates. The aim of the present investigation was to study the implications of this finding.An antiserum was raised against a fusion protein of E.coli β-galactosidase and amino acids 339-852 of the 126-kDa-protein. By immunoelectron microscopy of sections from systemically infected leaves the viral protein was not detectable in nuclei but, instead, was found to be present in cytoplasmic inclusions, characterized by the presence of tubules, and designated X- bodies. In embedded purified nuclear preparations from systemically infected leaves similar amorphous structures, most likely X-bodies,Iwere present and specifically labelled. In contrast, using antibodies against tobacco histones, only nuclei were labelled. Antibodies against viral coat protein labelled crystalline virus inclusions in the cytoplasm and did not react with nuclei. Light microscopic analysis indicated that Xbodies were almost always associated with nuclei. Thus, the presence of X- bodies in nuclear preparations seemed to result from adherence of the X-bodies to the nuclei and the viral 126-kDa protein appeared to be confined to the X-bodies (Chapter 2).To study the time course of X-body occurrence in relation to symptom development, a largely synchronized infection was employed. Plants were infected by differential temperature treatment and virus multiplication in systemically infected leaves was initiated by transfer of the plants from 4 to 25°C. An ELISA assay was developed to quantify the amount of viral 126-kDa protein present. In leaves up to 4 cm in length the accumulation of 126-kDa protein was followed in relation to viral multiplication, the development of X- bodies and the formation of symptoms. Both 126-kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h.By electron microscopy small X-bodies, weakly labelled with antibodies against the 126- kDa protein, were detected as early as 24 h after transfer but were not found to be associated with nuclei. Thereafter, X-bodies increased in size and 126-kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves developing mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations support the idea that association of X-bodies with nuclei is connected with symptom induction when the leaf is invaded by the virus early in its development (Chapter 3).TMV strains inducing symptoms ranging from severe mosaic to virtually none were used to further test this hypothesis. The RNAs of strains flavum , V4 and masked encoded in vitro proteins of similar sizes as those of the common strain W U1. Antisera against the coat and 126-kDa proteins of W U1. recognized corresponding proteins of the three strains. All virus strains accumulated to substantial levels in systemically infected leaves, as evidenced by the presence of coatprotein in virus crystals. The 126-kDa protein was localized in large X-bodies associated with nuclei in flavum -infected tissue, but in tiny X-bodies, free in the cytoplasm, in masked- infected plants. Neither X-bodies nor 126-kDa proteins were observed in V4-infected tissue. Since 126-kDa protein from V4 was - at least in vitro -not more sensitive to proteolysis than that from flavum , accumulation of the protein in X-bodies in the latter might have resulted from excess synthesis. These results are consistant with the hypothesis that the size and location of the X-bodies determine symptom expression (Chapter 4).Further TMV strains were used to probe the generality of this conclusion. Strains U2 and U5, inducing mild mosaic symptoms, were selected because of their presence in chloroplasts, and Holmes' ribgrass (HR) because it is a distantly related strain that produces a symptomless systemic infection at 30°C.Strains U2 and U5 coded for proteins of 126 kDa, while the strain HR coded for a protein of 130 kDa. Strikingly, these proteins were not recognized by antisera against the protein from W U1. Electron microscopic analysis of tissues infected with U2 and U5 established the presence of virus clusters in the cytoplasm as well as in chloroplasts. In scattered cells infected with HR virus clusters were found adjacent to nuclei and chloroplasts. X- bodies were not detected after infection with any of these strains, but were large and adjacent to nuclei in W U1-infected tomato displaying severe mosaic symptoms. The induction of X- bodies thus appears to be characteristic of some strains of TMV only. Since other strains induce mosaic symptoms without X-bodies being present, the occurrence of X-bodies can, at most, be associated with the severity of the symptoms induced by the former strains (Chapter 5).In Chapter 6 the significance of X-bodies in TMV infection is discussed. A function as the site of virus replication seems unlikely because some strains multiply to substantial levels without X-bodies being present. As discussed in chapter 4 their occurrence might be explained as resulting from a build up of 126-kDa proteins. Their close association with nuclei in systemically infected leaves suggests that they might influence nuclear activity. The rate at which association occurs and the developmental stage of the leaf at thattime appear to be factors that play a role in symptom expression in tobacco infected with M W U1. or flavum, and masked. Other mechanisms must also play a role, as evidenced by symptoms expressed by strains that do not induce X-bodies.

