Using a Viral Vector to Reveal the Role of MicroRNA159 in Disease Symptom Induction by a Severe Strain of Cucumber mosaic virus1[C][W][OPEN] Zhiyou Du*, Aizhong Chen, Wenhu Chen, Jack H. Westwood, David C. Baulcombe, and John P. Carr* College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China (Z.D., A.C., W.C.); Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom (Z.D., J.H.W., D.C.B., J.P.C.) ORCID ID: 0000-0003-0864-2246 (Z.D.).

In transgenic Arabidopsis (Arabidopsis thaliana), expression of the Cucumber mosaic virus (CMV) 2b silencing suppressor protein from the severe subgroup IA strain Fny disrupted microRNA (miRNA)-regulated development but orthologs from mild subgroup II strains (Q and LS) did not, explaining strain-specific differences in symptom severity. However, it is unknown which miRNAs affected by Fny2b critically affect viral symptoms. Observations that Fny2b-transgenic plants phenocopy microRNA159ab (mir159ab) mutant plants and that Fny2b altered miR159ab-regulated transcript levels suggested a role for miR159ab in elicitation of severe symptoms by Fny-CMV. Using restoration of the normal phenotype in transgenic plants expressing an artificial miRNA as a proof of concept, we developed a LS-CMV-based vector to express sequences mimicking miRNA targets. Expressing a miR159 target mimic sequence using LS-CMV depleted miR159 and induced symptoms resembling those of Fny-CMV. Suppression of Fny-CMV-induced symptoms in plants harboring mutant alleles for the miR159ab targets MYB DOMAIN PROTEIN33 (MYB33) and MYB65 confirmed the importance of this miRNA in pathogenesis. This study demonstrates the utility of a viral vector to express miRNA target mimics to facilitate functional studies of miRNAs in plants.

RNA silencing is a set of mechanisms by which small RNA molecules, including microRNAs (miRNAs) and short-interfering RNAs, guide RNA-induced silencing complexes (RISCs) to degrade transcripts, or in some cases hinder their translation, in a sequence-specific manner (Chapman and Carrington, 2007). Plant miRNAs range in size from 19 to 24 nucleotides (nt) and are encoded by nuclear genes that are transcribed to produce noncoding primary transcripts 1 This work was supported by the National Natural Science Foundation of China (grant nos. 30800043 and 31170141 to Z.D.), a Marie Curie International Incoming Fellowship (PIIF-GA-2009236443 to Z.D.), the 521 Talents Development Project (grant no. 11610032521303 to Z.D), the Leverhulme Trust (grant nos. F/09741/F and RPG-2012-667 to J.P.C.), and the UK Biotechnology and Biological Sciences Research Council (grant nos. BB/D014376/1 and BB/ J011762/1 to J.P.C.). * Address correspondence to [email protected] and jpc1005@ hermes.cam.ac.uk. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zhiyou Du ([email protected]). Z.D. and J.P.C. designed the experiments; Z.D., A.C., W.C., and J.H.W. performed the experiments; all authors analyzed the data; Z.D., J.P.C., and D.C.B. conceived the project. Z.D. and J.P.C. wrote the article. [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OPEN] Articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.113.232090

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called pri-miRNAs, which are processed into doublestranded RNAs (dsRNAs) with imperfect stem-loop structures called pre-miRNAs. Pre-miRNAs are cropped by Dicer-like1 (DCL1) endoribonuclease activity yielding miRNA/miRNA* (passenger strand of microRNA) duplexes and the guide strand associated with Argonaute1 to constitute a RISC, whereas the passenger strand miRNA*s are typically degraded (Kurihara and Watanabe, 2004; Voinnet, 2009). miRNAs play fundamental roles in the regulation of plant growth and development (Jones-Rhoades et al., 2006), as well as in adaptation to biotic and abiotic stresses (Sunkar et al., 2007, 2012; Ruiz-Ferrer and Voinnet, 2009). One potent antiviral mechanism is the RNA silencing directed by 22-nt and 21-nt short-interfering RNAs generated, respectively, by dicing of virus-derived dsRNA by DCL2 and DCL4 (Deleris et al., 2006). Many plant viruses counteract this antiviral defense by expressing viral suppressors of RNA silencing (VSRs) that inhibit or inactivate various components of the RNA silencing pathways (Voinnet et al., 1999; Roth et al., 2004; Li and Ding, 2006). In addition, many VSRs disrupt miRNA pathways, resulting in the appearance of developmental defects in VSR-transgenic and virusinfected plants (Mallory et al., 2002; Chapman et al., 2004; Dunoyer et al., 2004; Zhang et al., 2006; Lewsey et al., 2007; Yang et al., 2008). It has been over a decade since the first plant miRNAs were reported (Llave et al., 2002; Park et al., 2002; Rhoades et al., 2002) but traditional genetic approaches are not well suited to functional studies of plant miRNAs because many miRNA families comprise multiple members

Plant PhysiologyÒ, March 2014, Vol. 164, pp. 1378–1388, www.plantphysiol.org Ó 2014 American Society of Plant Biologists. All Rights Reserved.

