Deep learning image recognition enables efficient genome editing in zebrafish by automated injections

<div><p>One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 μm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.</p></div

Evolutionary change from induced to constitutive expression of an indirect plant resistance

Induced plant resistance traits are expressed in response to attack and occur throughout the plant kingdom1,2. Despite their general occurrence, the evolution of such resistances has rarely been investigated3. Here we report that extrafloral nectar, a usually inducible trait, is constitutively secreted by Central American Acacia species that are obligately inhabited by ants. Extrafloral nectar is secreted as an indirect resistance4, attracting ants that defend plants against herbivores5. Leaf damage induces extrafloral nectar secretion in several plant species6-8; among these are various Acacia species and other Fabaceae investigated here. In contrast, Acacia species obligately inhabited by symbiotic ants9 nourish these ants by secreting extrafloral nectar constitutively at high rates that are not affected by leaf damage. The phylogeny of the genus Acacia and closely related genera indicate that the inducibility of extrafloral nectar is the plesiomorphic or 'original' state, whereas the constitutive extrafloral nectar flow is derived within Acacia. A constitutive resistance trait has evolved from an inducible one, obviously in response to particular functional demands

Cloning and functional characterisation of an enzyme involved in the elongation of D6-polyunsaturated fatty acids from the moss Physcomitrella patens

The moss Physcomitrella patens contains high proportions of polyunsaturated very-long-chain fatty acids with up to 20 carbon atoms. Starting from preformed C18 polyunsaturated fatty acids, their biosynthesis involves a sequence of Delta6-desaturation, Delta6-elongation and Delta5-desaturation. In this report we describe for the first time the characterisation of a cDNA (PSE1) of plant origin with homology to the ELO-genes from Saccharomyces cerevisiae, encoding a component of the Delta6-elongase. Functional expression of PSE1 in S. cerevisiae led to the elongation of exogenously supplied Delta6-polyunsaturated fatty acids. By feeding experiments with different trienoic fatty acids of natural and synthetic origin, both substrate specificity and substrate selectivity of the enzyme were investigated. The activity of Pse1, when expressed in yeast, was not sensitive to the antibiotic cerulenin, which is an effective inhibitor of fatty acid synthesis and elongation. Furthermore, the PSE1 gene was disrupted in the moss by homologous recombination. This led to a complete loss of all C20 polyunsaturated fatty acids providing additional evidence for the function of the cDNA as coding for a component of the Delta6-elongase. The elimination of the elongase was not accompanied by a visible alteration in the phenotype, indicating that C20-PUFAs are not essential for viability of the moss under phytotron conditions

Pseudomonas aeruginosa Affects Airway Epithelial Response and Barrier Function During Rhinovirus Infection

Cystic fibrosis (CF) lung disease is aggravated by recurrent and ultimately chronic bacterial infections. One of the key pathogens in adult CF lung disease is P. aeruginosa (PA). In addition to bacteria, respiratory viral infections are suggested to trigger pulmonary exacerbations in CF. To date, little is known on how chronic infections with PA influence susceptibility and response to viral infection. We investigated the interactions between PA, human rhinovirus (HRV) and the airway epithelium in a model of chronic PA infection using differentiated primary bronchial epithelial cells (pBECs) and clinical PA isolates obtained from the respiratory sample of a CF patient. Cells were repeatedly infected with either a mucoid or a non-mucoid PA isolate for 16 days to simulate chronic infection, and subsequently co-infected with HRV. Key cytokines and viral RNA were quantified by cytometric bead array, ELISA and qPCR. Proteolytic degradation of IL-6 was analyzed by Western Blots. Barrier function was assessed by permeability tests and transepithelial electric resistance measurements. Virus infection stimulated the production of inflammatory and antiviral mediators, including interleukin (IL)-6, CXCL-8, tumor necrosis factor (TNF)-α, and type I/III interferons. Co-infection with a non-mucoid PA isolate increased IL-1β protein concentrations (28.88 pg/ml vs. 6.10 pg/ml), but in contrast drastically diminished levels of IL-6 protein (53.17 pg/ml vs. 2301.33 pg/ml) compared to virus infection alone. Conditioned medium obtained from co-infections with a non-mucoid PA isolate and HRV was able to rapidly degrade recombinant IL-6 in a serine protease-dependent manner, whereas medium from individual infections or co-infections with a mucoid isolate had no such effect. After co-infection with HRV and the non-mucoid PA isolate, we detected lower mRNA levels of Forkhead box J1 (FOXJ1) and Cilia Apical Structure Protein (SNTN), markers of epithelial cell differentiation to ciliated cells. Moreover, epithelial permeability was increased and barrier function compromised compared to single infections. These data show that PA infection can influence the response of bronchial epithelial cells to viral infection. Altered innate immune responses and compromised epithelial barrier function may contribute to an aggravated course of viral infection in PA-infected airways

