Plant genomes contain large numbers of cell surface leucine-rich repeat (LRR) and intracellular nucleotide binding (NB)-LRR immune receptors encoded by resistance (R) genes that recognize specific pathogen effectors and trigger resistance responses. The unregulated expression of NB-LRR genes can trigger autoimmunity in the absence of pathogen infection and inhibit plant growth. Despite the potential serious consequence on agricultural production, the mechanisms regulating R-gene expression are not well understood. We identified microRNA (miRNA) progenitor genes precursor transcripts, and two miRNAs [nta-miR6019 (22-nt) and ntamiR6020 (21-nt)] that guide cleavage of transcripts of the Toll and Interleukin-1 receptor-NB-LRR immune receptor N from tobacco that confers resistance to tobacco mosaic virus (TMV). We further showed that cleavage by nta-miR6019 triggers RNA-dependent RNA polymerase 6-and ribonuclease Dicer-like 4-dependent biogenesis of 21-nt secondary siRNAs "in phase" with the 22-nt miR6019 cleavage site. Furthermore, we found that processing of the 22-nt nta-miR6019 depended on an asymmetric bulge caused by mismatch in the nta-miR6019 precursor. Interestingly, coexpression of N with nta-miR6019 and nta-miR6020 resulted in attenuation of N-mediated resistance to TMV, indicating that these miRNAs have functional roles in NB-LRR regulation. Using a bioinformatics approach, we identified six additional 22-nt miRNA and two 21-nt miRNA families from three Solanaceae species-tobacco, tomato, and potato. We show that members of these miRNA families cleave transcripts of predicted functional R genes and trigger production of phased secondary 21-nt siRNAs. Our results demonstrate a conserved role for miRNAs and secondary siRNAs in NB-LRR/LRR immune receptor gene regulation and pathogen resistance in Solanaceae.

Imprinted gene expression occurs during seed development in plants and is associated with differential DNA methylation of parental alleles, particularly at proximal transposable elements (TEs). Imprinting variability could contribute to observed parent-of-origin effects on seed development. We investigated intraspecific variation in imprinting, coupled with analysis of DNA methylation and small RNAs, among three Arabidopsis strains with diverse seed phenotypes. The majority of imprinted genes were parentally biased in the same manner among all strains. However, we identified several examples of allele-specific imprinting correlated with intraspecific epigenetic variation at a TE. We successfully predicted imprinting in additional strains based on methylation variability. We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation. Our data demonstrate that epiallelic variation and genomic imprinting intersect to produce novel gene expression patterns in seeds.DOI: http://dx.doi.org/10.7554/eLife.03198.001

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.

The ecologically diverse genus Coniochaeta (Coniochaetaceae, Ascomycota) contains numerous endophytic strains that occur in healthy leaves and lichen thalli in temperate and boreal North America. These endophytes frequently represent undescribed species. Here we examine two endophytic isolates of Coniochaeta from healthy photosynthetic tissue of Platycladus orientalis (Cupressaceae), a conifer cultivated for horticultural use in Arizona, USA. On the basis of morphology, in vitro assays, phylogenetic analyses of two loci, and analyses of whole genome data, we designate these endophytes as a novel species, Coniochaeta endophytica sp. nov. Strains of C. endophytica are closely related to an isolate from a native lichen in North Carolina, which we also characterize here. We compare C. endophytica with two known species that appear to be close relatives: C. prunicola, associated with wood necrosis in stonefruit trees in South Africa, and C. cephalothecoides, isolated from soil in Asia. The new species is distinct in phylogenetic, in vitro, and whole-genome analyses from C. prunicola, and differs slightly in conidiophore morphology from that species. Although available sequence data for C. cephalothecoides are of uncertain relation to the type specimen for that species, our results support the distinctiveness of C. endophytica on the basis of morphology, perithecial formation, and phylogenetic analyses. We discuss the challenge of identifying new species in the context of fungal ecology surveys, such as those for endophytes, which often rely only on a single locus and can misidentify taxa based on their closest matches in public databases or simple comparisons of barcode sequences alone.

