RNA-Based Control of Fungal Pathogens in Plants.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Identification of transcription factors controlling cell wall invertase gene expression for reproductive development via bioinformatic and transgenic analyses.

Cell wall invertase (CWIN) hydrolyses sucrose into glucose and fructose in the extracellular matrix and plays crucial roles in assimilate partitioning and sugar signalling. However, the molecular regulators controlling CWIN gene transcription remain unknown. As the first step to address this issue, we performed bioinformatic and transgenic studies, which identified a cohort of transcription factors (TFs) modulating CWIN gene expression in Arabidopsis thaliana. Comprehensive bioinformatic analyses identified 18 TFs as putative regulators of the expression of AtCWIN2 and AtCWIN4 that are predominantly expressed in Arabidopsis reproductive organs. Among them, MYB21, ARF6, ARF8, AP3 and CRC were subsequently shown to be the most likely regulators of CWIN gene expression based on molecular characterization of the respective mutant of each candidate TF. More specifically, the obtained data indicate that ARF6, ARF8 and MYB21 regulate CWIN2 expression in the anthers and CWIN4 in nectaries, anthers and petals, whereas AP3 and CRC were determined primarily to regulate the transcriptional activity of CWIN4. TF-promoter interaction assays demonstrated that ARF6 and ARF8 directly control CWIN2 and CWIN4 transcription with AP3 activating CWIN4. The involvement of ARF8 in regulating CWIN4 expression was further supported by the finding that enhanced CWIN4 expression partially recovered the short silique phenotype displayed by the arf8-3 mutant. The identification of the five TFs regulating CWIN expression serves as a launching pad for future studies to dissect the upstream molecular network underpinning the transcription of CWINs and provides a new avenue, potentially, to engineer assimilate allocation and reproductive development for improving seed yield.

Genetic Variants Associated with Long-Terminal Repeats Can Diagnostically Classify Cannabis Varieties.

Cannabis sativa (Cannabis) has recently been legalized in multiple countries globally for either its recreational or medicinal use. This, in turn, has led to a marked increase in the number of Cannabis varieties available for use in either market. However, little information currently exists on the genetic distinction between adopted varieties. Such fundamental knowledge is of considerable value and underpins the accelerated development of both a nascent pharmaceutical industry and the commercial recreational market. Therefore, in this study, we sought to assess genetic diversity across 10 Cannabis varieties by undertaking a reduced representation shotgun sequencing approach on 83 individual plants to identify variations which could be used to resolve the genetic structure of the assessed population. Such an approach also allowed for the identification of the genetic features putatively associated with the production of secondary metabolites in Cannabis. Initial analysis identified 3608 variants across the assessed population with phylogenetic analysis of this data subsequently enabling the confident grouping of each variety into distinct subpopulations. Within our dataset, the most diagnostically informative single nucleotide polymorphisms (SNPs) were determined to be associated with the long-terminal repeat (LTRs) class of retroelements, with 172 such SNPs used to fully resolve the genetic structure of the assessed population. These 172 SNPs could be used to design a targeted resequencing panel, which we propose could be used to rapidly screen different Cannabis plants to determine genetic relationships, as well as to provide a more robust, scientific classification of Cannabis varieties as the field moves into the pharmaceutical sphere.

A novel role for milk fat globule-EGF factor 8 protein (MFGE8) in the mediation of mouse sperm-extracellular vesicle interactions.

Spermatozoa transition to functional maturity as they are conveyed through the epididymis, a highly specialized region of the male excurrent duct system. Owing to their transcriptionally and translationally inert state, this transformation into fertilization competent cells is driven by complex mechanisms of intercellular communication with the secretory epithelium that delineates the epididymal tubule. Chief among these mechanisms are the release of extracellular vesicles (EV), which have been implicated in the exchange of varied macromolecular cargo with spermatozoa. Here, we describe the optimization of a tractable cell culture model to study the mechanistic basis of sperm-extracellular vesicle interactions. In tandem with receptor inhibition strategies, our data demonstrate the importance of milk fat globule-EGF factor 8 (MFGE8) protein in mediating the efficient exchange of macromolecular EV cargo with mouse spermatozoa; with the MFGE8 integrin-binding Arg-Gly-Asp (RGD) tripeptide motif identified as being of particular importance. Specifically, complementary strategies involving MFGE8 RGD domain ablation, competitive RGD-peptide inhibition and antibody-masking of alpha V integrin receptors, all significantly inhibited the uptake and redistribution of EV-delivered proteins into immature mouse spermatozoa. These collective data implicate the MFGE8 ligand and its cognate integrin receptor in the mediation of the EV interactions that underpin sperm maturation.

