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.

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.

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.

Viral small interfering RNAs target host genes to mediate disease symptoms in plants.

The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5' RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.

Radiolabelling of steroids: the synthesis of 17α-[4-(14) C]trenbolone.

17ß-Hydroxyestra-4,9,11-trien-3-one or trenbolone is an anabolic steroid used in some meat producing countries where its use is licenced. In cattle it is metabolised into 17α-trenbolone. We were required to make 17α-[4-(14) C]trenbolone for use in environmental fate studies. At the same time we also had a request to make 17α-[4-(14) C]estradiol so we combined the two syntheses and made use of the synergy to allow us to make a batch of 17α-[4-(14) C]estradiol by known methodology and then elaborate a portion of this into 17α-[4-(14) C]trenbolone. The synthesis of 17α-[4-(14) C]trenbolone from 17α-[4-(14) C]estradiol was achieved in 8 steps and 3.1% overall yield to give material with a radiochemical purity of 99.5% and specific activity of 59 mCi/mmol.

RNA silencing and plant viral diseases.

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.

The presence of high-molecular-weight viral RNAs interferes with the detection of viral small RNAs.

Viral small interfering RNA (siRNA) accumulation in plants is reported to exhibit a strong strand polarity bias, with plus (+) strand siRNAs dominating over minus (-) strand populations. This is of particular interest, as siRNAs processed from double-stranded RNA would be expected to accumulate equivalent amounts of both species. Here, we show that, as reported, (-) strand viral siRNAs are detected at much lower levels than (+) strand-derived species using standard Northern hybridization approaches. However, when total RNA is spiked with in vitro-transcribed antisense viral genomic RNA, (-) strand viral siRNAs are detected at increased levels equivalent to those of (+) strand siRNA. Our results suggest that (+) and (-) strand viral siRNAs accumulate to equivalent levels; however, a proportion of the (-) strand siRNAs are sequestered from the total detectable small RNA population during gel electrophoresis by hybridizing to the high-molecular-weight sense strand viral genomic RNA. Our findings provide a plausible explanation for the observed strand bias of viral siRNA accumulation, and could have wider implications in the analysis of both viral and nonviral small RNA accumulation.

Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants.

Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine with thymine nucleotides, in a G:U hpRNA design, prevents self-silencing but still allows for the formation of hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90-96% of transgenic lines, compared to 57-65% for the traditional hpRNA design. While a traditional hpRNA transgene shows increasing self-silencing from cotyledons to true leaves, its G:U counterpart avoids this and induce RNAi throughout plant growth. Furthermore, siRNAs from G:U and traditional hpRNA show different characteristics and appear to function via different pathways to induce target DNA methylation.

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes.

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of double-stranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5' end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 5' thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.

Full-Length Hairpin RNA Accumulates at High Levels in Yeast but Not in Bacteria and Plants.

Hairpin-structured (hp) RNA has been widely used to induce RNA interference (RNAi) in plants and animals, and an in vivo expression system for hpRNA is important for large-scale RNAi applications. Bacterial expression systems have so far been developed for in vivo expression of hpRNA or double-stranded (ds) RNA, but the structure of the resulting RNAi molecules has remained unclear. Here we report that long hpRNAs expressed in the bacteria Escherichia coli and Sinorhizobium meliloti were largely processed into shorter dsRNA fragments with no or few full-length molecules being present. A loss-of-function mutation in the dsRNA-processing enzyme RNase III, in the widely used E. coli HT115 strain, did not prevent the processing of hpRNA. Consistent with previous observations in plants, the loop sequence of long hpRNA expressed in Agrobacterium-infiltrated Nicotiana benthamiana leaves was excised, leaving no detectable levels of full-length hpRNA molecule. In contrast to bacteria and plants, long hpRNAs expressed in the budding yeast Saccharomyces cerevisiae accumulated as intact, full-length molecules. RNA extracted from hpRNA-expressing yeast cells was shown to be capable of inducing RNAi against a ß-glucuronidase (GUS) reporter gene in tobacco leaves when applied topically on leaf surfaces. Our results indicate that yeast can potentially be used to express full-length hpRNA molecules for RNAi and perhaps other structured RNAs that are important in biological applications.