Upon infection with tobacco mosaic virus (TMV) sensitive tobacco varieties develop systemic mosaic symptoms in the developing leaves. These symptoms are the visible result of the interaction of the virus with its host and the nature and the severity of the symptoms are determined by the genetic constitutions of both partners. Their interaction involves several stages, including virus entry, replication and spread, the latter of which are dependent on viruscoded proteins. How the virus induces symptoms remains unclear.TMV codes for proteins of 183, 126, 30 and 17.5 kDa, that are necessary for replication (183 kDa and presumably 126 kDa), cell- to-cell transport (30 kDa) and long-distance transport, as well as for coating the viral RNA (17.5 kDa). Virus multiplication leads to cellular disturbances in the chloroplasts, cytoplasm and nucleus, as reviewed in chapter 1. Since viral multiplication most likely occurs in the cytoplasm, changes in the nucleus seem to be secondary and might be related to the developmentally- controlled appearance of mosaic symptoms. As shown by Van Telgen (Changes in chromatinassociated proteins of virus-infected tobacco leaves, thesis Agricultural University Wageningen, 1985), during the development of systemic mosaic symptoms the viral non-structural 126-kDa protein was present among the chromatin- associated proteins in fractionated leaf homogenates. The aim of the present investigation was to study the implications of this finding.An antiserum was raised against a fusion protein of E.coli β-galactosidase and amino acids 339-852 of the 126-kDa-protein. By immunoelectron microscopy of sections from systemically infected leaves the viral protein was not detectable in nuclei but, instead, was found to be present in cytoplasmic inclusions, characterized by the presence of tubules, and designated X- bodies. In embedded purified nuclear preparations from systemically infected leaves similar amorphous structures, most likely X-bodies,Iwere present and specifically labelled. In contrast, using antibodies against tobacco histones, only nuclei were labelled. Antibodies against viral coat protein labelled crystalline virus inclusions in the cytoplasm and did not react with nuclei. Light microscopic analysis indicated that Xbodies were almost always associated with nuclei. Thus, the presence of X- bodies in nuclear preparations seemed to result from adherence of the X-bodies to the nuclei and the viral 126-kDa protein appeared to be confined to the X-bodies (Chapter 2).To study the time course of X-body occurrence in relation to symptom development, a largely synchronized infection was employed. Plants were infected by differential temperature treatment and virus multiplication in systemically infected leaves was initiated by transfer of the plants from 4 to 25°C. An ELISA assay was developed to quantify the amount of viral 126-kDa protein present. In leaves up to 4 cm in length the accumulation of 126-kDa protein was followed in relation to viral multiplication, the development of X- bodies and the formation of symptoms. Both 126-kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h.By electron microscopy small X-bodies, weakly labelled with antibodies against the 126- kDa protein, were detected as early as 24 h after transfer but were not found to be associated with nuclei. Thereafter, X-bodies increased in size and 126-kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves developing mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations support the idea that association of X-bodies with nuclei is connected with symptom induction when the leaf is invaded by the virus early in its development (Chapter 3).TMV strains inducing symptoms ranging from severe mosaic to virtually none were used to further test this hypothesis. The RNAs of strains flavum , V4 and masked encoded in vitro proteins of similar sizes as those of the common strain W U1. Antisera against the coat and 126-kDa proteins of W U1. recognized corresponding proteins of the three strains. All virus strains accumulated to substantial levels in systemically infected leaves, as evidenced by the presence of coatprotein in virus crystals. The 126-kDa protein was localized in large X-bodies associated with nuclei in flavum -infected tissue, but in tiny X-bodies, free in the cytoplasm, in masked- infected plants. Neither X-bodies nor 126-kDa proteins were observed in V4-infected tissue. Since 126-kDa protein from V4 was - at least in vitro -not more sensitive to proteolysis than that from