G-U base-paired hpRNA confers potent inhibition of small RNA function in plants.

MicroRNA (miRNA) target mimicry technologies, utilizing naturally occurring miRNA decoy molecules, represent a potent tool for analyzing miRNA function. In this study, we present a highly efficient small RNA (sRNA) target mimicry design based on G-U base-paired hairpin RNA (hpG:U), which allows for the simultaneous targeting of multiple sRNAs. The hpG:U constructs consistently generate high amounts of intact, polyadenylated stem-loop (SL) RNA outside the nuclei, in contrast to traditional hairpin RNA designs with canonical base pairing (hpWT), which were predominantly processed resulting in a loop. By incorporating a 460-bp G-U base-paired double-stranded stem and a 312-576 nt loop carrying multiple miRNA target mimicry sites (GUMIC), the hpG:U construct displayed effective repression of three Arabidopsis miRNAs, namely miR165/166, miR157, and miR160, both individually and in combination. Additionally, a GUMIC construct targeting a prominent cluster of siRNAs derived from cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) effectively inhibited Y-Sat siRNA-directed silencing of the chlorophyll biosynthetic gene CHLI, thereby reducing the yellowing symptoms in infected Nicotiana plants. Therefore, the G-U base-paired hpRNA, characterized by differential processing compared to traditional hpRNA, acts as an efficient decoy for both miRNAs and siRNAs. This technology holds great potential for sRNA functional analysis and the management of sRNA-mediated diseases.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Silencing of transcription factor encoding gene StTCP23 by small RNAs derived from the virulence modulating region of potato spindle tuber viroid is associated with symptom development in potato.

Viroids are small, non-protein-coding RNAs which can induce disease symptoms in a variety of plant species. Potato (Solanum tuberosum L.) is the natural host of Potato spindle tuber viroid (PSTVd) where infection results in stunting, distortion of leaves and tubers and yield loss. Replication of PSTVd is accompanied by the accumulation of viroid-derived small RNAs (sRNAs) proposed to play a central role in disease symptom development. Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23, a member of the TCP (teosinte branched1/Cycloidea/Proliferating cell factor) transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation, especially in responses to gibberellic acid (GA). The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3' untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE) confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs. Expression of these VMR sRNA sequences as artificial miRNAs (amiRNAs) in transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants. Furthermore, the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23. In addition, virus-induced gene silencing (VIGS) of StTCP23 in potato also resulted in PSTVd-like phenotypes. Consistent with the function of TCP family genes, amiRNA lines in which StTCP23 expression was silenced showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes previously implicated in tuber development. Application of GA to the amiRNA plants minimized the PSTVd-like phenotypes. Taken together, our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression, thereby disrupting the signaling pathways regulating GA metabolism and leading to plant stunting and formation of small and spindle-shaped tubers.

Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms.

Tomato yellow leaf curl virus (TYLCV) and its related begomoviruses cause fast-spreading diseases in tomato worldwide. How this virus induces diseases remains largely unclear. Here we report a noncoding RNA-mediated model to elucidate the molecular mechanisms of TYLCV-tomato interaction and disease development. The circular ssDNA genome of TYLCV contains a noncoding intergenic region (IR), which is known to mediate viral DNA replication and transcription in host cells, but has not been reported to contribute directly to viral disease development. We demonstrate that the IR is transcribed in dual orientations during plant infection and confers abnormal phenotypes in tomato independently of protein-coding regions of the viral genome. We show that the IR sequence has a 25-nt segment that is almost perfectly complementary to a long noncoding RNA (lncRNA, designated as SlLNR1) in TYLCV-susceptible tomato cultivars but not in resistant cultivars which contains a 14-nt deletion in the 25-nt region. Consequently, we show that viral small-interfering RNAs (vsRNAs) derived from the 25-nt IR sequence induces silencing of SlLNR1 in susceptible tomato plants but not resistant plants, and this SlLNR1 downregulation is associated with stunted and curled leaf phenotypes reminiscent of TYLCV symptoms. These results suggest that the lncRNA interacts with the IR-derived vsRNAs to control disease development during TYLCV infection. Consistent with its possible function in virus disease development, over-expression of SlLNR1 in tomato reduces the accumulation of TYLCV. Furthermore, gene silencing of the SlLNR1 in the tomato plants induced TYLCV-like leaf phenotypes without viral infection. Our results uncover a previously unknown interaction between vsRNAs and host lncRNA, and provide a plausible model for TYLCV-induced diseases and host antiviral immunity, which would help to develop effective strategies for the control of this important viral pathogen.

miR159 Represses a Constitutive Pathogen Defense Response in Tobacco.

