Inhibition of tobacco mosaic virus replication in lateral roots is dependent on an activated meristem-derived signal

Summary. Viral invasion of the root system of Nicotiana benthamiana was studied noninvasively with a tobacco mosaic virus (TMV) vector expressing the green-fluorescent protein (GFP). Lateral root primordia, which developed from the pericycle of primary roots, became heavily infected as they emerged from the root cortex. However, following emergence, a progressive wave of viral inhibition occurred that originated in the lateral-root meristem and progressed towards its base. Excision of source and sink tissues suggested that the inhibition of virus replication was brought about by the basipetal movement of a root meristem signal. When infected plants were inoculated with tobacco rattle virus (TRV) expressing the red-fluorescent protein, DsRed, TRV entered the lateral roots and suppressed the host response, leading to a reestablishment of TMV infection in lateral roots. By infecting GFP-expressing transgenic plants with TMV carrying the complementary GFP sequence it was possible to silence the host GFP, leading to the complete loss of fluorescence in lateral roots. The data suggest that viral inhibition in lateral roots occurs by a gene-silencing-like mechanism that is dependent on the activation of a lateral-root meristem. Received July 23, 2001 Accepted October 11, 2001     

THE INFECTION OF PLANTS BY VIRUSES THROUGH ROOTS

Roots of young tomato plants became infected when inoculated with tomato bushy stunt, tobacco mosaic, and potato X viruses. Root infections also occurred when these viruses were added to soil or culture solutions in which plants were growing.
The viruses were sometimes localized around their initial entry points in roots; sometimes they invaded the root system but not the shoots, and sometimes they produced full systemic infection of roots and shoots. In some experiments, but not all, systemic infections were more frequent when the upper tap root or superficial roots were inoculated than when fibrous roots were inoculated.
In both tomato and potato, virus X spread from diseased to healthy plants sharing the same culture solution, if their roots were in contact, but not otherwise. Infection of the roots of potato plants by inoculation, produced only one plant with virus-infected haulms, although several had infected tubers.     

Concentration of virus and ultrastructural changes in oats at various stages of barley yellow dwarf virus infection

Enzyme-linked immunosorbent assay was used to monitor the concentration of barley yellow dwarf virus (BYDV) in roots and leaves of oats inoculated at the 1 - 2 leaf stage and at the 4 - 5 leaf stage, respectively. Virus was detectable 20 h after inoculation in the roots and after 48 h in the leaves of plants inoculated at the 1 - 2 leaf stage. The virus concentration reached a plateau in the roots after 7–8 days, and was 3–4 times higher than in the leaves. In plants inoculated at the 4 - 5 leaf stage virus was detectable in roots and leaves after 3 and 5 days, respectively. The concentration reached a maximum after 10 days in the roots and after 18 days in the leaves; the concentration in the leaves was 2–3 times higher than in the roots. Virus was readily detectable in seeds from infected plants, both fresh and old dried seeds. However, seed transmission could not be demonstrated. Virus-like particles were first observed in phloem cells of roots 4 days after inoculation, but no ultrastructural changes were detected at this stage. After 5–6 days, disintegrated nuclei and virus-induced vesicles were observed in many cells and abnormal production of callose was found after 10 days. Necrotic phloem cells were observed from day 13, shortly after the appearance of external symptoms.     

