Large Artificial microRNA Cluster Genes Confer Effective Resistance against Multiple Tomato Yellow Leaf Curl Viruses in Transgenic Tomato

Abstract

Tomato yellow leaf curl disease (TYLCD) has become the key limiting factor for the production of tomato in many areas because of the continuous infection and recombination of several tomato yellow leaf curl virus (TYLCV)-like species (TYLCLV) which produce novel and destructive viruses. Artificial microRNA (AMIR) is a recent and effective technology used to create viral resistance in major crops. This study applies AMIR technology in two ways, i.e., amiRNA in introns (AMINs) and amiRNA in exons (AMIEs), to express 14 amiRNAs targeting conserved regions in seven TYLCLV genes and their satellite DNA. The resulting pAMIN14 and pAMIE14 vectors can encode large AMIR clusters and their function in silencing reporter genes was validated with transient assays and stable transgenic N. tabacum plants. To assess the efficacy of conferring resistance against TYLCLV, pAMIE14 and pAMIN14 were transformed into tomato cultivar A57 and the resulting transgenic tomato plants were evaluated for their level of resistance to mixed TYLCLV infection. The results suggest that pAMIN14 transgenic lines have a more effective resistance than pAMIE14 transgenic lines, reaching a resistance level comparable to plants carrying the TY1 resistance gene.

Keywords: TY1; artificial miRNA; mixed infection; resistance; tomato yellow leaf curl viruses; whitefly.

Figure 1

Establishment of a method to design artificial miRNA targeting conserved viral sequences. (A) Method for designing AMIR precursor targeting conserved viral coding sequences. (B) Identification of conserved target sites in TYLCV-satellite-encoded βC1 gene. The nucleotide conservation profile of βC1 gene-coding sequences is shown in the top panel. Alignments between the designed amiRbC1a/b and the selected target sequences are shown in the middle and bottom panels.

Figure 2

Construction of an expression vector for artificial miRNA encoded in exon (AMIE) and in intron (AMIN). (A) Structure of dual miRNA cluster. Orange and green boxes represent MIR171 and MIR164 precursor backbones, respectively. Gray box represents connector sequences. Brown and green triangles represent mature artificial miRNA a and b, while the open triangles represent the corresponding miRNA*. (B) Structures of exon-encoded (AMIE, top) and intron-encoded (AMIN, bottom) dual miRNA clusters. Red boxes represent indicated enzyme restriction sites. Brown boxes represent dual miRNA cluster shown in A. Yellow box represents fragments of GUS-coding sequences. Gray boxes represent intron sequences from PDS gene while blue boxes represent short exon sequences flanking each intron. (C) Structure of expression vector for a dual miRNA cluster. Red box represents 35S promoter sequence. Blue box represents AMIE or AMIN targeting 7 TYLCV proteins, the structure of which is shown in B. Gray box represents 35S terminator sequence. (D) Method to clone eight-miRNA expression vectors pAMIN8 and pAMIE8. (E) Method for cloning six-miRNA expression vectors pAMIN6 and pAMIE6. (F) Method for cloning fourteen-miRNA expression vectors pAMIN14 and pAMIE14. At each cloning step, vector DNA was digested with XbaI and XhoI, while insert DNA was digested with SpeI and XhoI.

Figure 3

Validation of artificial miRNA expression in transient assay and transgenic tobacco plants using miRNA sensor constructs. (A) Diagram shows the combination of agrobacteria infiltration in each leaf of N. benthamiana (left) and the predicted outcome under UV light. (B) Green florescence image of N. benthamiana leaves under UV light. Each patch was infiltrated by agrobacteria harboring an empty vector (EV) or a miRNA expression vector (AMIN14 or AMIE14), as indicated on the top, and miRNA sensor constructs, as indicated to the left. (C) Expression of seven miRNA sensors in wild-type N. tabacum leaves (top; the # in the red circle corresponds to the # listed in the table in (D)). (D) Infiltration scheme on transgenic AMIN14 and AMIE14 leaves (bottom). (E) Green florescence image of infiltrated leaves of AMIN14 (left) and AMIE14 (right) transgenic plants.

Figure 4

TYLCV infectious clone construction and validation. (A) Workflow for TYLCLV infectious clone construction. (B) PCR analysis of different TYLCV species in inoculated leaves (I) and systemic leaves (S) of N. benthamiana plants.

Figure 5

AMIN14 transgenic plants confer better resistance to mixed TYLCLV infection than AMIE14 transgenic plants. (A) Overview (left) and close view (right) of disease symptoms in AMIN14 transgenic (1 and 2 from line 61-1) and wild-type (3 and 4) plants. (B) Overview (left) and close view (right) of disease symptoms in AMIE14 transgenic (5 and 6 from line 55-2) and wild-type (7 and 8) plants. (C) Plant height comparison of infected and non-infected tomato plants: 1 and 2, TYLCV-infected AMIN14 line 61-1 and line 61-2; 3 and 4, TYLCV-infected AMIE14 line 55-1 and line 55-2; 5, TYLCV-infected wild-type tomato plants; 6, mock-inoculated wild-type tomato plants. (D) Comparison of disease severity between infected transgenic plants and wild-type plants. Numbers 1–5 represents genotypes as in C. SY, portion of symptomatic plants; MS, portion of mild symptomatic plants; NS, portion of non-symptomatic plants.

Figure 6

Disease-resistance phenotyping in plastic house conditions via whitefly infestation. (A) Progression of infection in tomato infected by whitefly. Y axis is the rate of plants showing disease phenotype. X axis shows the days after infection (DAI). Yellow line, Line A57; gray line, Line TY1; orange line, Line AE-6; blue line, Line AN-12 (same for B). (B) Plant height at different days after infection. Y axis is the plant height in centimeter. (C) Disease index of plants at 60 days after inoculation. Y axis is the accumulative disease index. X axis: a, Line AN-12; b, Line AE-6; c, TY1; d, control tomato line A57. (D) Stem diameters measured at 60 days after infection. Y axis is diameter in centimeters. X axis is the same as in C. Age of plants at the time of inoculation via whitefly is indicated to the left of each row. Letters a, b, c on top of each bar indicate significant differences determined by Student’s test (p < 0.05) while ab indicates no significant difference to a or b.