Phenotypes and functional effects caused by various viral RNA silencing suppressors in transgenic Nicotiana benthamiana and N. tabacum

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.     

Role of a novel type of double infection in the geminivirus-induced epidemic of severe cassava mosaic in Uganda

To study the cause of the current epidemic of severe mosaic in Ugandan cassava, PCR analysis was used to detect and identify African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and the recently reported recombinant geminivirus (UgV), which is derived from ACMV and EACMV, in leaf extracts from cassava plants grown from cuttings in the glasshouse at Dundee. The cuttings were collected from plants showing symptoms of different severities and growing at different sites in Uganda inside, at the periphery of, and outside, the area affected by the epidemic. ACMV occurred throughout the nine districts sampled but UgV was detected only in the area affected by the epidemic. EACMV was not found in Uganda. Most plants containing ACMV alone expressed mild or moderate mosaic, whereas very severe mosaic developed in most plants containing UgV plus ACMV and a few of those containing UgV only. Very severe mosaic in cassava from southern Sudan was likewise associated with co-infection by UgV and ACMV. The very severe disease was reproduced by graft-inoculating geminivirus-free cassava with UgV plus ACMV; plants inoculated with either UgV or ACMV developed severe or moderate symptoms, respectively. Unlike ACMV, Malawian EACMV did not enhance the severity of symptoms induced by UgV. However, a very severely affected plant from Ukerewe Island, Tanzania, contained ACMV and EACMV but not UgV. UgV attained a much greater concentration in cassava than did ACMV but the opposite occurred in Nicotiana benthamiana. In neither host was total virus antigen concentration affected by co-infection. Factors affecting the genesis, selection and spread of UgV are discussed. The evidence indicates that UgV is probably of relatively recent origin, that such variants do not appear often, and that the current epidemic has resulted from the rapid spread of UgV to infect plants and to invade regions in which ACMV already occurred. The novel type of virus complex so produced, consisting of an interspecific recombinant virus (UgV) and one of its parents (ACMV), typically has even more severe effects than UgV alone.     

Antiviral RNA silencing is restricted to the marginal region of the dark green tissue in the mosaic leaves of tomato mosaic virus-infected tobacco plants

Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue ( approximately 0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.     

Studies on the spread of certain plant viruses in the field

Studies on the spread of potato viruses X and Y and cucumber mosaic virus in the field are described: tobacco was used as the experimental plant. The plants were set out in the form of a cross, one series with the leaves in contact and one with the leaves not touching. No spread of potato virus X was observed, but there was extremely rapid permeation of virus Y throughout the plots. The spread of cucumber mosaic virus was much slower than that of virus Y.     

Interaction between two synthetic pyrethroids and the spread of two non-persistent viruses in cowpea

The effectiveness of the synthetic pyrethroids deltamethrin and lambda-cyhal-othrin in preventing (i) aphid colonisation of four cowpea cultivars with different levels of aphid resistance and (ii) the introduction and subsequent spread of cowpea aphid-borne mosaic virus and cucumber mosaic virus was investigated under tropical field conditions. Sprays of these pyrethroids eight days apart prevent aphid colonisation and within crop spread of virus by the colonising Aphis craccivora. However, neither deltamethrin nor lambda-cyhalothrin prevented the initial introduction of virus into the cowpea crop and, when incoming alate incidence was high, virus incidence was higher in the sprayed than in the unsprayed plots. In addition, the degree of aphid resistance of each cultivar affected secondary virus spread within the crop, with greatest spread in the most resistant cultivar.     

Further studies on seed transmission in the ecology of some aphid-transmitted viruses

A small proportion (1–4%) of the seeds of Stellaria media extracted from fallow soil from three widely separated areas contained cucumber mosaic virus (CMV). S. media seeds buried for 21 months produced 5 % infected seedlings. S. media plants from Britain, N. America and Australia were least severely affected by the CMV strain obtained from their country of origin and showed more severe reactions when infected with two alien strains. Several weed species were experimentally infected with lettuce mosaic, turnip mosaic and cauliflower mosaic viruses but, although virus was detected in the seeds of some species, it was not transmitted to any of their seedlings.     