THE ANATOMIC RESPONSES OF DAUCUS CAROTA TO THE ASTER YELLOWS VIRUS

Struckmeyer, B. Esther. (U. Wisconsin, Madison.) The anatomic responses of Daucus carota to the aster yellows virus. Amer. Jour. Bot. 50(9): 959–963. Ilus. 1963.—The leaves, petioles, and roots of carrots (Daucus carota) displaying aster yellows virus in the field and those infected with the aster yellows virus inoculated by the 6-spotted leafhopper were examined anatomically. Compared to the uninoculated, the young infected leaves displayed fewer layers of palisade cells and larger spongy parenchyma cells with a more compact arrangement. Mature leaves of infected plants sometimes were undulated and had few chloroplasts, many of which appeared fragmented. Hypertrophy and hyperplasia in the phloem tissue were associated with some necrosis and obliteration of cells. Long, needle-shaped crystalline inclusion bodies were present in the phloem in the leaves and roots. Most of the vascular bundles of the petiole were abnormal. Malformations included proliferating phloem cells, which in some instances almost encircled the bundle, hyperplasia of the phloem, hypertrophy of the parenchyma, and considerable necrosis and obliteration of these cells. Other responses included the division into 3 or 4 rows of the large outer phloem parenchyma by parallel walls so that a cambium-like layer was simulated. The tissue enclosed by this layer divided and underwent considerable necrosis and gummosis. Lacunae were found between the phloem bundle cap and the older phloem. Some of the cells in the phloem tissue differentiated into bundles with xylem. Numerous, short, lateral roots were conspicuous on the main root. The most noticeable response of the root tissue to this malady was hyperplasia and hypertrophy of the phloem followed by cellular disorganization, necrosis, and obliteration of cells. Some sieve tubes, companion cells, and parenchyma contained a gumlike deposit.     

Movement of potato spindle tuber viroid reveals regulatory points of phloem-mediated RNA traffic

Increasing evidence indicates that the phloem mediates traffic of selective RNAs within a plant. How an RNA enters, moves in, and exits the phloem is poorly understood. Potato spindle tuber viroid (PSTVd) is a pathogenic RNA that does not encode proteins and is not encapsidated, and yet it replicates autonomously and traffics systemically within an infected plant. The viroid RNA genome must interact directly with cellular factors to accomplish these functions and is, therefore, an excellent probe to study mechanisms that regulate RNA traffic. Our analyses of PSTVd traffic in Nicotiana benthamiana yielded evidence that PSTVd movement within sieve tubes does not simply follow mass flow from source to sink organs. Rather, this RNA is transported into selective sink organs. Furthermore, two PSTVd mutants can enter the phloem to spread systemically but cannot exit the phloem in systemic leaves of tobacco (Nicotiana tabacum). A viroid most likely has evolved structural motifs that mimic endogenous plant RNA motifs so that they are recognized by cellular factors for traffic. Thus, analysis of PSTVd traffic functions may provide insights about endogenous mechanisms that control phloem entry, transport, and exit of RNAs.     

Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus

Using noninvasive imaging techniques, we compared phloem unloading of the membrane-impermeant, fluorescent solute carboxyfluorescein (CF) with that of potato virus X expressing the gene for the green fluorescent protein. Although systemic virus transport took considerably longer to occur than did CF transport, unloading of both solute and virus occurred predominantly from the class III vein network, a highly branched veinal system found between class II veins. The minor veins (classes IV and V) played no role in solute or virus import but were shown to be functional in xylem transport at the time of import by labeling with Texas Red dextran. After virus exit from the class III phloem, the minor veins eventually became infected by cell-to-cell virus movement from the mesophyll. During the sink/source transition, phloem unloading of CF was inhibited from class III veins before the cessation of phloem import through them, suggesting a symplastic isolation of the phloem in class III veins before its involvement in export. The progression of the sink/source transition for carbon was unaffected by the presence of the virus in the sink leaf. However, the virus was unable to cross the sink/source boundary for carbon that was present at the time of viral entry, suggesting a limited capacity for cell-to-cell virus movement into the apical (source) region of the leaf. A functional model of the sink/source transition in Nicotiana benthamiana is presented. This model provides a framework for the analysis of solute and virus movement in leaves.     

Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis

In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.     

Host-dependent requirement for the Potato leafroll virus 17-kda protein in virus movement

The requirement for the 17-kDa protein (P17) of Potato leafroll virus (PLRV) in virus movement was investigated in four plant species: potato (Solanum tuberosum), Physalis floridana, Nicotiana benthamiana, and N. clevelandii. Two PLRV P17 mutants were characterized, one that does not translate the P17 and another that expresses a P17 missing the first four amino acids. The P17 mutants were able to replicate and accumulate in agroinoculated leaves of potato and P. floridana, but they were unable to move into vascular tissues and initiate a systemic infection in these plants. In contrast, the P17 mutants were able to spread systemically from inoculated leaves in both Nicotiana spp., although the efficiency of infection was reduced relative to wild-type PLRV. Examination of virus distribution in N. benthamiana plants using tissue immunoblotting techniques revealed that the wild-type PLRV and P17 mutants followed a similar movement pathway out of the inoculated leaves. Virus first moved upward to the apical tissues and then downward. The P17 mutants, however, infected fewer phloem-associated cells, were slower than wild-type PLRV in moving out of the inoculated tissue and into apical tissues, and were unable to infect any mature leaves present on the plant at the time of inoculation.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