DEMETER plays a role in DNA demethylation and disease response in somatic tissues of Arabidopsis.

DNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNA methylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1) also known as DEMETER-LIKE 1 (DML1), DML2, and DML3. While ROS1, DML2, and DML3 were shown to function in disease response in somatic tissues, DME has been thought to function only in reproductive tissues to maintain the maternal-specific expression pattern of a subset of imprinted genes. Here we used promoter:ß-glucuronidase (GUS) fusion constructs to show that DME is constitutively expressed throughout the plant, and that ROS1, DML2, and DML3 have tissue-specific expression patterns. Loss-of-function mutations in DME cause seed abortion and therefore viable DME mutants are not available for gene function analysis. We knocked down DME expression in a triple ros1 dml2 dml3 (rdd) mutant background using green tissue-specific expression of a hairpin RNA transgene (RNAi), generating a viable 'quadruple' demethylase mutant line. We show that this rdd DME RNAi line has enhanced disease susceptibility to Fusarium oxysporum infection compared to the rdd triple mutant. Furthermore, several defence-related genes, previously shown to be repressed in rdd, were further repressed in the rdd DME RNAi plants. DNA methylation analysis of two of these genes revealed increased differential promoter DNA methylation in rdd DME RNAi plants compared to WT, beyond the difference observed in the parental rdd plants. These results indicate that DME contributes to DNA demethylase activity and disease response in somatic tissues.

The roles of plant dsRNA-binding proteins in RNAi-like pathways.

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway.

Root-Specific Expression of a Jacalin Lectin Family Protein Gene Requires a Transposable Element Sequence in the Promoter.

Transposable elements (TEs) are widespread in the plant genome and can impact on the expression of neighbouring genes. Our previous studies have identified a number of DNA demethylase-regulated defence-related genes that contain TE sequences in the promoter and show tissue-specific expression in Arabidopsis. In this study we investigated the role of the promoter TE insertions in the root-specific expression of a DNA demethylase-regulated gene, AT5G38550, encoding a Jacalin lectin family protein. Using a promoter:GUS fusion reporter gene approach, we first demonstrated that the full-length promoter fragment, carrying four TE sequences, contained the essential regulatory information required for root-specific expression and DNA demethylase regulation in Arabidopsis. By successive deletion of the four TE sequences, we showed that one of the four TE insertions, a 201-bp TE fragment of the hAT DNA transposon family, was required for root-specific expression: Deletion of this TE, but not the first two TE sequences, converted the root-specific expression pattern to a constitutive expression pattern in Arabidopsis plants. Our study provides an example indicating an important role of TE insertions in tissue-specific expression of plant defence-related genes.

The evolution and diversification of Dicers in plants.

Most multicellular organisms regulate developmental transitions by microRNAs, which are generated by an enzyme, Dicer. Insects and fungi have two Dicer-like genes, and many animals have only one, yet the plant, Arabidopsis, has four. Examining the poplar and rice genomes revealed that they contain five and six Dicer-like genes, respectively. Analysis of these genes suggests that plants require a basic set of four Dicer types which were present before the divergence of mono- and dicotyledonous plants ( approximately 200 million years ago), but after the divergence of plants from green algae. A fifth type of Dicer seems to have evolved in monocots.

Satellite RNA pathogens of plants: impacts and origins-an RNA silencing perspective.

Viral satellite RNAs (satRNAs) are among the smallest RNA pathogens in plants. They have little or no protein-coding capacity but can have a major impact on the host plants through trilateral interactions with helper viruses and host plants. Studies around the 1980s revealed much of what we know about satRNAs: they can affect helper virus accumulation, modulate helper virus-induced disease symptoms, and induce their own symptoms with the assistance of helper viruses which depend on specific nucleotide sequences of their genome and host species. The molecular basis of these satRNA-caused impacts and the origin of satRNAs have yet to be fully understood and revealed, but recent understanding of the antiviral RNA silencing pathways and advancement in RNA and DNA sequencing technologies have provided new avenues and opportunities to examine these unanswered questions. These RNA silencing-based studies have revealed the existence of cross silencing between some satRNAs and helper viruses, the downregulation of helper virus-encoded suppressor (VSR) of RNA silencing or inhibition/enhancement of VSR activity by satRNAs, the silencing of host-encoded genes by satRNA-derived small interfering RNA (siRNAs), and the presence of satRNA-like small RNAs in uninfected host plants. These findings have provided alternative RNA silencing-based models to explain the pathogenicity and origin of satRNAs. WIREs RNA 2016, 7:5-16. doi: 10.1002/wrna.1311 For further resources related to this article, please visit the WIREs website.

