Mechanical transmission of Potato leafroll virus

Mike Mayo1, Eugene Ryabov1, Gillian Fraser1 and Michael Taliansky1

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1

Author for correspondence: Mike Mayo. Fax +44 1382 562426. e-mail mail{at}scri.sari.ac.uk


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Like typical luteoviruses, Potato leafroll virus (PLRV) cannot be transmitted mechanically by rubbing plants with solutions containing virus particles. However, PLRV was found to be mechanically transmissible from extracts of plants that had been inoculated by viruliferous aphids and then post-inoculated with Pea enation mosaic virus-2 (PEMV-2). Unlike the asymptomatic infections induced by either virus alone, double infections in Nicotiana benthamiana induced necrotic symptoms with some line patterning and vein yellowing. Infective PLRV was recovered from a purified virus preparation by inoculating plants mechanically with purified virus particles mixed with PEMV-2. Similarly, Beet mild yellowing virus was readily transmitted mechanically from mixtures containing PEMV-2. PLRV was also transmissible from mixtures made with extracts of plants infected with Groundnut rosette virus, although less efficiently than from mixtures containing PEMV-2. This novel means of transmitting PLRV, and perhaps other poleroviruses, should prove very useful in a number of fields of luteovirus research.


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One of the principal characteristics of viruses that are members of the family Luteoviridae is that they cannot be transmitted mechanically by rubbing healthy plants with extracts made from virus-infected plants (Waterhouse et al., 1988 ). The explanation usually offered for this inability is that, although luteoviruses are able to multiply in mesophyll cells (at least in protoplasts prepared from mesophyll cells), transport of luteovirus infection between mesophyll cells is not possible (Taliansky & Barker, 1999 ). More recently, it was proposed that this inability to spread is because of an RNA-mediated defence in non-vascular tissues, similar to post-transcriptional gene silencing, that cannot be overcome by luteoviruses (Voinnet et al., 1999 ). Whatever the cause, the effect is that any primarily infected cell does not yield an infective centre from which infection can develop. Although productive infection starts when aphid-inoculated luteoviruses are delivered to cells in the phloem, cells in other tissues can become infected. For example, a few non-vascular cells in systemically infected Nicotiana clevelandii (Barker, 1987 ) and potato (van den Heuvel et al., 1995 ) have been found to contain Potato leafroll virus (PLRV, genus Polerovirus), epidermal and mesophyll cells of N. clevelandii leaves inoculated by viruliferous aphids carrying green fluorescent protein (GFP)-tagged PLRV accumulated PLRV (Nurkiyanova et al., 2000 ) and epidermal cells of tobacco leaves became infected with the luteovirus Tobacco necrotic dwarf virus after mechanical inoculation (Imaizumi & Kubo, 1980 ).

It is also possible to transmit luteoviruses to plants without using aphids, by agroinoculation. This is a form of mechanical inoculation in which plants are injected with Agrobacterium cells that carry a Ti plasmid containing a full-length DNA copy of the virus genome (Leiser et al., 1992 ; Mutterer et al., 1999 ). However, the infection that follows, presumably because of transformation of cells in the vascular system, resembles that initiated by viruliferous aphids. That is, virus is largely limited to phloem tissue and cannot be transmitted mechanically from the infected plants to healthy ones.

One apparently contradictory example of a mechanically transmissible member of the family Luteoviridae has been created by recent taxonomic revisions. These have added what was previously known as RNA-1 of pea enation mosaic virus to the family as a species [Pea enation mosaic virus-1 (PEMV-1)] in the revised genus Enamovirus (Mayo & D’Arcy, 1999 ). PEMV-1 is mechanically transmissible when inoculated together with Pea enation mosaic virus-2 (PEMV-2), now classified as an umbravirus, as well as being transmissible by aphids in the same circulative, non-propagative manner as other viruses in the family Luteoviridae. PEMV-1 is able to infect individual protoplasts but, unless ‘helped’ by PEMV-2, cannot spread in plants. This makes the pea enation mosaic complex unusual in that the luteoviruses that ‘help’ other umbraviruses to become aphid-transmissible are capable of independent multiplication, and they spread in infected plants in the absence of their umbravirus partners (Falk et al., 1999 ).