MicroR159 (miR159) regulation of GAMYB expression is highly conserved in terrestrial plants; however, its functional role remains poorly understood. In Arabidopsis (Arabidopsis thaliana), although GAMYB-like genes are constitutively transcribed during vegetative growth, their effects are suppressed by strong and constitutive silencing by miR159. GAMYB expression occurs only if miR159 function is inhibited, which results in detrimental pleiotropic defects, questioning the purpose of the miR159-GAMYB pathway. Here, miR159 function was inhibited in tobacco (Nicotiana tabacum) and rice (Oryza sativa) using miRNA MIM159 technology. Similar to observations in Arabidopsis, inhibition of miR159 in tobacco and rice resulted in pleiotropic defects including stunted growth, implying functional conservation of the miR159-GAMYB pathway among angiosperms. In MIM159 tobacco, transcriptome profiling revealed that genes associated with defense and programmed cell death were strongly activated, including a suite of 22 PATHOGENESIS-RELATED PROTEIN (PR) genes that were 100- to 1,000-fold upregulated. Constitutive expression of a miR159-resistant GAMYB transgene in tobacco resulted in phenotypes similar to that of MIM159 tobacco and activated PR gene expression, verifying the dependence of the above-mentioned changes on GAMYB expression. Consistent with the broad defense response, MIM159 tobacco appeared immune to Phytophthora infection. These findings suggest that the tobacco miR159-GAMYB pathway functions in the biotic defense response, which becomes activated upon miR159 inhibition. However, PR gene expression was not upregulated in Arabidopsis or rice when miR159 was inhibited, suggesting that miR159-GAMYB pathway functional differences exist between species, or factors in addition to miR159 inhibition are required in Arabidopsis and rice to activate this broad defense response.

Identification of Essential Sensitive Regions of the Aerolysin Nanopore for Single Oligonucleotide Analysis.

The aerolysin nanopore channel is one of the confined spaces for single molecule analysis which displays high spatial and temporal resolution for the discrimination of single nucleotides, identification of DNA base modification, and analyzing the structural transition of DNAs. However, to overcome the challenge of achieving the ultimate goal of the widespread real analytical application, it is urgent to probe the sensing regions of the aerolysin to further improve the sensitivity. In this paper, we explore the sensing regions of the aerolysin nanopore by a series of well-designed mutant nanopore experiments combined with molecular dynamics simulations-based electrostatic analysis. The positively charged lumen-exposed Lys-238, identified as one of the key sensing sites due to the presence of a deep valley in the electrostatic potentials, was replaced by different charged and sized amino acids. The results show that the translocation time of oligonucleotides through the nanopore can be readily modulated by the choice of the target amino acid at the 238 site. In particular, a 7-fold slower translocation at a voltage bias of +120 mV is observed with respect to the wild-type aerolysin, which provides a high resolution for methylated cytosine discrimination. We further determine that both the electrostatic properties and geometrical structure of the aerolysin nanopore are crucial to its sensing ability. These insights open ways for rationally designing the sensing mechanism of the aerolysin nanopore, thus providing a novel paradigm for nanopore sensing.

Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a ß-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

Incorporating Transgenerational Epigenetic Inheritance into Ecological Risk Assessment Frameworks.

