Regulatory activity is the default DNA state in eukaryotes.

Genomes encode for genes and non-coding DNA, both capable of transcriptional activity. However, unlike canonical genes, many transcripts from non-coding DNA have limited evidence of conservation or function. Here, to determine how much biological noise is expected from non-genic sequences, we quantify the regulatory activity of evolutionarily naive DNA using RNA-seq in yeast and computational predictions in humans. In yeast, more than 99% of naive DNA bases were transcribed. Unlike the evolved transcriptome, naive transcripts frequently overlapped with opposite sense transcripts, suggesting selection favored coherent gene structures in the yeast genome. In humans, regulation-associated chromatin activity is predicted to be common in naive dinucleotide-content-matched randomized DNA. Here, naive and evolved DNA have similar co-occurrence and cell-type specificity of chromatin marks, challenging these as indicators of selection. However, in both yeast and humans, extreme high activities were rare in naive DNA, suggesting they result from selection. Overall, basal regulatory activity seems to be the default, which selection can hone to evolve a function or, if detrimental, repress.

GIL: a python package for designing custom indexing primers.

SUMMARY: Generate Indexes for Libraries (GIL) is a software tool for generating primers to be used in the production of multiplexed sequencing libraries. GIL can be customized in numerous ways to meet user specifications, including length, sequencing modality, color balancing, and compatibility with existing primers, and produces ordering and demultiplexing-ready outputs. AVAILABILITY AND IMPLEMENTATION: GIL is written in Python and is freely available on GitHub under the MIT license: https://github.com/de-Boer-Lab/GIL and can be accessed as a web-application implemented in Streamlit at https://dbl-gil.streamlitapp.com.

Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.

Magnaporthe oryzae pathotype Triticum (MoT) can act as a heterologous expression system for fungal effectors with high transcript abundance in wheat.

Plant pathogens deliver effector proteins to reprogramme a host plants circuitry, supporting their own growth and development, whilst thwarting defence responses. A subset of these effectors are termed avirulence factors (Avr) and can be recognised by corresponding host resistance (R) proteins, creating a strong evolutionary pressure on pathogen Avr effectors that favours their modification/deletion to evade the immune response. Hence, identifying Avr effectors and tracking their allele frequencies in a population is critical for understanding the loss of host recognition. However, the current systems available to confirm Avr effector function, particularly for obligate biotrophic fungi, remain limited and challenging. Here, we explored the utility of the genetically tractable wheat blast pathogen Magnaporthe oryzae pathotype Triticum (MoT) as a suitable heterologous expression system in wheat. Using the recently confirmed wheat stem rust pathogen (Puccina graminis f. sp. tritici) avirulence effector AvrSr50 as a proof-of-concept, we found that delivery of AvrSr50 via MoT could elicit a visible Sr50-dependant cell death phenotype. However, activation of Sr50-mediated cell death correlated with a high transgene copy number and transcript abundance in MoT transformants. This illustrates that MoT can act as an effective heterologous delivery system for fungal effectors from distantly related fungal species, but only when enough transgene copies and/or transcript abundance is achieved.

Caveats of Using Bacterial Type Three Secretion Assays for Validating Fungal Avirulence Effectors in Wheat.

Functional characterization of effector proteins of fungal obligate biotrophic pathogens, especially confirmation of avirulence (Avr) properties, has been notoriously difficult, due to the experimental intractability of many of these organisms. Previous studies in wheat have shown promising data suggesting the type III secretion system (T3SS) of bacteria may be a suitable surrogate for delivery and detection of Avr properties of fungal effectors. However, these delivery systems were tested in the absence of confirmed Avr effectors. Here, we tested two previously described T3SS-mediated delivery systems for their suitability when delivering two confirmed Avr effectors from two fungal pathogens of wheat, Puccinia graminis f. sp. tritici and Magnaporthe oryzae pathotype tritici. We showed that both effectors (AvrSr50 and AvrRmg8) were unable to elicit a hypersensitive response on wheat seedlings with the corresponding resistance gene when expressed by the Pseudomonas fluorescens "Effector to Host Analyser" (EtHAn) system. Furthermore, we found the utility of Burkholderia glumae for screening Avr phenotypes is severely limited, as the wild-type strain elicits nonhost cell death in multiple wheat accessions. These results provide valuable insight into the suitability of these systems for screening fungal effectors for Avr properties that may help guide further development of surrogate bacterial delivery systems in wheat. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Peppermint Essential Oil for Nausea and Vomiting in Hospitalized Patients: Incorporating Holistic Patient Decision Making Into the Research Design.