flavum , accumulation of the protein in X-bodies in the latter might have resulted from excess synthesis. These results are consistant with the hypothesis that the size and location of the X-bodies determine symptom expression (Chapter 4).Further TMV strains were used to probe the generality of this conclusion. Strains U2 and U5, inducing mild mosaic symptoms, were selected because of their presence in chloroplasts, and Holmes' ribgrass (HR) because it is a distantly related strain that produces a symptomless systemic infection at 30°C.Strains U2 and U5 coded for proteins of 126 kDa, while the strain HR coded for a protein of 130 kDa. Strikingly, these proteins were not recognized by antisera against the protein from W U1. Electron microscopic analysis of tissues infected with U2 and U5 established the presence of virus clusters in the cytoplasm as well as in chloroplasts. In scattered cells infected with HR virus clusters were found adjacent to nuclei and chloroplasts. X- bodies were not detected after infection with any of these strains, but were large and adjacent to nuclei in W U1-infected tomato displaying severe mosaic symptoms. The induction of X- bodies thus appears to be characteristic of some strains of TMV only. Since other strains induce mosaic symptoms without X-bodies being present, the occurrence of X-bodies can, at most, be associated with the severity of the symptoms induced by the former strains (Chapter 5).In Chapter 6 the significance of X-bodies in TMV infection is discussed. A function as the site of virus replication seems unlikely because some strains multiply to substantial levels without X-bodies being present. As discussed in chapter 4 their occurrence might be explained as resulting from a build up of 126-kDa proteins. Their close association with nuclei in systemically infected leaves suggests that they might influence nuclear activity. The rate at which association occurs and the developmental stage of the leaf at thattime appear to be factors that play a role in symptom expression in tobacco infected with M W U1. or flavum, and masked. Other mechanisms must also play a role, as evidenced by symptoms expressed by strains that do not induce X-bodies.

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 0.75μg poly-D-lysine/ml. TMV was pre-incubated in the same buffer at room temperature at a concentration of 2μg TMV/mI and 2μg 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, 1μg TMV/ml and 1μg 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.).

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 0.75μg poly-D-lysine/ml. TMV was pre-incubated in the same buffer at room temperature at a concentration of 2μg TMV/mI and 2μg 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, 1μg TMV/ml and 1μg 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.).

This thesis concerns transmission of tobacco necrosis virus (TNV) by zoospores of Olpidiumbrassicae. Electron microscopic observations were made on: a. the fungus, the virus, and the outer layers of seedling roots of two host species (part I); b. ultrastructural aspects of the mode of virus transmission and specificity of transmission (part II). Summarized results of part I are:1. Uniflagellate zoospores of O.brassicae have a body plasmalemma that is continuous with the axonemal sheath (section 3.1).2. Upon encystment of the zoospore, the axoneme is withdrawn inside the body. This probably happens by a 'reeling-in' mode of withdrawal and later a structure that could be the axonemal sheath is present inside the cyst body (section 3.2).3. After encystment the cyst cytoplasm penetrates into the host cytoplasm through a hole in the host cell wall and through the papillum that develops between the host wall and the host plasmalemma at the attachment site. During the penetration the cyst plasmalemma and tonoplast remain behind in the empty cyst wall, but the young thallus is immediately surrounded by a new outer membrane (section 3.2).4. Up to 24h after penetration the young thallus is separated from the host cytoplasm by the thallus membrane only. Older thalli develop a wall (section 3.3).5. The epidermal cells in the zone of elongation of lettuce seedling roots rapidly change with increasing age. The relative amount of cytoplasm decreases and in the region between 5 and 15mm from the root tip very little cytoplasm remains and many cells seem to have died (section 4. 