Video_1_Transformation of Penicillium rubens 212 and Expression of GFP and DsRED Coding Genes for Visualization of Plant-Biocontrol Agent Interaction.MOV

<p>Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization.</p

Reductive stress leads to aggregation of peripheral ER strands.

<p><b>A.</b> Sec63-GFP images (inverted greyscale) of cells treated with DTT for the indicated time periods. Images correspond to maximal intensity projections of z-stacks processed with an ‘unsharp’ filter. In the 45 min GFP image, green arrowheads indicate aggregates of ER strands and magenta arrows indicate nuclei, whose NEs, unlike peripheral ER strands, appear unaltered. All images are shown at the same magnification. Bars, 5 µm. <b>B.</b> Examples of ring-shaped ER aggregates in cells treated with DTT for 130 min.</p

Table_1_Transformation of Penicillium rubens 212 and Expression of GFP and DsRED Coding Genes for Visualization of Plant-Biocontrol Agent Interaction.docx

<p>Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization.</p

Table_2_Transformation of Penicillium rubens 212 and Expression of GFP and DsRED Coding Genes for Visualization of Plant-Biocontrol Agent Interaction.PDF

<p>Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization.</p

The peripheral Sec63 ER is not disorganized during mitosis.

<p><b>A.</b> Nuclear division in a tip-distant region of a cell coexpressing Sec63-GFP and HhoA-mCherry (‘green’ and ‘red’ channels displayed in inverted greyscale). Time is indicated in min:sec. Whenever possible, the position of the intact NEs of the dividing nuclei is indicated with arrows. <b>B.</b> The peripheral ER does not undergo disorganization during mitosis. The top greyscale image is a kymograph displaying growth of the hyphal tip cell shown below. The kymograph was traced as schematized on the right. The series of images displayed below the kymograph (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067154#pone.0067154.s003" target="_blank">Video S3</a> should be consulted) show that the peripheral ER does not undergo rearrangements during mitosis.</p

The ER is brefeldin A-insensitive.

<p>A strain coexpressing Sec63-GFP with the late Golgi marker mRFP-PH^OSBP was treated with brefeldin A for 25 min. Under these conditions, late Golgi cisternae form aggregates. In contrast, the ER appears to be largely resistant to the drug. Images represent maximal intensity projections of z-stacks of deconvolved images.</p

The ER does not undergo major changes following microtubule depolymerization.

<p><b>A.</b> Control experiment demonstrating the depolymerization of MTs (green) upon treatment with benomyl. Unpolymerized TubA-GFP accumulates in the cytosol, leaving the nuclei empty (nuclei are labeled with HhoA-mCherry). The picture was taken after after 17 min of incubation in the presence of the drug. <b>B.</b> Effect of benomyl treatment on the Sec63-GFP-labeled ER. The representative example shown on the right corresponds to a cell incubated for 30 min in the presence of benomyl. The positions of the nuclei are revealed by the HhoA-mCherry fluorescence.</p

The ER visualized with Sec63-GFP.

<p>Different planes (z coordinates indicated in µm) of z-stack of images of a hyphal tip cell. The position of the nucleus is indicated in the middle plane. The asterisk indicates the apical border of the hypha, whereas the arrow indicates the apex-proximal ER protusion.</p

Image_2_Transformation of Penicillium rubens 212 and Expression of GFP and DsRED Coding Genes for Visualization of Plant-Biocontrol Agent Interaction.PDF

<p>Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization.</p

Table_3_Transformation of Penicillium rubens 212 and Expression of GFP and DsRED Coding Genes for Visualization of Plant-Biocontrol Agent Interaction.PDF

<p>Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization.</p