Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses

Abstract

Transcription activator-like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses.

Keywords: Antivirus; Begomovirus; Broad-spectrum resistance; DNA-binding protein; Transcription activator–like effectors.

Figure 1

Diagram illustrates of target sequences and artificial TALEs. (A) Locations of target sequences in TbCSV genome. TbCSV genome is shown as black circle, and TbCSV encoded genes are shown as green arrows. The two conserved sequence motifs (Motif_1 and Motif_2) were highlighted in red. AC1, replication-associated protein (Rep); AC2, transcriptional activator protein (TrAP); AC3, replication enhancer protein (REn); AC4, RNA-silencing suppressor; AV1, coat protein (CP); AV2, precoat protein; (B) Schematic representation of artificial TALE proteins. RVDs in the tandem repeats and their corresponding 12-bp DNA-binding targets were enlarged at the bottom. 35S, 35S promoter. NOS, NOS terminator. NLS, nuclear localization signal. Note that the TALE protein is not drawn in scale.

Figure 1

Diagram illustrates of target sequences and artificial TALEs. (A) Locations of target sequences in TbCSV genome. TbCSV genome is shown as black circle, and TbCSV encoded genes are shown as green arrows. The two conserved sequence motifs (Motif_1 and Motif_2) were highlighted in red. AC1, replication-associated protein (Rep); AC2, transcriptional activator protein (TrAP); AC3, replication enhancer protein (REn); AC4, RNA-silencing suppressor; AV1, coat protein (CP); AV2, precoat protein; (B) Schematic representation of artificial TALE proteins. RVDs in the tandem repeats and their corresponding 12-bp DNA-binding targets were enlarged at the bottom. 35S, 35S promoter. NOS, NOS terminator. NLS, nuclear localization signal. Note that the TALE protein is not drawn in scale.

Figure 2

Transgenic plants show resistance to TbCSV infection. (A) The symptoms induced by TbCSV on wild-type, TALE1, and TALE2 transgenic N. benthamiana plants. A detailed view of virus symptoms on leaves was shown at the bottom panel. Pictures were taken at 20 dpi; (B) Average plant height of wild-type, TALE1, and TALE2 transgenic N. benthamiana plants infected by TbCSV at 20 dpi. Numbers were calculated from two independent trials with each trial of 10 plants. ** indicates p < 0.01; (C) Average time for TbCSV inducing viral symptoms on systemic leaves of wild-type, TALE1, and TALE2 transgenic plants. Numbers were calculated from two independent trials with each trial of 10 plants. ** indicates p < 0.01; (D) Southern blot detection of TbCSV genomic DNA in wild-type, TALE1, and TALE2 transgenic plants. An ethidium bromide stained gel was shown at the bottom for indicating DNA loadings. scDNA, supercoiled DNA; ssDNA, single-stranded DNA.

Figure 3

Transgenic plants show resistance to TYCCNV and its associated betasatellite (TYCCNB). (A) Symptoms induced by TYCCNV and TYCCNB on wild-type and TALE1 and TALE2 transgenic plants at 20 dpi; (B) Average plant height wild-type and transgenic TALE1 and TALE2 plants after infected by TYCCNV and TYCCNB. Numbers were calculated from two independent trials with each trial of 10 plants. ** indicates p < 0.01; (C) Average time for TYCCNV and TYCCNB inducing viral symptom on systemic leaves of wild-type and transgenic plants. Numbers were calculated from two independent trials with each trial of 10 plants. * indicates p < 0.05, whereas ** indicates p < 0.01; (D) Southern blot detection of TYCCNV (upper panel) and TYCCNB (middle panel) genomic DNA. An ethidium bromide stained gel was shown at the bottom for indicating DNA loadings.