Session V - Virus-Vector associations: other
Identification and characterisation of virus genes implicated
in interactions with their fungal vectors
Pathology Department, IACR-Rothamsted, Harpenden, AL5 2JQ, UK.
The fungus vectors of plant viruses are all obligate root-infecting plant parasites and plant viruses have been shown to associate with these vectors in two distinct ways.
Some viruses, leached from plant roots into soil water, are adsorbed to the outside of fungus zoospores and resting spores and are transmitted to the plant host when the zoospore infects a root cell. With such "externally-borne" viruses, mostly members of the family Tombusviridae, there is evidence that the virus coat protein interacts with specific receptors on the zoospore membrane.
In the second type of association, viruses can only be acquired by the vector when it is growing inside a plant host and the virus is carried inside the fungus spores ("internally-borne"). Most of these viruses have particles that are rod-shaped (genera Benyvirus, Furovirus, Pecluvirus or Pomovirus) or filamentous (genus Bymovirus) and are transmitted by plasmodiophorid fungi. Mutants of rod-shaped viruses with deletions or mutations in the coat protein readthrough domain, or of filamentous viruses with deletions in the P2 protein, occur frequently and, when tested, have been shown to be deficient in fungus transmission. Following recent sequence analysis of some members of the genus Furovirus, we have identified two transmembrane regions in almost all CP-RTs and P2 proteins of the internally-borne viruses. These regions show evidence of compatibility between their amino acids, suggesting that they could be closely paired within a membrane. In non-transmissible mutants, the second of these regions is either missing or re-aligned in a way that could affect its function. It therefore seems possible that these regions become embedded in the zoosporangial plasmalemma and that their proteins assist virus particles to move between the cytoplasm of the plant host and that of the fungus vector.
Investigating the interaction between tobraviruses and their
Department of Virology, SCRI, Invergowrie, Dundee DD2 5DA,
Viruses belonging to only two genera, Tobravirus and Nepovirus, are transmitted between plants by nematodes. Recently, the application of recombinant DNA technology has made it possible to investigate the molecular basis of the interaction between virus and vector nematode. The tobraviruses (tobacco rattle virus [TRV], pea early-browning virus [PEBV] and pepper ringspot virus) have two positive sense, single stranded genomic RNAs. Studies with pseudorecombinant isolates showed that the smaller RNA, RNA2, most likely encoded the virus genes that were involved in transmission by Trichodorus spp. and Paratrichodorus spp. nematodes. Infectious cDNA clones of RNA2 have been constructed for four different nematode-transmissible tobravirus isolates. Mutagenesis studies have shown that part of the coat protein (CP) and the non-structural, 2b protein are intimately involved in the transmission process. In addition, a third (2c) protein is involved in the transmission of PEBV but not TRV. There is considerable specificity in the association between virus/virus isolate and particular vector nematode species. Current investigations aim to understand how the sequence diversity in the tobravirus RNA2 influences vector selection. Results will be presented of an electron microscopy study of the sites of virus retention in different species of vector nematode, and an "in vitro" study of CP:2b:2c interactions will be described.
Eriophyid mite transmitted viruses and virus-like agents
Scottish Crop Research Institute, Invergowrie,
Dundee DD2 5DA, Scotland, UK.
Eriophyid mites are the smallest known arthropods, measuring less than 0.2 mm. They are generally cryptic on plants, though some can induce serious feeding damage on crop plants. Most eriophyid mites have a very restricted host range, and often adapted to only a few species in a single plant genus. All eriophyid mites that are known, or believed to be, vectors of plant pathogens belong to the family Eriophyidae. Because of their tiny size, the mites have very short stylets (c. 20 m) that are capable of reaching only the epidermal cells of their plant hosts. They can therefore acquire disease agents from, and transmit them to, only these plant cells. By analogy with the properties of viruses with a similar distribution in their plant hosts, this would suggest that viruses transmitted by eriophyid mites should be transmitted mechanically using sap from infected plants. Indeed, more than 60% of the agents (viruses) of the 26 plant diseases with which eriophyid mites are associated have been transmitted in this way, although some only with great difficulty.
All the agents isolated and characterised from diseases associated with mites are viruses and all but one of them has filamentous particles. Most of the viruses infecting monocotyledonous plants are members of the genus Rymovirus or Tritimovirus in the family Potyviridae, or of the newly described genus, Allexivirus. Until very recently, none of the agents causing 9 different diseases in dicotyledonous plants had been characterised. However, two of these are now known to have filamentous particles c. 750 nm long, to be related serologically and to show some affinities with tricoviruses. A third has isometric particles c. 30 nm in diameter and is assigned to the genus Nepovirus, family Comoviridae, and a fourth has very narrow flexuous particles resembling tenuiviruses.
The location of these different viruses in epidermal cells of their plant host and their relationship with viruses in the four virus genera given above, might suggest that the mode of transmission by their mite vector should be of a non-persistent or semi-persistent type. However, the limited data available from studies made with some of these mite/virus associations indicate that some may be of a persistent (circulative) type.
Plant virus and insect interactions that determine the
specificity of virus transmission by leaf-feeding beetles
Department of Plant Pathology,
University of Arkansas, Fayetteville, Arkansas 72701, USA.
Leaf-feeding beetles in the order Coleoptera are a significant factor in the dissemination and occurrence of six genera of plant viruses. Viruses transmitted by beetles are all small (25-30 nm), icosahedral, RNA-containing viruses that are easily mechanically transmitted. Beetles acquire virus very quickly when feeding on an infected plant, and beetles transmit the virus immediately after acquisition suggesting that circulation within the beetle is not required for transmission to occur. The length of time a beetle retains virus is dependent on their feeding activity: actively feeding beetles lose their ability to transmit virus after several days, but quiescent beetles may retain virus for several months.
In contrast to other plant virus vectors such as aphids, leafhoppers, whiteflies and nematodes that have piercing-sucking mouthparts, beetles have chewing mouthparts that cut the leaf and remove entire sections of plant leaves during feeding. Plant viruses are acquired by beetles when the beetles ingest leaves of virus-infected plants, and these viruses are transmitted when they are deposited in regurgitant on the edges of feeding wounds. However, some stable plant viruses not vectored by beetles are also acquired by beetles and deposited in regurgitant on the edges of beetle feeding wounds. This suggests that the determinants of vector specificity function after acquisition and deposition of virus by viruliferous beetles, and that specificity is controlled by virus-plant interactions rather than the virus-vector interactions characteristic of other plant virus vectors.
An important step toward understanding the mechanism of vector specificity for beetle vectors was the development of the gross-wound inoculation technique. This technique produces a wound site and introduces the virus in a manner similar to viruliferous beetles. The addition of regurgitant or RNase to purified virus in gross-wound inocula demonstrated that regurgitant, and specifically the RNase in regurgitant, can selectively prevent the infection of those plant viruses not transmitted by beetles.
Ribonuclease is a powerful inhibitor of virus infection that prevents infection of wounded cells that are bathed in regurgitant at the edge of the beetle feeding wound. Fluorescent-antibody labelling to determine the location of virus deposited in beetle feeding wounds demonstrated that beetle-transmitted viruses translocate in the xylem of leaf veins surrounding feeding wounds, and that the site of virus infection is distant from the edge of the wound. We propose that beetle-transmitted viruses are unique in that they are mobile within the xylem of plants and are capable of infecting unwounded cells. These two virus interactions with a host plant are postulated to be the basis for vector specificity of virus transmission by leaf-feeding beetles.