1.12.1
ISOLATION OF SPECIFIC GENES EXPRESSED IN TMV-INJECTED CALLUS CELL AGGREGATES OF TOMATO BY MICRO-INJECTION IN SITU PCR

Y MATSUDA1, H TOYODA2, K KONAGAYA2 and S OUCHI2

1Institute for Comprehensive Agricultural Sciences, and 2Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631, Japan

Background and objectives
Using a micro-injection technique viral RNA could be successfully introduced into tomato callus cells, and the replicated RNA and multiplied viral particles were detected using an in situ Northern hybridization and in situ immunoassay, respectively. This method enabled us to introduce viral particles into tomato callus aggregates and analyse multiplication and cell-to-cell movement of the virus. In the present study, we attempted to micro-inject viral RNA or intact viral particles into target constituent cells of four- or five-cell aggregates of a TMV-resistant tomato cultivar (Tm22/+) and to detect mRNAs translated in aggregated cells which inhibited viral translocation, using micro-injection in situ PCR.

Results and conclusions
Firstly, TMV was micro-injected into one of the constituent cells of the callus aggregates derived from either susceptible (Fukuju No. 2) or resistant (Zuiko) tomato cultivars, and multiplication of injected TMV was examined using in situ detection methods. The result indicated that the viral multiplication was detected in all tandemly aligned constituent cells (TMV-injected and non-injected cells) in the aggregates of the susceptible cultivar. In the cell aggregates of the resistant cultivar, however, viral multiplication was detected only in the TMV-injected cells, but not in non-injected adjacent cells of the same aggregates. These results suggest that the resistance is due to inhibition of cell-to-cell movement of the virus in the callus cells originating from the resistant cultivar carrying the resistance gene Tm22. Since cells adjacent to the injected cells were expected to express specific genes for the resistance response, the aggregates were embedded in an agar-solidified medium and TMV was micro-injected into one of the constituent cells of the resistant aggregate in order to identify resistance-specific mRNAs in these cells. After incubation and fixation, the non-injected cells of the TMV-injected aggregates were pricked with a glass needle for an in situ cDNA synthesis of mRNAs and subsequent PCR amplification of synthesized cDNAs, according to the method described previously [1]. The same aggregates were used for detection of viral RNA and particles by in situ Northern hybridization and in situ immunoassay to confirm viral multiplication in the aggregate. Finally, the PCR products obtained were subcloned and used as probes for in situ Northern hybridization. The labelled PCR products were introduced into all of the constituent cells of the TMV-injected aggregates, and the clones specifically hybridized with mRNAs of the resistance-expressing cells were selected as candidates for genes involved in the expression of resistance; namely in the inhibition of the cell-to-cell movement of the virus.

References
1. Matsuda Y, Toyoda H, Kurita K, Ouchi S, 1997. Plant Cell Reports 16, 612-618.