Characterization and genomic analyses of Aeromonas hydrophilaphages AhSzq?1 and AhSzw?1, isolates representing new specieswithin the T5virus genus

AbstractIn this study, two bacteriophage isolates, AhSzq-1 and AhSzw-1 that specifically infect Aeromonas hydrophila strainKT998822, were isolated from seawater and characterized. One-step growth curves showed that the latent period of AhSzq-1and AhSzw-1 are 50 min and 60 min, respectively. The sequence similarities between AhSzq-1 and AhSzw-1 were 88% at theDNA and 83% at the protein level, suggesting that these two phages are representatives of two different species. The virionmorphology, DNA genome size and terminal repeats of these two phages are similar to those of viruses classified as T5virusphages. Both phylogenetic analyses and proteomic comparison show that AhSzq-1 and AhSzw-1 group with members ofthe T5virus genus. We thus propose these two phages as representative isolates of two new species within the T5virus genus

Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule

<div><p>In most plants, the female germline starts with the differentiation of one megaspore mother cell (MMC) in each ovule that produces four megaspores through meiosis, one of which survives to become the functional megaspore (FM). The FM further develops into an embryo sac. Little is known regarding the control of MMC formation to one per ovule and the selective survival of the FM. The ICK/KRPs (interactor/inhibitor of cyclin-dependent kinase (CDK)/Kip-related proteins) are plant CDK inhibitors and cell cycle regulators. Here we report that in the ovules of Arabidopsis mutant with all seven <i>ICK/KRP</i> genes inactivated, supernumerary MMCs, FMs and embryo sacs were formed and the two embryo sacs could be fertilized to form two embryos with separate endosperm compartments. Twin seedlings were observed in about 2% seeds. Further, in the mutant ovules the number and position of surviving megaspores from one MMC were variable, indicating that the positional signal for determining the survival of megaspore was affected. Strikingly, ICK4 fusion protein with yellow fluorescence protein was strongly present in the degenerative megaspores but absent in the FM, suggesting an important role of ICKs in the degeneration of non-functional megaspores. The absence of or much weaker phenotypes in lower orders of mutants and complementation of the septuple mutant by <i>ICK4</i> or <i>ICK7</i> indicate that multiple ICK/KRPs function redundantly in restricting the formation of more than one MMC and in the selective survival of FM, which are critical to ensure the development of one embryo sac and one embryo per ovule.</p></div

Frequency of surviving megaspores at each of the four positions in <i>ick</i> septuple mutant ovules with one MMC.

<p>Frequency of surviving megaspores at each of the four positions in <i>ick</i> septuple mutant ovules with one MMC.</p

Presentation_1_In Vitro Design and Evaluation of Phage Cocktails Against Aeromonas salmonicida.PDF

<p>As an alternative approach against multidrug-resistant bacterial infections, phages are now being increasingly investigated as effective therapeutic agents. Here, aiming to design an efficient phage cocktail against Aeromonas salmonicida infections, we isolated and characterized five lytic A. salmonicida phages, AS-szw, AS-yj, AS-zj, AS-sw, and AS-gz. The results of morphological and genomic analysis suggested that all these phages are affiliated to the T4virus genus of the Caudovirales order. Their heterogeneous lytic capacities against A. salmonicida strains were demonstrated by experiments. A series of phage cocktails were prepared and investigated in vitro. We observed that the cocktail combining AS-gz and AS-yj showed significantly higher antimicrobial activity than other cocktails and individual phages. Given the divergent genomes between the phages AS-yj and AS-gz, our results highlight that the heterogeneous mechanisms that phages use to infect their hosts likely lead to phage synergy in killing the host. Conclusively, our study described a strategy to develop an effective and promising phage cocktail as a therapeutic agent to combat A. salmonicida infections, and thereby to control the outbreak of relevant fish diseases. Our study suggests that in vitro investigations into phages are prerequisite to obtain satisfying phage cocktails prior to application in practice.</p

Silique and seed development in WT, <i>ick12567</i>, <i>ick123567</i> and septuple mutants.

<p><b>(A)</b> Silique length. The average and standard deviation are shown for the length of fully extended siliques (4 plants per line with 10 siliques from each plant measured). (<b>B</b>) Number of aborted ovules per silique. Fully extended siliques were opened and aborted ovules counted under a dissecting microscope (4 plants with 6 siliques from each plant counted). The averages and standard deviations are shown. (<b>C</b>) Opened siliques showing silique length and aborted ovules of the septuple mutant. (<b>D</b>) Number of seeds per silique (4 plants per line with 6 siliques from each plant). The averages and standard deviations are shown. (<b>E</b>) Seed weight. Three different seed lots were used. For each lot, seeds were harvested from four plants in one pot and one thousand seeds were counted. The averages and standard deviations are shown. Data in (<b>A, B, D, E</b>) were analyzed using one-way ANOVA and post-hoc Tukey test, and significant differences are indicated by different letters (upper case) at p<0.01 level.</p

Identification of megaspore mother cells (MMCs) in meiosis by DMC1 immunostaining.

<p>Developing ovules were prepared and immunostained with an antibody against DMC1, which is specifically expressed during meiosis. (<b>A—C</b>) WT ovules at MMC (<b>A</b>), meiosis (<b>B</b>) and FG1 (<b>C</b>) stages. (<b>D—I</b>) Mutant ovules at MMC (<b>D</b>), meiosis (<b>E—H</b>) and FG1 (<b>I</b>) stages. One to four cells in the mutant ovules could be stained with DMC1 (<b>E—H</b>). Scale bars equal 5 μm.</p

Functional megaspore (FM) development in WT and <i>ick</i> septuple mutant.

<p>(<b>A—F</b>) Ovules at functional megaspore stage and observed under a DIC microscope. (<b>A</b>) WT ovule with a typical FM. (<b>B–E</b>) <i>ick</i> septuple ovules with 1–4 FMs. A typical FM was present in (<b>B</b>). In (<b>C</b>), two pairs of nuclei appear to be derived from the second meiotic divisions. The two megaspores at the micropylar end are degenerating while the two close to the chalazal end were surviving. In (<b>D</b>), three megaspores of similar size and morphology were surviving, while in (<b>E</b>), four megaspores with different sizes and appearances were surviving. (<b>F</b>) No typical FM was observed in the ovule. (<b>G—L</b>) Callose staining and DIC images were obtained for the same mutant ovules at functional megaspore stage to determine which megaspore survived. The positions of megaspores from each MMC are indicated with a number, with position 1 indicating the megaspore closest to the chalazal end. (<b>G, I, K</b>) show callose staining while (<b>H, J, M</b>) show the corresponding DIC images with superimposed callose staining. (<b>G, H</b>) show that one MMC finished meiosis and two megaspores were surviving. (<b>I, J</b>) also show the result from one MMC, but megaspores at positions 1 and 3 (counting from the chalazal end) were surviving. Note that there are three callose bands plus the pointed callus deposition at the micropylar end. (<b>K, L</b>) indicate that two MMCs completed meiosis (<b>K</b>). While four megaspores derived from the MMC at one side were all surviving, no megaspore from the other MMC appeared to be surviving. Arrows in (<b>C–E</b>) indicate FMs, numbers in (<b>G–L</b>) indicate the megaspore positions counted from the chalazal end, and scale bars equal 10 μm.</p