ROGUING POTATO CROPS FOR VIRUS DISEASES

Removing virus-infected plants from plots of Majestic potatoes at Rothamsted on 2 July 1947 did not reduce the spread of leaf roll but reduced rugose mosaic (potato virus Y) to about one-fifth of that in plots rogued on 21 July or left unrogued. Roguing Arran Pilot potatoes on 16 June or 2 July reduced leaf roll to five-sixths of that in unrogued plots; roguing on 16 June reduced rugose mosaic to about half that in plots rogued on 2 July, and about a quarter of that in unrogued plots. Lifting Arran Pilot potatoes in mid-August reduced virus diseases to about two-thirds.
Roguing flattened the gradient (decrease in percentage plants diseased with increasing distance from the source of infection) with rugose mosaic, but had little effect with leaf roll. Evidently any plants prevented by roguing from contracting virus Y were near the initially infected plants.
In 1948, Majestic and King Edward potatoes at three places were rogued during 22–24 June and tubers were dug during 28–30 July and again at the end of the season. Leaf roll spread more in Majestic than in King Edward, and rugose mosaic spread more in King Edward. Roguing reduced the spread of both by about one-fifth at Rothamsted, but had no effect at Sutton Bonington. At Bretton, in the Derbyshire hills, roguing had no effect on leaf roll, but prevented the spread of rugose mosaic.
The small benefit occasionally achieved by roguing in the ware-growing districts of England does not make the practice economically worth while.     

Experiments on the spread of rugose mosaic and leaf roll in potato crops in 1946

A series of experiments on the spread of potato rugose mosaic (virus Y ), and leaf roll, which has been in progress on a uniform plan since 1943, was ended in 1946. Mean values for thirteen centres in England and Wales showed that in 1946 69% of the infections with virus Y and 48 % of those with leaf-roll virus reached the tubers of Majestic potatoes by the beginning of August. There was usually little subsequent increase of rugose mosaic, but a late increase of leaf roll was associated with a relatively high initial spread. Three-quarters of the virus Y and over half the leaf-roll infections occurred within five plants distance of the source. There was no close correlation between the spread of either virus and the maximum number of Myzus persicae , either apterous forms on the plants or alate forms caught on adhesive traps, but centres with high trap catches in July and August showed pronounced late season spread of leaf roll. There were marked differences at different centres in the relative spread of the two viruses. The amount of spread and the gradients from source of infection of the two viruses are compared over the period 1943–6.     

Cowpea Mosaic Virus-Encoded Protease Does Not Recognize Primary Translation Products of M RNAs from Other Comoviruses

The protease encoded by the large (B) RNA segment of cowpea mosaic virus was tested for its ability to recognize the in vitro translation products of the small (M) RNA segment from the comoviruses squash mosaic virus, red clover mottle virus, and cowpea severe mosaic virus (CPsMV, strains Dg and Ark), and from the nepovirus tomato black ring virus. Like M RNA from cowpea mosaic virus, the M RNAs from squash mosaic virus, red clover mottle virus, CPsMV-Dg, and CPsMV-Ark were all translated into two large polypeptides with apparent molecular weights which were different for each virus and even for the two CPsMV strains. Neither the in vitro products from squash mosaic virus, red clover mottle virus, and CPsMV M RNAs nor the in vitro product from tomato black ring virus RNA-2 were processed by the cowpea mosaic virus-encoded protease, indicating that the activity of this enzyme is highly specific.     