1).6. Epidermal cells of tomato roots undergo deterioration with increasing age, but at a slightly slower pace than lettuce. These cells are often covered with calyptra cells in the zone of elongation (section 4.2).7. Particles in sections of a TNV pellet have an apparent size of approximately 20nm after conventional double staining. For this reason TNV particles may be difficult to identify if present in cells that also contain ribosomes (chapter 5).Results published in part II of this thesis are also based partially on data obtained in part I. Summarized results of part II are:1. Zoospores of the lettuce isolate of O.brassicae, that transmit TNV after exposure to the virus, acquire TNV in vitro by a tight adsorption of the virus particles to their body plasmalemma and the axonemal sheath (section 7.1).2. Part of the adsorbed virus is taken into the encysting zoospore cytoplasm and this virus is eventually transmitted. Uptake of the virus probably takes place when the axoneme with its sheath is withdrawn. The fate of TNV not taken into the cyst by axonemal withdrawal is discussed and considered unimportant for transmission (section 7.2).3. TNV taken into the infecting zoospores is transmitted into the cytoplasm of the root epidermal cell by release from the young thallus. A possible mechanism for this virus release is proposed (section 7.3).4. Adsorption of TNV to zoospores of the lettuce isolate of O.brassicae is very specific in the sense that particles of other polyhedral viruses like cucumber necrosis virus (CNV), tomato bushy stunt virus (TBSV), and turnip yellow mosaic virus (TYMV) are not adsorbed under similar circumstances (section 8.1.1).5. Adsorption of TNV is also very specific in the sense that zoospores of the lettuce and tomato Olpidium adsorb many TNV particles, those of the oat Olpidium adsorb many fewer TNV particles, and those of the mustard Olpidium apparently do not adsorb any TNV under similar experimental conditions (section 8.1.1).6. A form of specificity of adsorption comparable to that between TNV and the lettuce Olpidium was encountered in the case of zoospores of O.cucurbitacearum. Zoospores of this species adsorb many particles of CNV, but they do not adsorb TNV when mixed in vitro with these viruses (section 8.1.2).7. Zoospores of lettuce Olpidium adsorb particles of satellite virus (SV) when mixed with a suspension of SV alone or in combination with either of two different isolates of TNV (NZ-TNV and AC-36) (section 8.1.2).8. Incompatibility in the fungus - host relationship is sometimes due to the absence or low incidence of encystment of a particular fungus isolate on that host plant, or to a break-down of the developing fungal thallus after a normal appearing infection process. The first form of incompatibility certainly influences transmission of TNV (section 8.2).9. Tomato roots seem to be good hosts for TNV inasmuch as they allow the build- up of a high TNV titer after infection and do not react necrotically but lettuce roots react with necrosis 18 h after TNV infection. Also, lettuce roots seem to be less favorable hosts for TNV in the sense that many virus particles have to be present at a given infection site in order for the infection to take place (sections 8.3 and 8.4).10. In epidermal cells of lettuce roots infected by TNV, thalli of O. brassicae do not mature. No virus was detected in lettuce epidermal cells that were not infected by Olpidium nor in the cortical cells. Nevertheless these cells often were necrotic. TNV-infected epidermal cells of tomato roots often contain growing thalli of O.brassicae but virus was not found in the thalli. These host cells also sometimes have granular or crystalline material between the cisternae of the endoplasmic reticulum. Virus could be detected in epidermal and cortical cells of tomato that had not been infected by Olpidium (section 8.3).11. It is possible to explain some forms of specificity of TNV transmission just by differences in fungus - virus relationship. Other examples of specificity of TNV transmission, however, can only be explained by taking into account simultaneously the combined effect of differences in fungus - virus, host fungus, and virus - host relationship (section 8.4).In conclusion, transmission of TNV by O.brassicae is explained by a sequence of events, all of which were shown to take place during exposure of zoospores to TNV and subsequent exposure of host roots to those zoospores. Furthermore, all known forms of specificity of TNV-transmission by different isolates of O.brassicae can be understood on the basis of specific fungus - virus, host fungus, and virus - host relationships.