Another unconventional result was obtained with the lettuce speckles complex. This is a mixed infection by a polerovirus, Beet western yellows virus (BWYV), and an umbravirus, Lettuce speckles mottle virus, and Falk et al. (1979 ) reported that it was sometimes possible to transmit BWYV mechanically from these doubly infected plants. Another interaction is in plants infected with PLRV that are post-inoculated with the umbravirus Carrot mottle virus; the umbravirus infection greatly increased the amount of PLRV extractable from the plants and the number of non-phloem cells that contained PLRV (Barker, 1989 ).

In similar experiments to those described by Barker (1987 , 1989 ), we have investigated the effect of co-infecting plants with PEMV-2 and PLRV. When plants were inoculated with extracts made from such doubly infected plants, all became infected with PEMV-2 and most became infected with PLRV. Moreover, plants that were mechanically inoculated with an extract of a PEMV-2-infected plant mixed with PLRV from a variety of sources also became infected with PLRV. The utility of this novel form of luteovirus transmission is discussed.

The PLRV inocula used in most of the experiments were extracts of plants (Physalis floridana or N. clevelandii) infected with strain 1 of PLRV (Mayo et al., 1989 ) following inoculation access feeds by Myzus persicae reared on PLRV-infected plants. Inocula of Beet mild yellowing virus (BMYV, genus Polerovirus) were obtained as infected Brassica napus plants (a gift from H. G. Smith, IACR, Broom’s Barn). Inocula of PEMV-2 were obtained initially as a transcript RNA from a full-length cDNA copy of the PEMV-2 genome cloned into a vector containing the T7 RNA polymerase promoter, prepared essentially as described by Demler et al. (1993 ). The template for the RT–PCR was RNA extracted from purified particles of PEMV. The sequences of the primers specific to PEMV-2 were either the 5'-terminal 19 nucleotides or the complement of the 3'-terminal 22 nucleotides of the sequence described by Demler et al. (1993 ). Groundnut rosette virus (GRV, YB strain) (Kumar et al., 1991 ) was obtained as an extract from infected N. benthamiana. For experimental work, inocula were water extracts either of N. clevelandii infected by allowing access of viruliferous aphids (luteoviruses) or Nicotiana benthamiana infected following mechanical inoculation (umbraviruses).

Initial experiments were made by inoculating N. clevelandii plants with PLRV by allowing viruliferous aphids (five per plant) to feed on young seedlings for 24 h prior to fumigation with nicotine. After 1–2 weeks culture at about 20 °C, the plants were inoculated with PEMV-2, either as transcript RNA or as extracts made from plants infected by prior inoculation with transcript RNA. PEMV-2 usually infected all of the plants inoculated and PLRV usually infected about 75% of the plants inoculated. When extracts of PEMV-2-inoculated plants that contained PLRV were inoculated to N. benthamiana, most plants became infected with PLRV. Typical results are shown in Table 1 (experiments 1 and 2). About 12–14 days after infection, upper leaves of these plants showed some chlorotic spots that became necrotic and developed vein yellowing and line patterns (Fig. 1). Plants infected with either PLRV or PEMV-2 alone showed no symptoms of infection. Similarly, double infection of N. clevelandii plants by PLRV and PEMV-2 induced more marked symptoms than were induced by infection by either virus alone. Most plants inoculated with extracts from these doubly infected plants became infected with both viruses (Table 1; ‘Further inoculated plants’). In one test, PLRV was maintained for up to five passages of mechanical transmission in N. benthamiana.


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Table 1. Detection of PLRV or BMYV in mechanically inoculated plants

 


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Fig. 1. The three leftmost leaves are those above the inoculated leaf taken from successive positions towards the tip of N. benthamiana plants 21 days after inoculating the lower leaves with an extract of a plant infected with both PLRV and PEMV-2. The leaf on the right was infected with PEMV-2 alone.

 
In other experiments, inocula were prepared by mixing extracts from plants infected singly with PLRV or PEMV-2. Plants mechanically inoculated with these mixtures also became infected with PLRV. However, although aphids fed for 24 h on plants infected by such mixed inocula transmitted PLRV to further test plants, none of these plants became infected with PEMV-2. This suggests that PEMV-2 was not encapsidated in the PLRV particles transmitted by the aphids.

