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People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.3 of SBOL Visual, which builds on the prior SBOL Visual 2.2 in several ways. First, the specification now includes higher-level “interactions with interactions,” such as an inducer molecule stimulating a repression interaction. Second, binding with a nucleic acid backbone can be shown by overlapping glyphs, as with other molecular complexes. Finally, a new “unspecified interaction” glyph is added for visualizing interactions whose nature is unknown, the “insulator” glyph is deprecated in favor of a new “inert DNA spacer” glyph, and the polypeptide region glyph is recommended for showing 2A sequences.

Summary: Viral nanoparticles (VNPs) are a new class of virus‐based formulations that can be used as building blocks to implement a variety of functions of potential interest in biotechnology and nanomedicine. Viral coat proteins (CP) that exhibit self‐assembly properties are particularly appropriate for displaying antigens and antibodies, by generating multivalent VNPs with therapeutic and diagnostic potential. Here, we developed genetically encoded multivalent VNPs derived from two filamentous plant viruses, potato virus X (PVX) and tobacco etch virus (TEV), which were efficiently and inexpensively produced in the biofactory Nicotiana benthamiana plant. PVX and TEV‐derived VNPs were decorated with two different nanobodies recognizing two different regions of the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein. The addition of different picornavirus 2A ribosomal skipping peptides between the nanobody and the CP allowed for modulating the degree of VNP decoration. Nanobody‐decorated VNPs purified from N. benthamiana tissues successfully recognized the RBD antigen in enzyme‐linked immunosorbent assays and showed efficient neutralization activity against pseudoviruses carrying the Spike protein. Interestingly, multivalent PVX and TEV‐derived VNPs exhibited a neutralizing activity approximately one order of magnitude higher than the corresponding nanobody in a dimeric format. These properties, combined with the ability to produce VNP cocktails in the same N. benthamiana plant based on synergistic infection of the parent PVX and TEV, make these green nanomaterials an attractive alternative to standard antibodies for multiple applications in diagnosis and therapeutics. [ABSTRACT FROM AUTHOR]

Summary: Plant Synthetic Biology aims to enhance the capacities of plants by designing and integrating synthetic gene circuits (SGCs). Quantitative reporting solutions that can produce quick, rich datasets affordably are necessary for SGC optimization. In this paper, we present a new, low‐cost, and high‐throughput reporter system for the quantitative measurement of gene expression in plants based on autonomous bioluminescence. This method eliminates the need for an exogenous supply of luciferase substrate by exploiting the entire Neonothopanus nambi fungal bioluminescence cyclic pathway to build a self‐sustained reporter. The HispS gene, the pathway's limiting step, was set up as the reporter's transcriptional entry point as part of the new system's design, which significantly improved the output's dynamic range and brought it on par with that of the gold standard FLuc/RLuc reporter. Additionally, transient ratiometric measurements in N. benthamiana were made possible by the addition of an enhanced GFP as a normalizer. The performance of new NeoLuc/eGFP system was extensively validated with SGCs previously described, including phytohormone and optogenetic sensors. Furthermore, we employed NeoLuc/eGFP in the optimization of challenging SGCs, including new configurations for an agrochemical (copper) switch, a new blue optogenetic sensor, and a dual copper/red‐light switch for tight regulation of metabolic pathways. [ABSTRACT FROM AUTHOR]

Summary: CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti‐CRISPR proteins as tools to prevent CRISPR/Cas‐mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti‐CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site‐directed mutagenesis in leaves when transiently co‐expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a‐mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas‐based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose‐dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin‐regulated activation of a downstream reporter gene. The strong anti‐Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants. [ABSTRACT FROM AUTHOR]

The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.

New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming. Here, we describe in detail the making of a multiplex DNA construct for genome editing in N. benthamiana using the GoldenBraid modular cloning platform. In this case, the procedure is adapted for the requirements of LbCas12a (Lachnospiraceae bacterium Cas12a), a nuclease whose cloning strategy differs from that of the more often used SpCas9 (Streptococcus pyogenes Cas9) enzyme. LbCas12a-mediated edition has several advantages, as its high editing efficiency, described for different plant species, and its T/A-rich PAM sequence, which expands the range of genomic loci that can be targeted by site-specific nucleases. The protocol also includes recommendations for the selection of protospacer sequences and indications for the analysis of editing results., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z) -11-hexadecenol (Z11-16OH) and (Z) -11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8  μ g g -1 FW) and high levels of Z11-16OH (111.4  μ g g -1 ). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g -1 FW per day for Z11-16OH and 9.44 ng g -1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements., Competing Interests: Ismael Navarro is employed by the company Ecología y Protección Agrícola S.L. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rubén Mateos-Fernández et al.)

GoldenBraid (GB) is a modular DNA assembly technology for plant multigene engineering based on type IIS restriction enzymes. GB speeds up the assembly of transcriptional units from standard genetic parts and facilitates the stacking of several genes within the same T-DNA in few days. GBcloning is software-assisted with a set of online tools. The GBDomesticator tool assists in the adaptation of DNA parts to the GBstandard. The combination of GB-adapted parts to build new transcriptional units is assisted by the GB TU Assembler tool. Finally, the assembly of multigene modules is simulated by the GB Binary Assembler. All the software tools are available at www.gbcloning.org . Here, we describe in detail the assembly methodology to create a multigene construct with three transcriptional units for polyphenol metabolic engineering in plants.