IRI-CCE: A Platform for Evaluating CRISPR-based Genome Editing Tools and Its Components

Rapid assessment of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based tools and validation of cloned CRISPR-plasmid vectors are critical aspects for the successful application of genome editing (GE) in different organisms, including plants. Also, the selection of active guide RNAs (gRNAs) is a determining factor to achieve efficient GE at the targeted locus. Incidentally, plasmid vectors capable of ectopic expression of Cas enzyme and sgRNAs in bacteria will facilitate the quick screening and reliability of cloned plasmids and the functionality of designed gRNAs. We report a platform for in vivo rapid investigation of CRISPR components in Escherichia coli (IRI-CCE), which is compatible with plant-transforming binary vectors comprising plant promoters/terminators. Besides, IRI-CCE analyses reveal distinct features of cytidine (PmCDA1, evoCDA1, APOBEC3A) and adenine (ABE8e) base editors. Finally, we show the IRI-CCE platform as a reliable and rapid method for screening functional gRNAs to achieve successful GE outcomes. Base editor-based CRISPR components expressed by promoters of different strengths led to the establishment of IRI-CCE platform for three major applications: optimization of novel CRISPR tools, validation of cloned CRISPR plasmids, and to know gRNA functionality.

Diversity of Endo and Exo-Bacteria Associated With Fungi Isolated From Plant Rhizospheres: A Pilot Study

Background: Over the past several years, the scientific community has described the diversity of microbial communities in a variety of soils associated with plants, but at present, little is known about the specific diversity of the soil fungal microbiome involving bacteria colonizing the surface of fungi (i.e., exo-bacteria) or existing within fungal hypha (i.e., endobacteria). This study aimed to collect, identify, and characterize several fungi and their associated (endo- and exo-) microbiome obtained from the rhizosphere of six different plants. Microcosm devices called fungal highway columns, containing one of four plant-based media as attractants, were placed in the rhizosphere of six different plants. The isolated fungi and their associated endo- and exo- bacteria were identified by sequencing of the ITS (fungi) or 16S (bacteria) rRNA regions, followed by Scanning Electron Microscope (SEM), and fluorescence in situ hybridization (FISH) imaging.Results: Most of the fungi recovered are known plant pathogens, such as Fusarium, Pleosporales, and Cladosporium together with species associated with the soil, e.g. Kalmusia. The exo-bacteria recovered were previously described as plant promoters, such as Bacillus, Rhizobium, Acinetobacter or Ensifer. The interactions between fungi and exo-bacteria recovered from fungal highway columns were further investigated via confrontation assays. From the reconstruction of the potential co-occurring bacterial-fungal associations in the rhizosphere, we discovered that the most promiscuous exo-bacterium group (associated with diverse fungi) was Bacillus. From the study of the endobacterial community, emerged a core of shared endosymbionts with a potential implication in the nitrogen cycle.Conclusions: The present study demonstrated the importance of selecting and studying cultivable fungi and bacteria from the rhizosphere. Our findings demonstrated that at the rhizosphere level, the range of interactions between fungi and bacteria, both internal and external to the fungal hypha, could vary even among closely related species.

Rational design of minimal synthetic promoters for plants

Promoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors. Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilize plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling the computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

Rational Design of Minimal Synthetic Promoters for Plants

ABSTRACTPromoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors (TFs). Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilise plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.