Chronic exposure to environmental contaminants can induce heritable "transgenerational" modifications to organisms, potentially affecting future ecosystem health and functionality. Incorporating transgenerational epigenetic heritability into risk assessment procedures has been previously suggested. However, a critical review of existing literature yielded numerous studies claiming transgenerational impacts, with little compelling evidence. Therefore, contaminant-induced epigenetic inheritance may be less common than is reported in the literature. We identified a need for multigeneration epigenetic studies that extend beyond what could be deemed "direct exposure" to F1 and F2 gametes and also include subsequent multiple nonexposed generations to adequately evaluate transgenerational recovery times. Also, increased experimental replication is required to account for the highly variable nature of epigenetic responses and apparent irreproducibility of current studies. Further, epigenetic end points need to be correlated with observable detrimental organism changes before a need for risk management can be properly determined. We suggest that epigenetic-based contaminant studies include concentrations lower than current "EC10-20" or "Lowest Observable Effect Concentrations" for the organism's most sensitive phenotypic end point, as higher concentrations are likely already regulated. Finally, we propose a regulatory framework and optimal experimental design that enables transgenerational epigenetic effects to be assessed and incorporated into conventional ecotoxicological testing.

Epigenetic Mechanisms: An Emerging Player in Plant-Microbe Interactions.

Plants have developed diverse molecular and cellular mechanisms to cope with a lifetime of exposure to a variety of pathogens. Host transcriptional reprogramming is a central part of plant defense upon pathogen recognition. Recent studies link DNA methylation and demethylation as well as chromatin remodeling by posttranslational histone modifications, including acetylation, methylation, and ubiquitination, to changes in the expression levels of defense genes upon pathogen challenge. Remarkably these inducible defense mechanisms can be primed prior to pathogen attack by epigenetic modifications and this heightened resistance state can be transmitted to subsequent generations by inheritance of these modification patterns. Beside the plant host, epigenetic mechanisms have also been implicated in virulence development of pathogens. This review highlights recent findings and insights into epigenetic mechanisms associated with interactions between plants and pathogens, in particular bacterial and fungal pathogens, and demonstrates the positive role they can have in promoting plant defense.

Ubiquitous miR159 repression of MYB33/65 in Arabidopsis rosettes is robust and is not perturbed by a wide range of stresses.

BACKGROUND: The microR159 (miR159) - GAMYB pathway is conserved in higher plants, where GAMYB, expression promotes programmed cell death in seeds (aleurone) and anthers (tapetum). In cereals, restriction of GAMYB expression to seeds and anthers is mainly achieved transcriptionally, whereas in Arabidopsis this is achieved post-transcriptionally, as miR159 silences GAMYB (MYB33 and MYB65) in vegetative tissues, but not in seeds and anthers. However, we cannot rule out a role for miR159-MYB33/65 pathway in Arabidopsis vegetative tissues; a loss-of-function mir159 Arabidopsis mutant displays strong pleiotropic defects and numerous reports have documented changes in miR159 abundance during stress and hormone treatments. Hence, we have investigated the functional role of this pathway in vegetative tissues. RESULTS: It was found that the miR159-MYB33/65 pathway was ubiquitously present throughout rosette development. However, miR159 appears to continuously repress MYB33/MYB65 expression to levels that have no major impact on rosette development. Inducible inhibition of miR159 resulted in MYB33/65 de-repression and associated phenotypic defects, indicating that a potential role in vegetative development is only possible through MYB33 and MYB65 if miR159 levels decrease. However, miR159 silencing of MYB33/65 appeared extremely robust; no tested abiotic stress resulted in strong miR159 repression. Consistent with this, the stress responses of an Arabidopsis mutant lacking the miR159-MYB33/65 pathway were indistinguishable from wild-type. Moreover, expression of viral silencing suppressors, either via transgenesis or viral infection, was unable to prevent miR159 repression of MYB33/65, highlighting the robustness of miR159-mediated silencing. CONCLUSIONS: Despite being ubiquitously present, molecular, genetic and physiological analysis failed to find a major functional role for the miR159-MYB33/65 pathway in Arabidopsis rosette development or stress response. Although it is likely that this pathway is important for a stress not tested here or in different plant species, our findings argue against the miR159-MYB33/65 pathway playing a major conserved role in general stress response. Finally, in light of the robustness of miR159-mediated repression of MYB33/65, it appears unlikely that low fold-level changes of miR159 abundance in response to stress would have any major physiological impact in Arabidopsis.

RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops.

Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3' UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops.

