Enrichment of human nasopharyngeal bacteriome with bacteria from dust after short-term exposure to indoor environment: a pilot study.

BACKGROUND: Indoor dust particles are an everyday source of human exposure to microorganisms and their inhalation may directly affect the microbiota of the respiratory tract. We aimed to characterize the changes in human nasopharyngeal bacteriome after short-term exposure to indoor (workplace) environments. METHODS: In this pilot study, nasopharyngeal swabs were taken from 22 participants in the morning and after 8 h of their presence at the workplace. At the same time points, indoor dust samples were collected from the participants' households (16 from flats and 6 from houses) and workplaces (8 from a maternity hospital - NEO, 6 from a pediatric hospital - ENT, and 8 from a research center - RCX). 16S rRNA sequencing analysis was performed on these human and environmental matrices. RESULTS: Staphylococcus and Corynebacterium were the most abundant genera in both indoor dust and nasopharyngeal samples. The analysis indicated lower bacterial diversity in indoor dust samples from flats compared to houses, NEO, ENT, and RCX (p < 0.05). Participants working in the NEO had the highest nasopharyngeal bacterial diversity of all groups (p < 0.05). After 8 h of exposure to the workplace environment, enrichment of the nasopharynx with several new bacterial genera present in the indoor dust was observed in 76% of study participants; however, no significant changes were observed at the level of the nasopharyngeal bacterial diversity (p > 0.05, Shannon index). These "enriching" bacterial genera overlapped between the hospital workplaces - NEO and ENT but differed from those in the research center - RCX. CONCLUSIONS: The results suggest that although the composition of nasopharyngeal bacteriome is relatively stable during the day. Short-term exposure to the indoor environment can result in the enrichment of the nasopharynx with bacterial DNA from indoor dust; the bacterial composition, however, varies by the indoor workplace environment.

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants.

We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

Protein expression and gene editing in monocots using foxtail mosaic virus vectors.

Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9-mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana. This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody-based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus-induced gene silencing (VIGS), virus-mediated overexpression (VOX), and virus-enabled gene editing (VEdGE) in monocots.

Conferring resistance to geminiviruses with the CRISPR-Cas prokaryotic immune system.

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR-Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR-Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.

Geographical gradient of the eIF4E alleles conferring resistance to potyviruses in pea (Pisum) germplasm.

BACKGROUND: The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the eIF4E gene to identify novel genetic diversity. METHODOLOGY/PRINCIPAL FINDINGS: Germplasm of 2803 pea accessions was screened for eIF4E intron 3 length polymorphism, resulting in the detection of four eIF4E(A-B-C-S) variants, whose distribution was geographically structured. The eIF4E(A) variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, eIF4E(B), was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The eIF4E(C) variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The eIF4E(S) variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (eIF4E(A-1-2-3-4-5-6-7), eIF4E(B-1), eIF4E(C-2)) conferred resistance to the P1 PSbMV pathotype. CONCLUSIONS/SIGNIFICANCE: This work identified novel eIF4E alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible eIF4E(S1) allele. Despite high variation present in wild Pisum accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis to study the co-evolution of potyviruses and the pea host.