THE DIFFERENT DISTRIBUTION OF TWO BRASSICA VIRUSES IN THE PLANT AND ITS INFLUENCE ON SPREAD IN THE FIELD

Previous knowledge provided no explanation for the greater prevalence of cauliflower mosaic than of cabbage black ring spot in field crops of cauliflower. Both viruses are spread principally by Myzus persicae and Brevicoryne brassicae , and both are transmitted equally readily from infected seedlings. Cabbage black ring spot virus has a much wider host range, and sap from infected leaves has a higher dilution end-point than sap from leaves infected with cauliflower mosaic virus.
At least part of the difference between the rate at which the two viruses spread in the field may be accounted for by the different manner in which they are distributed in old infected plants, and the effect this has on transmission by aphids. Cauliflower mosaic virus occurs in high concentration in all the new leaves produced by infected plants. Cabbage black ring spot virus, on the other hand, occurs mainly in the older leaves, and even there is localized in parts that show symptoms. Only in recently infected plants does cabbage black ring spot virus occur in young leaves.
After flying, most aphids alight on the upper parts of plants; they are therefore less likely to acquire cabbage black ring spot virus than cauliflower mosaic virus. It may be significant that cabbage, a host in which old leaves are in a more favourable position for alighting aphids than are those of cauliflower, is also often extensively infected with cabbage black ring spot virus.     

Genetic control of the reactions of raspberry to black raspberry necrosis, raspberry leaf mottle and raspberry leaf spot viruses

Black raspberry necrosis virus (BRNV) induces a severe apical necrosis in black raspberry (Rubus occidentalis) but fails to induce diagnostic symptoms in red raspberry. However, BRNV infection of F₁, F₂ and F₃ hybrids from the cross black raspberry × red raspberry induced mosaic symptoms of varying intensity but no typical apical necrosis. In a survey of 28 red raspberry cultivars, a few developed severe angular chlorotic leaf spots when infected with raspberry leaf mottle virus and a few others did so when infected with raspberry leaf spot virus. These reactions were determined by single dominant genes designated Lm and Ls respectively. The value of the different host reactions for controlling the effects and spread of these viruses is discussed.     

Effects of Salicylate on Systemic Invasion of Tobacco Plants by Various Viruses

Salicylate watered onto soil in which White Burley tobacco plants were grown represents a reversible stress characterized by stomatal closure, slight slackening of plant growth and low chlorophyll loss. Salicylate affected viral pathogenesis in opposite ways. It had no effect against local and systemic infections by potato virus X (PVX), potato virus Y⁰ (PVY⁰) or tobacco mosaic virus (TMV), whereas it completely prevented systemic infection by alfalfa mosaic virus (AIMV) or tobacco, rattle virus (TRV) in a high proportion of treated plants. When infection moved from leaves inoculated with AIMV or TRV, the tendency to limit systemic spread was shown by the restriction of systemic infection to very limited areas erratically distributed in some uninoculated leaves. The salicylate-induced restriction of AIMV or TRV infectivity to inoculated leaves did not appear due to inhibition of virus multiplication because the inoculation of potentially resistant leaves of salicylate-reated plants resulted in virus antigen accumulation comparable to that of untreated controls. Salicylate may therefore inhibit some long distance virus transport function. Salicylate appears able to evoke true hypersensitivity only against systemic viruses able to induce local necrotic lesions, probably by activating some genetic information for resistance that is normally not expressed.     

Genetic identification of multiple biological roles associated with the capsid protein of satellite panicum mosaic virus

Satellite panicum mosaic virus (SPMV), an 824-nucleotide, positive-sense, single-stranded RNA virus, depends on Panicum mosaic virus (PMV) for replication and spread in host plants. Compared with PMV infection alone, symptoms are intensified and develop faster on millet plants infected with SPMV and PMV. SPMV encodes a 157 amino acid capsid protein (CP) (17.5 kDa) to encapsidate SPMV RNA and form T = 1 satellite virions. The present study identifies additional biological activities of the SPMV CP, including the induction of severe chlorosis on proso millet plants (Panicum miliaceum cv. Sunup or Red Turghai). Initial deletion mutagenesis experiments mapped the chlorosis-inducing domain to amino acids 50 to 157 on the C-terminal portion of the SPMV CP. More defined analyses revealed that amino acids 124 to 135 comprised a critical domain associated with chlorosis induction and virion formation, whereas the extreme C-terminal residues 148 to 157 were not strictly essential for either role. The results also demonstrated that the absence of SPMV CP tended to stimulate the accumulation of defective RNAs. This suggests that the SPMV CP plays a significant role in maintaining the structural integrity of the full-length satellite virus RNA and harbors multiple functions associated with pathogenesis in SPMV-infected host plants.     