Disaggregated protein isolated from tobacco mosaic virus is hydrolysed by chymotrypsin and papain, but when aggregated into rod-shaped particles, the protein, like the original virus, resists attack by the enzymes. This suggests that only some walls of the protein subunit are susceptible to proteolysis and these walls become inaccessible to the enzymes when the protein aggregates in the orderly fashion required to form rods.

Tobacco rattle virus is a rod-shaped multiparticle virus.Short particles alone are not infectious, long ones are but give rise to the formation of incomplete virus. Mixtures of long and short particles induce the formation of complete virus. The interaction between long and short particles is not strain-specific: if long and short particles of different strains are inoculated together complete virus is also formed. These new strains have properties of both parent strains.The interaction between heterologous long and short particles explains why there are so many different tobacco rattle virus isolates and why no correlation can be found between classifications based on different characteristics.

Tobacco rattle virus is a rod-shaped multiparticle virus.Short particles alone are not infectious, long ones are but give rise to the formation of incomplete virus. Mixtures of long and short particles induce the formation of complete virus. The interaction between long and short particles is not strain-specific: if long and short particles of different strains are inoculated together complete virus is also formed. These new strains have properties of both parent strains.The interaction between heterologous long and short particles explains why there are so many different tobacco rattle virus isolates and why no correlation can be found between classifications based on different characteristics.

This thesis concerns transmission of tobacco necrosis virus (TNV) by zoospores of Olpidiumbrassicae. Electron microscopic observations were made on: a. the fungus, the virus, and the outer layers of seedling roots of two host species (part I); b. ultrastructural aspects of the mode of virus transmission and specificity of transmission (part II). Summarized results of part I are:1. Uniflagellate zoospores of O.brassicae have a body plasmalemma that is continuous with the axonemal sheath (section 3.1).2. Upon encystment of the zoospore, the axoneme is withdrawn inside the body. This probably happens by a 'reeling-in' mode of withdrawal and later a structure that could be the axonemal sheath is present inside the cyst body (section 3.2).3. After encystment the cyst cytoplasm penetrates into the host cytoplasm through a hole in the host cell wall and through the papillum that develops between the host wall and the host plasmalemma at the attachment site. During the penetration the cyst plasmalemma and tonoplast remain behind in the empty cyst wall, but the young thallus is immediately surrounded by a new outer membrane (section 3.2).4. Up to 24h after penetration the young thallus is separated from the host cytoplasm by the thallus membrane only. Older thalli develop a wall (section 3.3).5. The epidermal cells in the zone of elongation of lettuce seedling roots rapidly change with increasing age. The relative amount of cytoplasm decreases and in the region between 5 and 15mm from the root tip very little cytoplasm remains and many cells seem to have died (section 4. 1).6. Epidermal cells of tomato roots undergo deterioration with increasing age, but at a slightly slower pace than lettuce. These cells are often covered with calyptra cells in the zone of elongation (section 4.2).7. Particles in sections of a TNV pellet have an apparent size of approximately 20nm after conventional double staining. For this reason TNV particles may be difficult to identify if present in cells that also contain ribosomes (chapter 5).Results published in part II of this thesis are also based partially on data obtained in part I. Summarized results of part II are:1. Zoospores of the lettuce isolate