Ryan L. Walker and Dr. Mikel R. Stevens, Agronomy and Horticulture
Tomato Spotted Wilt Virus (TSWV) of the genus Tospovirus is able to infect both the plant and the thrips vector (an insect). This unique method of transmission has allowed the various Tospovirus species to rapidly spread throughout the world. This virus is very adaptable and currently infects over 1000 different monocot and dicot species.1 In tomato (Lycopersicon esculentum Mill.), TSWV causes stunting and yield reduction in infected plants. In parts of the United States, yield reductions have been reported of up to 38%.2 In Hawaii and South Africa, crop losses as high as 90% have occurred.3,4
Research efforts for broad genetic resistance to this pathogen have been conducted at Cornell University. Scientists there have inserted the nucleocapsid protein gene (N gene) from a specific isolate of TSWV into a nonresistant tomato cultivar.5 Transgenic plants were crossed with a wild species of tomato (L. pennellii (Corr.) D’Arcy) to increase diversity for molecular research purposes, then backcrossed to the original non-transformed cultivar (Geneva 80) to create a population segregating for the transformed N gene.
The objectives of this study were to identify the number of N gene insertions present in the transformed tomato line, and characterize the location that the insertions occurred. Using a technique called inverse polymerase chain reaction (IPCR), it would be possible to amplify the DNA adjacent to the inserted gene.6 The amplified product could then be analyzed to determine the number of inserts and to look for similarities in the insertion sites.
CLONTECH Laboratories supplied the sequence of the transformation vector. Two sets of primers (for the amplification of each end of the inserted gene) to use with IPCR were designed from this sequence. The two ends of the insert were designated as “left” and “right”. DNA from the two parents and from eight individuals of the segregating population was cut with a restriction enzyme known to cut near the left side of the insertion site. The DNA was cleaned, circularized, and ligated together. This circularized product was cleaned again and used as a template for an amplification reaction with the left primers. Included in the reaction was a test to verify the integrity of the primer design. The procedure just described is a brief summary of a previously published protocol.7
The amplification product was loaded into an agarose gel and separated by gel electrophoresis. Two to three bands per sample were obtained. No bands appeared in the negative control, thus showing that the primers were designed correctly. Although this pattern fit within expected ranges for a segregating product, the bands had a much smaller molecular weight than projected. The bands were cut out of the gel and the DNA isolated from them. This DNA was used as a template for the next amplification reaction.
This second reaction revealed that all but two of the previous 22 bands actually consisted of several bands of similar molecular weight. The two bands that amplified only one product were sequenced, but only one of the bands gave results. This particular sequence should have contained approximately 600 base pairs of DNA from the transformation vector. However, the resulting sequence showed no similarity to the known sequence of the transformation vector. The sequence was then compared to other sequences in GENBANK (a national database of known sequences from various organisms). The results showed that the sequence had 81% similarity with an exon associated with an Arabidopsis thaliana gene coding for a Prokaryotic membrane lipoprotein lipid attachment site. Although interesting with regards to plant-pathogen interactions, this has not yet been pursued further.
The primers were subjected to another test to determine their selectivity, and the left primers failed the test. Another set of left primers was designed, but they also failed the test. The right primers passed the test, proving that they were valid. However, an attempt to amplify circularized DNA prepared as mentioned above (except that a different restriction enzyme was used) failed. Because the possibility of contamination can be ruled out, it appears that the two primer sets are amplifying DNA from the Taq polymerase (the enzyme used to amplify the DNA in the reaction).
Due to the relatively short sequence on the left side of the insert, it is impossible to design another set of left primers. This makes it unlikely that the IPCR procedure will work for this side of the insert. However, there is a possibility of designing another set of right primers and attempting to amplify that end of the insert.
- Peters, D., and Goldbach, R. (1998) Recent progress in tospovirus and thrips research. Presented at the Fourth International Symposium on Tospoviruses and Thrips in Floral and Vegetable Crops, Wageningen, Netherlands.
- Paterson, R.G. (1987) Epidemiology and genetic resistance in tomato to the tomato spotted wilt virus in Arkansas. M.S. Thesis. University of Arkansas, Fayetteville, Arkansas, USA.
- Cho, J.J., Mau, R.F.L., German, T.L., Hartmann, R.W., Yudin, L.S., Gonsalves, D., and Provvidenti, R. (1989) A multidisciplinary approach to management of tomato spotted wilt virus in Hawaii. Plant Dis 73:375-383.
- van Zijl, J.J.B., Bosch, S.E., and Coetzee, C.P.J. (1986) Breeding tomatoes for processing in South Africa. Acta Hort 194:69-75.
- Pang, S.Z., Bock, J.H., Gonsalves, C., Slightom, J.L., and Gonsalves, D. (1994) Resistance of transgenic Nicotiana benthamiana plants to tomato spotted wilt and impatiens necrotic spot tospoviruses: Evidence of involvement of the N protein and N gene RNA in resistance. Mol Plant Pathol 3:243-249.
- Ochman, H., Gerber, A.S., Hartl, D.L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120:621-623.
- Thomas, C.M., Jones, D.A., English, J.J., Carroll, B.J., Bennetzen, J.L., Harrison, K., Burbidge, A., Bishop, G.J., Jones, J.D.J. (1994) Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction. Mol Gen Genet 242:573-585.