Cheng-Guo Duan

Artificial MicroRNAs Highly Accessible to Targets Confer Efficient Virus Resistance in Plants

ABSTRACT Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3′ untranslated region (UTR) of the Cucumber mosaic virus (CMV) genome. Transiently expressed 3′UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3′UTR of the CMV-infected wild-type Arabidopsis to those of the triple dcl2/3/4 mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3′UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.

Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein

This work identifies a nucleolar localization signal within the essential double-stranded RNA binding domain of the Cucumber mosaic virus silencing suppressor protein 2b. It also shows that direct 2b–ARGONAUTE interactions can redistribute both 2b and ARGONAUTE proteins in the nucleus but are not essential for 2b suppression of either posttranscriptional gene silencing or RNA-directed DNA methylation. Unique among the known plant and animal viral suppressors of RNA silencing, the 2b protein interacts directly with both small interfering RNA (siRNA) and ARGONAUTE1 (AGO1) and AGO4 proteins and is targeted to the nucleolus. However, it is largely unknown which regions of the 111-residue 2b protein determine these biochemical properties and how they contribute to its diverse silencing suppressor activities. Here, we identified a functional nucleolar localization signal encoded within the 61–amino acid N-terminal double-stranded RNA (dsRNA) binding domain (dsRBD) that exhibited high affinity for short and long dsRNA. However, physical interaction of 2b with AGOs required an essential 33-residue region C-terminal to the dsRBD and was sufficient to inhibit the in vitro AGO1 Slicer activity independently of its dsRNA binding activities. Furthermore, the direct 2b–AGO interaction was not essential for the 2b suppression of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) in vivo. Lastly, we found that the 2b–AGO interactions in vivo also required the nucleolar targeting of 2b and had the potential to redistribute both the 2b and AGO proteins in nucleus. These findings together suggest that 2b may suppress PTGS and RdDM in vivo by binding and sequestering siRNA and the long dsRNA precursor in a process that is facilitated by its interactions with AGOs in the nucleolus.

Application of RNA silencing to plant disease resistance

To reduce the losses caused by plant pathogens, plant biologists have adopted numerous methods to engineer resistant plants. Among them, RNA silencing-based resistance has been a powerful tool that has been used to engineer resistant crops during the last two decades. Based on this mechanism, diverse approaches were developed. In this review, we focus on the application of RNA silencing to produce plants that are resistant to plant viruses such as RNA and DNA viruses, viroids, insects, and the recent expansion to fungal pathogens.

The ABA Receptor PYL8 Promotes Lateral Root Growth by Enhancing MYB77-Dependent Transcription of Auxin-Responsive Genes

Plants use an alternative abscisic acid signaling pathway to promote lateral root growth. ABA Tells Roots to Stop and Then Grow Plants respond to environmental stresses, such as high salt or drought, by altering the organization and length of their roots. Plant hormones, such as auxins and abscisic acid, signal to cells to either promote or inhibit growth. In response to stress, plants use abscisic acid to halt the growth of lateral roots that extend horizontally from the main root. Later, the roots recover and these lateral roots start to grow. Whereas abscisic acid initially halted lateral root growth, Zhao et al. found that abscisic acid also signals through the receptor PYL8 to activate genes that stimulate the recovery of lateral root growth. Thus, PYL8 may represent a central molecule in abscisic acid–dependent patterning of root architecture in response to stress. The phytohormone abscisic acid (ABA) regulates plant growth, development, and abiotic stress responses. ABA signaling is mediated by a group of receptors known as the PYR1/PYL/RCAR family, which includes the pyrabactin resistance 1–like protein PYL8. Under stress conditions, ABA signaling activates SnRK2 protein kinases to inhibit lateral root growth after emergence from the primary root. However, even in the case of persistent stress, lateral root growth eventually recovers from inhibition. We showed that PYL8 is required for the recovery of lateral root growth, following inhibition by ABA. PYL8 directly interacted with the transcription factors MYB77, MYB44, and MYB73. The interaction of PYL8 and MYB77 increased the binding of MYB77 to its target MBSI motif in the promoters of multiple auxin-responsive genes. Compared to wild-type seedlings, the lateral root growth of pyl8 mutant seedlings and myb77 mutant seedlings was more sensitive to inhibition by ABA. The recovery of lateral root growth was delayed in pyl8 mutant seedlings in the presence of ABA, and the defect was rescued by exposing pyl8 mutant seedlings to the auxin IAA (3-indoleacetic acid). Thus, PYL8 promotes lateral root growth independently of the core ABA-SnRK2 signaling pathway by enhancing the activities of MYB77 and its paralogs, MYB44 and MYB73, to augment auxin signaling.

