MOLECULAR STUDIES OF THE TRANSMISSION OF PLANT VIRUSES BY NEMATODES
Scottish Crop Research Institute, lnvergowrie, Dundee DD2 5DA, Scotland. UK
Viruses belonging to the genera Tobravirus and Nepovirus are transmitted between plants by root-feeding nematodes. Tobraviruses are transmitted by nematodes belonging to the genera Trichodorus and Paratrichodorus, whereas nepoviruses are transmitted by nematodes belonging to the genera Longidorus, Paralongidorus and Xiphinema. These vector nematodes share similar feeding habits, they are ectoparasitic and probe from the outer surface of the root into tissues at or near the root tip. However, the tobravirus vector nematodes cannot transmit nepoviruses, nor can the nepovirus vector nematodes transmit tobraviruses. Also, the two types of virus differ greatly in their morphology and genetic organization. Nepoviruses have two positive-sense, single-strand genomic RNAs, which carry a small protein (VPg) covalently linked to their 5' end, and which are polyadenylated at the 3' end. The RNAs are packaged in isometric particles of approximately 28 nm diameter. Each nepovirus RNA is translated into a single 'polyprotein', which is then cleaved by virus-encoded proteases to produce individual viral proteins. Tobraviruses also possess two positive-sense, single-strand genomic RNAs, but these have a 5' cap and 3' end which is folded into a tRNA-like structure. The tobravirus RNAs are packaged in rod-shaped particles of 50-100 nm length (for RNA2) and approximately 200 nm (for RNA1). The viral proteins are translated either from the genomic RNA (to produce the viral replicase) or from subgenomic RNAs. Pseudorecombinant nepoviruses and tobraviruses have been produced which contain RNAs 1 and 2 from different virus isolates. Transmission tests of these recombinant viruses showed that, for both nepoviruses and tobraviruses, RNA2 contains the determinant(s) for nematode transmission. With the nepoviruses, RNA2 encodes the virus coat protein, a possible virus movement protein, and a third protein whose function has not been determined. With the tobraviruses, RNA2 encodes the coat protein and either two or three additional proteins (which are described below). More detailed investigation of the virus genes involved in nematode transmission requires the construction of full-length, infectious, nematode transmissible cDNA clones. Such clones are currently available only for the tobraviruses, which has made it possible to introduce defined mutations into the various virus genes and to test the mutants for transmission by nematodes.
One isolate of pea early-browning virus (PEBV) has been studied in this manner. Deletion of the COOH-terminus of the PEBV coat protein abolishes transmission (without affecting particle formation), and (some) mutations in the other three genes (9K, 29K and 23K) also affect transmission. Interestingly, deletion of the 29K gene abolishes transmission whereas deletion of the 3' proximal 23K gene reduces transmission frequency (to about 15%) but does not abolish it. Three nematode-transmissible isolates of TRV have been sequenced, only one of which carries a gene similar to the PEBV 9K gene, all three have a gene similar to the PEBV 29K gene, and all three have an additional, 3' proximal gene (these, however, have no similarity to each other or to the PEBV 23K gene). Coat protein mutants of TRV have not been tested for transmission; however, for two isolates the 29K-like gene has been shown to be essential for nematode transmission. In contrast to PEBV, the 3' proximal genes of these two TRV isolates have been found not to be required for efficient transmission. Antibodies have been raised against the PEBV 9K, 29K and 23K proteins. The 9K protein has not been detected in virus-infected plants whereas both the 29K and 23K proteins have been detected in leaves and roots of these plants. These experiments suggest that the 23K protein is glycosylated which may have a bearing on its function in the transmission process. Current experiments aim to determine the location of the PEBV 29K and 23K proteins in plants and in vector nematodes, information which might enable us better to understand the mechanism of transmission.