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having identical target sequences. Franco-Zorrilla et al. (2007) discovered a novel miRNA regulation mechanism in Arabidopsis (Arabidopsis thaliana), termed target mimicry (TM), by which microRNA399 (miR399) is inactivated by complementary pairing with the noncoding RNA transcribed from the gene, Induced by Phosphate Starvation1 (IPS1). The complementary base pairing raises a bulge at the expected miRNA cleavage site, preventing miR399-directed endonucleolytic cleavage of IPS1 by Argonaute1, which leads to increased accumulation of the miR399 target mRNA PHOSPHATE2. Subsequently, the natural phenomenon of TM was developed into a technique to dissect miRNA function by stable expression of artificial TM sequences in transgenic Arabidopsis plants (Wu et al., 2009; Todesco et al., 2010; Debernardi et al., 2012). This approach was further refined by transgenic expression of transcripts comprising two target mimics separated by a short spacer sequence, called a short tandem target mimic (STTM) sequence (Yan et al., 2012). Constitutive expression of STTM sequences was shown to be more effective at interfering with miRNA activity in transgenic Arabidopsis plants than the IPS1-based TM. We wondered whether it would be possible to further enhance the efficiency and speed of TM technology for probing miRNA function by expressing TM sequences from a viral vector. Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus. CMV is an economically important pathogen with a host range exceeding 1200 plant species and is a well-characterized model for plant-virus interaction studies (Palukaitis and GarcíaArenal, 2003; Jacquemond, 2012). The CMV genome comprises three positive-sense single-stranded RNAs and encodes five proteins (Palukaitis and García-Arenal, 2003; Jacquemond, 2012). The 1a methyltransferase/helicase replicase component is translated from RNA1. RNA2 is the mRNA for another replicase component, the 2a RNA-dependent RNA polymerase, and also encodes the 2b VSR, which is translated from the RNA2-derived subgenomic mRNA RNA4A. The 3a movement protein is translated directly from RNA3, whereas a second protein encoded by this genomic segment, the coat protein (CP), is translated from the RNA3-derived subgenomic mRNA RNA4. CMV 2b protein, which was one of the first VSRs identified, disrupts RNA silencing predominantly by sequestration of small dsRNA (Brigneti et al., 1998; Goto et al., 2007; González et al., 2010, 2012; Duan et al., 2012). In addition to inhibiting the establishment of antiviral silencing, the 2b protein also interferes with salicylic acid-induced CMV resistance (Brigneti et al., 1998; Ji and Ding, 2001; Lewsey and Carr, 2009), disrupts signaling mediated by jasmonic acid and abscisic acid (Lewsey et al., 2010; Westwood et al., 2013a), and modifies plant-aphid interactions (Ziebell et al., 2011; Westwood et al., 2013b). The 2b VSR also affects dynamics of local and systemic CMV movement and functions as a virulence determinant (Ding et al., 1995; Soards et al., 2002; Wang et al., 2004). Plant Physiol. Vol. 164, 2014

Numerous CMV strains and isolates have been characterized and can be divided into three subgroups: IA, IB, and II (Roossinck et al., 1999). Generally, subgroup IA and IB strains are more virulent than subgroup II strains (Wahyuni et al., 1992; Zhang et al., 1994; Cillo et al., 2009). Experiments with transgenic Arabidopsis plants showed that constitutive expression of the 2b gene from subgroup IA strain Fny, but not its orthologs from subgroup II strains Q or LS, impaired miRNA regulation on gene expression, leading to developmental defects, concomitant with increased steady-state accumulation of mature miRNA and miRNA* (Chapman et al., 2004; Zhang et al., 2006; Lewsey et al., 2007). This effect was proposed as an explanation of differences in viral symptoms between subgroup IA and subgroup II strains (Zhang et al., 2006; Lewsey et al., 2007). Because it still remains unknown which miRNA is associated with the induction of severe symptoms by subgroup IA strains, including Fny-CMV, we explored the problem using a viral vector carrying a TM sequence. RESULTS Constitutive Expression of CMV 2b Protein or CMV Infection Disrupted MicroRNA159 Functions

In line with previous findings, infection of Arabidopsis plants of ecotype Columbia with Fny-CMV caused severe distortion of systemically infected leaves, whereas infection with LS-CMV caused only mild stunting (Fig. 1A; Lewsey et al., 2007). We noted that Fny2b-transgenic lines exhibited developmental defects that phenocopied those previously observed by Allen et al. (2007) for microRNA159 (mir159ab) double mutant plants, which included severe stunting and upcurled rosette leaves (Fig. 1B). The Arabidopsis miR159 family contains three members, miR159a, miR159b, and miR159c, which are encoded by distinct MIR genes at separate loci (Park et al., 2002; Rhoades et al., 2002). The sequences of miR159a and miR159b differ by 1 nt but interact with the transcripts of the GIBBERELLIN-REGULATED MYB DOMAIN PROTEINlike genes MYB33 and MYB65 in a functionally redundant manner (Allen et al., 2007). Although miR159c differs in sequence from miR159a and miR159b only at nucleotides 2 and 1, respectively, it does not regulate MYB33 and MYB65 and mutation of its gene has no effect on Arabidopsis development (Allen et al., 2010). There is no documented investigation of miR159 accumulation or activity in 2b-transgenic or CMVinfected plants. Therefore, we examined steady-state levels of miR159 at 15, 30, and 50 d postgermination in Fny2b-transgenic as well as in Fny unt2b-transgenic plants, which express a nontranslatable Fny2b transcript and retain a normal phenotype (Lewsey et al., 2007). RNA gel blotting showed that compared with Fny unt2b-transgenic plants, Fny 2b-transgenic plants had a higher level of mature miR159 and miR159* at 15 and 50 d postinoculation (dpi; Fig. 1C). This increased 1379

