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
) 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 cucumber
mosaic 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 35
S- methionine and 14
C-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.