The key role of terminators on the expression and post-transcriptional gene silencing of transgenes.

Transgenes have become essential to modern biology, being an important tool in functional genomic studies and also in the development of biotechnological products. One of the major challenges in the generation of transgenic lines concerns the expression of transgenes, which, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). Several reasons have been put forward to explain why transgenes often trigger the production of sRNAs, such as the high level of expression induced by commonly used strong constitutive promoters, the lack of introns, and features resembling viral and other exogenous sequences. However, the relative contributions of the different genomic elements with respect to protecting genes from the silencing machinery and their molecular mechanisms remain unclear. Here, we present the results of a mutagenesis screen conceived to identify features involved in the protection of endogenes against becoming a template for the production of sRNAs. Interestingly, all of the recovered mutants had alterations in genes with proposed function in transcription termination, suggesting a central role of terminators in this process. Indeed, using a GFP reporter system, we show that, among different genetic elements tested, the terminator sequence had the greatest effect on transgene-derived sRNA accumulation and that a well-defined poly(A) site might be especially important. Finally, we describe an unexpected mechanism, where transgenes containing certain intron/terminator combinations lead to an increase in the production of sRNAs, which appears to interfere with splicing.

Post-testicular sperm maturation in the saltwater crocodile Crocodylus porosus: assessing the temporal acquisition of sperm motility.

Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this study we examined the development of motility during sperm transport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasing motility (percentage motile and rate of movement) as they transit the male reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMP levels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals.

Gross and microanatomy of the male reproductive duct system of the saltwater crocodile Crocodylus porosus.

Information on the morphology and histology of the male reproductive system of the Crocodylia species is necessary to determine the role of these tissues in the production of functional spermatozoa. Accordingly, in this study we examined the gross morphology and microanatomy of the testis and the male excurrent duct system through which spermatozoa pass before ejaculation. The data demonstrate that the reproductive system in male saltwater crocodiles comprises paired testes, which convey spermatozoa distally via the rete testis into an excurrent duct system comprising ductuli efferentes, ductuli epididymides, ductus epididymidis and ductus deferens. The epithelium delineating the male tract was dominated by non-ciliated and ciliated cells structured into a simple columnar lining of the ductuli efferentes and ductuli epididymides, through to the high pseudostratified columnar epithelium of the ductus epididymidis and ductus deferens. The morphology and histochemical staining of these ducts suggest their involvement in seminal fluid production and/or its modification, which likely contributes to the nourishment, protection and/or storage of crocodile spermatozoa. As a reflection of their common Archosaurs ancestry, the overall structural characteristics we describe for the crocodile male excurrent duct system share closer similarities to those of the Aves than other clades within the Reptilia class or Mammalia.

Molecular Manipulation of MicroRNA397 Abundance Influences the Development and Salt Stress Response of Arabidopsis thaliana.

Arabidopsis thaliana (Arabidopsis) has been used extensively as a heterologous system for molecular manipulation to genetically characterize both dicotyledonous and monocotyledonous plant species. Here, we report on Arabidopsis transformant lines molecularly manipulated to over-accumulate the small regulatory RNA microRNA397 (miR397) from the emerging C4 monocotyledonous grass model species Setaria viridis (S. viridis). The generated transformant lines, termed SvMIR397 plants, displayed a range of developmental phenotypes that ranged from a mild, wild-type-like phenotype, to a severe, full dwarfism phenotype. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based profiling of the SvMIR397 transformant population revealed a strong correlation between the degree of miR397 over-accumulation, repressed LACCASE (LAC) target gene expression, reduced lignin content, and the severity of the developmental phenotype displayed by SvMIR397 transformants. Further, exposure of SvMIR397 transformants to a 7-day regime of salt stress revealed the SvMIR397 transformant lines to be more sensitive to the imposed stress than were wild-type Arabidopsis plants. Taken together, the findings reported here via the use of Arabidopsis as a heterologous system show that the S. viridis miR397 small regulatory RNA is able to repress the expression of three Arabidopsis LAC genes which led to reduced lignin content and increased salt stress sensitivity.

The contribution of epididymosomes to the sperm small RNA profile.