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Figure 1. The effects of CMV 2b protein or CMV infection on miR159 function in Arabidopsis. A, Viral symptoms on Arabidopsis plants inoculated with Fny-CMV, LS-CMV, or sterile water (Mock) at 10 dpi. B, Phenotypes of nontransgenic, Fny unt2btransgenic (expressing a nontranslatable Fny2b transcript), Fny 2b-transgenic, and mir159ab mutant plants. The plants were photographed when they were aged approximately 35 d. C, RNA gel-blot analysis of accumulation of miR159 and its complementary strand miR159* in Fny unt2b-transgenic and Fny 2b-transgenic Arabidopsis plants at age 15, 30, and 50 d. U6 RNA served as a loading control. D, RT-qPCR analyses of accumulation of the miR159 target transcripts MYB33, MYB65, and MYB101 in Fny unt2b-transgenic (black bars) and Fny 2b-transgenic (white bars) in Arabidopsis plants at age 15, 30, and 50 d. E, RNA gel-blot analysis of accumulation of miR159 and miR159* in Arabidopsis plants inoculated with Fny-CMV, LS-CMV, or sterile water (Mock) at 10 dpi. F, RT-qPCR analyses of accumulation of MYB33, MYB65, and MYB101 in Arabidopsis plants inoculated with Fny-CMV (gray bars), LS-CMV (white bars), or sterile water (Mock, black bars) at 10 dpi. In (D) and (F), the Duncan new multiple range test was used to analyze statistical differences. Asterisk indicates statistically different values (P , 0.05) and error bars represent SE of the mean. Bar = 1 cm.

accumulation of miR159* and miR159 is likely to be the consequence of miR159/miR159* duplex stabilization by binding with Fny2b (Goto et al., 2007; González et al., 2010, 2012; Duan et al., 2012; Hamera et al., 2012). Duplex stabilization likely prevents recruitment of miR159 strands into RISCs, an idea supported by increased steady-state levels of the miR159 target transcripts MYB33 and MYB65 in Fny2b-transgenic plants (Fig. 1D). We next analyzed the effects of CMV infection on the steady-state levels of miR159 and its target transcripts. The clearest effect of Fny-CMV infection was elevated accumulation of miR159*, which was not seen in plants infected by LS-CMV (Fig. 1E). Analysis by reverse transcription-coupled quantitative PCR (RT-qPCR) showed that MBY65 was most markedly elevated by Fny-CMV infection (Fig. 1F), consistent with the increased miR159* accumulation. Thus, constitutive expression of Fny2b and Fny-CMV infection 1380

disrupted miR159 accumulation and activity. This led us to investigate whether miR159 was involved in induction of severe symptoms by Fny-CMV.

Development of a Mild Strain LS-CMV-Based Target Mimic System

We speculated that if the difference in viral symptoms between Fny-CMV and LS-CMV results specifically from differential effects of their respective 2b proteins on miR159ab, then engineering LS-CMV to carry a corresponding TM sequence would produce an agent that would induce disease symptoms resembling those caused by Fny-CMV. As a proof of concept for a viral TM vector, we engineered LS-CMV to carry a TM sequence to deplete a specific miRNA that has known and well-understood activity in Arabidopsis. For this, we Plant Physiol. Vol. 164, 2014