of O.brassicae, that transmit TNV after exposure to the virus, acquire TNV in vitro by a tight adsorption of the virus particles to their body plasmalemma and the axonemal sheath (section 7.1).2. Part of the adsorbed virus is taken into the encysting zoospore cytoplasm and this virus is eventually transmitted. Uptake of the virus probably takes place when the axoneme with its sheath is withdrawn. The fate of TNV not taken into the cyst by axonemal withdrawal is discussed and considered unimportant for transmission (section 7.2).3. TNV taken into the infecting zoospores is transmitted into the cytoplasm of the root epidermal cell by release from the young thallus. A possible mechanism for this virus release is proposed (section 7.3).4. Adsorption of TNV to zoospores of the lettuce isolate of O.brassicae is very specific in the sense that particles of other polyhedral viruses like cucumber necrosis virus (CNV), tomato bushy stunt virus (TBSV), and turnip yellow mosaic virus (TYMV) are not adsorbed under similar circumstances (section 8.1.1).5. Adsorption of TNV is also very specific in the sense that zoospores of the lettuce and tomato Olpidium adsorb many TNV particles, those of the oat Olpidium adsorb many fewer TNV particles, and those of the mustard Olpidium apparently do not adsorb any TNV under similar experimental conditions (section 8.1.1).6. A form of specificity of adsorption comparable to that between TNV and the lettuce Olpidium was encountered in the case of zoospores of O.cucurbitacearum. Zoospores of this species adsorb many particles of CNV, but they do not adsorb TNV when mixed in vitro with these viruses (section 8.1.2).7. Zoospores of lettuce Olpidium adsorb particles of satellite virus (SV) when mixed with a suspension of SV alone or in combination with either of two different isolates of TNV (NZ-TNV and AC-36) (section 8.1.2).8. Incompatibility in the fungus - host relationship is sometimes due to the absence or low incidence of encystment of a particular fungus isolate on that host plant, or to a break-down of the developing fungal thallus after a normal appearing infection process. The first form of incompatibility certainly influences transmission of TNV (section 8.2).9. Tomato roots seem to be good hosts for TNV inasmuch as they allow the build- up of a high TNV titer after infection and do not react necrotically but lettuce roots react with necrosis 18 h after TNV infection. Also, lettuce roots seem to be less favorable hosts for TNV in the sense that many virus particles have to be present at a given infection site in order for the infection to take place (sections 8.3 and 8.4).10. In epidermal cells of lettuce roots infected by TNV, thalli of O. brassicae do not mature. No virus was detected in lettuce epidermal cells that were not infected by Olpidium nor in the cortical cells. Nevertheless these cells often were necrotic. TNV-infected epidermal cells of tomato roots often contain growing thalli of O.brassicae but virus was not found in the thalli. These host cells also sometimes have granular or crystalline material between the cisternae of the endoplasmic reticulum. Virus could be detected in epidermal and cortical cells of tomato that had not been infected by Olpidium (section 8.3).11. It is possible to explain some forms of specificity of TNV transmission just by differences in fungus - virus relationship. Other examples of specificity of TNV transmission, however, can only be explained by taking into account simultaneously the combined effect of differences in fungus - virus, host fungus, and virus - host relationship (section 8.4).In conclusion, transmission of TNV by O.brassicae is explained by a sequence of events, all of which were shown to take place during exposure of zoospores to TNV and subsequent exposure of host roots to those zoospores. Furthermore, all known forms of specificity of TNV-transmission by different isolates of O.brassicae can be understood on the basis of specific fungus - virus, host fungus, and virus - host relationships.

