Triticale Isolated Microspore Culture for Doubled Haploid Production.

Here, we describe a method of triticale isolated microspore culture for production of doubled haploid plants via androgenesis. We use this method routinely because it is highly efficient and works well on different triticale genotypes. To force microspores into becoming embryogenic, we apply a 21-day cold pretreatment. The shock of cold facilitates redirecting microspores from their predestined pollen developmental program into the androgenesis pathway. Ovaries are included in our culture methods to help with embryogenesis, and the histone deacytelase inhibitor Trichostatin A (TSA) is added to further improve androgenesis and increase our ability to recover green doubled haploid plants.

Stress-responsive pathways and small RNA changes distinguish variable developmental phenotypes caused by MSH1 loss.

BACKGROUND: Proper regulation of nuclear-encoded, organelle-targeted genes is crucial for plastid and mitochondrial function. Among these genes, MutS Homolog 1 (MSH1) is notable for generating an assortment of mutant phenotypes with varying degrees of penetrance and pleiotropy. Stronger phenotypes have been connected to stress tolerance and epigenetic changes, and in Arabidopsis T-DNA mutants, two generations of homozygosity with the msh1 insertion are required before severe phenotypes begin to emerge. These observations prompted us to examine how msh1 mutants contrast according to generation and phenotype by profiling their respective transcriptomes and small RNA populations. RESULTS: Using RNA-seq, we analyze pathways that are associated with MSH1 loss, including abiotic stresses such as cold response, pathogen defense and immune response, salicylic acid, MAPK signaling, and circadian rhythm. Subtle redox and environment-responsive changes also begin in the first generation, in the absence of strong phenotypes. Using small RNA-seq we further identify miRNA changes, and uncover siRNA trends that indicate modifications at the chromatin organization level. In all cases, the magnitude of changes among protein-coding genes, transposable elements, and small RNAs increases according to generation and phenotypic severity. CONCLUSION: Loss of MSH1 is sufficient to cause large-scale regulatory changes in pathways that have been individually linked to one another, but rarely described all together within a single mutant background. This study enforces the recognition of organelles as critical integrators of both internal and external cues, and highlights the relationship between organelle and nuclear regulation in fundamental aspects of plant development and stress signaling. Our findings also encourage further investigation into potential connections between organelle state and genome regulation vis-á-vis small RNA feedback.

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development.

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

Arabidopsis MSH1 mutation alters the epigenome and produces heritable changes in plant growth.

Plant phenotypes respond to environmental change, an adaptive capacity that is at least partly transgenerational. However, epigenetic components of this interplay are difficult to measure. Depletion of the nuclear-encoded protein MSH1 causes dramatic and heritable changes in plant development, and here we show that crossing these altered plants with isogenic wild type produces epi-lines with heritable, enhanced growth vigour. Pericentromeric DNA hypermethylation occurs in a subset of msh1 mutants, indicative of heightened transposon repression, while enhanced growth epi-lines show large chromosomal segments of differential CG methylation, reflecting genome-wide reprogramming. When seedlings are treated with 5-azacytidine, root growth of epi-lines is restored to wild-type levels, implicating hypermethylation in enhanced growth. Grafts of wild-type floral stems to mutant rosettes produce progeny with enhanced growth and altered CG methylation strikingly similar to epi-lines, indicating a mobile signal when MSH1 is downregulated, and confirming the programmed nature of methylome and phenotype changes.

An immunity-triggering effector from the Barley smut fungus Ustilago hordei resides in an Ustilaginaceae-specific cluster bearing signs of transposable element-assisted evolution.

The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.

Temporal patterns of gene expression in developing maize endosperm identified through transcriptome sequencing.

Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development and seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed, and renewable resources. Little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step toward understanding these networks, we profiled all mRNAs in the maize kernel and endosperm at eight successive stages during the first 12 d after pollination. Analysis of these gene sets identified temporal programs of gene expression, including hundreds of transcription-factor genes. We found a close correlation of the sequentially expressed gene sets with distinct cellular and metabolic programs in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemporal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.

Do TE activity and counteracting genome defenses, RNAi and methylation, shape the sex lives of smut fungi?

The availability of three genomes from smut fungi differing in mating, TE load, and genome defense mechanisms, allowed a comparative analyses and a discussion on evolutionary forces shaping them. A complex balance of selective forces seems at play. A bipolar mating system in Ustilago hordei promotes selfing, advantageous for successful niche occupation but favoring accumulation of repetitive DNA, including TEs. TE activity may have caused genome variations necessary for these obligate parasites under high host selection pressures. Higher TE activity is balanced by genome defenses through recombination, RNAi, methylation and RIP mutagenesis. In tetrapolar U. maydis, lacking silencing and possibly methylation mechanisms, reduced inbreeding potential favors removal of repetitive DNA, presumably by its highly-efficient recombination system.

Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of transposable elements.

Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maize-infecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.

Computational dissection of Arabidopsis smRNAome leads to discovery of novel microRNAs and short interfering RNAs associated with transcription start sites.

The profiling of small RNAs by high-throughput sequencing (smRNA-Seq) has revealed the complexity of the RNA world. Here, we describe a computational scheme for dissecting the plant smRNAome by integrating smRNA-Seq datasets in Arabidopsis thaliana. Our analytical approach first defines ab initio the genomic loci that produce smRNAs as basic units, then utilizes principal component analysis (PCA) to predict novel miRNAs. Secondary structure prediction of candidates' putative precursors discovered a group of long hairpin double-stranded RNAs (lh-dsRNAs) formed by inverted duplications of decayed coding genes. These gene remnants produce miRNA-like small RNAs which are predominantly 21- and 22-nt long, dependent of DCL1 but independent of RDR2 and DCL2/3/4, and associated with AGO1. Additionally, we found two classes of transcription start site associated (TSSa) RNAs located at sense (+) and antisense (-) approximately 100-200 bp downstream of TSSs, but are differentially incorporated into AGO1 and AGO4, respectively.

Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis.

RNA interference (RNAi) acts through transcriptional and post-transcriptional gene silencing of homologous sequences. With the goal of using RNAi as a tool for studying gene function in the related basidiomycete cereal pathogens Ustilago hordei and Ustilago maydis, we developed a general purpose RNAi expression vector. Tandem, inverted fragments of the GUS gene were inserted into this vector flanking an intron and used to transform engineered GUS-expressing haploid cells. Down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei, even though corresponding GUS double-stranded RNA was detected in both species. Similarly, when the endogenous bW mating-type gene was targeted by RNAi, mating was reduced only in U. hordei. Our work demonstrates the feasibility of using RNAi in U. hordei and provides experimental support for the observed lack of RNAi components in the U. maydis genome. We hypothesize that the sharply limited transposon complement in U. maydis is a biological consequence of this absence.