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selected the Arabidopsis enhanced miRNA activity1-1 (ema1-1) mutant (amiR-triOX-transgenic) line, plants of which have an easily observable phenotype (enhanced trichome clustering on its leaves) that results from transgenic expression of an artificial miRNA (amiRNA), the amiR-trichome (Wang et al., 2011). We designed an amiR-trichome TM sequence (MIM-trichome) according to the previously described design rule (Todesco et al., 2010), and in parallel designed an arbitrary sequence (MIM-CK) unrelated to any known Arabidopsis miRNA sequence to use as a control (Fig. 2A). To take advantage of the relatively high accumulation levels of RNA3 and its subgenomic RNA (RNA4) that occur in LS-CMVinfected plants, we introduced MIM-trichome or MIMCK between the CP gene and 39 untranslated region (UTR) of RNA3 as shown in Figure 2A, to generate the LS-CMV-derived variants LS-MIM-trichome and LS-MIM-CK, respectively. Infection with LS-MIM-CK or wild-type LS-CMV had no discernible effect on the extreme trichome clustering phenotype on leaves of ema1-1 plants (Fig. 2B). By contrast, LS-MIM-trichome infection strongly inhibited trichome clustering, demonstrating that expression of the TM sequence interfered with the activity of the amiRNA expressed in ema1-1 plants (Fig. 2B). RNA-blot analysis of viral RNAs showed that the viruses carrying either MIM-trichome or MIM-CK sequences on RNA3 replicated as efficiently as wild-type LS-CMV (Fig. 2C). Recent studies demonstrate that TM expression reduced stability of targeted miRNAs in transgenic Arabidopsis plants (Wu et al., 2009; Todesco et al., 2010; Debernardi et al., 2012). Therefore, we used RNA gel blotting to analyze accumulation of amiR-trichome in the infected plants. Infection with LS-CMV or LS-MIM-CK had no obvious effect on accumulation of either amiR-trichome or its corresponding star strand amiR-trichome*. Infection with LS-MIM-trichome caused only a modest decrease in mature amiR-trichome, with the 24-nt form affected more than the 21-nt molecule; however, it did not decrease amiR-trichome* accumulation (Fig. 2D). The target transcripts of amiR-trichome are CAPRICE (CPC), TRIPTYCHON (TRY), and ENHANCER of TRIPTYCHON AND CAPRICE2 (ETC2; Wang et al., 2011). RT-qPCR analysis of steady-state levels of these mRNAs in ema1-1 plants showed that infection with LS-CMV or LS-MIM-CK markedly increased accumulation of TRY, and had a limited effect on ETC2 accumulation. Plants infected with LS-MIM-trichome had higher levels of CPC and TRY (Fig. 2E), demonstrating that MIM159 had indeed reversed the inhibition of CPC and TRY expression by amiR-trichome. By contrast, infection with LS-MIM159 reduced ETC2 expression (Fig. 2E). To determine whether changes in accumulation of ETC2 and TRY in LS-CMV- or LSMIM-CK-infected ema1-1 plants resulted from disruption of amiR-trichome regulation by these viruses, we tested the responses of amiR-trichome targets to LS-CMV infection in wild-type and ema1-1 mutant plants. RT-qPCR results showed that infection with Plant Physiol. Vol. 164, 2014

LS-CMV had no significant effect on accumulation of these three transcripts in wild-type plants (Fig. 2F). Consistent with the data shown above, accumulation of TRY, but not CPC or ETC2, was altered significantly in the ema1-1 plants when infected with LS-CMV (Fig. 2F). This demonstrated that infection with LS-CMV or LS-MIM-CK disrupted repression by amiR-trichome of its targets. To some extent, this may have been a result of the silencing suppression activity of its 2b protein. In addition, we noticed that in contrast with CPC and TRY, ECT2 was up-regulated in mock-inoculated ema1-1 plants compared with levels seen in mock-inoculated wild-type plants (Fig. 2F). This demonstrated that in ema1-1 plants, CPC and TRY but not ETC2, were negatively regulated by the amiRNA amiR-trichome, and suggested that there could be a CPC-TRY-ETC2 triple interaction at the transcriptional level. This may explain the decreased accumulation of ETC2 in LS-MIM159infected ema1-1 plants, in which CPC and TRY were up-regulated (Fig. 2E). Although expression of the MIM-trichome sequence from LS-CMV had limited effects on the level of 21-nt amiR-trichome, which is responsible for target cleavage, it clearly altered expression levels of the amiRtrichome targets and completely inhibited trichome clustering. This implies that MIM-trichome functions mainly by inactivating amiR-trichome, rather than by reducing its stability. Such an effect has been observed in the case of the endogenous IPS1-miR399 interaction (Franco-Zorrilla et al., 2007). To further demonstrate the efficacy of LS-CMVbased TM expression we selected the endogenous Arabidopsis miR165/miR166 as a model because inhibition of miR165/miR166 functions by a STTM leading to alteration of developmental (morphological) phenotypes (Yan et al., 2012). We adopted a previously described target mimic sequence for miR165/miR166 (MIM165/166; Todesco et al., 2010) for introduction into LS RNA3 at the same position between the CP gene and 39 UTR used to generate LS-MIM-CK and LSMIM-trichome (Fig. 2A), to generate LS-MIM165/166. We compared the effects of this construct with those of LS-CMV and LS-MIM-CK in wild-type Arabidopsis plants. Infection with either LS-CMV or LS-MIM-CK caused mild distortion of rosette leaves and mild stunting, which had become apparent by 28 dpi (Fig. 3A). Strikingly, the upper, young leaves of plants infected with LS-MIM165/166 grew more vertically than the equivalent leaves on wild-type plants, which were apparent by 14 dpi, and leaves were shaped like trumpets by 28 dpi. The majority of trumpet-like leaves had outgrowths (enations) from their abaxial sides. These disease symptoms are typical phenotypes of the phabulosa-1d (phb-1d) mutant, which has a gain of function mutation disrupting miR165/miR166 binding sites in the PHB gene (McConnell and Barton, 1998). Expression of the TM sequence MIM165/166 has been shown to modestly reduce miR165/miR166 levels in transgenic Arabidopsis (Todesco et al., 2010; Yan et al., 2012). Using RNA gel blotting, we found that although 1381

