Search

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.

Diverse strains of Luteibacter ( Gammaproteobacteria ) have been isolated from a variety of environments, most frequently in association with both plants and fungi. Motivated by the lack of genomic information for strains throughout the genus Luteibacter, we report here a complete genome sequence for Luteibacter pinisoli strain MAH-14.

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., Author summary The contribution of genetic variation to phenotypic variation is well-established. By contrast, it is unknown how frequently epigenetic variation causes differences in organismal phenotypes. Epigenetic information is closely associated with but not encoded in the DNA sequence. In practice, it is challenging to disentangle genetic variation from epigenetic variation, as what appears to be epigenetic variation might have an underlying genetic basis. DNA methylation is one form of epigenetic information. HDG3 encodes an endosperm specific transcription factor that exists in two states in A. thaliana natural populations: methylated and expressed and hypomethylated and repressed. We show that pure epigenetic variation is sufficient to explain expression variation of HDG3 - a naturally lowly expressed allele can be switched to a higher expressed state by adding DNA methylation. We also show that expression of HDG3 in strains where it is normally hypomethylated and relatively repressed causes a seed development phenotype. These data indicate that naturally circulating epialleles have consequences for seed phenotypic variation.

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.

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.

1. RNAse-treated DNA (at least 1 μg, volume should not exceed 130 μl) 2. AMPure XP beads (Beckman Coulter, catalog number: A63880) 3. 200 proof Ethyl alcohol (Sigma-Aldrich, catalog number: 459844-500ML) 4. Illumina TruSeq kit (Illumina, catalog number: FC-121-2001) 5. Methylcode Invitrogen kit (Life Technologies, Invitrogen TM , catalog number: MECOV50) Note: Other bisulfite conversion kits can be used, but were not tested in this protocol. 6. T4 DNA ligase (NEB, catalog number: M0202T) 7. Pfu Cx Hotstart DNA polymerase (Agilent, catalog number: 600412) 8. TOPO Blunt cloning kit (Life Technologies, Invitrogen TM , catalog number: 450245) 9. TOPO TA cloning kit (Life Technologies, Invitrogen TM , catalog number: 450030) 10. Ex Taq DNA polymerase (TaKaRa, Clonetech, catalog number: RR001B) 11. 1 Kb-plus DNA ladder (Life Technologies, Invitrogen TM , catalog number: 10787-018) 12. Agarose 13. 1x TAE buffer (see Recipes)

Until recently, only a handful of imprinted genes, or genes with parent-of-origin dependent expression patterns, were known in plants. Study of these genes yielded key insights into mechanisms of monoallelic expression and imprinted gene function. The recent application of high throughput sequencing to the study of imprinting has confirmed that many previous findings are relevant on a genome-wide scale. The catalogue of imprinted genes in monocots and dicots now includes a large number of transcription factors, chromatin related genes, and metabolic or hormone biosynthesis enzymes. Interpretation of allele specific expression data remains a challenge, with careful validation of candidate imprinted genes necessary., Pew Charitable Trusts (Pew Scholar in the Biomedical Sciences’ grant), National Science Foundation (U.S.)

The merger of evolutionarily diverged genomes to form a new polyploid genetic system can involve extensive remodeling of gene regulation. A recent paper in BMC Biology provides important insights into regulatory events that have affected the evolution of allopolyploid cotton.

Author(s): Pignatta, Daniela; Dilkes, Brian; Wroblewski, Tadeusz; Michelmore, Richard W; Comai, Luca | Abstract: BackgroundWhole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids) of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species) are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana.Methodology/principal findingsDiploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS) inverted repeat (hairpin) constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA.Conclusions/significanceOur results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species.

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., Pew Charitable Trusts (Biomedical Scholars Award), Alexander and Margaret Stewart Trust (Scholars Award)