Aims Phase I

Interim Report Phase I
   - Studies on viruses in
     Thailand
   - RNA-mediated
      resistance

Aims and planned studies Phase II

Publications

Scientists:

Prof. E. Maiss
D. Knierim
S. Attathom

last update:
january 2008

Transgenic tomato plants conferring resistance to Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) and Cucumber mosaic virus (CMV) will be produced. For this purpose RNA-mediated-resistance and protein-mediated-resistance strategies will be applied. To establish RNA-mediated-resistance, non-translatable fragments of viral genomes will be expressed in tomato plants. For generation of protein-mediated-resistance, inhibitory peptides identified from a phage-display-library, will be introduced into plants. An advantage of using only short genomic viral fragments in transgenic plants is the reduction of the likelihood for the generation of altered viruses due to possible recombination events. In a second strategy the potential of protein-mediated resistance will be elucidated. A phage-display library, expressing peptides, is screened for the presence of biologically active molecules interfering with virus infection in vitro. The corresponding sequences of peptides will be subsequently cloned and expressed in plants to verify their potential of virus suppression in vivo. The concept of transgenic virus resistant plants in the frame of IPM will be addressed in close co-operation with project 1.


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Studies on viruses in Thailand

During the experiments in Thailand viruses were recognized in the greenhouses by heavy symptom expression in infected tomato plants (see figure 1). The symptoms indicated the presence of Tomato yellow leaf curl virus (TYLCV) and a Tospovirus e.g. Tomato spotted wilt virus and/or Groundnut bud necrosis virus (GBNV, synonym Peanut bud necrosis virus).

Fig. 1.: Symptoms of a Tospovirus affecting tomato fruits and leaves

Serological tests confirmed TYLCV infections (Antiserum: DSMZ AS-0546/2) and the presence of a Tospovirus belonging to serogroup IV (Antiserum: Agdia SRA 61500/0500 for GBNV and Watermelon silver mottle virus; WSMV).
To get more detailed information about TYLCV (named TYLCV-AIT) and the Tospovirus affecting the tomatoes in the AIT greenhouses further molecular characterisations were done.

Using TYLCV specific primers fragments of the TYLCV-AIT named virus were amplified. The entire nucleotide sequence of component A of TYLCV-AIT comprises of 2747 nts. A nucleotide BLAST at GenBank (NCBI) reveals 94% sequence homology to the TYLCV-Thailand segment A DNA (Accession: X63015). Also the phylogenetic tree reveals a clear position of TYLCV-AIT in the cluster of other TYLCV isolates from Thailand.

In addition PCR fragments were generated, using different primers designed according to sequences of Tospoviruses from GenBank. Approximately 1100 nts of a S-RNA sequence containing the complete 3`terminus, an entire N-gene and a part of an intergenic region were determined. The alignment of the new N-gene amino acid sequence with described viruses of the tospovirus serogroup IV reveals the highest identity with Capsicum chlorosis virus (CCV, Accession: AY036058). However, the alignment of the intergenic region of about 270 bp, starting from the end of the N-gene sequence, shows a greater distance to CCV. Because of these differences and further descriptions of the biology of this virus (Chiemsombat, pers. communication) the name Tomato necrotic spot virus (TNSV) is proposed.

RNA-mediated resistance

Transformation cassettes were established according to sequence information gathered from analysis of PCR fragments generated from Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV) and Tomato spotted wilt virus (TSWV). To minimize putative risks of virus resistant transgenic plants, only a part of each coat protein (CP) gene or the N-gene, respectively, was used as a non translatable form and without the 3`termini. For CMV and ToMV the core region of the CP was chosen, for TSWV the core region of the N-gene was selected. All the cassettes were constructed as inverted repeats separated by an intron. The entire construct was assembled under control of an enhanced 35S Cauliflower mosaic virus (CaMV) promoter. Four different cassettes were transformed. For each of the viruses a single construct consisting only of an enhanced 35S promoter, an inverted repeat of the viral sequence separated by an intron and terminated by a termination signal of CaMV was cloned. In addition a triple construct with all three virus sequences in the order TSWV, CMV and ToMV was created. As binary transformation vectors pGreen0229 and pLX222 (bar-gene respectively nptII-gene as selection marker) were chosen.

Transformation with the binary vectors containing the different cassettes was done with Nicotiana benthamiana as a model plant to check the suitability of the constructs in conferring resistance. The resistance tests were done on the T1 generation. For this purpose the seeds of each regenerated line were harvested.
Each resistance test was done with 15 plants per line. Visual inspection and recoding of symptoms starts 7-10 dpi. For comparison 15 non transgenic N. benthamiana plants act as control plants. The lines containing the single cassettes were tested with the corresponding virus. For transgenic lines containing the triple constructs, 15 plants each were separately inoculated with each virus. Not all lines have been tested so far for resistance. The single inverted repeat constructs confer resistance to TSWV, CMV and ToMV. Several plants of the tested lines show either a delay in symptom expression or reacted immune, without any visible symptoms. No plants were observed with a recovery type of resistance. Unfortunately the construct consisting of an inverted repeat of all three viruses confers resistance only to TSWV in 3 lines but none of the lines was also resistant to CMV and ToMV.


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RNA-mediated virus resistance will be established against Tomato necrotic spot virus (TNSV) and Tomato yellow leaf curl virus (TYLCV) in tobacco and tomato. The aim is to generate multiple virus resistance against both viruses within only one transformation step. For this purpose transformation vectors will be constructed with inverted-repeat cassettes of both viruses. These constructs will be transformed with Agrobacterium-mediated gene transfer into N. benthamiana and Lycopersicum esculentum. The proof of principle will be checked with transgenic N. benthamiana plants. Tomatoes will be transformed in close co-operation with the project partner in Thailand. The plants will also be investigated within project P1 to evaluate the performance under conditions of IPM.
TNSV is the prevalent virus occurring in Thailand. The complete sequences of L-, M- and S-RNA will be determined. Comparisons of N-genes from different TNSV will indicate the diversity of the virus. Molecular data taken together with data concerning vector transmission and virus-host interaction will allow a definite characterisation of this tospovirus within serogroup IV of the tospoviruses.


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