The Duckbot: A system for automated imaging and manipulation of duckweed.

Laboratory automation can boost precision and reproducibility of science workflows. However, current laboratory automation systems are difficult to modify for custom applications. Automating new experiment workflows therefore requires development of one-off research platforms, a process which requires significant time, resources, and experience. In this work, we investigate systems to lower the threshold to automation for plant biologists. Our approach establishes a direct connection with a generic motion platform to support experiment development and execution from a computational notebook environment. Specifically, we investigate the use of the open-source tool-changing motion platform Jubilee controlled using Jupyter notebooks. We present the Duckbot, a machine customized for automating laboratory research workflows with duckweed, a common multicellular plant. The Duckbot comprises (1) a set of end-effectors relevant for plant biology, (2) software modules which provide flexible control of these tools, and (3) computational notebooks which make use of these tools to automate duckweed experiments. We demonstrate the Duckbot's functionality by automating a particular laboratory research workflow, namely, duckweed growth assays. The Duckbot supports setting up sample plates with duckweed and growth media, gathering image data, and conducting relevant data analysis. We discuss the opportunities and limitations for developing custom laboratory automation with this platform and provide instructions on usage and customization.

Open-source workflow design and management software to interrogate duckweed growth conditions and stress responses.

Duckweeds, a family of floating aquatic plants, are ideal model plants for laboratory experiments because they are small, easy to cultivate, and reproduce quickly. Duckweed cultivation, for the purposes of scientific research, requires that lineages are maintained as continuous populations of asexually propagating fronds, so research teams need to develop optimized cultivation conditions and coordinate maintenance tasks for duckweed stocks. Additionally, computational image analysis is proving to be a powerful duckweed research tool, but researchers lack software tools to assist with data collection and storage in a way that can feed into scripted data analysis. We set out to support these processes using a laboratory management software called Aquarium, an open-source application developed to manage laboratory inventory and plan experiments. We developed a suite of duckweed cultivation and experimentation operation types in Aquarium, which we then integrated with novel data analysis scripts. We then demonstrated the efficacy of our system with a series of image-based growth assays, and explored how our framework could be used to develop optimized cultivation protocols. We discuss the unexpected advantages and the limitations of this approach, suggesting areas for future software tool development. In its current state, our approach helps to bridge the gap between laboratory implementation and data analytical software for duckweed biologists and builds a foundation for future development of end-to-end computational tools in plant science.

Broadening Participation: 21st Century Opportunities for Amateurs in Biology Research.

The modern field of biology has its roots in the curiosity and skill of amateur researchers and has never been purely the domain of professionals. Today, professionals and amateurs contribute to biology research, working both together and independently. Well-targeted and holistic investment in amateur biology research could bring a range of benefits that, in addition to positive societal benefits, may help to address the considerable challenges facing our planet in the 21st century. We highlight how recent advances in amateur biology have been facilitated by innovations in digital infrastructure as well as the development of community biology laboratories, launched over the last decade, and we provide recommendations for how individuals can support the integration of amateurs into biology research. The benefits of investment in amateur biology research could be many-fold, however, without a clear consideration of equity, efforts to promote amateur biology could exacerbate structural inequalities around access to and benefits from STEM. The future of the field of biology relies on integrating a diversity of perspectives and approaches-amateur biology researchers have an important role to play.

Aquarium: open-source laboratory software for design, execution and data management.

Automation has been shown to improve the replicability and scalability of biomedical and bioindustrial research. Although the work performed in many labs is repetitive and can be standardized, few academic labs can afford the time and money required to automate their workflows with robotics. We propose that human-in-the-loop automation can fill this critical gap. To this end, we present Aquarium, an open-source, web-based software application that integrates experimental design, inventory management, protocol execution and data capture. We provide a high-level view of how researchers can install Aquarium and use it in their own labs. We discuss the impacts of the Aquarium on working practices, use in biofoundries and opportunities it affords for collaboration and education in life science laboratory research and manufacture.

From Reductionism to Reintegration: Solving society's most pressing problems requires building bridges between data types across the life sciences.

Decades of reductionist approaches in biology have achieved spectacular progress, but the proliferation of subdisciplines, each with its own technical and social practices regarding data, impedes the growth of the multidisciplinary and interdisciplinary approaches now needed to address pressing societal challenges. Data integration is key to a reintegrated biology able to address global issues such as climate change, biodiversity loss, and sustainable ecosystem management. We identify major challenges to data integration and present a vision for a "Data as a Service"-oriented architecture to promote reuse of data for discovery. The proposed architecture includes standards development, new tools and services, and strategies for career-development and sustainability.

A Plant Pathogen Type III Effector Protein Subverts Translational Regulation to Boost Host Polyamine Levels.

