Establishment Of Virus-induced Gene Silencing System And Functional Analysis Of LeEIN2 Gene In Tomato

In recent years, whole genome studies in plant have produced a massive amount of sequence information. It is necessary for us to find large-scale functional genomic approaches. Antisense-mediated inhibition of gene expression was commonly used to down regulate gene expression in plants. However, the approach relies on the stable transformation that is difficult in many plant species and loss-of-function of some genes results in the death at early stages of plant development. Virus-induced gene silencing (VIGS) offers recently an attractive and quick alternative for knocking out expression of a gene of interest and avoids the need for processes of transformation.Although the efficient VIGS induced by TRV-VIGS vectors has been achieved in the seedlings of tomato plants, there is no report about the flowers and fruits of tomato, which is the most studied model for fruit ripening. We have found that the symptoms of VIGS were hardly observed in flower and fruit when the inoculated tomato seedlings were cultured under the conditions for optimum plant growth (22 ℃-28℃, 60-70 % RH), but the TRV could be efficiently transformed to the flowers and the fruit when the inoculated seedlings were cultivated at relatively low temperature at 15℃-18℃ and low humidity 30-40% RH respectively. This paper has shown that the systemic silencing of a phytoene desaturase gene was preserved in TRV-PDS-inoculated tomato plant and maintained throughout flowers and fruits by the stems. Silencing of PDS gene results in a dramatic reduction of the levels of PDS mRNA and the accumulation of phytoene in the leaves, flowers and fruits of PDS-silenced tomato plants. In addition, the contents of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants were reduced by more than 90% in comparison with the control plant inoculated with only TRV. The lycopene content in the yellow pulp of the PDS-silenced fruit was only about 2% of that in the red pulp of control fruit at breaker and red stage. The result suggests that this approach will facilitate large-scale functional analysis of genes involved in the development and ripening of tomato fruit, which is also a more rapid alternative to the stable transformation.We report that either by syringe-infiltrating the TRV-vector into the surface, stem or carpopodium of a tomato fruit attached to the plant or by vacuum-infiltrating into a tomato fruit detached from the plant, TRV can efficiently spread and replicate in the tomato fruit. Although VIGS can be performed in tomato fruit by all of the means mentioned above, the most effective method is to inject the TRV-vector into the carpopodium of young fruit attached to the plant about 10 days after pollination. For vacuum-infiltration, the content of ACC (the precursor compound of ethylene biosynthesis) and the rate of ethylene production in the LeACS2-silenced fruit was significantly lower than those observed the in control fruit (infiltrated with TRV alone). About 65% of the vacuum-infiltrated fruit (total 100 pieces) were discarded because of decay. For the infiltration from carpopodium, two reporter genes related toethylene responses and fruit ripening, including LeCTR1 and LeEILs genes, were also successfully silenced during fruit development and ripening. The suppression of the LeCTRl gene promotes the ripening of tomato fruit. The silencing of LeEILs genes inhibit fruit ripening and further supported the view that expression of LeEILs genes is necessary for tomato fruit ripening.The silencing of LeEIN2 gene results in the suppression of fruit ripening. As the fruit developed and ripened, we observed that the silenced sector of the LeEIN2-silenced fruit remained green and showed no ripening because of the suppression of the LeEIN2 gene, while the other sector turned red. The control fruit turned red and softened. In addition, it was found that the E4 gene was poorly expressed in the green tissue but highly expressed in the red tissue of the LeEIN2-silenced fruit. These results indicate that the LeEIN2 gene may play a critical...