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation of the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/015941 doi: bioRxiv preprint first posted online Mar. 4, 2015; Author SummaryOrganisms must adapt to dynamic and variable internal and external environments. Maintaining homeostasis in core biological processes is crucial to minimizing the deleterious consequences of environmental fluctuations. Genomes are also dynamic and variable, and must be robust against stresses, including the invasion of genomic parasites, such as transposable elements (TEs). In this work we present the discovery of an epigenetic rheostat in plants that maintains homeostasis in levels of DNA methylation. DNA methylation typically silences transcription of TEs. Because there is positive feedback between existing and de novo DNA methylation, it is critical that methylation is not allowed to spread and potentially silence transcription of genes. To maintain homeostasis, methylation promotes the production of a demethylase enzyme that removes methylation from gene-proximal regions. The demethylation of genes is therefore always maintained in concert with the levels of methylation suppressing TEs. In addition, this DNA demethylating enzyme also represses its own production in a negative feedback loop. Together, these feedback mechanisms shed new light into how the conflict between gene expression and genome defense is maintained in homeostasis. The presence of this rheostat in multiple species suggests it is an evolutionary conserved adaptation.

Summary• Two fundamental types of polyploids are known: allopolyploids, in which different parental chromosome sets were combined by ancestral hybridization and duplication; and autopolyploids, which derive from multiplication of the same chromosome set. In autopolyploids, changes to the nuclear environment are not as profound as in allopolyploids, and therefore the effects of genome doubling on gene regulation remain unclear.• To investigate the consequences of autopolyploidization per se, we performed a microarray analysis in three equivalent lineages of matched diploids and autotetraploids of Arabidopsis thaliana. Additionally, we compared the expression levels of GFP transgenes driven by endogenous enhancer elements (enhancer traps) in diploids and autotetraploid of 16 transgenic lines.• We expected that true ploidy-dependent changes should occur in independently derived autopolyploid lineages. By this criterion, our microarray analysis detected few changes associated with polyploidization, while the enhancer-trap analysis revealed altered GFP expression at multiple plant life stages for 25% of the lines tested. Genes on individual traps were coordinately regulated while endogenous gene expression was not affected except for one line.• The unique sensitivity of enhancer traps to ploidy, in contrast to the observed stability of genes, could derive from lower complexity of regulatory pathways acting on traps versus endogenous genes.

The contribution of epigenetic variation to phenotypic variation is unclear. Imprinted genes, because of their strong association with epigenetic modifications, represent an opportunity for the discovery of such phenomena. In mammals and flowering plants, a subset of genes are expressed from only one parental allele in a process called gene imprinting. Imprinting is associated with differential DNA methylation and chromatin modifications between parental alleles. In flowering plants imprinting occurs in a seed tissue - endosperm. Proper endosperm development is essential for the production of viable seeds. We previously showed that in Arabidopsis thaliana intraspecific imprinting variation is correlated with naturally occurring DNA methylation polymorphisms. Here, we investigated the mechanisms and function of allele-specific imprinting of the class IV homeodomain leucine zipper (HD-ZIP) transcription factor HDG3. In imprinted strains, HDG3 is expressed primarily from the methylated paternally inherited allele. We manipulated the methylation state of endogenous HDG3 in a non-imprinted strain and demonstrated that methylation of a proximal transposable element is sufficient to promote HDG3 expression and imprinting. Gain of HDG3 imprinting was associated with earlier endosperm cellularization and changes in seed weight. These results indicate that epigenetic variation alone is sufficient to explain imprinting variation and demonstrate that epialleles can underlie variation in seed development phenotypes.

Tomato bushy stunt virus (TBSV) coat protein (CP) replacement vectors have been used previously to silence transgenes (e.g., the green fluorescent protein gene) but have not been effective for silencing endogenous plant genes. New TBSV vectors which retained the CP gene were developed by engineering an XhoI restriction site in three positions (3f, CEB, and CEA) of the pTBSV-100 infectious clone. Magnesium chelatase (ChlH) and phytoene desaturase (PDS) were chosen as targets for endogenous gene silencing. Initial experiments using CP replacement vectors with a 230-bp sense or antisense ChlH insert gave a silencing phenotype prominent only in the first new leaves above those inoculated. No silencing phenotype was apparent beyond these leaves whereas, for PDS, no silencing phenotype was observed. When plants were inoculated with the XhoI insert vectors containing ChlH and PDS sequences, plants showed a silencing phenotype extensively throughout the challenged plant, indicating an improved ability for virus movement and silencing in Nicotiana benthamiana host plants. Silencing efficiencies were quantified using realtime reverse-transcription polymerase chain reaction, indicating specific silencing effects of each individual silencing vector. Only one recombinant vector (pPD-3f5), where the XhoI insert was at the 3' end of the CP gene, failed to give effective silencing. Here, we show that our new CP-retaining TBSV vectors (CEA-CEB) form typical TBSV virions, retain silencing inserts of variable lengths (110 to 260 nucleotides), and can systemically silence endogenous genes in N. benthamiana.