The nature and the severity of the reaction of a plant to infection by a virus are determined by the genetic properties of both virus and hostplant. The genetic information of both is expressed in the formation of proteins. Our aim was to gain insight into the specificity of the interaction between virus and hostplant by investigating the biochemical mechanism of pathogenesis and symptom expression. Hence, we attempted to show if differences in protein constitution between noninfected and infected plants could indeed be established and whether such differences can be regarded as characteristic of the infecting virus or of the hostplant. In this study we made use of tobacco varieties that differ in reaction type to tobacco mosaic virus (TMV) and of strains of this virus that are distinguished by the symptoms they induce in tobacco. As the multiplication of TMV most probably takes place in the cytoplasm, the soluble protein fraction was investigated. Previous investigations have emphasized alterations in enzyme activities as a result of infection. In contrast to this, our attention was focused on changes in the electrophoretic pattern of protein bands induced by infection, not manifesting themselves immediately under the form of enzymatic activity.By using electrophoresis in 5, 7.5 and 10% polyacrylamide gel, over fifty different protein components in the soluble protein fraction from leaves of Nicotiana tabacum could be distinguished. No differences in protein patterns were observed between noninfected plants of the varieties Samsun and Samsun NN, although Samsun plants react to infection with TMV W U1 by formation of systemic mosaic symptoms, while Samsun NN plants - which contain the factor N from N. glutinosa - develop local lesions at temperatures below 25°. However, in the two varieties different and characteristic changes in protein patterns appeared upon infection. Apart from a number of quantitative changes, one new band was present in mosaic-diseased Samsun plants four weeks after infection. This band was identified as the free coat protein of the virus by co-electrophoresis and serology. A reduction in the electrophoretic mobility of the major band was also recorded.In the inoculated leaves of Samsun NN plants four new protein components (I-IV) were present one week after infection. These new components are not related to TMV coat protein. In N. glutinosa one new band was induced and two bands increased markedly after infection with TMV W U1, while one other band disappeared. These bands differed in electrophoretic mobility from the new bands observed after infection of Samsun NN plants. Therefore, none of the new components I-IV can be regarded as product of the Hg chromosomes, that are derived from N. glutinosa and contain the factor N. This was further substantiated by patterns from Samsun plants showing semi-systemic yellow ringspot symptoms as a result of infection with TMV HR. In this combination, both free TMV HR coat protein and the new components I-IV were apparent.In Samsun plants infected by TMV W U1 or TMV HR only a limited number of quantitative changes was observed. Contrary to this, Samsun NN plants infected by TMV W U1 showed a considerable number of quantitative changes, most of which did not appear in the two combinations mentioned earlier. The extent of these changes correlated with the lesion density on the leaves.When systemic mosaic symptoms were induced in both Samsun and Samsun NN plants as a result of infection with TMV W U1 at 30°, identical changes in protein patterns were observed for both varieties. These changes were the same as those in the combination TMV W U1 - Samsun at 20° and those in the combination TMV W U1 - Samsun EN, in which identical symptoms are induced. on the other hand, the formation of local lesions on the variety Samsun EN upon infection with TMV HR led to the appearance of the new components I- IV. It follows that in the combination TMV - tobacco the changes in soluble proteins are connected with the type of symptoms - either mosaic or local lesions produced, and that in all cases they are hostplant dependent.The induction of local or systemic necrosis on Samsun and Samsun NN tobacco with tobacco necrosis virus (TNV), tobacco rattle virus (TRV) and potato virus Y n(PVY n) always led to the appearance of the four new components in both varieties,but the relative proportions of the bands varied with the variety used, and with the characteristic of the virus to remain local or become systemic. Relatively low concentrations of the four new components were observed after infection with cucumbermosaic virus, although no necrosis developed in these combinations. Bands I and II were present after infection with PVY o, that only causes mild mottling. Although potato virus X (PVX) induces a similar mottling, the new components were not detected in plants infected by this virus. In none of the combinations the presence of virus-specific proteins could be established.Many of the quantitative changes observed in the various combinations occurred under different conditions and evidently represented general reactions, as similar changes were detected after cutting or freezing of the leaves. Same changes, however, were characteristic of the type of symptoms produced after virus infection. Cutting or freezing of the leaves or production of artificial necrosis by spraying with HgCl 2 induced no new components. The greater part of the quantitative changes occurring as a result of necrosis induced by virus infection