RNA-Dependent RNA Polymerase 1 from Nicotiana tabacum Suppresses RNA Silencing and Enhances Viral Infection in Nicotiana benthamiana

This work analyzes the surprising result that Nicotiana benthamiana transformed with RNA-dependent RNA polymerase 1 from Nicotiana tabacum (Nt-RDR1) is hypersusceptible to several viruses. It provides evidence supporting a dual role for RDR1 in contributing to salicylic acid–mediated antiviral defense at the same time as it suppresses RDR6-mediated antiviral RNA silencing. Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid–mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.

DCL4 Targets Cucumber Mosaic Virus Satellite RNA at Novel Secondary Structures

ABSTRACT It has been reported that plant virus-derived small interfering RNAs (vsiRNAs) originated predominantly from structured single-stranded viral RNA of a positive single-stranded RNA virus replicating in the cytoplasm and from the nuclear stem-loop 35S leader RNA of a double-stranded DNA (dsDNA) virus. Increasing lines of evidence have also shown that hierarchical actions of plant Dicer-like (DCL) proteins are required in the biogenesis process of small RNAs, and DCL4 is the primary producer of vsiRNAs. However, the structures of such single-stranded viral RNA that can be recognized by DCLs remain unknown. In an attempt to determine these structures, we have cloned siRNAs derived from the satellite RNA (satRNA) of Cucumber mosaic virus (CMV-satRNA) and studied the relationship between satRNA-derived siRNAs (satsiRNAs) and satRNA secondary structure. satsiRNAs were confirmed to be derived from single-stranded satRNA and are primarily 21 (64.7%) or 22 (22%) nucleotides (nt) in length. The most frequently cloned positive-strand satsiRNAs were found to derive from novel hairpins that differ from the structure of known DCL substrates, miRNA and siRNA precursors, which are prevalent stem-loop-shaped or dsRNAs. DCL4 was shown to be the primary producer of satsiRNAs. In the absence of DCL4, only 22-nt satsiRNAs were detected. Our results suggest that DCL4 is capable of accessing flexibly structured single-stranded RNA substrates (preferably T-shaped hairpins) to produce satsiRNAs. This result reveals that viral RNA of diverse structures may stimulate antiviral DCL activities in plant cells.

The unique mode of action of a divergent member of the ABA-receptor protein family in ABA and stress signaling

Proteins in the PYR/PYL/RCAR family (PYLs) are known as receptors for the phytohormone ABA. Upon ABA binding, PYL adopts a conformation that allows it to interact with and inhibit clade A protein phosphatase 2Cs (PP2Cs), which are known as the co-receptors for ABA. Inhibition of the PP2Cs then leads to the activation of the SnRK2 family protein kinases that phosphorylate and activate downstream effectors in ABA response pathways. The PYL family has 14 members in Arabidopsis, 13 of which have been demonstrated to function as ABA receptors. The function of PYL13, a divergent member of the family, has been enigmatic. We report here that PYL13 differs from the other PYLs in three key residues that affect ABA perception, and mutations in these three residues can convert PYL13 into a partially functional ABA receptor. Transgenic plants overexpressing PYL13 show increased ABA sensitivity in seed germination and postgermination seedling establishment as well as decreased stomatal conductance, increased water-use efficiency, accelerated stress-responsive gene expression, and enhanced drought resistance. pyl13 mutant plants are less sensitive to ABA inhibition of postgermination seedling establishment. PYL13 interacts with and inhibits some members of clade A PP2Cs (PP2CA in particular) in an ABA-independent manner. PYL13 also interacts with the other PYLs and antagonizes their function as ABA receptors. Our results show that PYL13 is not an ABA receptor but can modulate the ABA pathway by interacting with and inhibiting both the PYL receptors and the PP2C co-receptors.

