Competitiveness as a function of local and regional growth and development

Each economic entity, institution and individual has the responsibility of contributing to the economic development in its region. Creating conditions for the development and empowerment of the business sector are activities that in the long run lead to the strengthening of not only certain economic sectors, but the entire region. In the recent period sources from EU funds for co-financing of capital projects have become available to investors. Given the uncertainty in business conditions, investors’ poor capitalization, lack of business profitability and, in terms of profitability and risk, lack of high-quality capital projects, the benefits of these resources are insufficient and/or inadequately used. The aim of this paper is to analyse the strength and capabilities of Croatian companies for financing and implementation of high-quality capital projects. For this purpose, this paper will present the results of research of financial position of selected companies in 2012. Also, it presents the results of research from 2011 that examined the reality of projections of later activated investment projects. These results are the basis for a conclusion about the ability of management, in the analysed region, to make realistic plans and carry out high-quality capital projects

Presentation_1_Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System.PDF

<p>Ustilaginoidea virens is the causal agent of rice false smut, one of the major fungal diseases of rice. However, there are only limited molecular studies with this important pathogen due to the lack of efficient approaches for generating targeted gene disruption mutants. In this study, we used the CRISPR-Cas9 system to efficiently generate mutants deleted of the USTA ustiloxin and UvSLT2 MAP kinase genes. Three gRNA spacers of USTA, UA01, UA13, and UA21, were expressed with the RNAP III promoter of Gln-tRNA. For all of them, the homologous gene replacement frequency was higher when the Cas9 and gRNA constructs were transformed into U. virens on the same vector than sequentially. UA01, the spacer with the highest on-target score, had the highest knockout frequency of 90%, which was over 200 times higher than that of Agrobacterium tumefaciens-mediated transformation (ATMT) for generating ustA mutants. None of these USTA spacers had predicted off-targets with 1 or 2-nt variations. For predicted off-targets with 3 or 4-nt variations, mutations were not detected in 10 ustA mutants generated with spacer UA13 or UA21, indicating a relatively low frequency of off-target mutations in U. virens. For UvSLT2, the homologous gene replacement frequency was 50% with CRISPR-Cas9, which also was significantly higher than that of ATMT. Whereas ustA mutants had no detectable phenotypes, Uvslt2 mutants were slightly reduced in growth rate and reduced over 70% in conidiation. Deletion of UvSLT2 also increased sensitivity to cell wall stresses but tolerance to hyperosmotic or oxidative stresses. Taken together, our results showed that the CRISPR-Cas9 system can be used as an efficient gene replacement or editing approach in U. virens and the UvSlt2 MAP kinase pathway has a conserved role in cell wall integrity.</p

MAP kinase phosphorylation assays.

<p>Total proteins were isolated from the wild-type (PH-1) and <i>amt1</i> mutant (M2) strains. When detected with an anti-TpEY antibody, the phosphorylation levels of Mgv1 had no significant changes in the <i>amt1</i> mutant in comparison with the wild type. The phosphorylation of FgHog1 detected with an anti-TpGY antibody also appeared to be normal in the <i>amt1</i> mutant. Detection with a monoclonal anti-actin antibody showed equal amount of proteins.</p

Assays for the effects of Δ<i>amt1</i> mutation on other PRMT genes and genes adjacent to the telomere.

<p>RNA samples were isolated from germlings of the wild-type (PH-1) and Δ<i>amt1</i> mutant strains grown in liquid YEPD for 6 h. The expression levels of (<b>A</b>) three other PRMT genes, <i>AMT2</i>, <i>AMT3</i>, and <i>AMT4</i>, and (<b>B</b>) three predicted genes located in the telomeric region of chromosome 4 (FGSG_14027, FGSG_11614, and FGSG_11613) were assayed by qRT-PCR.</p

The <i>AMT1</i> gene replacement construct and deletion mutants.

<p><b>A.</b> The <i>AMT1</i> locus and gene replacement construct. The <i>AMT1</i> and <i>hph</i> genes are marked with empty and black arrows, respectively. 1F, 2R, 3F, and 4R are primers used to amplify the flanking sequences. <i>Bam</i>HI (B). <b>B.</b> Southern blot analysis with the wild type (PH-1) and Δ<i>amt1</i> transformants (M1, M2, and M3). All DNA samples were digested with <i>Bam</i>HI. The blots were hybridized with probe A (left) amplified with primers AMT1/5F and AMT1/6R and probe B (right) amplified with H852 and H850. <b>C.</b> Colony morphology of the PH-1, Δ<i>amt1</i> mutant M2, and Δ<i>amt1/AMT1</i> transformant C2 cultures grown on CM. Photographs were taken after incubation for 3 days.</p

Assays for defects of the Δ<i>amt1</i> mutant to different stresses.

<p>Cultures of the wild-type (PH-1), Δ<i>amt1</i> mutant (M2), and Δ<i>amt1/AMT1</i> (C2) strains grown on regular PDA or PDA with 0.7 M NaCl, 0.05% H₂O₂, or 0.01% SDS. Photographs were taken after incubation at 25°C for 3 days.</p

Infection assays with flowering wheat heads and corn silks.

<p><b>A.</b> Flowering wheat heads were drop-inoculated with conidia from the wild-type (PH-1), Δ<i>amt1</i> mutant (M2), and Δ<i>amt1/AMT1</i> (C2) strains. Black dots mark the inoculated spikeletes. Photographs were taken 14 days post-inoculation (dpi). <b>B.</b> Corn silks were inoculated with blocks of cultures of PH-1, Δ<i>amt1</i> mutant M2, and Δ<i>amt1/AMT1</i> transformant C2. Photographs were taken 6 dpi.</p

Infection assays and stress response tests with mutants deleted of other PRMT genes.

<p><b>A</b>. Corn silks inoculated with the wild type (PH-1), M2 (Δ<i>amt1</i>), KS2 (Δ<i>amt2</i>), KT3 (Δ<i>amt3</i>), KF4 (Δ<i>amt4</i>), and DM7 (Δ<i>amt1</i> Δ<i>amt2</i>). <b>B</b>. Colonies of PH-1, DM7, M2, and KS2 grown on PDA plates with 0.05% H₂O₂ or 0.01% SDS.</p

The wild-type and mutant strains of <i>Fusarium graminearum</i> used in this study.

<p>The wild-type and mutant strains of <i>Fusarium graminearum</i> used in this study.</p

Expression and subcellular localization of FgHog1-GFP.

<p><b>A.</b> Conidia harvested from the <i>Fghog1</i>/<i>FgHOG1</i>-GFP transformant HGC1 were re-suspended in sterile distilled water or 0.3 M NaCl and examined by DIC or epifluorescence microscopy (GFP). <b>B.</b> GFP signals in germlings of FGC1 were incubated in the liquid YEPD medium with or without 0.3M NaCl. Nuclei were stained with DAPI. Bar = 20 µm.</p