Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus

Abstract

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.

Figure 1

Construction of artificial microRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV). (A) The conserved motifs A, B, C, D and E are shown on L RNA. (B) Six amiRNAs (amiR‐LA, amiR‐LB1, amiR‐LB2, amiR‐LC, amiR‐LD and amiR‐LE) targeting individual motifs of L RNA viral strand were designed. The sequences targeted by individual amiRNAs are shown in base pairs.

Figure 2

Construction of artificial microRNAs (amiRNAs) targeting the conserved motifs of the tospoviral replicase sequence in the pre‐miR159a backbone. (A) Construction of single amiRNAs. MicroRNA159a (miR159a) processed from the pre‐miRNA159a backbone is presented as a hairpin. Forward primer amiR‐L‐F1 and reverse primer amiR‐L‐R, containing the complementary sequence to an individual amiRNA, were used to replace the miRNA159a and miRNA159a* sequences with an amiRNA targeting the A, B1, B2, C, D or E conserved motif of the L gene (replicase) of Watermelon silver mottle virus by first polymerase chain reaction (PCR). BglII‐F2 primer containing a BglII site and R primer containing a SmaI site were used in nested PCR. The pre‐amiRNA159a was digested with BglII/SmaI, subcloned into the pENTR vector and placed downstream of the 35S promoter in a binary destination vector (destination cassette, DC) by the Gateway recombination system. (B) Strategy for triple amiRNA construction. The primer pair M13‐F/amiR159Xba‐R was used to amplify amiR‐LA (or amiR‐LB2). The primer pairs amiR159AvrII‐F/amiLB1XbaAscI‐R and amiR159AvrII‐F/amiLDXbaAscI‐R were used to amplify amiR‐LB1 and amiR‐LD, respectively. The primer pair amiR159AvrII‐F/M13‐R was used to amplify amiR‐LE or amiR‐LC. From the amplified products, the amiR‐LA or amiR‐LB2 fragment was digested by XbaI and ligated to AvrII‐digested amiR‐LB1 or amiR‐LD fragment, respectively. After ligation, the double amiRNA segment was amplified by the primer pair M13‐F/amiLB1XbaAscI‐R or M13‐F/amiLDXbaAscI‐R. The fragment was digested by XbaI and ligated to AvrII‐digested amiR‐LE or amiR‐LC fragment. The triple amiRNA segment was amplified by M13II‐F/pENTRAscI. The triple amiRNA fragment was released from the amplified product by NotI/AscI digestion and subcloned into the pENTR vector to generate pENTR‐amiR‐LAB1E and pENTR‐amiR‐LB2DC, and then into binary DC vector by Gateway recombination to form triple amiRNA constructs pre‐amiR‐LAB1E and pre‐amiR‐LB2DC.

Figure 3

Expression of artificial microRNAs (amiRNAs) in transgenic Nicotiana benthamiana plants. (A–H) Detection of amiRNA expression in T₁ plants of different lines carrying amiR‐LA, amiR‐LB1, amiR‐LB2, amiR‐LC, amiR‐LD, amiR‐LE, amiR‐LAB1E and amiR‐LB2DC constructs (10 plants as one sample for each line). Total RNAs extracted from N. benthamiana leaves agro‐infiltrated with individual constructs were used as positive controls (+). rRNA was used as loading control. Line numbers are indicated on the top. The amiRNA expression levels in agro‐infiltrated plants were arbitrarily set as 100 for each construct. The numbers on the panels represent the percentage amiRNA expression levels.

Figure 4

Symptoms and virus accumulation detected by enzyme‐linked immunosorbent assay (ELISA) of different transgenic Nicotiana benthamiana lines, 14 days post‐inoculation with Watermelon silver mottle virus (WSMoV). (A) Typical symptoms were observed on a nontransgenic (NT) plant (i), mild mosaic symptoms on a T₁ plant of the amiR‐LD‐27 line (ii) and no symptoms on a T₁ plant of the amiR‐LD‐20 line (iii). (B) WSMoV was detected by indirect ELISA with six plants/line. One representative line was used for pre‐amiR‐LA, pre‐amiR‐LB1, pre‐amiR‐LC, pre‐amiR‐LE and pre‐amiR‐LAB1E constructs, three lines were used for pre‐amiR‐LB2 and pre‐amiR‐LD, and four lines were used for pre‐amiR‐LB2DC transgenic plants. WSMoV‐infected NT and mock‐inoculated plants (Mock) of N. benthamiana were used as positive and negative controls, respectively.