RNA-binding protein regulates plant DNA methylation by controlling mRNA processing at the intronic heterochromatin-containing gene IBM1

Significance Transposons in higher eukaryotes are subject to epigenetic silencing. Some transposons have found residence in introns of genes. It is unclear how intronic transposon-containing genes are regulated. This paper reports a cellular factor, ANTI-SILENCING 1 (ASI1), which is required for proper expression of intronic transposon-containing genes. ASI1 plays an important role in regulating genome DNA methylation patterns. The work represents a major advancement in cellular antisilencing mechanisms. A previously undescribed mechanism for higher eukaryotes to cope with the collateral effects of silencing intronic transposon elements is discovered. This work has implications beyond the plant epigenetics field because studies in mammalian systems have suggested important roles of intronic heterochromatin on 3′ distal polyadenylation, although the underlying mechanism is not known. DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1. Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3′ distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements.

Arabidopsis EDM2 promotes IBM1 distal polyadenylation and regulates genome DNA methylation patterns

Significance The histone 3 lysine 9 demethylase Increase in BONSAI Methylation 1 (IBM1) is critical for preventing CHG (H=A, T, or C) methylation in plant genes. We found that the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) controls genome CHG methylation and transgene silencing by regulating alternative polyadenylation of IBM1. EDM2 contains a composite plant homeo domain that simultaneously recognizes both repressive and active histone marks, allowing it to associate specifically with the intronic heterochromatin of IBM1 and other plant genes so that it may affect polyadenylation by methylating the RNA through an N6-adenine methyltransferase-like activity. This work significantly advances our understanding of the regulation of DNA methylome and mechanisms of antisilencing and alternative polyadenylation of intronic heterochromatin-containing genes. DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin.

Satellite RNA reduces expression of the 2b suppressor protein resulting in the attenuation of symptoms caused by Cucumber mosaic virus infection

Satellite RNAs (satRNAs) depend on cognate helper viruses for replication, encapsidation, movement and transmission. Many satRNAs with different symptom modulation effects have been reported. The pathogenicity of satRNAs is thought to be the result of a direct interaction among the satRNA, helper viruses and host factors by unknown mechanisms. To understand the effect of satRNA of Cucumber mosaic virus (a severe field ShanDong strain, SD-CMV) on pathogenicity, and the possible involvement of host RNA silencing pathways in pathogenicity, we constructed biologically active CMV cDNA clones and a CMV-Δ2b mutant lacking the open reading frame of 2b, a silencing suppressor protein, in order to infect Nicotiana benthamiana and Arabidopsis with or without SD-satRNA. We found that SD-satRNA reduced the accumulation of the 2b protein and its coding RNA4A and attenuated the yellowing caused by SD-CMV infection. Small RNA analysis indicated that the 2b protein interfered with RNA silencing, specifically in the synthesis of CMV RNA3-derived small interfering RNAs (R3-siRNAs). The accumulation of R3-siRNAs in CMV-Δ2b infection was reduced in the presence of satRNA, for which greater accumulation of satRNA-derived siRNAs (satsiRNAs) was detected. Our results suggest that abundant SD-satRNA serving as target for RNA silencing may play a role in protecting helper CMV RNA, especially, subgenomic RNA4, from being targeted by RNA silencing. This compensates for the increase in RNA silencing resulting from the reduction in expression of the 2b suppressor in the presence of satRNA. Our data provide evidence that a plant silencing mechanism is involved in the pathogenicity of satRNA.