Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus

Abstract

Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns.

Figure 1. Construction of different transgenes in pK2T binary vector and analysis of transcript splicing in transgenic tobacco plants.

(a) Physical map of individual constructs. LB: T-DNA left border; 2X35S-P: Cauliflower mosaic virus (CaMV) double 35S promoter; ^★★: stop codons; MY-: 5′ part of MYSV-NP coding sequence; AT-In-: 5′ part of the intron of gene At3947160 of Arabidopsis thaliana; IGR: fragment of the intergenic region (IGR) flanked by 54 bp of C1 gene at the right (shaded in dark gray) and 56 bp of V2 gene at the left (shaded in light gray) of Ageratum yellow vein virus (AYVV); spacer: a 96 bp fragment of the middle part of the At3947160 intron; Inverted IGR: the fragment of the inverted IGR repeat of AYVV; -tron: 3′ part of the At3947160 intron; -SV-NP: 3′ part of the MYSV-NP coding sequence; 35S-T: CaMV 35S terminator; RB: T-DNA right border; nos-P: nopaline synthase gene promoter; nptII: neomycin phosphotransferase gene; nos-T: nos terminator. pK2T-MY-intron -NP: A positive control for confirmation of the action of the splicing process. (b) RT-PCR analysis for the splicing of transcripts from individual constructs, using primers targeting regions flanking the intron sequence. RNAs extracted at 3 dpi from tobacco leaves agroinfiltrated with individual constructs of pK2T-MYSV-NP, pK2T-MY-intron -NP and pK2T-MY-int-hpIGR-NP were analyzed by RT-PCR to examine the splicing of the intron. Total DNAs extracted from the leaf tissues agroinfiltrated with individual constructs were analyzed by PCR as un-spliced controls.

Figure 2. Greenhouse evaluation of transgenic tobacco lines against Ageratum yellow vein virus (AYVV) and Melon yellow spot virus (MYSV).

(a) Transgenic lines showed significant delay in symptom development as compared to non-transgenic (NT) plants when challenged with AYVV. Mock, a NT plant inoculated with buffer. MY-int-hpIGR-NP-6, hpIGR-3 and IGR-2 indicate transgenic lines transformed with the constructs pK2T-MY-int-hpIGR-NP, pK2T-hpIGR and pK2T-IGR, respectively. All the inoculated NT plants developed leaf curl symptom 10 days post-agroinfection (dpa). The hpIGR-3, IGR-2 and NT plants showed leaf curl symptom at 27 dpa, while the plants of MY-int-hpIGR-NP-6 line was symptomless. (b) Northern blot analysis for the detection of IGR siRNAs in MY-int-IGR-NP, hpIGR and IGR lines. Ribosomal RNA (5S) and tRNA were used as loading controls. Different resistant (R) lines showed different days (the number in parentheses) of delay in symptom development were used for siRNA analysis. S: susceptible line. (c) Evaluation of transgenic MY-int-hpIGR-NP and MYSV-NP lines by mechanical inoculation with MYSV. All NT plants developed leaf yellow spot and mosaic symptoms at 8 dpi. The photographs were taken at 20 dpi. HR: highly resistant, all inoculated plants did not show symptoms and were ELISA negative to MYSV NP. MR: moderately resistant, symptom development delayed up to 10 days. S: susceptible. (d) Northern blot analysis for the detection of MYSV-NP transcript and siRNAs in MY-int-hpIGR-NP and MYSV-NP lines before inoculation. (e) Southern blotting analyses of MY-int-hpIGR-NP transgenic lines. Susceptibility (S) and resistance (R) were analyzed comparing to the negative control of a NT plant. The putative insert numbers of the transgene are indicated.

Figure 3. Methylation of the transgene analyzed by bisulfite sequencing and fluorescence assay of rightward promoter activity of an auto-replicating vector of Ageratum yellow vein virus (AYVV) after agroinfiltration on transgenic tobacco lines.

(a) Physical map of the MY-int-hpIGR-NP transgene, in which the regions analyzed by bisulfite sequencing are indicated. The 247 bp (IGR-247) and the 350 bp (IGR-350) fragments were analyzed by the primer pairs BisP1/BisM1and BisP2/BisM2, respectively. Circles indicate sites of methylation identified from resistant MY-int-hpIGR-NP lines (6 and12), and squares indicate sites of methylation identified from the susceptible MY-int-hpIGR-NP lines (2 and19). Open circles (for resistant lines) and squares (for susceptible lines) represent 0–25% methylation of cytosines, half-filled symbols 26–50%, three-quarters-filled symbols 51–75% and full-filled symbols 76–100%. Sequencing data were averaged from three repeats. TAATATT↓AC: the conserved replication origin, TATA box: an essential motif of predicted promoter. Translation start sites of C1and V2 ORFs are also boxed. (b) Analysis of the transcriptional activity of an AYVV-derived auto-replicating GFP expression vector pAY-ST (described in Supplemental Experimental Procedure). AYVV shutter vector carrying GFP driven by the IGR rightward promoter was used to infect MY-int-hpIGR-NP, hpIGR and non-transgenic (NT) lines by agroinfiltration, and GFP expression was examined at 1.5 and 2.5 days. (c) Relative fluorescence intensity of GFP in transgenic MY-int-hpIGR and hpIGR using pAY-ST vector at 1.5 days post-agroinfiltration. The ANOVA analysis was conducted by measuring four-time repeats for each sample. The significant differences in fluorescence intensity are indicated (P < 0.05).