Analysis Of Artificial TALEs Against Geminiviruses

Geminiviruses are circular single-stranded DNA viruses, which have caused severe damages in many crops throughout the world. While the existing control strategies against geminiviruses are defective and inefficient, it is important to explore new control strategies. Transcription-like effector (TALE) is a class of specific DNA-binding proteins secreted by plant pathogenic bacteria in the genus Xanthomonas, which has been developed as effective tools for genome engineering in recent years. Thus, artificial TALEs was engineered and used to inhibit geminivirus infection.While Cabbage leaf curl virus (CaLCuV) is a bipartite begomovirus, Tomato yellow leaf curl virus (TYLCV) is a single-component geminiviruses the most severe damage and wide distribution in China. According to the design guidelines based on naturally occurring TALE and important elements involved in geminiviruses replication, we designed the target TALE against the binding sites of replication protein (Rep) with online tools, https://boglab.plp.iastate.edu/. Then we assembled the plasmids of pTAL1-L4and pTAL1-L5which against TYLCV and CaLCuV with Golden-Gate cloning methods. The recombinant plasmids are confirmed via enzyme digestion, sequencing and BLAST comparison.In order to check the activity of the artificial TALE proteins in vitro, a high efficiency expression and purification system was established to produce the TALE proteins in prokaryote. Firstly, truncated TALE fragment with152amino acids deleted in N terminal and46amino acids retained in C terminal were obtained and subsequently cloned into five different expressing vectors carrying different tags to compare the expression levels of TALE proteins. We found that pET-28a、pET-32a and pMBP were the most appreciate vectors for TALE protein expression. Then the recombinant plasmids pET-32a-L4was transformed into four different expression strains. We identified that BL21(DE3)、BL21(DE3)-pLus and BL21(DE3)-Tf2could expressed more proteins. Considering the sizes of tags and operation simplicity, pET-28a vector and BL21(DE3) strain were chosen for further study.In order to explore the antiviral activity of the artificial TALEs, electrophoretic mobility shift assay with artificial TALE proteins and target DNA probes were performed. We confirmed that the artificial TALE protein L4and L5could bind to the probe designed according to TYLCV and CaLCuV, respectively. Then the TALE fragment which carrying nuclear location signals were cloned to pCAMBIA2300vector with flag tag. We found the artificial TALE protein could expressed in plants through agrobacterium infiltration and Western blot analysis. After that, we inoculated the recombinant pCAMBIA2300plasmids and their target geminiviruses together, extracted the soluble proteins and genome DNAs of inoculated plants at three different time periods. We found that the artificial TALE protein L4could depress TYLCV DNA accumulation while L5could depress CaLCuV DNA accumulation by Southern blot analysis. In order to obtain stable expression TALE transgenic plants, we transformed the artificial TALE gene into N.benthamiana and A.thaliana by using leaf disk and floral dip transformation method. Three transgenic N.benthamiana and seven transgenic A.thaliana plants which could overexpress the artificial TALE protein L5were obtained.