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

Mutation of the RDR1 gene caused genome-wide changes in gene expression, regional variation in small RNA clusters and localized alteration in DNA methylation in rice.

BACKGROUND: Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown. RESULTS: We characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions. CONCLUSIONS: Our results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response.

Long noncoding RNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana.

Short noncoding RNAs have been demonstrated to play important roles in regulation of gene expression and stress responses, but the repertoire and functions of long noncoding RNAs (lncRNAs) remain largely unexplored, particularly in plants. To explore the role of lncRNAs in disease resistance, we used a strand-specific RNA-sequencing approach to identify lncRNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana. Antisense transcription was found in c. 20% of the annotated A. thaliana genes. Several noncoding natural antisense transcripts responsive to F. oxysporum infection were found in genes implicated in disease defense. While the majority of the novel transcriptionally active regions (TARs) were adjacent to annotated genes and could be an extension of the annotated transcripts, 159 novel intergenic TARs, including 20 F. oxysporum-responsive lncTARs, were identified. Ten F. oxysporum-induced lncTARs were functionally characterized using T-DNA insertion or RNA-interference knockdown lines, and five were demonstrated to be related to disease development. Promoter analysis suggests that some of the F. oxysporum-induced lncTARs are direct targets of transcription factor(s) responsive to pathogen attack. Our results demonstrated that strand-specific RNA sequencing is a powerful tool for uncovering hidden levels of transcriptome and that IncRNAs are important components of the antifungal networks in A. thaliana.

Stress-induced early flowering is mediated by miR169 in Arabidopsis thaliana.

Plants interact with their environment and they often flower earlier under stress conditions, but how such stress-induced flowering is regulated remains poorly understood. Here evidence is presented that the miR169 family plays a key role in stress-induced flowering in plants. The microRNA (miRNA) miR169 family members are up-regulated in Arabidopsis, maize, and soybean under abiotic stresses. Overexpression of miR169d in Arabidopsis results in early flowering, and overexpression of the miR169d target gene, AtNF-YA2, especially a miR169d-resistant version of AtNF-YA2, results in late flowering. The results suggest that the miR169 family regulates stress-induced flowering by repressing the AtNF-YA transcription factor, which in turn reduces the expression of FLOWERING LOCUS C (FLC), allowing for the expression of FLC target genes such as FLOWERING LOCUS T (FT) and LEAFY (LFY) to promote flowering. It was shown that the expression of genes or miRNAs involved in the other flowering pathways, namely the photoperiod (CO), ambient temperature (SVP), ageing (miR156), and gibberelin (SOC1) pathways, was not affected in miR169d-overexpressing plants, suggesting that stress-induced early flowering is a novel signalling pathway mediated by miR169.

Characterizing RNA-protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation.

RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4-siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein-RNA interactions in plants.

C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms.

Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.

A simple method for comparing fungal biomass in infected plant tissues.

Plant phenotypes resistant and susceptible to fungal pathogens are usually scored using qualitative, subjective methods that are based upon disease symptoms or by an estimation of the amount of visible fungal growth. Given that plant resistance genes often confer partial resistance to fungal pathogens, a simple, sensitive, nonsubjective quantitative method for measuring pathogen growth would be highly advantageous. This report describes an in planta quantitative assay for fungal biomass based upon detection of chitin using wheat germ agglutinin conjugated to a fluorophore. Using this assay, the growth of wheat rust pathogens on wheat was assayed and the additivity of several adult plant and seedling resistance genes to Puccinia striiformis, P. graminis, and P. triticina was assayed on both glasshouse- and field-grown material. The assay can discriminate between individual rust pustules on a leaf segment or, alternatively, compare fungal growth on field plots. The quantification of Erysiphe necator (powdery mildew) growth on Vitis vinifera (grapevine) is also demonstrated, with resistant and susceptible cultivars readily distinguished. Given that chitin is a major cell wall component of many plant fungal pathogens, this robust assay will enable simple and accurate measurement of biomass accumulation in many plant-fungus interactions.

Construction of a genomewide RNAi mutant library in rice.

Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification-mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down-regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross-silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs. Our results indicate that RNAi mutant library is a high-efficient approach for genomewide gene identification in plants.