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Figure 2. Expression of the target mimic sequence MIM-trichome using LS-CMV inactivated the amiRNA causing trichome clustering in Arabidopsis. A, Diagram of LS-CMV RNA3 harboring amiR-trichome target mimic (MIM-trichome) or mimic control (MIM-CK) sequences inserted between the CP gene and the 39 UTR. Nucleotides in red indicate nucleotides in the MIMtrichome sequence introduced to create a bulge to prevent miRNA mediated cleavage. Asterisks indicate complementary nucleotides between amiR-trichome and MIM-trichome or MIM-CK. MP indicates the open reading frame for the CMV movement protein. B, Alteration of trichome clustering on Arabidopsis ema1-1 mutant plants inoculated with LS-CMV, LSMIM-CK, LS-MIM-trichome, or sterile water (Mock). RNA3 segments of LS-MIM-CK and LS-MIM-trichome harbor MIM-CK and MIM-trichome, respectively. Photographs were taken at 14 dpi. Bar = 1 cm. C, RNA gel-blot analysis of accumulation of viral progeny RNAs in the infected plants. A biotin-labeled DNA oligonucleotide complementary to the conserved sequence common to the 39 UTRs of the CMV genomic and subgenomic RNAs was used to detect viral RNA. Equal loading was confirmed by ethidium bromide staining. D, RNA gel-blot analysis of accumulation of mature amiR-trichome and its opposite strand amiR-trichome*. U6 RNA served as a loading control. E, RT-qPCR analyses of accumulation of amiR-trichome target transcripts CPC, ETC2, and TRY in ema1-1 plants. F, RT-qPCR analysis of accumulation of CPC, ETC2, and TRY in wild-type (WT) and ema1-1 plants inoculated with LS-CMV (white bars) or sterile water (Mock; black bars). The ANOVA Duncan new multiple range test was used to analyze statistical differences. Different letters in (F) indicate statistically different values (P , 0.05), and error bars represent SE of the mean. Total RNA samples used in the experiments shown in (C) to (F) were prepared from pooled upper, noninoculated leaves harvested from five individual plants at 14 dpi.

all three viruses accumulated to similar levels (Fig. 3B), miR165/miR166 was substantially reduced only in the plants infected with LS-MIM165/166 but not in mockinoculated plants or plants infected with LS-CMV or the control virus LS-MIM-CK (Fig. 3C). LS-MIM165/166 had no effect on miR167 levels, showing that the effect of TM expression was specific (Fig. 3C). RT-qPCR 1382

experiments showed that the miR165/miR166 targets PHB and PHV were strongly up-regulated in the plants infected with LS-MIM165/166 but not in plants infected with either the control virus LS-MIM-CK or the parental virus LS-CMV (Fig. 3D). These data strongly supported the conclusion that expression of MIM165/166 in LS-CMV effectively reduced miR165/miR166 levels Plant Physiol. Vol. 164, 2014

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Figure 3. Expression of a miR165/miR166 target mimic sequence using LS-CMV substantially reduced miR165/miR166 accumulation, leading to dramatic alteration to viral symptoms. A, Arabidopsis plants showing viral symptoms induced by the infection with LS-CMV, LS-MIM-CK, and LS-MIM165/166. Mock plants were mock-inoculated with sterile water. Photographs were taken at 14 dpi and 28 dpi as indicated to the left of the images. A close-up view of LS-MIM165/166-infected plants showed outgrowths (enations) from leaf abaxial sides indicated by arrowheads in red. Bar = 1 cm. B, RNA-blot analysis of accumulation of viral progeny RNAs in the infected plants. A biotin-labeled DNA oligonucleotide complementary to the conserved sequence common to the 39 UTRs of the CMV genomic and subgenomic RNAs was used to detect viral RNA. Equal loading was confirmed by ethidium bromide staining. C, RNA-blot analysis of accumulation of miR165/miR166 and miR167 in the infected plants and mockinoculated plants at 14 dpi. U6 RNA was used as a loading control. D, RT-qPCR analysis of relative accumulation of miR165/ miR166 target transcripts PHB and PHV at 14 dpi. Total RNA samples used in the experiments from (B) to (D) were prepared from pooled upper, noninoculated leaves harvested from five individual plants at 14 dpi.

and derepressed expression of its target transcripts, leading to appearance of disease symptoms resembling phb-1d mutant plant phenotypes.