In another experiment, a sample from a purified preparation of PLRV (strain V) that had been stored for several years at -70 °C was mixed at 4 µg/ml with an extract of N. clevelandii infected with PEMV-2 and inoculated to test plants (Table 1, experiment 4). One of the four inoculated N. benthamiana plants was infected 2 weeks after inoculation and another was found to be infected only 3 weeks after inoculation. Further plants inoculated with extracts from the plant first showing evidence of infection became infected and ELISAs on these plants showed the PLRV to resemble PLRV-V in that it failed to bind monoclonal antibody SCR 8 (Massalski & Harrison, 1987 ).

To determine whether the effect we observed was peculiar to PLRV and PEMV-2, experiments were done with another umbravirus, GRV, and with another polerovirus, BMYV. The results (Table 1, experiments 2, 3 and 5) show that in both instances co-infection of plants with the polerovirus and an umbravirus resulted in the polerovirus becoming mechanically transmissible. BMYV was mechanically transmissible from plants that were infected first by viruliferous aphids and then after 2 weeks with PEMV-2 (experiment 3), and also from plants inoculated simultaneously with a mixture of extracts from plants infected with each virus (experiment 5). The amounts of BMYV that accumulated in the doubly infected plants, as judged by ELISA, were comparable to those that accumulated in singly infected plants.

Post-inoculation of aphid-inoculated plants with GRV resulted in doubly infected plants from which PLRV (experiment 2) or BMYV (experiment 3) could be mechanically transmitted to further plants. As with the combination of PLRV and PEMV-2, the results of ELISA of plants doubly infected by mechanical transmission of BMYV and PEMV-2 were similar to those of plants singly infected by BMYV. In contrast, ELISA of plants infected by mechanical transmission from plants doubly infected by PLRV and GRV gave much lower values than for plants infected with PLRV and PEMV-2 or PLRV alone. The ELISA signals were confirmed as positive by protracted exposure to substrate in the ELISA test (Table 1). Perhaps because of the small amount of virus in the plants, PLRV was much less readily transmitted from plants infected with PLRV and GRV than from those infected with PLRV and PEMV-2. In one experiment, N. benthamiana plants inoculated with a mixture of extracts made from PLRV-infected leaves and leaves infected with the umbravirus Tobacco mottle virus did not become infected with PLRV. Also, PLRV could not be transmitted mechanically from plants doubly infected with the umbravirus Carrot mottle virus and PLRV (Barker, 1989 ). Thus, the effect of assisting mechanical transmission of PLRV may be confined mainly to PEMV-2.

Our results show that it is possible to transmit PLRV and BMYV mechanically by mixing virus particles with extracts from plants infected with PEMV-2. Only PLRV was aphid-transmissible from such doubly infected plants, suggesting that no PEMV-2 RNA became encapsidated in PLRV coat protein. Such plants are therefore a potential source of PLRV or BMYV particles, because the procedures used to purify virus particles would quickly eliminate the umbravirus component from extracts of the doubly infected plants. Our results also show that PLRV-V, which is only poorly transmissible by aphids (Massalski & Harrison, 1987 ), could be readily transmitted mechanically when mixed with a source of PEMV-2. The use of mixed mechanical inoculation therefore offers the possibility of avoiding the need to maintain such poorly transmissible virus isolates in plants, with its attendant risk of genetic drift in the culture. Moreover, it is now possible to recover infective PLRV (and presumably other poleroviruses) from stored purified virus or sap inocula without using aphids. The other obvious advantage of the use of mixed inocula is in the propagation of mutant isolates that have low infectivity and can only accumulate to relatively low levels in infected plants. Agroinfection with mutants such as this rarely results in more than a few infections, greatly limiting the scope for experimentation with preparations of purified virus (M. Mayo, unpublished observations).

The results described here extend observations made previously about the effects of mixed infections with luteoviruses and other viruses. As shown previously (Barker, 1987 , 1989 ), the effects are most marked with umbraviruses and our results suggest that, of the umbraviruses, PEMV-2 seems especially well suited to this ‘helper’ role. The effects we report have considerable practical implications for assisting the study of luteoviruses. The specific effect of assisting the spread of PLRV between cells may be especially relevant to the study of cell-to-cell movement, in particular transport into and out of phloem tissues, or the suppression of the host defence response, or both.


   Acknowledgments
 
We thank David Robinson for the inocula of the pea enation mosaic complex and of Tobacco mottle virus. The authors acknowledge the financial support of the Scottish Executive Rural Affairs Department.


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Received 27 June 2000; accepted 15 August 2000.