Robert W. Fulton

The specific involvement of coat protein in tobacco mosaic virus cross protection

Nicotiana sylvestris infected by strains of tobacco mosaic virus (TMV) causing mosaic can be superinfected in the dark green leaf tissue, but not light green tissue, by necrotizing strains of TMV. The dark green tissue, however, is much less susceptible than healthy tissue, to some extent, even to unrelated viruses. The RNA of necrotizing strains of TMV was relatively more infectious than intact virus on mosaic than on healthy leaves and caused lesions in both light and dark green tissues. The same relationship was found in Nicotiana longiflora and, when the protecting strain in N. sylvestris could be used as a challenge, in Capsicum baccatum. The efficiency of superinfection by RNA was not found with viruses unrelated to TMV. When bentonite at 1 mg/ml, which is known to strip protein from TMV, was included in the inoculum of intact TMV it superinfected in the same manner as RNA. RNA of a necrotizing strain of TMV, encapsidated in brome mosaic virus protein and used as a challenge, superinfected in the same manner as RNA. When encapsidated in common TMV protein, however, it behaved as native virus. Cross protection apparently results from the prevention of uncoating of related challenge virus in light green tissue of N. sylvestris. Locally inoculated N. sylvestris leaves were insusceptible to challenge RNA or intact virus when the protecting virus was increasing. After increase ceased, RNA was more infectious than intact virus.

Purification of sour cherry necrotic ringspot and prune dwarf viruses

Abstract Sour cherry necrotic ringspot and prune dwarf viruses were purified from cucumber ( Cucumis sativus L.) cotyledons harvested 3–6 days after inoculation. Infected tissue was homogenized in 0.02 M pH 8.0 phosphate buffer containing 0.01 M sodium diethyldithiocarbamate and 0.02 M sodium thioglycolate. After low speed centrifugation the supernatant was mixed with hydrated calcium phosphate. This adsorbed chloroplastic material and cell debris, which then was removed by low speed centrifugation. Two cycles of low and high speed centrifugation of the clarified homogenate produced clear glassy pellets which were highly infectious. Suspensions of necrotic ringspot virus were composed of round particles about 23 mμ in diameter; prune dwarf virus particles were about 22 mμ in diameter. Purifed preparations had ultraviolet absorption spectra typical of nucleoproteins. Healthy cucumber cotyledons processed in the same manner as infected tissue yielded no visible material. Both infectivity and typical particles adsorbed to hydrated calcium phosphate in 0.002 M buffer and failed to adsorb in 0.03 M buffer. Purified virus in 0.02 M phosphate buffer was much more stable than virus in crude extracts. Purified necrotic ringspot virus lost infectivity rather rapidly in distilled water and was quickly inactivated by vigorously shaking the suspension.

Purification and some properties of tobacco streak and tulare apple mosaic viruses

Abstract Tobacco streak virus (TSV) was stabilized in extracts by 2-mercaptoethanol and, after clarification of the extracts with hydrated calcium phosphate, concentrated by high speed centrifugation. Host protein was removed from concentrated preparations by acid precipitation and by precipitation with γ-globulin of antiserum to host protein. Purification was completed by two cycles of high (40,000 rpm) and low (10,000) speed centrifugation. TSV measured about 28 mμ in diameter. Centrifugation of purified virus on sucrose density gradients resulted in two closely spaced zones, only the lower of which was infective. Material from the upper zone enhanced infectivity of infective virus; enhancement was more pronounced on the host on which the infectivity-dilution curve was abnormally steep. Tulare apple mosaic virus was purified by the procedure used for TSV. It was slightly larger (32–34 mμ in diameter) than TSV, reacted with homologous antiserum but not with TSV antiserum, and was separable on sucrose density gradients into infectious and noninfectious zones.

The relation of polyphenol oxidase to instability in vitro of prune dwarf and sour cherry necrotic ringspot viruses.