Replication in the phloem is not necessary for efficient vascular transport of tobacco mosaic tobamovirus

Plant viruses move systemically from one leaf to another via phloem. However, the viral functions needed for systemic movement are not fully elucidated. An experimental system was designed to study the effects of low temperature on the vascular transport of the tobacco mosaic tobamovirus (TMV). Vascular transport of TMV from lower inoculated leaves to upper non-inoculated leaves via a stem segment kept at low temperature (4 degrees C) was not affected. On the other hand, several experiments were performed on tobacco leaves to demonstrate that virus replication did not occur at the same temperature. The data suggest that replication of TMV in the phloem of wild-type tobacco plants is not necessary for the vascular transport of TMV, and that the virus moves with photoassimilates as suggested previously.     

Potassium in the Apoplast of the Root Sink Region

Using carboxyfluorescein, a fluorochrome transported along the phloem, we demonstrated that symplasmic phloem unloading in the watermelon root occurred in the basal zone of the meristem adjusting to the elongation zone. In the similar zones of maize and pumpkin roots, a high level of potassium was detected by X-ray microanalysis in the cell walls and intercellular spaces. Potassium concentration in these compartments comprised two-thirds of that in the cytoplasm. Such proportion between potassium concentrations in the cytoplasm and apoplast was characteristic of both the cortex and stele. Since potassium is a dominant osmotically active component in root tissues, such a proportion between its intracellular and apoplastic concentrations provides for a low turgor pressure in the cells of the sink region, in the phloem in particular. This might increase a turgor pressure gradient along the translocation route between source and sink tissues, which is a driving force for phloem assimilate transport.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 591–599.Original Russian Text Copyright © 2005 by Krasavina, Burmistrova, Feshchenko, Nosov.     

The influence of primary infection date and establishment of vector populations on the spread of yellowing viruses in sugar beet

In three field experiments in 1985 and 1986, we studied the effect of the date of primary infection on the spread of beet yellows closterovirus (BYV) and beet mild yellowing luteovirus (BMW) from artificially inoculated sugar beet plants. Laboratory-reared vector aphids, Myzus persicae, were placed on these sources of virus. There was no substantial natural immigration of vectors or viruses. In two experiments, one with BMYV in 1985 and the other in BYV in 1986, populations of vector aphids remained low and there was little virus spread, i.e. c. 50 infected plants from one primarily infected source. The cause of this small amount of spread was the low number of vector aphids. In the third experiment, with BYV in 1986, large populations of M. persicae developed and there was substantial virus spread: c. 2000 infected plants in the plots which were inoculated before canopy closure. In later-inoculated plots in the same experiment, there was much less spread: c. 100 infected plants per virus source plant. Differences between fields in predator impact are implicated as the most probable factor causing differences in vector establishment and virus spread between these three experiments. Virus spread decreased with later inoculation in all three experiments. A mathematical model of virus spread incorporating results from our work has been used to calculate how the initial proportion of infected plants in a crop affects the final virus incidence. This model takes into account the effect of predation on the development of the aphid populations. The processes underlying the spread and its timing are discussed.     

Spread of beet necrotic yellow vein virus in infected seedlings and plants of sugar beet (Beta vulgaris)

Summary Infection of sugar beet roots by beet necrotic yellow vein virus (BNYVV) was investigated with transmission electron microscopy, immunogold labelling and enzyme linked immuno sorbent assay (ELISA). Here we show that infection of sugar beet roots is very fast, occurring during germination. Seedlings grown directly in infected soil showed higher BNYVV infection than plants transplanted into infected soil after seven days of initial growth in sterilized soil. The earlier the initial infection, the faster was its spread. The study showed that a few differentiated cells of the cortex and of the xylem parenchyma were the preferred sites of viral multiplication. The spread of viral infection was slow through differentiated tissues. Intact virions were frequently found in undifferentiated and mature vessel elements and xylem parenchyma, whereas they were rare in sieve elements. Virus particle number in the differentiating tracheary elements was high, suggesting that infection of the vessel elements preceded their differentiation. This would explain increased infection after early inoculation. Even the xylem tissue of the primary root was highly infected, the seedlings lacked virus particles in their hypocotyls and leaves.     