Infection with an LS-CMV Variant Expressing a miR159 Target Mimic Sequence Induced Symptoms Resembling Those Induced by the Severe Strain Fny-CMV

Having validated the LS-CMV-based TM expression system, we investigated the effect of disrupting miR159 functions on viral symptoms by expressing a miR159 TM sequence from the virus. The MIM159 sequence was introduced into LS-CMV RNA3 as before, generating LS-MIM159. We compared viral symptoms on Arabidopsis plants infected with LS-CMV, LS-MIM-CK, Plant Physiol. Vol. 164, 2014

LS-MIM159, and Fny-CMV. Infection with LS-MIM159 caused disease symptoms that were similar to those induced by Fny-CMV, whereas those induced by LS-CMV or LS-MIM-CK were very mild, as observed in the previous experiments (Fig. 4A). Viral infection was confirmed by RNA-blot analysis as shown in Figure 4B. Next, we conducted RNA gel blotting to analyze miRNA accumulation in the plants at 10 dpi. Mockinoculated plants and plants infected with LS-CMV or LS-MIM-CK possessed similar levels of miR159, which were markedly higher than in plants infected with LS-MIM159 (Fig. 4C). By contrast, levels of miR167 were comparable in all of these plants, showing that the effect of the MIM159 sequence on miR159 accumulation was specific (Fig. 4C). We also analyzed relative accumulation of the miR159 targets MYB33, MYB65, and 1383

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Figure 4. Infection with an LS-CMV variant expressing a miR159 target mimic sequence caused viral symptoms resembling those induced by the severe strain, Fny-CMV. A, Viral symptoms in Arabidopsis plants inoculated with LS-CMV, LS-MIM-CK, LS-MIM159, or Fny-CMV. Mock plants were inoculated with sterile water. Photographs were taken at 10 dpi. Bar = 1 cm. B, RNA gel-blot analysis of viral progeny RNAs in the infected plants. A biotin-labeled DNA oligonucleotide complementary to the conserved sequence common to the 39 UTRs of the CMV genomic and subgenomic RNAs was used to detect viral RNA. Equal loading was confirmed by ethidium bromide staining. C, RNA gel-blot analysis of accumulation of miR159 and miR167 in the infected plants as well as mock plants. U6 RNA was used as a loading control. D, RT-qPCR analysis of relative accumulation of selected miR159 target transcripts MYB33, MYB65, and MYB101 in the infected plants as well as mock plants. Total RNA samples used in the experiments from (B) to (D) were prepared from pooled upper, noninoculated leaves harvested from five individual plants at 10 dpi.

MYB101 using RT-qPCR. As expected, infection with LS-CMV or LS-MIM-CK had no obvious effect on steady-state levels of these target transcripts; however, infection with LS-MIM159 markedly increased accumulation of MYB33 and MYB65 but had no effect on MYB101 (Fig. 4D), which was consistent with the decreased level of miR159. Previous studies demonstrated that miR159a and miR159b only target MYB33 and MYB65, and deregulation of MYB33 and MYB65 is responsible for the phenotypes exhibited by mir159ab mutant plants (Allen et al., 2007). Our results showed that disease symptoms induced by LS-MIM159 were not identical to the phenotypes of mir159ab mutant plants (compare Fig. 4A with Fig. 1B). To further ascertain that the disease symptoms were specifically a consequence of disrupting miR159 functions by the MIM159 sequence, we compared viral symptoms on Arabidopsis wild-type and myb33/myb65 double mutant plants infected with LS-CMV, LS-MIM-CK, and LS-MIM159. As expected, severe disease symptoms seen in wild-type plants 1384

infected with LS-MIM159 were largely ameliorated in the myb33/myb65 mutant plants infected with the same virus. LS-MIM159 infection caused mild leaf distortion in the mutant plants, which was comparable with that elicited by LS-CMV or LS-MIM-CK (Fig. 5). This was convincing evidence that the severe disease symptoms were the outcome of deregulating the miR159 targets MYB33 and MYB65.

The myb33/myb65 Alleles Moderate Disease Symptoms Caused by Infection with the Severe Strain Fny-CMV

As shown above, constitutive expression of Fny2b or infection with Fny-CMV negatively regulated miR159 activity, leading to an increase of miR159 targets MYB33 and MYB65 (Fig. 1). To determine whether derepression of MYB33 and MYB65 by impairing miR159 activity is responsible for induction of severe symptoms by Fny-CMV, we inoculated Arabidopsis wild-type and myb33/myb65 mutant plants with Plant Physiol. Vol. 164, 2014

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Figure 5. Mutant alleles for the miR159ab targets MYB33 and MYB65 diminished disease symptoms caused by LS-MIM159. Arabidopsis wild-type and myb33/myb65 mutant plants were inoculated with LS-CMV, LS-MIM-CK, LS-MIM159, or sterile water (Mock), and photographed at 14 dpi. Bar = 1 cm.