Abstract The viruses that cause prune dwarf (PDV), sour cherry recurrent necrotic ringspot (RNRSV), and necrotic ringspot (NRSV) lose infectivity in tissue extracts within a few hours. RNRSV and NRSV were stabilized in cucurbit extracts by sodium diethyldithiocarbamate (NaDIECA), a copper-chelating inhibitor of polyphenol oxidase, and by two competitive inhibitors of this enzyme. PDV was stabilized by a combination of NaDIECA and cysteine hydrochloride. PDV and NRSV, stabilized by partial purification, were inactivated by o -quinone, but not by reduced polyphenols. PDV and NRSV inactivated by o -quinone could not be reactivated by chemically reducing the o -quinone or by centrifugally sedimenting the virus. Serological activity and gross structure of the viruses were not altered by o -quinone. Polyphenol oxidase activity was higher in cucurbit tissues infected with NRSV and PDV than in healthy tissue. Spraying inoculated plants with a polyphenol oxidase inhibitor (4-chlororesorcinol) resulted in higher concentrations of NRSV and PDV than in control plants, presumably because of in vivo inhibition of the enzyme. Similar treatment with an oxidizable substrate of polyphenol oxidase (catechol) had the reverse effect on virus concentration.

The effect of dilution on necrotic ringspot virus infectivity and the enhancement of infectivity by noninfective virus.

Abstract The infectivity dilution curves of sour cherry necrotic ringspot (NRSV) and prune dwarf (PDV) viruses on local-lesion hosts were significantly steeper than a theoretical one-hit curve. The slope of the curve varied with the host; on Dolichos biflorus each twofold dilution of NRSV resulted in a three- to fourfold decrease in infectivity. On Momordica balsamina twofold dilution resulted in eight- to tenfold decreases in infectivity. Dilution curves of other viruses on these hosts resembled typical one-hit curves. Supplementing dilute NRSV extracts with additional serologically related virus caused disproportionate increases in infectivity, but the serologically unrelated PDV had no effect. A strain of NRSV noninfective to D. biflorus markedly increased infectivity when added to dilute extracts of a strain causing lesions on this host. UV- or heat-inactivated virus also increased infectivity of dilute extracts of active virus, but virus inactivated by formalin or freezing did not. These results suggest that a dose of two or more particles of NRSV at an infection site is necessary to initiate infection and that the dose requirement may be satisfied partly by virus that is unable to infect.

Resistance in tobacco to cucumber mosaic virus.

Abstract Neither tolerance nor hypersensitivity to cucumber mosaic virus suitable for disease control was found in the genus Nicotiana . Therefore the nature and inheritance of the tendency to escape infection (klendusity) was investigated. Relative susceptibility of tobacco varieties was determined by comparing numbers of lesions induced by a strain of cucumber mosaic virus that had been selected for its ability to incite necrotic local lesions on tobacco. The ratio of lesion counts on the most resistant variety (T.I. 245), on a standard type, and on the most susceptible variety (T.I. 787) was approximately 1:5:20. Similar low lesion counts were obtained on T.I. 245 tobacco with the viruses of tobacco mosaic, tobacco necrosis, tobacco streak, and turnip mosaic. No evidence was found that inhibitors or a lower rate of virus multiplication were associated with this type of resistance. Darkness 24 hours prior to inoculation increased susceptibility of T.I. 245 tobacco much more than it increased susceptibility of the standard type, indicating that resistance was conditioned by physiological factors. Genetically, resistance appeared to depend upon multiple factors. F 2 generation plants from crosses between resistant and susceptible varieties ranged from very susceptible to very resistant. In field tests a lower incidence of disease was observed on T.I. 245 tobacco than on a standard type except in one instance when infection on both types reached 100%. The possibility of obtaining higher degrees of resistance was suggested by the occurrence in hybrid progenies of plants apparently more resistant than the resistant parent.

Superinfection by strains of tobacco streak virus

Abstract Eight selected strains of tobacco streak virus varied in their reactions in cross-protection tests. Of the 28 possible pairs of strains, eight reciprocally protected tobacco against each other, eight unilaterally protected, and with 12 pairs, neither member protected against the other. When infected tobacco was susceptible to superinfection it was less susceptible than healthy tobacco except with two of the strains, which reduced susceptibility very little. Susceptibility to other viruses was retained, however. The capacity of strain WC to superinfect was not increased by selection. Nonsuperinfecting strains added to superinfecting inoculum decreased lesion numbers. Top particles seemed not to function in superinfection. The determinant for superinfection was carried in bottom particles in heterologous mixtures. Recombination of superinfecting capacity and lesion type occurred upon superinfection with heterologous mixtures of middle and bottom particles.