Isolation and Partial Characterization of Peanut Top Paralysis Virus (PTPV), a Destructive Virus Causing a New Disease in Peanut (Arachis hypogaea L.)

A destructive virus, causing top paralysis to peanut, was discovered in the wild germplasm collection growing in the USDA-ARS greenhouses, Stillwater, Oklahoma, USA. The symptoms observed on the wild plant were restricted to a few leaves as green batches in a light green to yellow background with some leaflets having lost most of the basal part of the laminae leaving the top portion rolling upwards forming a cone. The virus was mechanically transmitted to cultivated peanut ( Arachis hypogaea L,.) where it caused more severe and destructive symptoms including stunting, severe malformation of leaves and partial or complete disappearance of leaflet laminae. This virus differed in symptomology, host range, and/or serological reactivity from allpeanut viruses reported in the literature, particularly those causing leaf malformation and stunting. The virus induced necrotic local lesions on Phaseolus vulgaris L. cv. "Topcrop" and chlorotic local lesions with necrotic centres bordered withvery bright intense red color on Chenopodium amaranticolor. In both passive indirect enzyme-linked immunosorbent assay (PAS-ELISA) and Ouchterlony double immunodiffusion test, the virus did not react with antisera against brome mosaic, bean yellow mosaic, peanut stripe, potato Y, tobacco mosaic, watermelon mosaic 1, watermelon mosaic 2, wheat soilborne mosaic, wheat streak mosaic, and zucchini yellow mosaic viruses.
However, in reciprocal cross reactions the virus seemed to share a common antigenic determinant with a peanut mottle virus isolate from Oklahoma (PMV-OK). The virus had flexuous filamentous particles with a length of 750–850 nm, falling within the range reported for the potyvirus group. The virus was successfully purified and the molecular weight of its protein subunit was found to be 30000 d. A polyclonal antiserum was raised in rabbits against the virus and used for reciprocal serological tests.     

Aphid-transmitted viruses from lilies in Britain

Three aphid-transmitted viruses, tulip breaking, lily symptomless and cucumber mosaic, were obtained from lilies in Britain. Tulip breaking virus was detected by the leaf mottle produced in Lilium formosanum, cucumber mosaic virus by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and lily symptomless virus by electron microscopy of crude leaf extracts from symptomless L. formosanum. Liiy symptomless virus was transmitted by Myzus persicae, Macrosiphum euphorbiae, Aulacorthum solani and Aphis fabae. M. persicae, which in a small experiment appeared a more efficient vector than A. fabae, transmitted the virus in a non-persistent manner. This conflicts with the original report of transmission in a persistent manner by A. gossypii but no transmission by M. persicae. The possibility that there are two distinct viruses with similar pathological effects is discussed. Tulip breaking and lily symptomless viruses spread to bait plants of L. formosanum within a field planting of lilies in Scotland especially during July to September; lily symptomless virus was the more prevalent. No spread of cucumber mosaic virus was detected.     