were not observed in HgCl 2 induced necrosis either. So necrosis due to virus infection and artificially induced necrosis can be clearly distinguished by the accompanying changes in the soluble protein fraction. Since cutting or freezing of the leaves induces changes that are only partly similar to those observed when necrosis is induced by virus infections, ageing and injury seem to be only minor facets of the metabolic alterations underlying this type of symptom.In the combination TMV W U1 - Samsun NN the four new components first appeared at the onset of necrosis, and the bands increased in intensity with time. By five clays after inoculation band I ceased to increase, whereas bands II, III and IV increased up to day 14. From day 7 onward, the four bands were also present in the young leaves that had developed after inoculation and neither showed symptoms nor contained virus. In these leaves also, the bands increased in intensity with time. These bands were also present in the young leaves that had developed after inoculation with TNV or TRV.In the combination TMV W U1 - Samsun NN the amount of the four new components correlated with lesion density. The increase in intensity of the bands was reduced by treatment of the leaves with actinomycin D (MD) two days after inoculation. AMD inhibited the incorporation of 35S- methionine and 14C-leucine in the soluble protein fraction by 56-60%. Infection with TMV in itself also strongly inhibited synthesis of soluble proteins. These two effects appeared to be at least additive. However, the inhibition of the amount of the new components by AMD always amounted to less than 50%. Although preferential synthesis of the new components could not be demonstrated by electrophoresis of labeled proteins, the ratios of the radioactivities incorporated into protein from infected and noninfected plants point to de novo synthesis which can be only partly inhibited by AMD.The new components are not isoenzymes; of thirty enzymes studied, and do not contain carbohydrate, lipid or RNA. Their strong colouration with coomassieblue may indicate a high content of basic amino acids.A possible relation between the occurrence of these new components in young, developing leaves not containing virus, and the ability of these leaves to react with the formation of small lesions after (a second) inoculation with a virus that induces local lesions, was further investigated. There appeared to be a distinct correlation between the presence of the new components and the state of systemic acquired resistance. However, when eluates from gel slices that contained the four new components were applied to the plants simultaneously or 24 hours before inoculation with TMV, no effect on number or size of the lesions could be demonstrated. On the other hand, the multiplication of TMV in leaves that developed after inoculation of Samsun plants with TNV did appear to be inhibited to a considerable extent as a result of the first infection.Ammonium sulfate fractionation of purified protein fractions from noninfected and TMV IV U1-infected Samsun NN plants revealed two other new components. Upon gelfiltration on Sephadex G 100, in addition to the new components I-IV, another eight, more slowly migrating components were detected. These twelve new components all have molecular weights between 10,000 and 20,000. A number of these were observed as quantitative changes upon electrophoresis of unfractionated extracts. During electrophoresis of extracts from noninfected plants separation due to differences in molecular size prevailed. Therefore, the new components differ from the majority of the other soluble proteins from tobacco leaves by their relatively small charges.In addition to these twelve new components, a new ribonuclease and a new peroxidase isoenzyme were detected. The peroxidase isoenzyme was distinguished by a low pH optimum and a far greater affinity towards guaiacol than towards o -diphenols. It was induced in all combinations in which necrosis due to virus infection occurs, and to a small extent after infection with PVX and PVY o. In the combination TMV W U1 - Samsun NN maximal activity was reached when symptom development could be considered complete. The new isoenzymes were not present in young, symptomless leaves not containing virus.On the basis of data given in the literature, it can be envisaged that the new components might also be present in tobacco plants which, upon TMV infection, react with systemic mosaic symptoms, but in that case only after the multiplicaticn of the virus has stopped. Therefore, it seems possible that both in hypersensitively reacting tobacco plants and in plants that react with systemic mosaic symptoms, the appearance of the new components is connected with a slowing down of viral synthesis through a direct or indirect inhibition of virus multiplication. Their presence, together with the occurrence of increased peroxidase activity - that is correlated with a decreased rate of lesion enlargement - might be responsible for the expression of acquired resistance to subsequent inoculation. However, this phenomenon could also be explained by inhibition of the formation of a specific protein which may not, or in turn may result in the induction of new proteins and enzymes.The induction of the hypersensitive reaction and of necrosis by viruses seems to be governed by a common mechanism. Presumably TMV W U1 in Samsun tobacco represses this mechanism. This would enable the virus to multiply and spread throughout the plant.