AIMS: This study examined nausea and vomiting (N/V) in hospitalized patients following the use of inhaled peppermint essential oil (aromatherapy) compared to combined aromatherapy/antiemetics or antiemetics alone. METHOD AND MATERIALS: A total of 103 hospitalized patients were offered one of three options to control N/V. Patient choice was considered in the holistic trial design so that patients were not denied either the essential oil or antiemetics. Patients rated nausea 0 to 10 on the Edmonton Symptom Assessment Scale at symptom onset and within 60 minutes of the intervention. RESULTS: Only three subjects enrolled in the antiemetic arm; thus this arm was eliminated from analysis, resulting in 100 evaluable patients. Mean nausea score improved significantly for the entire sample following the aromatherapy or aromatherapy/antiemetic intervention (p < .0001). Patients in the aromatherapy arm had significant improvement in nausea compared to the combined aromatherapy/antiemetic arm (p < .0001). Patient perception that peppermint oil relieves N/V significantly improved for the entire sample. Notable is that 65% of patients used peppermint essential oil alone. CONCLUSIONS: Peppermint essential oil is an effective independent or complementary modality for relief of N/V in hospitalized patients. Research designs that incorporate patient decision making should be considered for studies in which placebos do not contribute to holistic care.

N-terminal ß-strand underpins biochemical specialization of an ATG8 isoform.

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal ß-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal ß-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.

Evolutionary reversals in Bossiella (Corallinales, Rhodophyta): first report of a coralline genus with both geniculate and nongeniculate species.

This is the first report of a coralline genus with both geniculate (upright fronds with non-calcified joints) and nongeniculate species that has been verified by DNA sequence data. Two nongeniculate (crustose) species of Bossiella are recognized, B. mayae sp. nov. and B. exarticulata sp. nov. DNA sequencing of the lectotype specimen of Pseudolithophyllum whidbeyense revealed that this name had been misapplied and instead belongs to an undescribed coralline species in the Hapalidiales. Phylogenetic analyses of concatenated DNA sequences (psbA, rbcL, COI-5P) indicate that B. mayae and B. exarticulata represent phenotypic reversals from the geniculate character state back to the nongeniculate character state. Secondary loss of genicula has occurred three times in the subfamily Corallinoideae, once to generate the entirely nongeniculate genus Crusticorallina and twice in the now morphologically heterotypic Bossiella. Since phenotypic reversals have occurred several times during the evolution of coralline algae, we speculate about the putative mechanism and adaptive significance of this phenomenon.

Crusticorallina gen. nov., a nongeniculate genus in the subfamily Corallinoideae (Corallinales, Rhodophyta).