Application of a Simplified Molecular Protocol to Reveal Mixed Infections with Begomoviruses in Cassava

Samples of cassava leaves exhibiting severe symptoms of cassava mosaic disease (CMD) were collected with the PhytoPASS kit in fields surrounding the city of Bujumbura (Burundi). These materials were then sent to Belgium for polymerase chain reaction determination of the CMD begomoviruses inducing the observed symptoms. Different pairs of specific primers were used to amplify DNA sequences specific to African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV), East African cassava mosaic Zanzibar virus (EACMZV), the Uganda variant of East African cassava mosaic virus (EACMV-UG) and South African cassava mosaic virus (SACMV). It was revealed that mixed infections were prevailing in the analyzed materials. Most of the samples submitted to this analysis were found to be co-infected by three different begomoviruses (ACMV + EACMV + EACMV-UG). The so revealed mixed infections could explain the high severity of CMD symptoms noticed on cassava in the region of Bujumbura while the diversity within the CMD causal agents illustrates the importance to take this parameter into consideration for a successful use of plant genetic resistance to control the disease.     

Natural infections ofSisymbrium loeselii Jusl. with cabbage black ringspot and tobacco mosaic viruses

Investigating weeds for viruses in ruderal localities of Greater Prague two forms of mosaic diseases inSisymbrium loeselii Jusl. were demonstrated (green and yellowish-green mosaic). Transmission tests carried out on differential host plants showed that the green mosaic is caused by cabbage black ringspot virus (CBRV) and the yellowish green by mixed infection of CBRV and tobacco mosaic virus (TMV). TMV—isolate is characterized as an unusual necrotic strain; its capability to reproduce in cruciferous plant in nature is unique. It was ascertained that green mosaic was commonly spread overSisymbrium plants in ruderal ***DIRECT SUPPORT *** A01GP029 00004 associations on Prague territory; epidemiological significance of this discovery is discussed.     

The appearance and spread of mosaic infection in the tomato crop and the relation to seed transmission of the virus

Appearance and spread of infection with mosaic-inducing viruses were studied for three seasons in tomato crops under glass. Comparison was made between the reactions of plants raised from virus-free seed and those of plants raised from virus-infected seed, on plots distributed at random in a house in which no precautions against entry and spread of virus were taken. Freedom from mosaic infection was maintained longest in plants raised from virus-free seed. An experiment was carried out after steam sterilization of the soil and under exceptionally favourable weather conditions. Appearance of mosaic symptoms occurred later in the life of the plants in this season and plants raised from virus-free seed did not react differently from other plants.
The location of plants first showing mosaic symptoms was related to the depth and texture of soil beneath those plants.
Tests were made of the apparent virus content of infected tomato seed during germination and differences were found in the persistence of virus during germination in seeds of differing origin.
Apparent, 'delayed' seed transmission of mosaic-inducing viruses occurs in the tomato crop, but as yet, this condition can only be interpreted in terms of differences in the resistance of plants raised from seed of differing origin to the multiplication and systemic spread of those viruses. The use of virus-free seed taken from well-nourished vigorous plants is essential to the production of a virus-free tomato crop under commercial conditions.     

Tobacco-mosaic-virus-induced increase in abscisic-acid concentration in tobacco leaves:

The concentrations of free and bound abscisic acid (ABA and the presumed ABA glucose ester) increased three- to fourfold in leaves of White Burley tobacco (Nicotiana tabacum L.) systemically infected with tobacco mosaic virus. Infected leaves developed a distinct mosaic of light-green and dark-green areas. The largest increases in both free and bound ABA occurred in dark-green areas. In contrast, virus accumulated to a much higher concentration in light-green tissue. Free ABA in healthy leaves was contained predominantly within the chloroplasts while the majority of bound ABA was present in non-chloroplastic fractions. Chloroplasts from light-green or dark-green tissues were able to increase stromal pH on illumination by an amount similar to chloroplasts from healthy leaf. It is unlikely therefore that any virus-induced diminution of pH gradient is responsible for increased ABA accumulation. Tobacco mosaic virus infection had little effect on free ABA concentration in chloroplasts; the virus-induced increase in free ABA occurred predominantly out-side the chloroplast. The proportional distribution of bound ABA in the cell was not changed by infection. Treatment of healthy plants with ABA or water stress increased chlorophyll concentration by an amount similar to that induced by infection in dark-green areas of leaf. A role for increased ABA concentration in the development of mosaic symptoms is suggested.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein

Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus.