[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a class of endogenous small RNAs (sRNAs) that play pivotal roles in plant development, abiotic stress response, and pathogen response. miRNAs have been extensively studied in plants, but rarely in Nicotiana benthamiana, despite its wide use in plant virology studies, particularly for studying N protein–tobacco mosaic virus (TMV) interactions. We report an efficient method using high-throughput sequencing and bioinformatics to identify genome-wide miRNAs in N. benthamiana. A total of 30 conserved miRNA families and 113 novel miRNAs belonging to 93 families were identified. Some miRNAs were clustered on chromosomes, and some were embedded in host gene introns. The predicted miRNA targets were involved in diverse biological processes, such as metabolism, signaling, and responses to stimuli. miRNA expression profiling revealed that most of them were differentially expressed during N-mediated immunity to TMV. This study provides a framework for further analysis of miRNA functions in plant immunity.
[Show abstract][Hide abstract] 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, functions of these miRNAs remain largely unknown due to the lack of effective genetic tools. Here, we report a virus-based miRNA silencing (VbMS) system that can be used for functional analysis of plant miRNAs. VbMS is performed through Tobacco rattle virus (TRV)-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 compound leaves. These results demonstrate that TRV-based VbMS is a powerful tool to silence endogenous miRNAs and to dissect their functions in different plant species.
[Show abstract][Hide abstract] ABSTRACT: Virus-induced gene silencing using artificial microRNAs (MIR VIGS) is a newly developed technique for plant reverse genetic studies. Traditional virus-induced gene silencing (VIGS) assays introduce a large gene fragment, which is expressed and then converted into small RNAs by the endogenous siRNA-based gene silencing machinery of the plant host. By contrast, MIR VIGS uses well-designed miRNAs to induce RNA-mediated silencing of the target gene. Using a single artificial miRNA can provide greater specificity by reducing off-target effects. Here, we describe a detailed protocol for MIR VIGS in Nicotiana benthamiana using a modified Cabbage leaf curl virus (CaLCuV)-based vector.
[Show abstract][Hide abstract] ABSTRACT: The hypersensitive response (HR), a form of programmed cell death (PCD), is a tightly regulated innate immune response in plants that is hypothesized to restrict pathogen growth and disease development. Although considerable efforts have been made to understand HR PCD, it remains unknown whether the retrograde pathway from the Golgi to the endoplasmic reticulum (ER) is involved. Here we provide direct genetic evidence that two Nicotiana benthamiana homologs, ERD2a and ERD2b, function as ER luminal protein receptors and participate in HR PCD. Virus-induced gene silencing (VIGS) of ERD2a and/or ERD2b caused escape of ER-resident proteins from the ER, and resulted in plants that were more sensitive to ER stress. Silencing of ERD2b delayed HR PCD induced by the non-host pathogens Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. tomato DC3000. However, both silencing of ERD2a and co-silencing of ERD2a and ERD2b exacerbated HR PCD. Individual and combined suppression of ERD2a and ERD2b exaggerated R gene-mediated cell death. Nevertheless, silencing of ERD2a and/or ERD2b had no detectable effects on bacterial growth. Furthermore, VIGS of several putative ligands of ERD2a/2b, including the ER quality control (ERQC) component genes BiP, CRT3 and UGGT, had different effects on HR PCD induced by different pathogens. This indicates that immunity-related cell death pathways are separate with respect to the genetic requirements for these ERQC components. These results suggest that ERD2a and ERD2b function as ER luminal protein receptors to ensure ERQC and alleviate ER stress, thus affecting HR PCD during the plant innate immune response.
The Plant Journal 05/2012; 72(1):57-69. DOI:10.1111/j.1365-313X.2012.05053.x · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: *The hairpin-based RNA interference (RNAi) technique plays an important role in exploring gene function in plants. Although there are several methods for making hairpin RNA (hpRNA) constructs, these methods usually need multiple relatively laborious, time-consuming or high-cost cloning steps. Here we describe a one-step, zero-background ligation-independent cloning (OZ-LIC) method for making intron-containing hpRNA (ihpRNA) constructs by our vector pRNAi-LIC. *To generate the ihpRNA constructs with zero-background, this method only requires treating two PCR products of target gene flanked with different LIC sequences and SmaI-linearized pRNAi-LIC vector by T4 DNA polymerase respectively, and then transforming these treated DNA mixture into Escherichia coli. *The ihpRNA constructs generated with our OZ-LIC RNAi vector can efficiently induce not only transient silencing of the exogenous marker genes and the endogenous resistance-related Nicotiana benthamiana SGT1 gene, but also stable transgenic suppression of Arabidopsis SGT1b gene. *Our new OZ-LIC method and RNAi vector will represent a powerful tool for gene knockdown in plants and may facilitate high-throughput determination of plant gene function.
New Phytologist 07/2010; 187(1):240-50. DOI:10.1111/j.1469-8137.2010.03253.x · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Traditional virus-induced gene silencing (VIGS) is a powerful virus-based short interfering RNA-mediated RNA silencing technique for plant functional genomics. Besides short interfering RNAs, microRNAs (miRNAs) have also been shown to regulate gene expression by RNA silencing in various organisms. However, plant virus-based miRNA silencing has not been reported. In addition, a number of plant miRNAs have been identified or predicted, while their functions are largely unknown. Thus, there is an urgent need for the development of new technologies to study miRNA function. Here, we report that a modified cabbage leaf-curl geminivirus vector can be used to express artificial and endogenous miRNAs in plants. Using this viral miRNA expression system, we demonstrate that VIGS using artificial miRNAs, dubbed as "MIR VIGS," was effective to silence the expression of endogenous genes, including PDS, Su, CLA1, and SGT1, in Nicotiana benthamiana. Silencing of SGT1 led to the loss of N-mediated resistance to Tobacco mosaic virus. Furthermore, using this viral miRNA expression system, we found that viral ectopic expression of endogenous miR156 and miR165 but not their mutants in N. benthamiana resulted in earlier abnormal developmental phenotypes, and expression of miR165 induced abnormal chlorotic spots on leaves. These results demonstrate that the cabbage leaf-curl geminivirus-based miRNA expression system can be utilized not only to specifically silence genes involved in general metabolism and defense but also to investigate the function of endogenous miRNAs in plants.