Molecular phylogenetic analyses of 18S rDNA (SSU) gene sequences confirm the placement of Crusticorallina gen. nov. in Corallinoideae, the first nongeniculate genus in an otherwise geniculate subfamily. Crusticorallina is distinguished from all other coralline genera by the following suite of morpho-anatomical characters: (i) sunken, uniporate gametangial and bi/tetrasporangial conceptacles, (ii) cells linked by cell fusions, not secondary pit connections, (iii) an epithallus of 1 or 2 cell layers, (iv) a hypothallus that occupies 50% or more of the total thallus thickness, (v) elongate meristematic cells, and (vi) trichocytes absent. Four species are recognized based on rbcL, psbA and COI-5P sequences, C. painei sp. nov., the generitype, C. adhaerens sp. nov., C. nootkana sp. nov. and C. muricata comb. nov., previously known as Pseudolithophyllum muricatum. Type material of Lithophyllum muricatum, basionym of C. muricata, in TRH comprises at least two taxa, and therefore we accept the previously designated lectotype specimen in UC that we sequenced to confirm its identity. Crusticorallina species are very difficult to distinguish using morpho-anatomical and/or habitat characters, although at specific sites, some species may be distinguished by a combination of morpho-anatomy, habitat and biogeography. The Northeast Pacific now boasts six coralline endemic genera, far more than any other region of the world.

Arabidopsis TAF15b Localizes to RNA Processing Bodies and Contributes to snc1-Mediated Autoimmunity.

In both animals and plants, messenger (m)RNA export has been shown to contribute to immune response regulation. The Arabidopsis nuclear protein MOS11, along with the nucleoporins MOS3/Nup96/SAR3 and Nup160/SAR1 are components of the mRNA export machinery and contribute to immunity mediated by nucleotide binding leucine-rich repeat immune receptors (NLR). The human MOS11 ortholog CIP29 is part of a small protein complex with three additional members: the RNA helicase DDX39, ALY, and TAF15b. We systematically assessed the biological roles of the Arabidopsis homologs of these proteins in toll interleukin 1 receptor-type NLR (TNL)-mediated immunity using reverse genetics. Although mutations in ALY and DDX39 did not result in obvious defects, taf15b mutation partially suppressed the autoimmune phenotypes of a gain-of-function TNL mutant, snc1. An additive effect on snc1 suppression was observed in mos11-1 taf15b snc1 triple mutant plants, suggesting that MOS11 and TAF15b have independent functions. TAF15b-GFP fusion protein, which fully complemented taf15b mutant phenotypes, localized to nuclei similarly to MOS11. However, it was also targeted to cytosolic granules identified as processing bodies. In addition, we observed no change in SNC1 mRNA levels, whereas less SNC1 protein accumulated in taf15b mutant, suggesting that TAF15b contributes to SNC1 homeostasis through posttranscriptional mechanisms. In summary, this study highlights the importance of posttranscriptional RNA processing mediated by TAF15b in the regulation of TNL-mediated immunity.

Resolving cryptic species of Bossiella (Corallinales, Rhodophyta) using contemporary and historical DNA.

PREMISE OF THE STUDY: Phenotypic plasticity and convergent evolution have long complicated traditional morphological taxonomy. Fortunately, DNA sequences provide an additional basis for comparison, independent of morphology. Most importantly, by obtaining DNA sequences from historical type specimens, we are now able to unequivocally match species names to genetic groups, often with surprising results. METHODS: We used an integrative taxonomic approach to identify and describe Northeast Pacific pinnately branched species in the red algal coralline genus Bossiella, for which traditional taxonomy recognized only one species, the generitype, Bossiella plumosa. We analyzed DNA sequences from historical type specimens and modern topotype specimens to assign species names and to identify genetic groups that were different and that required new names. Our molecular taxonomic assessment was followed by a detailed morphometric analysis of each species. KEY RESULTS: Our study of B. plumosa revealed seven pinnately branched Bossiella species. Three species, B. frondescens, B. frondifera, and B. plumosa, were assigned names based on sequences from type specimens. The remaining four species, B. hakaiensis, B. manzae, B. reptans, and B. montereyensis, were described as new to science. In most cases, there was significant overlap of morphological characteristics among species. CONCLUSIONS: This study underscores the pitfalls of relying upon morpho-anatomy alone to distinguish species and highlights our likely underestimation of species worldwide. Our integrative taxonomic approach can serve as a model for resolving the taxonomy of other plant and algal genera.