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Quantitative labeling of biomolecules is necessary to advance areas of antibody–drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein. Here, we demonstrate that reduction of on-protein Tet ncAAs impacts their reactivity, while the leading cause of the unreactive protein is near-cognate suppression (NCS) of UAG codons by endogenous aminoacylated tRNAs. To overcome incomplete conjugation due to NCS, we developed a more catalytically efficient tRNA synthetase and developed a series of new machinery plasmids harboring the aminoacyl tRNA synthetase/tRNA pair (aaRS/tRNA pair). These plasmids enable robust production of homogeneously reactive Tet-protein in truncation-free cell lines, eliminating the contamination caused by NCS and protein truncation. Furthermore, these plasmid systems utilize orthogonal synthetic origins, which render these machinery vectors compatible with any common expression system. Through developing these new machinery plasmids, we established that the aaRS/tRNA pair plasmid copy-number greatly affects the yields and quality of the protein produced. We then produced quantitatively reactive soluble Tet-Fabs, demonstrating the utility of this system for rapid, homogeneous conjugations of biomedically relevant proteins. 

This is the data archive for: Meyer et al. 2022. Plant neighborhood shapes diversity and reduces interspecific variation of the phyllosphere microbiome. ISME-J. Please cite this article when using these archived data.</div>DOI: 10.1038/s41396-021-01184-6</div></div>Included are raw genetic sequences of the V5-V7 region of the 16S rRNA gene derived from experimental leaf surfaces of tomato, pepper, and bean plants.</div></div>Included in this archive are:</div>Raw sequence data (RawFASTQ.zip)</div>Reproducible R scripts (MeyerEtAl2021_RScript.R, VarPartSupplement.R)</div>R objects corresponding to archived scripts (.RDS)</div>Data for generating certain plots (PermanovaRValues.txt, PermanovaValuesByHost.txt, NeutralModelRValuesByHarvest.txt, VarPartHostEffects.txt)</div>Sample metadata (NeighborhoodMetaData.txt)</div>Phylogenetic Tree file for sample ASVs (PhyloTree.tre)</div>Geographic distance matrix for distances between plots (GeodistNeighborhood.txt)</div>ddPCR (microbial abundance) data (ddPCR_Neighborhood.csv)</div>R script for rarefication function (Rarefy_mean.R)</div>Taxonomic assignments for all ASVs in study (Taxonomy_Neighborhood.txt)</div>R image files to load R environment instead of running script (MeyerEtAl2021_RScript.RData, VarPartSupplement.RData)</div></div></div></div> 

Abstract Microbial communities associated with plant leaf surfaces (i.e., the phyllosphere) are increasingly recognized for their role in plant health. While accumulating evidence suggests a role for host filtering of its microbiota, far less is known about how community composition is shaped by dispersal, including from neighboring plants. We experimentally manipulated the local plant neighborhood within which tomato, pepper, or bean plants were grown in a 3-month field trial. Focal plants were grown in the presence of con- or hetero-specific neighbors (or no neighbors) in a fully factorial combination. At 30-day intervals, focal plants were harvested and replaced with a new age- and species-matched cohort while allowing neighborhood plants to continue growing. Bacterial community profiling revealed that the strength of host filtering effects (i.e., interspecific differences in composition) decreased over time. In contrast, the strength of neighborhood effects increased over time, suggesting dispersal from neighboring plants becomes more important as neighboring plant biomass increases. We next implemented a cross-inoculation study in the greenhouse using inoculum generated from the field plants to directly test host filtering of microbiomes while controlling for directionality and source of dispersal. This experiment further demonstrated that focal host species, the host from which the microbiome came, and in one case the donor hosts’ neighbors, contribute to variation in phyllosphere bacterial composition. Overall, our results suggest that local dispersal is a key factor in phyllosphere assembly, and that demographic factors such as nearby neighbor identity and biomass or age are important determinants of phyllosphere microbiome diversity.