Herbivory-responsive calmodulin-like protein CML9 does not guide jasmonate-mediated defenses in <i>Arabidopsis thaliana</i>

<div><p>Calcium is an important second messenger in plants that is released into the cytosol early after recognition of various environmental stimuli. Decoding of such calcium signals by calcium sensors is the key for the plant to react appropriately to each stimulus. Several members of Calmodulin-like proteins (CMLs) act as calcium sensors and some are known to mediate both abiotic and biotic stress responses. Here, we study the role of the <i>Arabidopsis thaliana</i> CML9 in different stress responses. CML9 was reported earlier as defense regulator against <i>Pseudomonas syringae</i>. In contrast to salicylic acid-mediated defense against biotrophic pathogens such as <i>P</i>. <i>syringae</i>, defenses against herbivores and necrotrophic fungi are mediated by jasmonates. We demonstrate that <i>CML9</i> is induced upon wounding and feeding of the insect herbivore <i>Spodoptera littoralis</i>. However, neither different <i>CML9</i> loss-of-function mutant lines nor overexpression lines were impaired upon insect feeding. No difference in herbivore-induced phytohormone elevation was detected in <i>cml9</i> lines. The defense against the spider mite <i>Tetranychus urticae</i> was also unaffected. In addition, <i>cml9</i> mutant lines showed a wild type-like reaction to the necrotrophic fungus <i>Alternaria brassicicola</i>. Thus, our data suggest that CML9 might be a regulator involved only in the defense against biotrophic pathogens, independent of jasmonates. In addition, our data challenge the involvement of CML9 in plant drought stress response. Taken together, we suggest that CML9 is a specialized rather than a general regulator of stress responses in <i>Arabidopsis</i>.</p></div

Genetic differences between <i>cml9-a</i> and <i>cml9-b</i>.

<p>(<i>a</i>) Schematic overview of <i>CML9</i> with T-DNA insertions and the used primers for RT- and qRT-PCR. Exons are indicated with E, introns with I. Light gray triangles indicate T-DNA insertions. RT primers used are indicated by black arrows, qRT primers by grey arrows (qRT FP1 and qRT RP1 are published as CML9 primers in Vadassery, Scholz [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197633#pone.0197633.ref016" target="_blank">16</a>]). Total length of <i>CML9</i> gDNA without insertions is 1137 bp. (<i>b</i>) Verification of T-DNA insertions in <i>CML9</i> by genotyping. The expected product length is indicated on the right sites of the respective pictures. (<i>c</i>) Semi quantitative RT-PCR analysis of <i>CML9</i> expression in wild type and knock-out mutants. Plants were treated with a pattern wheel and either water (WW) or <i>S</i>. <i>littoralis</i> oral secretion (WOS) was applied for 30 min. Untreated plants were used as controls (CON). Besides full length expression, expression of the E1-E3 fragment, upstream of the intronic T-DNA insertions, is shown. Expression of <i>ACTIN</i> was used as quantitative control. The expected product length is written on the right sites of the respective pictures. Asterisks indicate unspecific bands in the <i>cml9-a</i> mutant. (<i>d</i>) Normalized fold expression of <i>CML9</i> E1-E3 fragment in wild type and <i>cml9-a</i> and <i>cml9-b</i> lines. Plants were treated as described in (<i>c</i>). Expression level was normalized with respect to the <i>RPS18B</i> transcript level. Bars represent the means ± SE (n ≥ 5). Experiments were repeated two times independently. Statistically significant changes were determined by two-way ANOVA. Results of statistical analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197633#pone.0197633.t001" target="_blank">Table 1</a>.</p