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For most farmers, the production of maize grain is the ultimate goal of the entire field season. From the point of view of plant microbiome studies, seeds are particularly interesting in that they are the only avenue for vertical transmission of microbes from parent to offspring, though microbes can also enter maize seeds via wounds or silks. Although the presence of seed endophytes is well documented, their role, if any, in seed health and their effects on the next generation of plants are largely unknown. This protocol describes the isolation of seed endophytes. Its primary focus is properly sterilizing the seed surface, followed by grinding to release the endophytes. The end product is a cell suspension suitable for either culturing or DNA analysis.
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Maize (Zea mays) is a multifaceted cereal grass used globally for nutrition, animal feed, food processing, and biofuels, and a model system in genetics research. Studying the maize microbiome sometimes requires its manipulation to identify the contributions of specific taxa and ecological traits (i.e., diversity, richness, network structure) to maize growth and physiology. Due to regulatory constraints on applying engineered microorganisms in field settings, greenhouse-based experimentation is often the first step for understanding the contribution of root-associated microbiota-whether natural or engineered-to plant phenotypes. In this protocol, we describe methods to inoculate maize with a specific microbiome as a tool for understanding the microbiota's influence on its host plant. The protocol involves removal of the native seed microbiome followed by inoculation of new microorganisms; separate protocols are provided for inoculations from pure culture, from soil slurry, or by mixing in live soil. These protocols cover the most common methods for manipulating the maize microbiome in soil-grown plants in the greenhouse. The methods outlined will ultimately result in rhizosphere microbial assemblages with varying degrees of microbial diversity, ranging from low diversity (individual strain and synthetic community [SynCom] inoculation) to high diversity (percent live inoculation), with the slurry inoculation method representing an "intermediate diversity" treatment.
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[This corrects the article DOI: 10.3389/fpls.2023.1279231.].
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Introduction: Gene expression is often controlled via cis-regulatory elements (CREs) that modulate the production of transcripts. For multi-gene genetic engineering and synthetic biology, precise control of transcription is crucial, both to insulate the transgenes from unwanted native regulation and to prevent readthrough or cross-regulation of transgenes within a multi-gene cassette. To prevent this activity, insulator-like elements, more properly referred to as transcriptional blockers, could be inserted to separate the transgenes so that they are independently regulated. However, only a few validated insulator-like elements are available for plants, and they tend to be larger than ideal. Methods: To identify additional potential insulator-like sequences, we conducted a genome-wide analysis of Utricularia gibba (humped bladderwort), one of the smallest known plant genomes, with genes that are naturally close together. The 10 best insulator-like candidates were evaluated in vivo for insulator-like activity. Results: We identified a total of 4,656 intergenic regions with expression profiles suggesting insulator-like activity. Comparisons of these regions across 45 other plant species (representing Monocots, Asterids, and Rosids) show low levels of syntenic conservation of these regions. Genome-wide analysis of unmethylated regions (UMRs) indicates ~87% of the targeted regions are unmethylated; however, interpretation of this is complicated because U. gibba has remarkably low levels of methylation across the genome, so that large UMRs frequently extend over multiple genes and intergenic spaces. We also could not identify any conserved motifs among our selected intergenic regions or shared with existing insulator-like elements for plants. Despite this lack of conservation, however, testing of 10 selected intergenic regions for insulator-like activity found two elements on par with a previously published element (EXOB) while being significantly smaller. Discussion: Given the small number of insulator-like elements currently available for plants, our results make a significant addition to available tools. The high hit rate (2 out of 10) also implies that more useful sequences are likely present in our selected intergenic regions; additional validation work will be required to identify which will be most useful for plant genetic engineering.