It is now well established that mature spermatozoa harbour a rich and diverse profile of small non-protein-coding regulatory RNAs (sRNAs). There is also growing appreciation that this sRNA profile displays considerable plasticity, being altered in response to paternal exposure to a variety of environmental stressors. Coupled with evidence that upon delivery to the oocyte at the moment of fertilisation, sperm-borne sRNAs are able to influence both early embryonic development and the subsequent health of the offspring, there is now interest in both the timing and degree of change in the composition of the sRNA cargo of sperm. Models in which such epigenetic changes are linked to the spermatogenic cycle are seemingly incompatible with the lack of overt phenotypic changes in the spermatozoa of affected males. Rather, there is mounting consensus that such changes are imposed on sperm during their transit and storage within the epididymis, a protracted developmental window that takes place over several weeks. Notably, since spermatozoa are rendered transcriptionally and translationally silent during their development in the testes, it is most likely that the epididymis-documented alterations to the sperm sRNA profile are driven extrinsically, with a leading candidate being epididymosomes: small membrane enclosed extracellular vesicles that encapsulate a complex macromolecular cargo of proteins and RNAs, including the sRNAs. Here, we review the role of epididymosome-sperm communication in contributing to the establishment of the sperm sRNA profile during their epididymal transit.

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula.

Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi-allelic double mutant for the two soya bean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer-like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole-genome sequencing to reveal a spectrum of non-germ-line-targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development.

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development.

Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa.

Post-testicular sperm maturation and storage within the epididymis is a key determinant of gamete quality and fertilization competence. Here we demonstrate that mouse spermatozoa possess a complex small non-protein-coding RNA (sRNA) profile, the composition of which is markedly influenced by their epididymal transit. Thus, although microRNAs (miRNAs) are highly represented in the spermatozoa of the proximal epididymis, this sRNA class is largely diminished in mature spermatozoa of the distal epididymis. Coincident with this, a substantial enrichment in Piwi-interacting RNA (piRNA) abundance in cauda spermatozoa was detected. Further, features of cauda piRNAs, including; predominantly 29-31 nts in length; preference for uracil at their 5' terminus; no adenine enrichment at piRNA nt 10, and; predominantly mapping to intergenic regions of the mouse genome, indicate that these piRNAs are generated by the PIWIL1-directed primary piRNA production pathway. Accordingly, PIWIL1 was detected via immunoblotting and mass spectrometry in epididymal spermatozoa. These data provide insight into the complexity and dynamic nature of the sRNA profile of spermatozoa and raise the intriguing prospect that piRNAs are generated in situ in maturing spermatozoa. Such information is of particular interest in view of the potential role for paternal sRNAs in influencing conception, embryo development and intergenerational inheritance.

MicroRNA Regulatory Mechanisms Play Different Roles in Arabidopsis.

Plant microRNAs (miRNAs) operate by guiding the cleavage or translational inhibition of mRNA targets. They act as key gene regulators for development and environmental adaptation, and Dicer-partnering proteins DRB1 and DRB2 govern which form of regulation plays the dominant role. Mutation of Drb1 impairs transcript cleavage, whereas mutation of Drb2 ablates translational inhibition. Regulation of gene expression by miRNA-guided cleavage has been extensively studied, but there is much less information about genes regulated through miRNA-mediated translation inhibition. Here, we compared the proteomes of drb1 and drb2 mutants to gain insight into the indirect effect of the different miRNA regulatory mechanisms in Arabidopsis thaliana. Our results show that miRNAs operating through transcript cleavage regulate a broad spectrum of processes, including catabolism and anabolism, and this was particularly obvious in the fatty acid degradation pathway. Enzymes catalyzing each step of this pathway were upregulated in drb1. In contrast, DRB2-associated translational inhibition appears to be less ubiquitous and specifically aimed toward responses against abiotic or biotic stimuli.

A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis.

It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families.

Assessment of the impact of direct in vitro PFAS treatment on mouse spermatozoa.

Abstract: Poly- and per-fluoroalkyl substances (PFAS) are synthetic environmentally persistent chemicals. Despite the phaseout of specific PFAS, their inherent stability has resulted in ubiquitous and enduring environmental contamination. PFAS bioaccumulation has been reported globally with omnipresence in most populations wherein they have been associated with a range of negative health effects, including strong associations with increased instances of testicular cancer and reductions in overall semen quality. To elucidate the biological basis of such effects, we employed an acute in vitro exposure model in which the spermatozoa of adult male mice were exposed to a cocktail of PFAS chemicals at environmentally relevant concentrations. We hypothesized that direct PFAS treatment of spermatozoa would induce reactive oxygen species generation and compromise the functional profile and DNA integrity of exposed cells. Despite this, post-exposure functional testing revealed that short-term PFAS exposure (3 h) did not elicit a cytotoxic effect, nor did it overtly influence the functional profile, capacitation rate, or the in vitro fertilization ability of spermatozoa. PFAS treatment of spermatozoa did, however, result in a significant delay in the developmental progression of the day 4 pre-implantation embryos produced in vitro. This developmental delay could not be attributed to a loss of sperm DNA integrity, DNA damage, or elevated levels of intracellular reactive oxygen species. When considered together, the results presented here raise the intriguing prospect that spermatozoa exposed to a short-term PFAS exposure period potentially harbor an alternate stress signal that is delivered to the embryo upon fertilization. Lay summary: PFAS are synthetic chemicals widely used in non-stick cookware, food packaging, and firefighting foam. Such extensive use has led to concerning levels of environmental contamination and reports of associations with a spectrum of negative health outcomes, including testicular cancer and reduced semen quality. To investigate the effects of PFAS on male reproduction, we incubated mouse sperm in a cocktail of nine PFAS at environmentally relevant concentrations before checking for a range of functional outcomes. This treatment strategy was not toxic to the sperm; it did not kill them or reduce their motility, nor did it affect their fertilization capacity. However, we did observe developmental delays among pre-implantation embryos created using PFAS-treated sperm. Such findings raise the intriguing prospect that PFAS-exposed sperm harbor a form of stress signal that they deliver to the embryo upon fertilization.

