Andrew J. Hamilton

An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus

Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.

Two classes of short interfering RNA in RNA silencing

RNA silencing is a eukaryotic genome defence system that involves processing of double‐stranded RNA (dsRNA) into 21–26 nt, short interfering RNA (siRNA). The siRNA mediates suppression of genes corresponding to the dsRNA through targeted RNA degradation. In some plant systems there are additional silencing processes, involving systemic spread of silencing and RNA‐directed methylation/transcriptional suppression of homologous genomic DNA. We show here that siRNAs produced in plants from a green fluorescent protein (GFP) transgene are in short (21–22 nt) and long (24–26 nt) size classes, whereas those from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these classes of siRNA have different roles. The long siRNA is dispensable for sequence‐specific mRNA degradation, but correlates with systemic silencing and methylation of homologous DNA. Conversely, the short siRNA class correlates with mRNA degradation but not with systemic signalling or methylation. These findings reveal an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals.

RNA–DNA Interactions and DNA Methylation in Post-Transcriptional Gene Silencing

Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transgene. In each instance, homology-based, RNA-directed methylation was associated with silencing. When the GFP-transcribed region was targeted, PTGS affected both transgene and viral RNA levels. When methylation was targeted to a promoter region, transgene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) by inoculation with potato virus X–rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP-transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transgene associated with systemic spread of a gene-silencing signal from the infiltrated part of the plant. This transgene methylation was not affected when systemic PTGS was blocked by suppressors of silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epigenetic model of PTGS in which transgene methylation is associated with an RNA–DNA interaction that ensures that PTGS is maintained.

Improved northern blot method for enhanced detection of small RNA.

This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.

Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40 nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.

Virus-Induced Silencing of a Plant Cellulose Synthase Gene

Specific cDNA fragments corresponding to putative cellulose synthase genes (CesA) were inserted into potato virus X vectors for functional analysis in Nicotiana benthamiana by using virus-induced gene silencing. Plants infected with one group of cDNAs had much shorter internode lengths, small leaves, and a “dwarf” phenotype. Consistent with a loss of cell wall cellulose, abnormally large and in many cases spherical cells ballooned from the undersurfaces of leaves, particularly in regions adjacent to vascular tissues. Linkage analyses of wall polysaccharides prepared from infected leaves revealed a 25% decrease in cellulose content. Transcript levels for at least one member of the CesA cellulose synthase gene family were lower in infected plants. The decrease in cellulose content in cell walls was offset by an increase in homogalacturonan, in which the degree of esterification of carboxyl groups decreased from ∼50 to ∼33%. The results suggest that feedback loops interconnect the cellular machinery controlling cellulose and pectin biosynthesis. On the basis of the phenotypic features of the infected plants, changes in wall composition, and the reduced abundance of CesA mRNA, we concluded that the cDNA fragments silenced one or more cellulose synthase genes.

Taking the “Waste” Out of “Wastewater” for Human Water Security and Ecosystem Sustainability

Humans create vast quantities of wastewater through inefficiencies and poor management of water systems. The wasting of water poses sustainability challenges, depletes energy reserves, and undermines human water security and ecosystem health. Here we review emerging approaches for reusing wastewater and minimizing its generation. These complementary options make the most of scarce freshwater resources, serve the varying water needs of both developed and developing countries, and confer a variety of environmental benefits. Their widespread adoption will require changing how freshwater is sourced, used, managed, and priced.

Quantitative Microbial Risk Assessment Models for Consumption of Raw Vegetables Irrigated with Reclaimed Water

ABSTRACT Quantitative microbial risk assessment models for estimating the annual risk of enteric virus infection associated with consuming raw vegetables that have been overhead irrigated with nondisinfected secondary treated reclaimed water were constructed. We ran models for several different scenarios of crop type, viral concentration in effluent, and time since last irrigation event. The mean annual risk of infection was always less for cucumber than for broccoli, cabbage, or lettuce. Across the various crops, effluent qualities, and viral decay rates considered, the annual risk of infection ranged from 10−3 to 10−1 when reclaimed-water irrigation ceased 1 day before harvest and from 10−9 to 10−3 when it ceased 2 weeks before harvest. Two previously published decay coefficients were used to describe the die-off of viruses in the environment. For all combinations of crop type and effluent quality, application of the more aggressive decay coefficient led to annual risks of infection that satisfied the commonly propounded benchmark of ≤10−4, i.e., one infection or less per 10,000 people per year, providing that 14 days had elapsed since irrigation with reclaimed water. Conversely, this benchmark was not attained for any combination of crop and water quality when this withholding period was 1 day. The lower decay rate conferred markedly less protection, with broccoli and cucumber being the only crops satisfying the 10−4 standard for all water qualities after a 14-day withholding period. Sensitivity analyses on the models revealed that in nearly all cases, variation in the amount of produce consumed had the most significant effect on the total uncertainty surrounding the estimate of annual infection risk. The models presented cover what would generally be considered to be worst-case scenarios: overhead irrigation and consumption of vegetables raw. Practices such as subsurface, furrow, or drip irrigation and postharvest washing/disinfection and food preparation could substantially lower risks and need to be considered in future models, particularly for developed nations where these extra risk reduction measures are more common.

Potato Virus X Amplicons in Arabidopsis Mediate Genetic and Epigenetic Gene Silencing

Amplicon transgenes from potato virus X (PVX) are based on a modified version of the viral genome and are efficient activators of post-transcriptional gene silencing (PTGS). To determine whether PVX amplicons activate PTGS in Arabidopsis, we used constructs based on the genome of PVX carrying a green fluorescent protein (GFP) reporter gene. Our analysis of the transgene phenotype exploited previous observations indicating that PTGS is associated with short 25-nucleotide RNA species, transgene methylation, and homology-dependent virus resistance. We also used the ability of turnip mosaic virus to suppress gene silencing as a means of dissecting stages of the mechanism. The results showed that a PVX:GFP amplicon induces weak PTGS and that this PTGS was enhanced in the presence of a GFP reporter gene. Our interpretation of these data is that the PTGS induced by the amplicon was genetically determined and equivalent to the initiation stage of the PTGS mechanism. The PTGS induced by the combined amplicon and reporter gene was equivalent to the maintenance stage and was associated with an epigenetic conversion of the transgene. The distinction between genetic and epigenetic PTGS explains the well-characterized effects of transgene dosage on PTGS that have been previously interpreted in terms of RNA expression thresholds.

Quantifying Uncertainty in Estimation of Tropical Arthropod Species Richness

There is a bewildering range of estimates for the number of arthropods on Earth. Several measures are based on extrapolation from species specialized to tropical rain forest, each using specific assumptions and justifications. These approaches have not provided any sound measure of uncertainty associated with richness estimates. We present two models that account for parameter uncertainty by replacing point estimates with probability distributions. The models predict medians of 3.7 million and 2.5 million tropical arthropod species globally, with 90% confidence intervals of [2.0, 7.4] million and [1.1, 5.4] million, respectively. Estimates of 30 million or greater are predicted to have <0.00001 probability. Sensitivity analyses identified uncertainty in the proportion of canopy arthropod species that are beetles as the most influential parameter, although uncertainties associated with three other parameters were also important. Using the median estimates suggests that in spite of 250 years of taxonomy and around 855,000 species of arthropods already described, approximately 70% await description.