Fny-CMV. Observation of viral symptoms showed that a majority (80%) of myb33/myb65 plants infected with Fny-CMV showed less deformation of the upper, young systemically infected leaves than wild-type plants with the virus infection at 10 dpi (Fig. 6A). RNA gel blotting showed that there was little difference in accumulation of Fny-CMV RNA between the wild-type and mutant plants (Fig. 6B). This strongly suggests that during FnyCMV infection, MYB33 and MYB65 expression is deregulated by the Fny 2b protein through inhibition of miR159 activity; although this contributes to viral symptom induction, it does not enhance virus titer. DISCUSSION

This work demonstrates the biological significance of miR159 in the induction of viral symptoms by a severe subgroup IA strain of CMV by developing a

mild strain LS-CMV-based TM system and subsequently using the system to express a miR159 target mimic sequence. This study also demonstrates the potential of a virus-based TM system to investigate miRNA function not only in plant responses to stress (in this case, infection by a highly virulent virus strain) but also in the control of plant development. Previous studies showed that CMV 2b is not the only factor mediating CMV virulence. For example, viral mutants that are unable to express the 2b protein (CMVD2b) are symptomless in wild-type Arabidopsis but induce disease in Arabidopsis dcl2/dcl4 or dcl2/dcl3/ dcl4 mutant plants, coincident with a marked increase in the titer of these CMV mutants (Diaz-Pendon et al., 2007; Ziebell and Carr, 2009). Our data provide strong evidence that 2b proteins from severe CMV strains contribute directly to pathogenicity in wild-type plants by perturbing miR159 activity. This is consistent with

Figure 6. myb33/myb65 mutant alleles moderate disease symptoms induced by the severe strain Fny-CMV. A, Arabidopsis wild-type and myb33/myb65 double mutant plants were inoculated with Fny-CMV or sterile water (Mock). The plants were photographed at 10 dpi. Bar = 1 cm. B, RNA gel-blot analysis of viral accumulation in Arabidopsis wild-type and myb33/myb65 mutant plants. Three virus-infected individual Arabidopsis wild-type or myb33/myb65 mutant plants were tested. Total RNA samples were prepared from upper, noninoculated leaves at 10 dpi. A biotin-labeled DNA oligonucleotide complementary to the conserved sequence common to the 39 UTRs of the CMV genomic and subgenomic RNAs was used to detect viral RNA. Equal RNA loading was confirmed by ethidium bromide staining. [See online article for color version of this figure.] Plant Physiol. Vol. 164, 2014

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findings for other VSRs, including the helper componentproteinase of Turnip mosaic virus, which contributes to viral symptom induction by inhibiting miR167-mediated effects on the transcript AUXIN RESPONSIVE FACTOR8 (Jay et al., 2011). There is a high sequence similarity between the miR159 and miR319 families, and the expression of MIM159 by LS-CMV may have disrupted regulation by miR319 of its target transcripts encoding TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1 transcription factors (Palatnik et al., 2007). Our data demonstrated that the exacerbated viral symptoms caused by MIM159 expression were associated with derepression of miR159ab targets MYB33 and MYB65. Thus, despite whatever effect LS-MIM159 has on miR319 functions, MIM159 expression by the vector derived from LS-CMV substantially reduced miR159 levels and elevated accumulation of MYB33 and MYB65, leading to production of severe viral symptoms resembling the disease symptoms caused by the severe strain Fny-CMV. The use of LS-CMV-based TM is an effective technology to assess miRNA functions in planta. It is worth noting that 21-nt and 24-nt amiR-trichome molecules produced from the primary transcript of amiR-trichome had different stabilities when exposed to the target mimic MIM-trichome (Fig. 2D). The MIM-trichome was originally designed to affect the 21-nt miRNA, but worked better in reducing stability of 24-nt molecules. The 24-nt miRNA has three more nucleotides at its termini than a 21-nt miRNA molecule. In Arabidopsis, mature miRNA is degraded by SMALL RNA DEGRADATION NUCLEASEs, one of which (SMALL RNA DEGRADATION NUCLEASE1) was shown to specifically degrade single-stranded miRNAs using its 39 to 59 exonuclease activity (Ramachandran and Chen, 2008). This implies that base pairing of 39 terminal nucleotides of miRNA with its target mimic largely determines miRNA fate (survival or degradation), and this could be one of the reasons that various miRNA-TM interactions exhibit different effects on miRNA stability. By optimizing mimic sequences corresponding to miR165 and miR166, Yan and colleagues (2012) recently found that STTM165/166 with a 48-nt spacer is most effective in destabilization of miR165/miR166. The greater effectiveness was suggested to be a result of STTM transcripts being more stable than IPS1-based TM transcripts in vivo. However, when IPS-based TM was expressed from LS-CMV, its efficacy was apparently greater than that of STTM, as exemplified by its effects on miR165/miR166. The increased efficacy of TM when expressed from a CMV vector could be attributed to be either increased stability of TM sequences when present in CMV RNAs or the higher expression levels achieved by expression from a virus, or both. This demonstrates that CMV-based TM is more effective than transgenic TM in impairing miRNA functions. The fact that LS-CMV 2b has no or little effect on miRNAs in planta and that CMV has an extremely broad host range would make LS-CMV-based TM a potentially useful 1386

technology for analysis of miRNA functions in a variety of plant species, not just in Arabidopsis. In addition, virus-based TM has other advantages because it is time-saving (compared with generation of stably transformed plants) and has the potential for highthroughput investigation of miRNA function. Therefore, we suggest that virus-based TM technology could be used as a high-throughput method to initially identify the effects of specific miRNAs and would be followed up by focused studies using the transgenic plant miRNA decoy approach in combination with specific knockouts (or virus-induced gene silencing for silencing specific mRNAs) to identify the mRNA target responsible for a given phenotype.

