The Intragenesis and Synthetic Biology Approach towards Accelerating Genetic Gains on Strawberry: Development of New Tools to Improve Fruit Quality and Resistance to Pathogens

Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines

Reporting antimicrobial susceptibilities and resistance phenotypes in Acinetobacter spp: a nationwide proficiency study.

To evaluate the proficiency of Spanish microbiology laboratories with respect to the antimicrobial susceptibility testing (AST) of Acinetobacter spp. Eight Acinetobacter spp. with different resistance mechanisms were sent to 48 Spanish centres which were asked to report: (i) the AST system used; (ii) MICs; (iii) breakpoints used (CLSI versus EUCAST); (iv) clinical category; and (v) resistance mechanisms inferred. Minor, major and very major errors (mE, ME and VME, respectively) were determined. The greatest percentages of discrepancies were: (i) by AST method: 18.5% Etest, 14.3% Vitek 2 and Sensititre; (ii) by breakpoints: 20.5% (CLSI) and 10.8% (EUCAST); and (iii) by antimicrobial agent: ampicillin/sulbactam (56.2% CLSI), minocycline (40.7% CLSI), tobramycin (38.7% CLSI, 16.8% EUCAST), imipenem (27.8% CLSI, 30.0% EUCAST) and meropenem (25.4% CLSI, 20.8% EUCAST). Categorical error rates: (i) by AST method ranged from 30.0% (Phoenix) to 100% (Sensititre and disc diffusion) for mE, 0.0% (Etest, Sensititre, disc diffusion) to 40% (Phoenix) for ME, and 0.0% (Sensititre and disc diffusion) to 30% (Phoenix) for VME; (ii) by breakpoints: mE (80.1% CLSI, 58.4% EUCAST), ME (3.5% CLSI, 12.4% EUCAST) and VME (16.4% CLSI, 29.2% EUCAST); and (iii) by antimicrobial agent: mE (100% levofloxacin/CLSI, 100% levofloxacin and meropenem/EUCAST), ME (35.3% colistin/CLSI, 25.0% colistin/EUCAST) and VME (64.7% colistin/CLSI, 86.7% gentamicin/EUCAST). Clinical microbiology laboratories must improve their ability to determine antimicrobial susceptibilities of Acinetobacter spp. isolates. Higher discrepancies using CLSI when compared with EUCAST are mainly due to mE and to a much lesser extent to ME or VME

Reduced susceptibility to biocides in Acinetobacter baumannii: association with resistance to antimicrobials, epidemiological behaviour, biological cost and effect on the expression of genes encoding porins and efflux pumps

<p><strong>Objectives</strong></p><p>The objective of this study was to analyse whether there is an association between reduced susceptibility to biocides in <i>Acinetobacter baumannii</i> and (i) antimicrobial resistance (co-resistance), (ii) prevalent (epidemic) clones, (iii) changes in the fitness or (iv) expression of genes related to efflux pumps and porins.</p><p><strong>Methods</strong></p><p>Susceptibility to biocides and antimicrobials was determined in 49 clonally unrelated isolates of <i>A. baumannii</i>. Biological cost, in terms of mean generation time, was determined by spectrophotometry. Quantitative real-time RT–PCR was used to determine the relative expression of genes encoding several efflux pumps and porins.</p><p><strong>Results</strong></p><p>Reduced susceptibility to chlorhexidine digluconate, benzalkonium chloride and Irgasan® was associated with resistance to aminoglycosides, tetracycline and ciprofloxacin (<i>P</i> < 0.05). The MICs of carbapenems, aminoglycosides, doxycycline and ciprofloxacin for isolate Ab70 (epidemic clone) exposed to these biocides increased by ≥2 dilutions. Reduced susceptibility to Orsan® was more frequent among prevalent clones than non-prevalent clones (<i>P</i> < 0.05). Mean generation times for Ab70 before and after exposure to benzalkonium chloride were 57.8 and 78.1 min, respectively (<i>P</i> = 0.02). Relative expression of <i>abeS</i> and <i>adeB</i> was increased in Ab46 and Ab70 after exposure to chlorhexidine digluconate, but was decreased for <i>ompA</i> and <i>carO</i> after exposure to Irgasan®.</p><p><strong>Conclusions</strong></p><p>Reduced susceptibility to biocides is associated with co-resistance to carbapenems, aminoglycosides, tetracycline and ciprofloxacin. Reduced susceptibility to Orsan® may be a marker of prevalent clones. Acquisition of reduced susceptibility to benzalkonium chloride has a biological cost. Exposure to biocides affects the relative expression of genes related to some efflux pump genes (increased expression) or porins (reduced expression).</p&gt

Resistencia frente a Colletotrichum acutatum en frutos de fresa: prueba de función de genes mediante ensayos de expresión transitoria