Pathogenic bacteria inject effector proteins into host cells to manipulate cellular processes and facilitate the infection. Transcription-activator-like effectors (TALEs), an effector class in plant pathogenic bacteria, transcriptionally activate host genes to promote disease. We identify arginine decarboxylase (ADC) genes as the host targets of Brg11, a TALE-like effector from the plant pathogen Ralstonia solanacearum. Brg11 targets a 17-bp sequence that was found to be part of a conserved 50-bp motif, termed the ADC-box, upstream of ADC genes involved in polyamine biosynthesis. The transcribed ADC-box attenuates translation from native ADC mRNAs; however, Brg11 induces truncated ADC mRNAs lacking the ADC-box, thus bypassing this translational control. As a result, Brg11 induces elevated polyamine levels that trigger a defense reaction and likely inhibits bacterial niche competitors but not R. solanacearum. Our findings suggest that Brg11 may give R. solanacearum a competitive advantage and uncover a role for bacterial effectors in regulating ternary microbe-host-microbe interactions.

Synthetic genetic circuits in crop plants.

The love affair between crop breeding and genetics began over a century ago and has continued unabated, from mass selection programs to targeted genome modifications. Synthetic genetic circuits, a recent development, are combinations of regulatory and coding DNA introduced into a crop plant to achieve a desired function. Genetic circuits could accelerate crop improvement, allowing complex traits to be rationally designed and requisite DNA parts delivered directly into a genome of interest. However, there is not yet a standardized pipeline from exploratory laboratory testing to crop trials, and bringing transgenic products to market remains a considerable barrier. We highlight successes so far and future developments necessary to make genetic circuits a viable crop improvement technology over this century.

Exploiting the sequence diversity of TALE-like repeats to vary the strength of dTALE-promoter interactions.

Designer transcription activator-like effectors (dTALEs) are programmable transcription factors used to regulate user-defined promoters. The TALE DNA-binding domain is a tandem series of amino acid repeats that each bind one DNA base. Each repeat is 33-35 amino acids long. A residue in the center of each repeat is responsible for defining DNA base specificity and is referred to as the base specificying residue (BSR). Other repeat residues are termed non-BSRs and can contribute to TALE DNA affinity in a non-base-specific manner. Previous dTALE engineering efforts have focused on BSRs. Non-BSRs have received less attention, perhaps because there is almost no non-BSR sequence diversity in natural TALEs. However, more sequence diverse, TALE-like proteins are found in diverse bacterial clades. Here, we show that natural non-BSR sequence diversity of TALEs and TALE-likes can be used to modify DNA-binding strength in a new form of dTALE repeat array that we term variable sequence TALEs (VarSeTALEs). We generated VarSeTALE repeat modules through random assembly of repeat sequences from different origins, while holding BSR composition, and thus base preference, constant. We used two different VarSeTALE design approaches combing either whole repeats from different TALE-like sources (inter-repeat VarSeTALEs) or repeat subunits corresponding to secondary structural elements (intra-repeat VarSeTALEs). VarSeTALE proteins were assayed in bacteria, plant protoplasts and leaf tissues. In each case, VarSeTALEs activated or repressed promoters with a range of activities. Our results indicate that natural non-BSR diversity can be used to diversify the binding strengths of dTALE repeat arrays while keeping target sequences constant.

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between RipTAL repeats allows for a reconstruction of repeat array biogenesis, for example through slipped strand mispairing or gene conversion. Using these studies we show how RipTALs of broad host range strains evolved convergently toward a shared target sequence. Finally, we discuss the differences between TALE-likes of plant pathogens in the context of disease ecology.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

From dead leaf, to new life: TAL effectors as tools for synthetic biology.

Whether rice, yeast or fly there is barely a model organism not yet reached by transcription activator-like effectors (TALEs) and their derivative fusion proteins. Insights into fundamental biology are now arriving on the back of work in recent years to develop these proteins as tools for molecular biology. This began with the publication of the simple cipher determining base-specific DNA recognition by TALEs in 2009, and now encompasses a huge variety of established fusion proteins mediating targeted modifications to transcriptome, genome and, recently, epigenome. Straightforward design and exquisite specificity, allowing unique sites to be targeted even within complex eukaryote genomes, are key to the popularity of this system. Synthetic biology is one field that is just beginning to make use of these properties, with a number of recent publications demonstrating TALE-mediated regulation of synthetic genetic circuits. Intense interest has surrounded the CRISPR/Cas9 system within the last 12 months, and it is already proving its mettle as a tool for targeted gene modifications and transcriptional regulation. However, questions over off-target activity and means for independent regulation of multiple Cas9-guide RNA pairs must be resolved before this method can be included in the synthetic biology toolbox. TALEs are already showing promise as regulators of synthetic biological systems, a role that is likely to be developed further in the coming years.

Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.