MATERIALS AND METHODS Plant Materials and Plant Growth Conditions Arabidopsis (Arabidopsis thaliana Heyn) ecotype Columbia plants, mutant plants, and transgenic plants were grown under an 8-h photoperiod with a light intensity of 150 to 200 mE m22 s21 at 22°C. Arabidopsis mutants ema1-1 and myb33/myb65 were previously described (Allen et al., 2007; Wang et al., 2011). Nicotiana glutinosa and Nicotiana tabacum plants were grown under a 16-h photoperiod with a light intensity of 150 to 200 mE m22 s21 at 25°C.

DNA Constructs Infectious clones pLS109, pLS209, and pLS309 corresponding to RNA1, RNA2, and RNA3 of LS-CMV were previously described (Zhang et al., 1994). To generate pLS309-derived infectious RNAs expressing miRNA TM sequences or an arbitrary sequence as a control (CK; Fig. 2A), we introduced the desired DNA sequence immediately downstream of the CP sequence in pLS309 by overlapping PCR using partially complementary oligonucleotides. Briefly, DNA fragment I was amplified using pLS309 as a template with a forward primer (LSR3F1393) and a reverse primer complementary to the 39 end sequence of CP and target mimic. DNA fragment II was amplified using pLS309 as template with a reverse primer (LSR3R2198) and a forward primer corresponding to target mimic and the 59 end of 39 UTR. Fragments I and II were mixed together as overlapping PCR template to generate a DNA fragment that was subsequently digested using HindIII and PstI. The restriction product was ligated into predigested pLS309 to generate infectious clones, pLS309-MIM-trichome, pLS309-MIM165/166, pLS309-MIM159, and pLS309MIM-CK (control). All primers used for making constructs are shown in Supplemental Table S1.

Viruses and Viral Inoculation LS-CMV variants were constituted by coinoculating Nicotiana glutinosa plants with in vitro-synthesized transcripts of pLS109 and pLS209 together with transcripts of pLS309-derived constructs carrying TM sequences or the control sequence (MIM-CK). Genetic stability of these mutants was confirmed by DNA sequencing of reverse transcription PCR products of viral RNA3 extracted from systemically infected leaves of the inoculated plants. These viruses were passaged from the infected Nicotiana glutinosa to Nicotiana tabacum plants for virus propagation and virus purification. Virion purification was carried out as described by Ng and Perry (2004). Purified virions at a concentration of 100 ng/ml were mechanically inoculated onto Arabidopsis seedlings at the 5-6 true-leaf stage using Carborundum as an abrasive.

RNA Gel Blotting Total RNA was extracted from a pool of leaf tissues harvested from five plant individuals using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA gel blotting for analysis of CMV genomic RNAs was performed as previously described (Xu et al., 2013). For analysis of low-Mr RNAs, including miRNAs and U6, 15 mg total RNA was used for RNA-blot Plant Physiol. Vol. 164, 2014

MicroRNA159 and Cucumber mosaic virus Symptoms*

analysis according to the protocol described in the instructions of the miRVana miRNA Isolation Kit (Ambion). DNA oligonucleotides completely complementary to mature miRNA, miRNA*, or U6 were labeled with digoxigenin (DIG) at their 39 ends using the DIG Oligonucleotide Tailing Kit (second generation; Roche) following the manufacturer’s instructions, and were purified using a G25 Sephadex column (GE) according to the manufacturer’s instructions. The purified probe was used for detection of corresponding miRNA or miRNA* using the DIG Luminescence Detection Kit (Roche) according to the manufacturer’s instructions. The sequence of the U6 probe was previously described (Wu et al., 2006).

RT-qPCR For analysis of relative accumulation of miRNA target transcripts, RT-qPCR was conducted using a previously described protocol (Westwood et al., 2013a, 2013b). Prior to reverse transcription, total RNA preparations were incubated with Turbo DNase (Ambion). The primers and PCR conditions for detection of amiR-trichome targets CPC, ETC2, and TRY, miR165/166 targets PHB and PHV, and miR159 targets MYB33, MYB65, and MYB101 were previously described (Allen et al., 2007; Wang et al., 2011; Yan et al., 2012).

Supplemental Data The following materials are available in the online version of this article. Supplemental Table S1. Primers used for generation of DNA constructs.

ACKNOWLEDGMENTS We thank Dr. Anthony Millar for providing seeds for the myb33/myb65 mutant, Dr. Yijun Qi for providing seeds for the ema1-1 mutant, and Drs. Alex M. Murphy, Simon C. Groen, and Qiansheng Liao for useful discussions. Received November 5, 2013; accepted January 30, 2014; published February 3, 2014.

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