Ulrike Schumann

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

BackgroundDNA 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.ResultsWe 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.ConclusionsOur 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 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.

RNA silencing in fungi

RNA silencing is an evolutionarily conserved mechanism in eukaryotic organisms induced by double-stranded RNA (dsRNA) and plays an essential role in regulating gene expression and maintaining genome stability. RNA silencing occurs at both posttranscriptional levels through sequence-specific RNA degradation or translational repression and at transcriptional levels through RNA-directed DNA methylation and/or heterochromatin formation. RNA silencing pathways have been relatively well characterized in plants and animals, and are now also being widely investigated in diverse fungi, some of which are important plant pathogens. This review focuses primarily on the current understanding of the dsRNA-mediated posttranscriptional gene silencing processes in fungi, but also discusses briefly the known gene silencing pathways that appear to be independent of the RNA silencing machineries. We review RNA silencing studies for a variety of fungi and highlight some of the mechanistic differences observed in different fungal organisms. As RNA silencing is being exploited as a technology in gene function studies in fungi as well as in engineering anti-fungal resistance in plants and animals, we also discuss the recent progress towards understanding dsRNA uptake in fungi.

Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum

BackgroundHairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing.ResultsHere we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum.ConclusionsThese results indicate that F. oxysporum possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage, but hpRNA transgenes may induce transcriptional self-silencing due to its inverted-repeat structure. Our results suggest that F. oxysporum possesses a similar gene silencing pathway to other fungi like fission yeast, and indicate a need for developing more effective RNA silencing technology for gene function studies in this fungal pathogen.

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 β-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.

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

BackgroundFungal 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.FindingsHere 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.ConclusionsThis 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.

Simple quantification of in planta fungal biomass.

An accurate assessment of the disease resistance status of plants to fungal pathogens is an essential requirement for the development of resistant crop plants. Many disease resistance phenotypes are partial rather than obvious immunity and are frequently scored using subjective qualitative estimates of pathogen development or plant disease symptoms. Here we report a method for the accurate comparison of total fungal biomass in plant tissues. This method, called the WAC assay, is based upon the specific binding of the plant lectin wheat germ agglutinin to fungal chitin. The assay is simple, high-throughput, and sensitive enough to discriminate between single Puccinia graminis f.sp tritici infection sites on a wheat leaf segment. It greatly lends itself to replication as large volumes of tissue can be pooled from independent experiments and assayed to provide truly representative quantification, or, alternatively, fungal growth on a single, small leaf segment can be quantified. In addition, as the assay is based upon a microscopic technique, pathogen infection sites can also be examined at high magnification prior to quantification if desired and average infection site areas are determined. Previously, we have demonstrated the application of the WAC assay for quantifying the growth of several different pathogen species in both glasshouse grown material and large-scale field plots. Details of this method are provided within.

DNA-Demethylase Regulated Genes Show Methylation-Independent Spatiotemporal Expression Patterns

Recent research has indicated that a subset of defense-related genes is downregulated in the Arabidopsis DNA demethylase triple mutant rdd (ros1 dml2 dml3) resulting in increased susceptibility to the fungal pathogen Fusarium oxysporum. In rdd plants these downregulated genes contain hypermethylated transposable element sequences (TE) in their promoters, suggesting that this methylation represses gene expression in the mutant and that these sequences are actively demethylated in wild-type plants to maintain gene expression. In this study, the tissue-specific and pathogen-inducible expression patterns of rdd-downregulated genes were investigated and the individual role of ROS1, DML2, and DML3 demethylases in these spatiotemporal regulation patterns was determined. Large differences in defense gene expression were observed between pathogen-infected and uninfected tissues and between root and shoot tissues in both WT and rdd plants, however, only subtle changes in promoter TE methylation patterns occurred. Therefore, while TE hypermethylation caused decreased gene expression in rdd plants it did not dramatically effect spatiotemporal gene regulation, suggesting that this latter regulation is largely methylation independent. Analysis of ros1-3, dml2-1, and dml3-1 single gene mutant lines showed that promoter TE hypermethylation and defense-related gene repression was predominantly, but not exclusively, due to loss of ROS1 activity. These data demonstrate that DNA demethylation of TE sequences, largely by ROS1, promotes defense-related gene expression but does not control spatiotemporal expression in Arabidopsis. Summary: Ros1-mediated DNA demethylation of promoter transposable elements is essential for activation of defense-related gene expression in response to fungal infection in Arabidopsis thaliana.

RNAModR: Functional analysis of mRNA modifications in R

Motivation Research in the emerging field of epitranscriptomics is increasingly generating comprehensive maps of chemical modifications in messenger RNAs (mRNAs). A computational framework allowing a reproducible and standardised analysis of these mRNA modification data is missing, but will be crucial for reliable functional meta-gene analyses and cross-study comparisons. Results We have developed RNAModR, an open-source and R-based set of methods, to analyse and visualise the transcriptome-wide distribution of mRNA modifications. RNAModR allows the statistical evaluation of the mRNA modification site distribution relative to null sites on a meta-gene level, providing insight into the functional role of these mRNA modifications on e.g. mRNA structure and stability. Availability and implementation RNAModR is available under the GNU General Public License (GPL) as an R-package from https://github.com/mevers/RNAModR. Contact maurits.evers@anu.edu.au