Cloning and profiling of small RNAs from cucumber mosaic virus satellite RNA.

RNA silencing is not only a gene regulation mechanism that is conserved in a broad range of eukaryotes but also an adaptive immune response against foreign nucleic acids including viruses in plants. A major feature of RNA silencing is the production of small RNA (sRNA) of 21-24 nucleotides (nt) in length from double-stranded (ds) or hairpin-like (hp) RNA by Dicer-like (DCL) proteins. These sRNAs guide the binding and cleavage of cognate single-stranded (ss) RNA by an RNA silencing complex. Like all plant viruses and subviral agents, replication of viral satellite RNAs (satRNAs) is associated with the accumulation of 21-24 nt viral small interfering RNA (vsiRNA) derived from the whole region of a satRNA genome in both plus and minus-strand polarities. These satRNA-derived siRNAs (satsiRNAs) have recently been shown to play an important role in the trilateral interactions among host plants, helper viruses and satRNAs. Here, we describe the cloning and profile analysis of satsiRNAs from satRNAs of Cucumber mosaic virus (CMV). We also describe a method to minimize the strand bias that often occurs during vsiRNA cloning and sequencing.

Satellite RNAs interfere with the function of viral RNA silencing suppressors.

Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a ß-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.

In Nicotiana species, an artificial microRNA corresponding to the virulence modulating region of Potato spindle tuber viroid directs RNA silencing of a soluble inorganic pyrophosphatase gene and the development of abnormal phenotypes.

Potato spindle tuber viroid (PSTVd) is a small non-protein-coding RNA pathogen that can induce disease symptoms in a variety of plant species. How PSTVd induces disease symptoms is a long standing question. It has been suggested that PSTVd-derived small RNAs (sRNAs) could direct RNA silencing of a targeted host gene(s) resulting in symptom development. To test this, we expressed PSTVd sequences as artificial microRNAs (amiRNAs) in Nicotiana tabacum and Nicotiana benthamiana. One amiRNA, amiR46 that corresponds to sequences within the PSTVd virulence modulating region (VMR), induced abnormal phenotypes in both Nicotiana species that closely resemble those displayed by PSTVd infected plants. In N. tabacum amiR46 plants, phenotype severity correlated with amiR46 accumulation and expression down-regulation of the bioinformatically-identified target gene, a Nicotiana soluble inorganic pyrophosphatase (siPPase). Taken together, our phenotypic and molecular analyses suggest that disease symptom development in Nicotiana species following PSTVd infection results from sRNA-directed RNA silencing of the host gene, siPPase.

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis.

BACKGROUND: DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear. RESULTS: We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection. CONCLUSIONS: Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

A fast and efficient method for preparation of high-quality RNA from fungal mycelia.

BACKGROUND: Fungal RNA samples are usually isolated from fungal mycelia grown in liquid culture, which relies on prolific growth of the fungus in liquid media. The fungal biomass is then collected by vacuum filtration, which can result in low recovery for samples with reduced biomass due to poor growth in liquid media. FINDINGS: Here we report an alternative culturing method, based on growth on solid media which is independent of the ability of a fungus to grow in liquid culture. We show that growth on solid media overlayed with a nylon membrane is superior to other culturing methods, producing large amounts of biomass and allowing for easy harvesting of fungal mycelia. Furthermore, we show that mycelium harvested with this method yielded high-quality RNA, superior to RNA isolated from liquid grown mycelium. We also show that inclusion of a second chloroform extraction step in the procedure significantly increases RNA yield. CONCLUSIONS: This method is particularly useful for fungal species that show poor or no growth in liquid media, but are easily cultured on solid media. Culturing can be performed on small petri dishes, which significantly reduces handling and therefore allowing growth and isolation of RNA from multiple strains in a high throughput manner. The obtained RNA samples are of high quality in sufficient quantities for several northern blot experiments or quantitative RT-PCR experiments.