1. スポロトリコーシス5例(第443回千葉医学会例会 第16回千葉皮膚科臨床談話会)

A potential stable stem-loop structure in the 5’-terminal sequence (left) and a triple stem-loop structure in 3’-terminal sequences (right) were predicted with a RNA structure software. (PDF 60 kb

Festividades sazonais e comunitárias no currículo em educação de infância

Relatório apresentado para a obtenção do grau de Mestre em Educação Pré-EscolarO presente relatório engloba o percurso formativo ao longo da prática de ensino supervisionada em contexto de Creche e Jardim de Infância, refletindo as aprendizagens realizadas e as dificuldades sentidas, assim como a emergência de questões decorrentes da prática. A problemática estudada, centrada nas valências de Creche e de Jardim de Infância, refere-se à Importância das festividades sazonais e comunitárias num currículo em Educação de Infância. A partir da abordagem do significado e importância das festividades feita por alguns autores, o presente estudo tem como objetivo geral compreender a importância atribuída pelas educadoras de infância à celebração das festividades, quais as datas que privilegiam, os motivos que justificam essas escolhas, assim como as implicações que identificam para as aprendizagens e para o desenvolvimento infantil. O estudo, qualitativo e de carácter exploratório, utiliza para a recolha de dados a técnica do inquérito por entrevista. Conclui-se que nas duas valências as profissionais integram no trabalho educativo a comemoração de determinados dias festivos, garantindo a sua importância e considerando que promovem aprendizagens em todas as áreas do desenvolvimento.The present report covers the formative path along the supervised teaching practice in the context of Nursery and Kindergarten, reflecting the learning achieved and difficulties experienced as well as the emergence of issues arising from practice. The studied problem, focusing on the valences of Nursery and Preschool, refers to the importance of seasonal and community festivities in a curriculum in Childhood Education. From the approach to the meaning and importance of the festivities by some authors, this study has the overall objective of understanding the importance given by the kindergarten teachers to the celebration of the festivities, which dates that privilege, the reasons for these choices, as well as identifying the implications for learning and child development. The study, of qualitative and exploratory nature, uses for data collection technique interview survey. It is concluded that on both valences, the kindergarten teachers integrate on the educational work the celebration of certain festive days, ensuring their importance as promoters of learning in all areas of development.info:eu-repo/semantics/publishedVersio

A Small Secreted Virulence-Related Protein Is Essential for the Necrotrophic Interactions of <i>Sclerotinia sclerotiorum</i> with Its Host Plants

<div><p>Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen <i>Sclerotinia sclerotiorum</i> was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1^∆SP could induce significant plant cell death and targeted silencing of <i>SsSSVP1</i> resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1^∆SP interacted with QCR8, a subunit of the cytochrome b-c₁ complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C³⁸ and C⁴⁴) in SsSSVP1^∆SP had significant effects on its homo-dimer formation, SsSSVP1^∆SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1^∆SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1^∆SP might be caused by the SsSSVP1^∆SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of <i>S</i>. <i>sclerotiorum</i>. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction.</p></div

Construction of an Ss-Sl2 RNAi vector (pSisl2) and functional analysis of <i>Ss-Sl2</i> silenced strains.

<p>(A) A 310 bp fragment of <i>Ss-Sl2</i> was inserted in the sense orientation between an <i>Aspergillus nidulans trpC</i> promoter and an intron from <i>Gibberella zeae</i>, and the same fragment of <i>Ss-Sl2</i> was inserted in antisense orientations between this intron and the <i>trpC</i> terminator. (B) Expression level of <i>Ss-Sl2</i> in isolates containing pSisl2 and in the wild type strain were determined by real-time RT-PCR. The expression level of <i>Ss-Sl2</i> cDNA was normalized to that of <i>actin</i> cDNA in extracts from each strain. The abundance of cDNA from the wild type was assigned a value of 1. Bars indicate standard error. (C) Phenotype of the wild type strain and <i>Ss-Sl2</i> gene-silenced transformants.</p

Full SsSSVP1 could still induce plant cell death and it can be internalized into plant cells in the absence of a pathogen.

<p>(<b>A</b>) SsSSVP1 with SP still can induce cell death in leaves. Upper leaves from above the infiltrated sites were taken photos 10 days after <i>A</i>. <i>tumefaciens</i> infiltration. (<b>B</b>) SsSSVP1 with SP still can induce cell death in stems. GFP alone was used as control. Photos were taken 10 days after <i>A</i>. <i>tumefaciens</i> infiltration. (<b>C</b>) Both SP-GFP (which was used as control) and SsSSVP1 with SP localized in ER-like structure in plant cells (details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005435#ppat.1005435.s002" target="_blank">S2 Fig</a>), however, only full SsSSVP1 could also localize in cytoplasmic compartments in a particle like form. No particle-like form of SP-GFP was observed in cytoplasm. The SP refers in particular to the SP of SsSSVP1. Red particles show chloroplast autofluorescence. White solid arrows indicate the internalized particle-like form of SsSSVP1-GFP; White hollow arrows show the endocytic vesicle-like structure near plasma membrane. Photos were taken 3 days after agroinfiltration.</p

SsSSVP1 is a <i>Sclerotinia-</i> and <i>Botryotinia-</i>specific, cysteine-rich, small, secreted protein.

