Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA

Abstract

Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

Enhanced Host-Parasite Resistance Based on Down-Regulation of Phelipanche aegyptiaca Target Genes Is Likely by Mobile Small RNA

“…The binary pBINPLUS35: pma construct ( Figure 3A ) harboring fragments of PaPrx1 , PaM6PR , and PaACS in a hairpin configuration was transformed into tomato Solanum lycopersicum ‘MP-1’as described by Leibman et al (2015) . Several independent lines of transgenic tomato containing pBINPLUS:pma were developed through Agrobacterium -mediated transformation.…”

“…The above three fragment genes (799 nt) were also cloned in a hairpin configuration in a binary vector pBINPLUS under the control of the CaMV35S promoter, as described by Leibman et al (2015) . The transgenic tomato plants were generated using kanamycin as a selection marker ( Barg et al, 1997 ; Leibman et al, 2015 ). T1 seeds were collected and sterilized with 70% ethanol for 1 min and 1% sodium hypochlorite and 0.01% Tween for 15–20 min, and then rinsed three times with sterilized distilled water.…”

“…Gene-silencing in plants has been shown to effectively control nematodes ( Atkinson et al, 2003 ; Huang et al, 2006 ) and viruses ( Prins et al, 2008 ; Leibman et al, 2015 ), and evidence is available to suggest a natural antiviral role for RNA silencing in vertebrates, fungi, worms, and flies ( Fire et al, 1998 ; Denli and Hannon, 2003 ). RNAi strategies have also been tried for the control of parasitic plants such as Triphysaria pusilla (Benth.)…”

Plant Responses to Pathogen Attack: Small RNAs in Focus

“…Tomato Ty-1 and Ty-3 , TYLCV resistance genes, encodes RDRs with sequence similarity to A. thaliana RDR3, -4, and -5. The susceptible Lycopersicum lines without these loci witness lower levels of TYLCV vsiRNAs and accrue high viral titers [ 126 , 127 , 128 ]. Still in Arabidopsis , the complete antiviral attributes of RDR3, RDR4, and RDR5 have not yet been unveiled.…”

Evaluation the virus resistance stability of T1 transgenic tobacco lines carrying RNAi TMV and RNAi TCYS construct

“…Cho đến nay, trên thế giới, đã có nhiều loại cây trồng kháng virus được tạo thành công dựa trên công nghệ RNAi, trong đó có nhóm cây kháng được một loại virus như: lúa [16], khoai tây [10], cà chua [7]... và nhóm cây kháng đồng thời với nhiều loại virus khác nhau như: đậu tương [15], dưa hấu [8]... Trong nghiên cứu trước của chúng tôi, cơ chế RNAi cũng được ứng dụng trong tạo các dòng thuốc lá T0 chuyển gen kháng virus khảm thuốc lá (Tobacco mosaic virus-TMV) và kháng virus phổ rộng (gồm 4 loại virus là TMV, CMVCucumber mosaic virus, TYLCV-Tomato yellow leaf curl virus và TSWV-Tomato spotted wilt virus). Kết quả đánh giá tính kháng sơ bộ cho thấy, tỷ lệ kháng TMV thu được ở 2 giống K326 và C9-1 lần lượt là 64,3% và 58,3%, tỷ lệ kháng virus phổ rộng là 30,3% [11,12].…”