Trabajo presentado en el XVIII Congreso de la Sociedad Española de Fitopatología (SEF), celebrado en Palencia del 20 al 23 de septiembre de 2016.La fresa (Fragaria ananassa) es fuente de vitaminas, antioxidantes y metabolitos de múltiples beneficios para la salud. Su producción anual en España (tercer productor mundial) supera 200.000 toneladas (>300 millones euros) y sufre la acción de gran variedad de organismos fitopatógenos que causan importantes pérdidas. En general, las plantas han desarrollado mecanismos de defensa naturales frente al ataque de patógenos. En plantas modelo, existe gran evidencia de que miembros de las familias génicas NPR, WRKY o E3-Ubiquitin ligasas, entre otras, son clave para la activación de rutas moleculares que conducen a la defensa. En fresa, hemos identificados genes homólogos a miembros de dichas familias. Para conocer la verdadera función de dichos genes hemos obtenido construcciones plasmídicas, mediante sistema Gateway, para sobreexpresión/silenciamiento de los mismos en la fresa y hemos desarrollado un sistema de ensayo fitopatológico tras su expresión transitoria en fruto. Así, a frutos de fresa se inyectó (a una mitad del fruto) una suspensión de Agrobacterium tumefaciens portadora de la construcción de interés. A las 48h estos frutos se inocularon con Colletotrichum acutatum en ambas mitades, mediante filtros-disco (5 mm) estériles previamente sumergidos en suspensión de 104conidias/mL. Tras 5 días se realizaron cortes longitudinales desde los puntos de inoculación, evaluándose la penetración fúngica según escala 1-3 (1 fruto sano, 2 penetración moderada y 3 penetración profunda) en cada una de las 2 caras (agroinyectada/no agroinyectada), obteniéndose de esta forma un ratio (valor zona-agroinyectada/valor zona-no agroinyectada) para cada fruto. Análisis preliminares muestran diferencias entre frutos control (no agroinyectados y agroinyectados medio Murashige-Skoog) y frutos agroinyectados con construcciones de genes tipo NPR y WRKY, que permiten implicarlos de forma inequívoca en defensa. Según estos resultados actualmente poseemos un sistema fiable de ensayo de susceptibilidad a C. acutatum, lo que permitirá evaluar la implicación de genes en defensa a este patógeno.Peer Reviewe

Effect of the application of the ABA biosynthesis inhibitor NDGA on the expression of the strawberry ripening-related <i>NAC</i> genes determined by qRT-PCR.

<p>After treatment, relative expression values were calculated in relation to untreated control C_t, which was assigned an arbitrary value equal to the unity. Control: G-W fruit injected with H₂O; NDGA: G-W fruits injected with NDGA (100 μM). Both samples were harvested 8 days after beginning of treatment. Mean values ± SD of three independent experiments are shown. Student’s t-test determined statistical significance with respect to reference sample. (***) p-value < 0.001.</p

Developmental expression of the strawberry ripening-related <i>NAC</i> genes in fruit receptacles.

<p>qRT-PCR results were obtained using specific primers for <i>NAC</i> genes. Quantification is based on C_t values. Relative expression values were calculated in relation to G1 fruits receptacles C_t value in all cases, which was assigned an arbitrary value equal to unity. G1, green stage 1; G3, green stage 3; W, white stage; R, red stage; OR, overripe stage and SE, senescent stage. Data are a mean of three independent experiments. One-way ANOVA determined statistical significance. Letters indicate significant differences (p < 0.05, Scheffe post-hoc test).</p

Ligation Group (FvH) distribution of the curated collection of the strawberry <i>NAC</i> genes.

<p>The putative <i>NAC</i> genes were renamed from <i>FvNAC001</i> to <i>FvNAC112</i>, based on their placement into the Ligation Groups (FvH) or pseudo-chromosomes. The lengths of the drawings are proportional to the actual pseudo-chromosome lengths. FvH1 has a length of 24.25Mbp nucleotides and this can be used as a reference. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196953#pone.0196953.s007" target="_blank">S7 Table</a> shows the correspondence between the current gene names provided in the <i>Fragaria</i> genome database and these newly assigned and ordered names, and the actual length in bases of all pseudo-chromosomes and contigs of the current V4.0.a1 of the genome.</p

Auxin regulated expression of the strawberry ripening-related <i>NAC</i> genes determined by qRT-PCR.

<p>After auxin treatment, relative expression values were calculated in relation to the C_t of control fruits at the G3 stage, which was assigned an arbitrary value equal to the unity. G3: fruit at the G3 stage; G3 –Ach: G3 fruit receptacle without achenes for 5 days; G3 -Ach + NAA: G3 fruit receptacle without achenes plus NAA for 5 days (added at day zero). Data are a mean of three independent experiments. One-way ANOVA determined statistical significance. Letters indicate significant differences (p<0.05, Scheffe post-hoc test).</p

Genome-wide analysis of the NAC transcription factor family and their expression during the development and ripening of the <i>Fragaria</i> × <i>ananassa</i> fruits

<div><p>NAC proteins are a family of transcription factors which have a variety of important regulatory roles in plants. They present a very well conserved group of NAC subdomains in the N-terminal region and a highly variable domain at the C-terminus. Currently, knowledge concerning NAC family in the strawberry plant remains very limited. In this work, we analyzed the NAC family of <i>Fragaria vesca</i>, and a total of 112 NAC proteins were identified after we curated the annotations from the version 4.0.a1 genome. They were placed into the ligation groups (pseudo-chromosomes) and described its physicochemical and genetic features. A microarray transcriptomic analysis showed six of them expressed during the development and ripening of the <i>Fragaria</i> x <i>ananassa</i> fruit. Their expression patterns were studied in fruit (receptacle and achenes) in different stages of development and in vegetative tissues. Also, the expression level under different hormonal treatments (auxins, ABA) and drought stress was investigated. In addition, they were clustered with other NAC transcription factor with known function related to growth and development, senescence, fruit ripening, stress response, and secondary cell wall and vascular development. Our results indicate that these six strawberry NAC proteins could play different important regulatory roles in the process of development and ripening of the fruit, providing the basis for further functional studies and the selection for NAC candidates suitable for biotechnological applications.</p></div

Phylogenetic relationship and conserved subdomain composition of the 112 NAC strawberry proteins using MEME.

<p>The phylogenetic tree was constructed with the protein sequences with the IQ-tree program using 1000 bootstrappings. Domain analysis was performed with the MEME web service as indicated in Material and methods. A colored box represents each individual subdomain. Grey lines represent sequences that do not share common domains. Detailed amino acid sequence information for each subdomain is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196953#pone.0196953.s008" target="_blank">S8 Table</a>.</p