Virus-based microRNA silencing in plants

Abstract

MicroRNAs (miRNAs) play pivotal roles in various biological processes across kingdoms. Many plant miRNAs have been experimentally identified or predicted by bioinformatics mining of small RNA databases. However, the functions of these miRNAs remain largely unknown due to the lack of effective genetic tools. Here, we report a virus-based microRNA silencing (VbMS) system that can be used for functional analysis of plant miRNAs. VbMS is performed through tobacco rattle virus-based expression of miRNA target mimics to silence endogenous miRNAs. VbMS of either miR172 or miR165/166 caused developmental defects in Nicotiana benthamiana. VbMS of miR319 reduced the complexity of tomato (Solanum lycopersicum) compound leaves. These results demonstrate that tobacco rattle virus-based VbMS is a powerful tool to silence endogenous miRNAs and to dissect their functions in different plant species.

Figure 1.

Schemes of TRV-based VbMS vectors. pTRV1 is the TRV RNA1 T-DNA vector. pTRV2e is a PEBV CP subgenomic promoter (sgP)-containing TRV RNA2 T-DNA vector; the complementary DNA of TRV RNA2 is cloned between a cauliflower mosaic virus 35S promoter with the duplicated enhancers (2x35S) and NOS terminator (Nos). pTRV2e contains the LIC cassette for the insertion of the target gene sequences. LB, Left border of T-DNA; RdRP, RNA-dependent RNA polymerase; MP, movement protein; 16K, 16-kD Cys-rich protein; Rz, self-cleaving ribozyme; RB, right border of T-DNA. IPS1-based MIM and STTM sequences can be cloned into pTRV2e by the LIC reaction. MIM contains an AtIPS1 backbone, but the target mimic motif of miR399 is changed to that of corresponding miRNAs. STTM contains two tandem target mimics separated by a 48-nucleotide imperfect stem-loop linker (48nt). [See online article for color version of this figure.]

Figure 2.

Visualization of GFP expressed by the modified TRV vector in N. benthamiana. A, The TRV-GFP-infiltrated plants were photographed at 4 d post inoculation under white light (left) or UV illumination (right). Arrows indicate the upper uninfiltrated leaves, and “I” indicates the infiltrated leaves. Green color under UV light indicates the GFP signal. B, RT-PCR detection of TRV RNA in upper uninfiltrated leaves. RNA samples were extracted from TRV-GFP and TRV control plants, and RT-PCR was performed with GFP- and TRV CP- specific primers. [See online article for color version of this figure.]

Figure 3.

VbMS of miR172 using ISP1-based miRNA target mimicry caused flower defects in N. benthamiana. A, Diagrammatic representation of MIM172. B, The flowers of plants infected with TRV control (left) and with TRV-MIM172 (right) were photographed at 12 d post flowering. Shown are typical flowers with sepal (top row) and sepal removed (bottom row). Bars = 1 cm. C, Stem-loop RT-PCR detection of miR172 level in plants infected with TRV control and with TRV-MIM172. D, Real-time RT-PCR analysis of mRNA levels of miR172 target NbAP2L1 in TRV control and plants expressing MIM172. Error bars show sd. [See online article for color version of this figure.]

Figure 4.

VbMS of miR172 using the STTM approach caused varied floral defects in N. benthamiana. A, Diagram of STTM172. 48nt, 48-nucleotid imperfect stem-loop linker. B, The varied morphology of flowers caused by TRV-based expression of STTM172. Bars = 1 cm. Each flower was photographed with sepal (top row) and sepal removed (bottom row). C, In some TRV-STTM172-infected plants, there are abnormal petals that could not enclose interior flower organs (top row, arrow) and ectopic genesis of petal-like tissues (bottom row, arrowheads). Bars = 1 cm. D, Stem-loop RT-PCR detection of miR172 level in plants infected with TRV control and with TRV-STTM172. E, Real-time RT-PCR analysis of miR172 target NbAP2L1 in VbMS plants. Error bars show sd. [See online article for color version of this figure.]

Figure 5.

VbMS of miR319 using an ISP1-based target mimicry approach converted large compound leaves into small simple ones in tomato. A, Diagram of MIM319. B, The plants (left column) and the third leaf excised from the left-sided plants (right column) of TRV control (bottom row) and plants expressing MIM319 (top row) were photographed at 20 dpi. Leaf orders are indicated with numbers. Bars = 1 cm. C, Stem-loop RT-PCR detection of miR319 level in plants infected with TRV control and with TRV-MIM319. D, Real-time RT-PCR analysis of miRNA levels of the miR319 target LA in TRV and plants expressing MIM319. Error bars show sd. [See online article for color version of this figure.]

Figure 6.

VbMS of miR319 using the STTM approach caused smaller and simpler leaves in tomato plants. A, Diagram of STTM319. 48nt, 48-nucleotid imperfect stem-loop linker. B, The whole stature and the third to sixth leaves of plants infected with TRV (left column) and with TRV-STTM319 (right column) were photographed at 10 dpi. L1 to L7 indicate leaves 1 to 7. Bars = 1 cm. C, Stem-loop RT-PCR detection of miR319 level in plants infected with TRV control and with TRV-STTM319. D, Real-time RT-PCR analysis of miR319 target LA. Error bars show sd. [See online article for color version of this figure.]

Figure 7.

VbMS of miR165/166 using the STTM approach caused the loss of apical dominance in N. benthamiana. A, Diagram of STTM165/166. 48nt, 48-nucleotid imperfect stem-loop linker. B, Whole plants infected with TRV-STTM165/166 (right column) or TRV (left column) were photographed at 28 dpi. Photographs were captured from top view (bottom row) and side view (top row). Arrows indicate the branched shoot apex. C, Outgrowth of an ectopic leaf. The ectopic leaf was photographed in front and side views. Arrows indicate ectopic leaf tissues, and “mv” and arrowhead indicate the middle vein. Bars = 1 cm. D, Stem-loop RT-PCR detection of miR165/166 level in plants infected with TRV control and with TRV-STTM165/166. E, Real-time RT-PCR analysis of relative mRNA levels of the putative miR165/166 target gene TC21810 in TRV control plants and TRV-STTM165/166 plants. Error bars show sd. [See online article for color version of this figure.]