<p>(<b>A</b>) A predicted structure diagram of SsSSVP1 which comprises 163 aa. A putative N-terminal SP (aa 1 to 17) and the position of the eight cysteine residues of SsSSVP1 are present (C³⁸, C⁴⁴, C⁵⁴, C⁷⁹, C⁸¹, C⁹², C⁹⁵ and C¹¹⁷). (<b>B</b>) Multiple alignments indicate the homologs of SsSSVP1 are only present in <i>Sclerotinia</i>- and <i>Botryotinia</i> in the organisms sequenced so far and the eight cysteine residues are conserved in these homologs. Red rectangle labels the sites of the eight cysteine residues in multiple alignments. Protein sequences from top to bottom are derived from <i>B</i>. <i>cinerea</i> T4, <i>B</i>. <i>cinerea</i> B05.10, <i>B</i>. <i>cinerea</i> BcDW1, <i>Sclerotinia borealis</i> F-4157 and <i>S</i>. <i>sclerotiorum</i> Ep-1PNA367 respectively. The protein sequences of SsSSVP1 in <i>S</i>. <i>sclerotiorum</i> Ep-1PNA367 and 1980 are the same. (<b>C</b>) Western blot analysis with total proteins isolated from the liquid CM culture of the wild-type strain and SsSSVP1-FLAG engineered strains. SDS-polyacrylamide gel electrophoresis shows the equal loading amount of proteins used for the west blot analysis. Horseradish peroxidase conjugated secondary antibody detected an approximate 17 kDa band in SsSSVP1-FLAG engineered strains, but not in the wild-type strain.</p

SsMYRV4-mediated enhancement of horizontal transmission of unrelated mycoviruses among <i>S</i>. <i>sclerotiorum</i> individuals belonging to different VCGs.

<p>(A) Horizontal transmission of three mycoviruses SsDRV, SsRVL and SsMV1 using a dual-culture method on PDA plates (15-cm in diameter). SsDRV and SsRVL co-infect hypovirulent strain Ep-1PN, whereas SsMV1 naturally infects hypovirulent strain HC025. SsMV1 was horizontally transmitted into strain Ep-1PNA367 under laboratory conditions and the newly obtained SsMV1-infected strain was named HC-A367. Ep-A367T1 harbors three mycoviruses (SsDRV, SsRVL, and SsMYRV4). HC-A367T1 carries two mycoviruses (SsMV1 and SsMYRV4). After the mycelia of two different strains (a hypovirulent strain and a virulent strain) contact each other for 7 days, the new isolates (indicated by blue stars) were picked up from colony margin of the virulent strain, and the designations of newly obtained isolates are shown at the bottom of each plate. (B) Mycovirus content was assessed by dsRNA extraction and RT-PCR amplification. Primers SsMYRV4F and SsMYRV4R, SsDRV-F and SsDRV-R, SsRVLF and SsRVLR, and SsMV1-F and SsMV1-R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006234#ppat.1006234.s011" target="_blank">S5 Table</a>) were used for detection of SsMYRV4, SsDRV, SsRVL, and SsMV1, respectively. The <i>actin</i> gene served as an internal control; sizes of molecular mass standards (M) are indicated to the left of each panel.</p

Additional file 2: Figure S2. of Co-infection of a hypovirulent isolate of Sclerotinia sclerotiorum with a new botybirnavirus and a strain of a mitovirus

The 5’- (A) and 3’ - (B) terminus sequence alignment of the two L-dsRNA segments. (PDF 384 kb

Summary of Ss-Sl2 interacting proteins identified by Ss-Sl2 antibodies co-immunoprecipitation and LC/MS/MS analysis.

a<p># of peptides refers to the combined number of peptides identified for the protein in two samples.</p>b<p>Percent coverage refers to percent of MS/MS peptide coverage of identified protein seen over the entire amino-acid sequence.</p

Ss-Sl2 was detected in cytoplasm and cell wall of <i>S. sclerotiorum</i>.

<p>The cell wall protein and cytoplasm protein in mycelium were extracted respectively (50 μg) and subjected to western blot analysis with anti-Ss-Sl2 polyclonal antibodies.</p