Open Access Research article

PacC and pH–dependent transcriptome of the mycotrophic fungus Trichoderma virens

Naomi Trushina1, Michal Levin1, Prasun K Mukherjee23 and Benjamin A Horwitz1*

Author Affiliations

1 Department of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel

2 Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 440085, India

3 Present address: Central Institute for Cotton Research, Nagpur 440010, India

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BMC Genomics 2013, 14:138  doi:10.1186/1471-2164-14-138

Published: 28 February 2013

Additional files

Additional file 1:

List of primers not included in Tables 1and 2. The primers listed here are referred to in the text. Degenerate primers, gene-specific primers (GSP) and adaptors were used to clone and sequence pacC prior to release of the genome sequence. Actin primers were used for normalization (housekeeping gene); “standard” primers were used in construction and validation of transformants. In the degenerate primers, I indicates inosine.

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Additional file 2:

Alignment of PacC protein from T. virens IMI 304061 and T. virens Gv29.8, the published reference strain. Protein sequence alignment.

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Additional file 3:

Verification of double-crossover events in transformants. This is a set of data confirming gene successful double crossover integration events. The linear map shows the pacC genomic region, with the relevant genes and markers indicated. Primer names are shown, with their locations and directions indicated by small arrows. PCR products are shown in the gel images; the source of the template DNA is shown in bold text below each lane, and the primer pairs used for the amplification are noted below the image, referring to the corresponding set of lanes. The primer names are also listed in the table (Expected sizes of PCR products from mutant validation), along with the predicted ampicon sizes.

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Additional file 4:

Split-marker gene replacement strategy. This figure illustrates the split-marker strategy. In the first round of PCR reactions, the flanking regions are amplified; in the second round, they are each fused to part of the hygromycin resistance cassette (HYG). For primer sequences see Additional file 14; the strategy and diagram are adapted from [46].

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Additional file 5:

Growth rate of wild type and ΔpacC. This graph shows growth rate of the ΔpacC strain relative to the wild type. Growth was measured by placing a 5-mm-diameter mycelial disk of the fungus in the center of a PDA plate and measuring the colony diameter at the indicated times. Values represent an average of 4 replicates. Error bars represent the standard deviation.

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Additional file 6:

Morphology of wild type and ΔpacC mutant T. virens. This figure is a photo of cultures, showing colony morphology. A 5-mm-diameter mycelial disk of the fungus was inoculated in the center of a PDA plate. The plates were incubated in the light and dark and colonies were photographed at the indicated times.

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Additional file 7:

Lists of differentially expressed genes from statistical analysis (Figure 5). List of protein ID numbers for each category of the genes shown in Figure 5 of the main text.

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Additional file 8:

Expression profiles of PacC-dependent, alkaline pH upregulated genes, and available annotation. Details of regulated genes are given here. Secreted proteins are indicated by a + if predicted at a SignalP score of at least 0.7. The number of predicted PacC binding sites (GCCARG, see text) in the 1 kb region upstream of the start codon is given in the last column. Expression profiles (fold expression at pH 8 compared to pH 4, and wt compared to ΔpacC at pH 8) are given. Annotation by homology: entries are best hits by BLAST, E values are given in the following column.

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Additional file 9:

Mutants in P type ATPase Δena1 are similar to wt in plate assays for growth and confrontation. Photos of cultures grown on PDA plates, taken at 11 days from above to show overgrowth and sporulation (A), or below to show pH indicator color (B). The wt and Δena1 strains were grown in confrontation with two plant pathogens.

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Additional file 10:

Expression profiles of PacC-dependent, acid pH upregulated genes, and available annotation. Details of regulated genes are given here. Secreted proteins are indicated by a + if predicted at a SignalP score of at least 0.7. The number of predicted PacC binding sites (GCCARG, see text) in the 1 kb region upstream of the start codon is given in the last column. Expression profiles (fold expression at pH 8 compared to pH 4, and wt compared to ΔpacC at pH 8) are given. Annotation by homology: entries are best hits by BLAST, E values are given in the following column.

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Additional file 11:

Disruption of pacC by homologous integration. The diagram shows the scheme used for replacement of the entire pacC coding sequence. HYG indicates selectable marker cassette (for full details, see Methods, main text).

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Additional file 12:

Primers for gene knock-out. Sequences and names of the primers used for knock-out of pacC and confirmation of the integration event are given.

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Additional file 13:

Primers for split-marker gene replacement strategy. This is a primer list for split-marker gene replacement. In the Table, lower case indicates sequences that are not complementary to the template in the first PCR step. In the second PCR step, these sequences, which are complementary to the ends of the selectable marker, allow joining of the fragments.

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Additional file 14:

Complete microarray data. The complete microarray data are provided as a comma delimited file (Excel compatible). The first column gives the Protein ID. This number, when entered into the “search” box in the T. virens v1.0 web page, leads conveniently to all the available gene model, transcript and protein information. The second column gives the number of (identical) oligonucleotides spotted on the microarray for this gene. The following columns give the signal values for each independent experiment, after background subtraction and normalization (see Methods, main text).

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