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.

Miniature Inverted-Repeat Transposable Elements: Small DNA Transposons That Have Contributed to Plant MICRORNA Gene Evolution.

Angiosperms form the largest phylum within the Plantae kingdom and show remarkable genetic variation due to the considerable difference in the nuclear genome size of each species. Transposable elements (TEs), mobile DNA sequences that can amplify and change their chromosome position, account for much of the difference in nuclear genome size between individual angiosperm species. Considering the dramatic consequences of TE movement, including the complete loss of gene function, it is unsurprising that the angiosperms have developed elegant molecular strategies to control TE amplification and movement. Specifically, the RNA-directed DNA methylation (RdDM) pathway, directed by the repeat-associated small-interfering RNA (rasiRNA) class of small regulatory RNA, forms the primary line of defense to control TE activity in the angiosperms. However, the miniature inverted-repeat transposable element (MITE) species of TE has at times avoided the repressive effects imposed by the rasiRNA-directed RdDM pathway. MITE proliferation in angiosperm nuclear genomes is due to their preference to transpose within gene-rich regions, a pattern of transposition that has enabled MITEs to gain further transcriptional activity. The sequence-based properties of a MITE results in the synthesis of a noncoding RNA (ncRNA), which, after transcription, folds to form a structure that closely resembles those of the precursor transcripts of the microRNA (miRNA) class of small regulatory RNA. This shared folding structure results in a MITE-derived miRNA being processed from the MITE-transcribed ncRNA, and post-maturation, the MITE-derived miRNA can be used by the core protein machinery of the miRNA pathway to regulate the expression of protein-coding genes that harbor homologous MITE insertions. Here, we outline the considerable contribution that the MITE species of TE have made to expanding the miRNA repertoire of the angiosperms.

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.

DEFECTIVE EMBRYO AND MERISTEMS1 (DEM1) Is Essential for Cell Proliferation and Cell Differentiation in Tomato.

Most flowering plant species contain at least two copies of the DEFECTIVE EMBRYO AND MERISTEMS (DEM) gene with the encoded DEM proteins lacking homology to proteins of known biochemical function. In tomato (Sl; Solanum lycopersicum), stable mutations in the SlDEM1 locus result in shoot and root meristem defects with the dem1 mutant failing to progress past the cotyledon stage of seedling development. Generation of a Somatic Mutagenesis of DEM1 (SMD) transformant line in tomato allowed for the characterization of SlDEM1 gene function past the seedling stage of vegetative development with SMD plants displaying a range of leaf development abnormalities. Further, the sectored or stable in planta expression of specific regions of the SlDEM1 coding sequence also resulted in the generation of tomato transformants that displayed a range of vegetative development defects, which when considered together with the dem1 mutant seedling and SMD transformant line phenotypic data, allowed for the assignment of SlDEM1 gene function to early embryo development, adaxial epidermis cell development, lateral leaf blade expansion, and mesophyll cell proliferation and differentiation.

Quantitative proteomic dataset of mouse caput epididymal epithelial cells exposed to acrylamide in vivo.

This article reports the proteomic legacy of in vivo exposure to the xenobiotic, acrylamide, on the epithelial cell population of the proximal segments of the mouse epididymis. Specifically, adult male mice were administered acrylamide (25 mg/kg bw/day) or vehicle control for five consecutive days before dissection of the epididymis. Epididymal epithelial cells were isolated from the proximal (caput) epididymal segment and subjected to quantitative proteomic analysis using multiplexed tandem mass tag (TMT) labeling coupled to mass spectrometry. Here, we report the data generated by this strategy, including the identification of 4405 caput epididymal epithelial cell proteins, approximately 6.8% of which displayed altered expression in response to acrylamide challenge. Our interpretation and discussion of these data features in the article "Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development".