Background:Canola is an agro-economically oilseed crop. Yield loss due to fungal disease of stem rot caused by Sclerotinia sclerotiorum is a serious problem in canola cultivation. Thaumatin-like proteins are large groups of the pathogenesis-related proteins which provide resistance to the fungal infection in response to invading pathogens and play a key role in plant defense system. Objectives: Transformation of the rice tlp into canola via Agrobacterium-mediated transformation and evaluation of the antifungal activity of the expressed TLP in the transgenic events on the S. sclerotiorum growth was subject to investigation.
Materials and methods:The canola (R line Hyola308) was used for transformation experiment. The vector, pBITLPRA1, was used for the stable transformation. The PCR and southern blotting techniques were used to confi rm transgene's presence in the transgenic canola events. Antifungal activity of transgenic plants was evaluated by the radial diff usion and spore germination assays. T 2 transgenic plants were evaluated by the intact leaf inoculation method in greenhouse assay. Results: In this study, pBITLPRA1 construct containing tlp gene was introduced into canola and the transformed plants were verifi ed by PCR. The glucanase activity of tlp gene in T 0 generation was measured and transgenic plants with high activity were assessed by Southern blot analysis to confi rm the copy number of the gene. Also, antifungal activity of the single copy T 0 transgenic plants against Sclerotinia sclerotiorum was evaluated by radial diff usion and spore germination assays. In greenhouse assay, evaluation of T 2 transgenic plants by the intact leaf inoculation method demonstrated that following the infection with S. sclerotiorum, there was a signifi cant reduction in the lesion's diameter in transgenic lines compared to the non-transgenic ones. Conclusions: These results revealed that expression of TLP has an inhibitory eff ect against fungus compared to nontransgenic plants both in vitro and in vivo (i.e., greenhouse condition). These transgenic lines could be used as the additional sources of disease resistance for canola breeding program.
Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T2 generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.
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