Correlation of ectodesmata number with nonspecific resistance to initial virus infection

Abstract The resistance of T.I. 245 tobacco to initial infection by necrotic strains of cucumber mosaic or tobacco mosaic viruses was markedly decreased by darkening plants before inoculation, by immersing leaves in water at 45° for 2 minutes, or by keeping plants at 16° for 3 days before inoculation. There was more chlorogenic acid oxidase and peroxidase in the resistant T.I. 245 tobacco than in the susceptible T.I. 787 tobacco. Treatments decreasing resistance, however, did not significantly alter the content of these two enzymes. In each variety virus became resistant at about the same rate, after inoculation, to external application of infection-inhibiting Phytolacca extract. Bentonite stimulated infectivity of TMV-RNA to about the same extent on each variety, indicating that resistance was not due to destruction of virus by leaf ribonuclease. Varietal differences in susceptibility and alterations in susceptibility due to age were correlated with numbers of ectodesmata demonstrable in the outer walls of epidermal cells. Similarly, darkening and temperature treatments that increased susceptibility of T.I. 245 tobacco also increased numbers of ectodesmata. These treatments had little effect on ectodesmata numbers or susceptibility of T.I. 787 tobacco. It was concluded that ectodesmata function as infection sites, and their number or condition determine relative susceptibility.

Cross-protection between sunn-hemp mosaic and tobacco mosaic viruses

Summary Cross-protection reactions of two tobamoviruses, sunn-hemp mosaic virus (SHMV) and common tobacco mosaic virus (TMV-C), were investigated and compared. A mutant of SHMV (SHMV-n), produced by nitrous acid treatment, induced necrotic lesions in bean. SHMV protected completely against this mutant and against SHMV-n RNA. SHMV in bean protected only weakly, however, against TMV-C. To determine whether the coat protein of these viruses affected the ability to superinfect, RNA of each virus was encapsidated in the coat protein of the other. TMV-C RNA encapsidated in SHMV coat protein was five- to 27-fold less infectious on SHMV-infected bean leaves than TMV-C RNA re-encapsidated in TMV-C coat protein. When homologous or heterologous coat protein was added to inocula, infectivity for healthy plants was diminished markedly more by homologous protein, suggesting that extraneous homologous protein diminished infectivity by inhibiting viral uncoating. SHMV-n RNA encapsidated in TMV-C coat protein did not superinfect SHMV-infected bean leaves. Thus, although coat protein was shown to be a factor in cross-protection in some situations, other factors must also be involved.

The role of particle heterogeneity in infection by tobacco streak virus

Abstract Tobacco streak virus was separable by density gradient centrifugation into four types of nueleoprotein particles. These were designated, from the slowest to the most rapidly sedimenting, T, a , b , and c . T and c were noninfectious; a and b were weakly infectious. When mixed in pairs the a plus b pair was highly infective; the other pairs were not. T, when mixed with a plus b mixtures markedly enhanced infectivity, c did not. This pattern of enhanced infectivity was also typical of mixtures of components from different strains. In heterologous mixtures of a and b , the lesions were typical of the strain from which the a component was derived. In three-component mixtures of homologous a and b plus heterologous T, enhanced lesion numbers were due to lesions typical of the strain from which T was derived. To determine whether infectivity of a or b was intrinsic, heterologous mixtures were prepared and reseparated. The separated b component produced lesions typical of the strain which produced it, not the strain which supplied the a component with which it had been mixed. Apparently infectivity of a or b was not due to contamination with the other particle type. Infectivity dilution curves of most isolated a or b components were typical of multiple-hit curves, suggesting that at some infection sites several particles cooperate in supplying a complete complement of factors necessary for infection. T enhanced infectivity of a and b separately when concentrations of each were sufficiently high, but the effect was less marked than when a was mixed with b .