Figure 5

Correlation of the levels of resistance to Watermelon silver mottle virus (WSMoV) of artificial microRNA (amiRNA) transgenic Nicotiana benthamiana plants with the expression levels of amiRNA. (A) Expression levels of amiRNA before inoculation of amiR‐LD (1, 27 and 33) and amiR‐LB2DC (8, 9 and 20) transgenic lines. The relative expression levels of amiRNAs in transgenic lines were compared with the expression level in agro‐infiltrated tissue, arbitrarily set as 100. (B) Symptoms at 14 days after WSMoV inoculation. In each section of (B), a WSMoV‐inoculated transgenic plant (centre) is flanked by a WSMoV‐infected nontransgenic (NT) control plant (left) and a mock‐inoculated transgenic plant (right). I/T (as defined in Table 2) represents the results at 14 days post‐inoculation.

Figure 6

Infectivity assay of artificial microRNA (amiRNA) transgenic Nicotiana benthamiana plants challenged with different serogroup tospoviruses. (A) Alignment of amiR‐LB2 and amiR‐LD with the targeting sequences of five Watermelon silver mottle virus (WSMoV) serogroup viruses [WSMoV, Peanut bud necrosis virus (PBNV), Watermelon bud necrosis virus (WBNV), Capsicum chlorosis virus (CaCV) and Calla lily chlorotic spot virus (CCSV)] and Tomato spotted wilt virus (TSWV). The underlined letters indicate nucleotide mismatches and the grey letters indicate G:U paring between viral RNA and amiRNA. The sequences of L RNAs of WSMoV (accession number NC_003832), TSWV (NC_002052), PBNV (NC_003614), CaCV (NC_008302) and CCSV (FJ822962) were obtained from the National Center for Biotechnology Information (NCBI) GenBank database. The L RNA of WBNV was sequenced by T‐CC in our laboratory. (B) The symptoms of T₁ plants 14 days post‐inoculation (dpi) with WSMoV (i), CaCV (iii), CCSV (iv) and TSWV (v), and 10 dpi with PBNV (ii). Three T₁ plants from each line were used for challenge inoculation.

Figure 7

Evaluation of transgenic resistance of Nicotiana benthamiana plants expressing amiR‐LA, amiR‐LB or amiR‐LD by challenge with recombinant Turnip mosaic viruses (TuMV) carrying the corresponding artificial microRNA (amiRNA) target sequences. (A) Construction of infectious clones of TuMV recombinants. The 21‐nucleotide P69 targeting sequence of TuMV‐GP69 was replaced with the amiR‐LA, amiR‐LB1 or amiR‐LD targeting sequence to generate TuMV‐GLA, TuMV‐GLB1 or TuMV‐GLD, respectively. The additional three G nucleotides (italic) in TuMV‐GLA or TuMV‐GLB1 were used to avoid stop codons (UGA or UAG, underlined) and frameshifts of the viral reading frame. (B) Symptoms of transgenic lines expressing amiR‐LA (22 and 27), amiR‐LB (16 and 32) or amiR‐LD (33 and 35) at 10 days post‐inoculation with TuMV‐GFP, TuMV‐GLA, TuMV‐GLB1 or TuMV‐GLD. NT, nontransgenic N. benthamiana plants. (C) Virus accumulation was detected by indirect enzyme‐linked immunosorbent assay (ELISA). For each line, 10 plants were used for challenge inoculation. TuMV‐GFP‐infected NT and mock‐inoculated plants were used as positive and negative controls, respectively. Crowning of two adjacent bars of the histograms with the same letter is indicative of a nonsignificant difference, according to Fisher's least‐significant difference (LSD) test (P < 0.05) (SAS Institute Inc., Cary, NC, USA).