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Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.

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Plant-specific Rho-type GTPases (ROPs) are master regulators of cell polarity and development. Over the past 30 years, their localization and dynamics have been largely examined with fluorescent proteins fused at the amino terminus without investigating their impact on protein function. The moss Physcomitrium patens genome encodes four rop genes. In this study, we introduce a fluorescent tag at the endogenous amino terminus of ROP4 in wild-type and rop1,2,3 triple mutant via homologous recombination and demonstrate that the fluorescent tag severely impairs ROP4 function and inhibits its localization on the plasma membrane. This phenotype is exacerbated in mutants lacking ROP-related GTPase-activating proteins. By comparing the localization of nonfunctional and functional ROP4 fusion reporters, we provide insight into the mechanism that governs the membrane association of ROPs.

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Fluorescent protein (FP) tagging is a key method for observing protein distribution, dynamics and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells.

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Exploration of endophytic bacteria with multiple plant growth promoting (PGP) attributes is considered as an eco-friendly and cost-effective alternative to agricultural chemicals for increasing crop productivity. In the present endeavor, healthy chickpea plants (Cicer arietinum L.) collected from district Birbhum, West Bengal, India were subjected for the isolation of endophytic bacteria having multifarious PGP properties. One potent endophytic Gram positive bacterial strain CNE6 was isolated from the nodule of chickpea and was identified as Bacillus siamensis based on 16S rDNA sequence homologies. The isolate showed a number of PGP properties like phosphate solubilization, IAA production, nitrogen fixation, hydroxamate type of siderophore production and ACC deaminase activities. The isolate CNE6 produced 33.27 ± 2.16 µg/mL of IAA in the presence of tryptophan. Production of IAA was also confirmed by HPLC analysis and it was found effective for inducing lateral root branching in chickpea. In addition, the isolate displayed significant antagonistic activity against a number of plant pathogenic fungi when tested by dual culture overlay and agar well diffusion assay. 50 % cell free supernatant of CNE6 was found effective for 60-80 % inhibition of radial growth of pathogenic fungi tested. Scanning electron microscopic observation revealed massive degradation of pathogenic fungal mycelia by the antifungal metabolites of CNE6. LC-MS analysis of bacterial lipopeptides suggested the production of antifungal antibiotics like surfactin, fengycin and iturin by the isolate. The presence of genes encoding antifungal lipopeptides was also confirmed by PCR amplification using specific primers. Green fluorescent protein (GFP) tagging of CNE6 using broad host range plasmid vector (pDSK-GFPuv) followed by colonization study indicated very good host colonization potential of the isolate and its probable movement through xylem vessels. Enhanced shoot and root length and chlorophyll content upon treatment with CNE6 as observed in in vivo pot experiments also supported the positive role of the endophytic isolate on overall development and growth of the chickpea plants. This is the first report of Bacillus siamensis as an endophyte of Cicer arietinum L. which can be successfully applied for improving the productivity of this crop plant.

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The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

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During the last 25 y, fluorescent protein tagging has become a tool of choice to investigate protein function in a cellular context. The information gathered with this approach is not only providing insights into protein subcellular localization but also allows contextualizing protein function in multicellular settings. Here we illustrate the power of this method by commenting on the recent successful localization of the large membrane DEK1 protein during three-dimensional body formation in the moss Physcomitrella patens. But as many approaches, protein tagging is not exempt of caveats. The multiple infructuous (failed) attempts to detect DEK1 using a fluorescent protein tag present a good overview of such potential problems. Here we discuss the insertion of different fluorescent proteins at different positions in the PpDEK1 protein and the resulting unintended range of mutant phenotypes. Albeit none of these mutants generated a detectable fluorescent signal they can still provide interesting biological information about DEK1 function.

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The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.

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Vaccinia virus produces two distinct infectious virions; the single-enveloped intracellular mature virus (IMV), which remains in the cell until cell lysis, and the double-enveloped extracellular enveloped virus (EEV), which mediates virus spread. The latter is derived from a triple-enveloped intracellular enveloped virus (IEV) precursor, which is transported to the cell periphery by the kinesin-1 motor complex. This transport involves the viral protein A36 as well as F12 and E2. A36 is an integral membrane protein associated with the outer virus envelope and is the only known direct link between virion and kinesin-1 complex. Yet in the absence of A36 virion egress still occurs on microtubules, albeit at reduced efficiency. In this paper double-fluorescent labelling of the capsid protein A5 and outer-envelope protein F13 was exploited to visualize IEV transport by live-cell imaging in the absence of either A36 or F12. During the generation of recombinant viruses expressing both A5-GFP and F13-mCherry a plaque size defect was identified that was particularly severe in viruses lacking A36. Electron microscopy showed that this phenotype was caused by abnormal wrapping of IMV to form IEV, and this resulted in reduced virus egress to the cell surface. The aberrant wrapping phenotype suggests that the fluorescent fusion protein interferes with an interaction of F13 with the IMV surface that is required for tight association between IMVs and wrapping membranes. The severity of this defect suggests that these viruses are imperfect tools for characterizing virus egress.

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In Neurospora, genes not paired during meiosis are targeted by meiotic silencing by unpaired DNA (MSUD). Here, our bimolecular fluorescence complementation (BiFC) study suggests that RNA-directed RNA polymerase, Dicer, Argonaute, and others form a silencing complex in the perinuclear region, with intimate interactions among the majority of them. We have also shown that SAD-2 is likely the anchor for this assembly.

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