Phytoplasma distribution in coconut palms affected by lethal yellowing disease

Lethal yellowing (LY), the most devastating disease affecting the coconut palm in America, is caused by phytoplasmas known to be distributed in different parts of infected plants. However, no comprehensive reports exist on the phytoplasma distribution. This study refers to the detection of LY phytoplasma DNA using PCR in different coconut plant parts, throughout the development of the disease. Sample analysis of positive palms taken at different stages of disease development (either symptomatic or symptomless) showed differences in the percentage of LY detection between plant parts. Some parts showed a very high level of LY DNA (stem, young leaves, inflorescences, stem apex and root apex), low levels were found in the intermediate leaves and roots without apex, whereas no LY phytoplasma DNA was detected in mature leaves. The detection percentage of LY phytoplasma DNA was lowest in symptomless‐infected palms for all parts, except the stem, where phytoplasma accumulations were consistently detected. This pattern of detection among parts is consistent with the hypothesis that phytoplasmas move from photosynthate source tissues to sink tissues via the phloem mass flow process. The accumulations in the (lower) stem, prior to the appearance of symptoms, suggest that this part of the palm is where phytoplasmas first move from leaves after foliar feeding by vectors and in which they probably multiply and distribute to other palm parts, including roots. Embryos from infected palms were analysed by nested‐PCR and 28% of 394 embryos were positive. Phytoplasma DNA was detected in embryos from fruit on any of the fruiting bunches regardless the age, but no pattern of quantitative distribution throughout the bunch developmental stages was observed. Germination of seeds from LY‐positive symptomatic palms was 58% and from LY‐negative symptomless palms were 71%. No phytoplasma was detected in seedlings tested from both symptomatic and non‐symptomatic palms. Seedlings tested after 2 years did not develop LY symptoms or eventually died.     

The multibasic cleavage site in H5N1 virus is critical for systemic spread along the olfactory and hematogenous routes in ferrets

The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space-including along the olfactory route-and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.     

SOME HISTOLOGICAL OBSERVATIONS ON VIRUS-INFECTED THEOBROMA CACAO L.

Although the five cacao viruses studied produce different external symptoms in infected plants, they affect internal anatomy similarly. Symptoms on leaves occur only if these are still developing when they become infected, and the viruses seem to produce their effects usually by preventing differentiation of the cells. The tissues of chlorotic areas of infected leaves are undifferentiated and similar in structure to young unexpanded leaves. In stem and root swellings xylem and phloem are both increased, but they occur in the same proportions as in normal secondary thickening. The anatomical effects of infection seem insufficient alone to account for the death of cacao trees, but they may well be complementary to the serious necrosis of the root system which results from virus infections.     

The epidemiology of tomato mosaic: Sources of TMV in commercial tomato crops under glass

Of the several possible sources of tomato mosaic virus, seeds and root debris in the soil are considered to be of greatest importance. A survey of 374,000 seedlings on ten commercial holdings found 0.05% of them infected, and although these were removed virus had been spread to other young plants which did not show infection when transplanted into the growing houses, seven of twenty-two of which contained a few infected plants when sampled shortly after planting. Virus overwintering on clothing, and debris on structures, are thought to be of minor importance, and smoking tobacco is seldom a source of infection for the tomato crop. A further survey of seventy-eight samples from tomato crops in Britain confirmed the 1960-61 survey: all were infected with tomato strains of TMV, none with tobacco strains, but one of the 187 infected seedlings referred to above was carrying a tobacco strain. Petunia was not as satisfactory as a special cultivar of White Burley tobacco for distinguishing between the tobacco and tomato TMV isolates. Observations and tests on a commercial holding showed that TMV was readily carried from plants in infected glasshouses into clean ones by workers, and once introduced, spread rapidly within the crop.