Agrobacterium-Mediated Transformation of the Oryza sativa Thaumatin-Like Protein to Canola (R Line Hyola308) for Enhancing Resistance to Sclerotinia sclerotiorum

Abstract

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.

Comparative Transcriptome Analyses Revealed Conserved and Novel Responses to Cold and Freezing Stress in Brassica napus L

“…As shown in Figure 8b, PR1 and PR5 were induced by cold acclimation and cold shock. Overexpression of a rice PR5 gene in rapeseed enhanced the resistance to Sclerotinia sclerotiorum , a fungal pathogen that caused Sclerotinia stem rot in rapeseed (Aghazadeh et al 2017). We propose that overexpression of PR genes in rapeseed might produce rapeseed germplasm with tolerance to cold and S. sclerotiorum .…”

Molecular Identification and Antifungal Properties of Four Thaumatin-like Proteins in Spruce (Picea likiangensis)

“…It has been reported that both Pru-TLP from cherry and Mal-TLP from apple exhibit a reasonably high endo-β-1,3-glucanase activity [29]. TLP proteins extracted from the leaves of multiple transgenic rice plants have been shown to possess a wide range of β-1,3-glucanase activities [32]. In this work, four recombinant PlTLPs were found to have β-1,3-glucanase activity, and the optimal reaction pH of these recombinant PlTLPs was determined to be 4.…”

“…TLP proteins can bind and degrade β-1,3-glucan, which is the main component of the fungal cell wall [30], laying the foundation for TLP proteins to further damage the fungal cell membrane [31]. For instance, a rice TLP protein with glucanase activity showed a significant inhibitory effect on the growth of Sclerotinia sclerotiorum and could change the permeability of the hyphal membrane [32]. Furthermore, many TLP proteins with antifungal activity can penetrate the fungal cell membrane and increase the content of reactive oxygen species (ROS) in the cell, leading to oxidative damage [18,33].…”

The Use of Genetic and Gene Technologies in Shaping Modern Rapeseed Cultivars (Brassica napus L.)

“…An increase of up to 15-fold in glyphosate herbicide use in cultivation compared to 1974 [ 166 ] was recorded, contributed by the rapid spread of RR crops globally [ 156 , 160 ]. Overreliance on a few HT crops and single active herbicidal ingredients has caused leaking of HT traits [ 160 , 167 , 168 , 169 ], with cases relating to all of the introduced HT systems being reported worldwide [ 170 ].…”