Hui-Shan Guo

MicroRNA Directs mRNA Cleavage of the Transcription Factor NAC1 to Downregulate Auxin Signals for Arabidopsis Lateral Root Development

Although several plant microRNAs (miRNAs) have been shown to play a role in plant development, no phenotype has yet been associated with a reduction or loss of expression of any plant miRNA. Arabidopsis thaliana miR164 was predicted to target five NAM/ATAF/CUC (NAC) domain–encoding mRNAs, including NAC1, which transduces auxin signals for lateral root emergence. Here, we show that miR164 guides the cleavage of endogenous and transgenic NAC1 mRNA, producing 3′-specific fragments. Cleavage was blocked by NAC1 mutations that disrupt base pairing with miR164. Compared with wild-type plants, Arabidopsis mir164a and mir164b mutant plants expressed less miR164 and more NAC1 mRNA and produced more lateral roots. These mutant phenotypes can be complemented by expression of the appropriate MIR164a and MIR164b genomic sequences. By contrast, inducible expression of miR164 in wild-type plants led to decreased NAC1 mRNA levels and reduced lateral root emergence. Auxin induction of miR164 was mirrored by an increase in the NAC1 mRNA 3′ fragment, which was not observed in the auxin-insensitive mutants auxin resistant1 (axr1-12), axr2-1, and transport inhibitor response1. Moreover, the cleavage-resistant form of NAC1 mRNA was unaffected by auxin treatment. Our results indicate that auxin induction of miR164 provides a homeostatic mechanism to clear NAC1 mRNA to downregulate auxin signals.

SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals

The plant hormone indole-3 acetic acid (IAA or auxin) controls many aspects of plant development, including the production of lateral roots. Ubiquitin-mediated proteolysis has a central role in this process. The genes AXR1 and TIR1 aid the assembly of an active SCF (Skp1/Cullin/F-box) complex that probably promotes degradation of the AUX/IAA transcriptional repressors in response to auxin. The transcription activator NAC1, a member of the NAM/CUC family of transcription factors, functions downstream of TIR1 to transduce the auxin signal for lateral root development. Here we show that SINAT5, an Arabidopsis homologue of the RING-finger Drosophila protein SINA, has ubiquitin protein ligase activity and can ubiquitinate NAC1. This activity is abolished by mutations in the RING motif of SINAT5. Overexpressing SINAT5 produces fewer lateral roots, whereas overexpression of a dominant-negative Cys49 → Ser mutant of SINAT5 develops more lateral roots. These lateral root phenotypes correlate with the expression of NAC1 observed in vivo. Low expression of NAC1 in roots can be increased by treatment with a proteasome inhibitor, which indicates that SINAT5 targets NAC1 for ubiquitin-mediated proteolysis to downregulate auxin signals in plant cells.

A viral protein inhibits the long range signaling activity of the gene silencing signal

Post‐transcriptional gene silencing (PTGS) provides protection against viruses in plants by homology‐dependent RNA degradation. PTGS initiated locally produces a mobile signal that instructs specific RNA degradation at a distance. Here we show that this signal‐mediated intercellular spread of PTGS does not occur after PTGS initiation in cells expressing cucumber mosaic virus 2b protein (Cmv2b), a nucleus‐localized plant viral PTGS suppressor. Silencing spread via the signal was also effectively blocked in independent assays by expressing Cmv2b only in tissues through which the signal must travel to induce PTGS in the target cells. Furthermore, the signal imported externally into the Cmv2b‐expressing cells was not active in triggering degradation of the target RNA and loss of signal activity in these cells was associated with a significantly reduced transgene DNA methylation. These findings indicate that Cmv2b inhibits the activity of the mobile signal and interferes with DNA methylation in the nucleus. Signal inactivation provides a mechanistic basis for the known role of Cmv2b in facilitating virus spread to tissues outside of the primarily infected sites.

SDIR1 Is a RING Finger E3 Ligase That Positively Regulates Stress-Responsive Abscisic Acid Signaling in Arabidopsis

Ubiquitination plays important roles in plant hormone signal transduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1), is involved in abscisic acid (ABA)-related stress signal transduction. SDIR1 is expressed in all tissues of Arabidopsis and is upregulated by drought and salt stress, but not by ABA. Plants expressing the ProSDIR1–β-glucuronidase (GUS) reporter construct confirmed strong induction of GUS expression in stomatal guard cells and leaf mesophyll cells under drought stress. The green fluorescent protein–SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDIR1 is an E3 ubiquitin ligase and that the RING finger conservation region is required for its activity. Overexpression of SDIR1 leads to ABA hypersensitivity and ABA-associated phenotypes, such as salt hypersensitivity in germination, enhanced ABA-induced stomatal closing, and enhanced drought tolerance. The expression levels of a number of key ABA and stress marker genes are altered both in SDIR1 overexpression and sdir1-1 mutant plants. Cross-complementation experiments showed that the ABA-INSENSITIVE5 (ABI5), ABRE BINDING FACTOR3 (ABF3), and ABF4 genes can rescue the ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDIR1 acts upstream of those basic leucine zipper family genes. Our results indicate that SDIR1 is a positive regulator of ABA signaling.

Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus.

Post‐transcriptional gene silencing (PTGS) is a homology‐dependent RNA degradation process that may target RNA exclusively in the cytoplasm. In plants, PTGS functions as a natural defense mechanism against viruses. We reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (CMV) is a virulence determinant and a suppressor of PTGS initiation in transgenic Nicotiana benthamiana. By fusion with the green fluorescent protein, we now show that the CMV 2b protein localizes to the nuclei of tobacco suspension cells and whole plants via an arginine‐rich nuclear localization signal, 22KRRRRR27. We further demonstrate that the nuclear targeting of the 2b protein is required for the efficient suppression of PTGS, indicating that PTGS may be blocked in the nucleus. In addition, our data indicate that the PTGS suppressor activity is important, but not sufficient, for virulence determination by the 2b protein.

Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism.

The 2b protein encoded by cucumber mosaic cucumovirus (Cmv2b) acts as an important virulence determinant by suppressing post‐transcriptional gene silencing (PTGS), a natural plant defence mechanism against viruses. We report here that the tomato aspermy cucumovirus 2b protein (Tav2b), when expressed from the unrelated tobacco mosaic tobamovirus (TMV) RNA genome, activates strong host resistance responses to TMV in tobacco which are typical of the gene‐for‐gene disease resistance mechanism. Domain swapping between Cmv2b, which does not elicit these responses, and Tav2b, revealed functional domains in Tav2b critical for triggering virus resistance and hypersensitive cell death. Furthermore, substitution of two amino acids from Tav2b by those found at the same positions in Cmv2b, Lys21→Val and Arg28→Ser, abolished the ability to induce hypersensitive cell death and virus resistance. However, in Nicotiana benthamiana, a species related to tobacco, Tav2b functions as a virulence determinant and suppresses PTGS. Thus, a viral suppressor of the host gene silencing defence mechanism is the target of another independent host resistance mechanism. Our results provide new insights into the complex molecular strategies employed by viruses and their hosts for defence, counter‐defence and counter counter‐defence.

Characterization of conserved and novel microRNAs and their targets, including a TuMV-induced TIR-NBS-LRR class R gene-derived novel miRNA in Brassica

Nine conserved miRNA families and three potential novel miRNAs in Brassica rapa were identified from a small RNA library. The expression patterns of some conserved miRNAs had different tissue specificity in Brassica and Arabidopsis. One of the three potential miRNAs, named bra‐miR1885, was verified as a true functional miRNA. It could be induced specifically by Turnip mosaic virus (TuMV) infection, and target TIR–NBS–LRR class disease‐resistant transcripts for cleavage. Based on the hypothesis for de novo generation of new miRNA genes and the sequence similarity between bra‐MIR1885 precursor loci and target transcript sequences, we suggest that bra‐MIR1885 is a new miRNA gene that originated through inverted duplication events from TIR–NBS–LRR class disease‐resistant protein‐coding gene sequences, which became bra‐miR1885 targets.

An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana.

The ubiquitination proteasome pathway has been demonstrated to regulate all plant developmental and signaling processes. E3 ligase/substrate-specific interactions and ubiquitination play important roles in this pathway. However, due to technical limitations only a few instances of E3 ligase-substrate binding and protein ubiquitination in plants have been directly evidenced. An efficient in vivo and in vitro ubiquitination assay was developed for analysis of protein ubiquitination reactions by agroinfiltration expression of both substrates and E3 ligases in Nicotiana benthamiana. Using a detailed analysis of the well-known E3 ligase COP1 and its substrate HY5, we demonstrated that this assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, as well as the effects of MG132 and substrate ubiquitination and degradation. We were able to differentiate between the original and ubiquitinated forms of the substrate in vivo with antibodies to ubiquitin or to the target protein. We also demonstrated that the substrate and E3 ligase proteins expressed by agroinfiltration can be applied to analyze ubiquitination in in vivo or in vitro reactions. In addition, we optimized the conditions for different types of substrate and E3 ligase expression by supplementation with the gene-silencing suppressor p19 and by time-courses of sample collection. Finally, by testing different protein extraction buffers, we found that different types of buffer should be used for different ubiquitination analyses. This method should be adaptable to other protein modification studies.

BSCTV C2 Attenuates the Degradation of SAMDC1 to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis

This work shows that a geminivirus-encoded silencing suppressor C2 interacts with a plant host cellular component SAMDC1 and attenuates its fast turnover mediated by the 26S proteasome. As a result, it interferes with the host plant’s DNA methylation-related gene silencing mechanism and facilitates geminivirus infection. Plant viruses are excellent tools for studying microbial–plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus–plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein–directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1.

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.