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Pleurotus ostreatus manganese-dependent peroxidase silencing impairs decolourization of Orange II.

Salame TM, Yarden O, Hadar Y - Microb Biotechnol (2009)

Bottom Line: Relative real-time PCR quantification analysis confirmed that all the nine genes are transcribed, and that Mn(2+) amendment results in a drastic increase in the transcript levels of the predominantly expressed MnP genes (mnp 3 and mnp 9), while decreasing versatile peroxidase gene transcription (mnp 4).Knock-down of mnp 3 resulted in the reduction of fungal OII decolourization capacity, which was co-linear with marked silencing of the Mn(2+)-dependent peroxidase genes mnp 3 and mnp 9.This is the first direct genetic proof of an association between MnP gene expression levels and azo dye decolourization capacity in P. ostreatus, which may have significant implication on understanding the mechanisms governing lignin biodegradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

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Relative transcript abundance of P. ostreatus PC9 MnP gene family members in RNA extracted from a 7‐day‐old liquid culture in GP medium amended with 27 µM Mn2+ (+Mn treatment), relative to that measured in a non‐amended medium (−Mn treatment), which served as the calibrator treatment. The expression of mnp1–9 gene transcript abundance, relative to β‐tubulin transcript abundance (endogenous control), was measured by real‐time PCR relative quantification analysis using the ΔΔCT method. Data represent the average of three biological replicates. Bars denote the standard deviation; when not visible, the standard deviation is included within the graph line width. Note the log10 scale.
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f2: Relative transcript abundance of P. ostreatus PC9 MnP gene family members in RNA extracted from a 7‐day‐old liquid culture in GP medium amended with 27 µM Mn2+ (+Mn treatment), relative to that measured in a non‐amended medium (−Mn treatment), which served as the calibrator treatment. The expression of mnp1–9 gene transcript abundance, relative to β‐tubulin transcript abundance (endogenous control), was measured by real‐time PCR relative quantification analysis using the ΔΔCT method. Data represent the average of three biological replicates. Bars denote the standard deviation; when not visible, the standard deviation is included within the graph line width. Note the log10 scale.

Mentions: The P. ostreatus genome sequencing project has revealed the existence of at least nine non‐allelic genes coding for MnP gene family members (Table 1; http://genome.jgi‐psf.org/PleosPC15‐1). To date, only four of these genes (mnp1–4) have been studied (Cohen et al., 2001; 2002b). Of these known genes, only mnp3 encodes a Mn2+‐dependent peroxidase, whereas the others encode VPs (Mn2+‐independent peroxidases). We designated the additional five genes mnp5–9 (Table 1). The deduced protein sequences of mnp5–9 indicate that MnP6, 7, 8 and 9 are Mn2+‐dependent peroxidases, whereas MnP5 is most likely a VP (Asada et al., 1995; Ruiz‐Dueñas et al., 1999; Giardina et al., 2000; Irie et al., 2000; Cohen et al., 2001). In order to study the effect of Mn2+ on the transcription profile of P. ostreatus MnP gene family members we used relative real‐time PCR quantification analysis. The fungus was grown for 7 days in either a liquid medium amended with 27 µM Mn2+ (+Mn treatment) or a non‐amended medium (−Mn treatment). The results presented in Fig. 2 show the relative expression of the nine different MnP gene family members. The primers used for real‐time PCR analyses (Table 1) were verified to be gene‐specific, and examination of melting curves indicated highly specific amplification of the respective cDNAs (data not shown). The endogenous control gene used was β‐tubulin, and the calibrator was the −Mn treatment. Transcripts of all the nine mnp genes were detected in both Mn2+‐amended and non‐amended cultures. However, Mn2+ in the medium affected the transcript abundance level of the mnp genes analysed in different manners. The transcripts levels of mnp3 and mnp9 were about 200‐fold higher when Mn2+ was present in the medium; conversely, mnp4 transcript abundance was about 70‐fold higher in the non‐amended medium. The transcript levels of all the other genes were increased by only 1.4‐ to 3‐fold in the presence of Mn2+, and were therefore not considered to be substantially induced. In addition, absolute quantification of the basal expression levels of the MnP gene family members, based on evaluation deduced from the real‐time PCR amplification plots, indicate that, relative to the treatment, the transcript levels of mnp3, 4 and 9 were at least eightfold higher than those of the other mnp genes. Similar results were observed on the basis of semi‐quantitative RT‐PCR. The abundance of the β‐tubulin transcript was not affected by the presence of Mn2+. We therefore concluded that the presence of Mn2+ can affect different mnp genes in opposing ways.


Pleurotus ostreatus manganese-dependent peroxidase silencing impairs decolourization of Orange II.

Salame TM, Yarden O, Hadar Y - Microb Biotechnol (2009)

Relative transcript abundance of P. ostreatus PC9 MnP gene family members in RNA extracted from a 7‐day‐old liquid culture in GP medium amended with 27 µM Mn2+ (+Mn treatment), relative to that measured in a non‐amended medium (−Mn treatment), which served as the calibrator treatment. The expression of mnp1–9 gene transcript abundance, relative to β‐tubulin transcript abundance (endogenous control), was measured by real‐time PCR relative quantification analysis using the ΔΔCT method. Data represent the average of three biological replicates. Bars denote the standard deviation; when not visible, the standard deviation is included within the graph line width. Note the log10 scale.
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Related In: Results  -  Collection

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f2: Relative transcript abundance of P. ostreatus PC9 MnP gene family members in RNA extracted from a 7‐day‐old liquid culture in GP medium amended with 27 µM Mn2+ (+Mn treatment), relative to that measured in a non‐amended medium (−Mn treatment), which served as the calibrator treatment. The expression of mnp1–9 gene transcript abundance, relative to β‐tubulin transcript abundance (endogenous control), was measured by real‐time PCR relative quantification analysis using the ΔΔCT method. Data represent the average of three biological replicates. Bars denote the standard deviation; when not visible, the standard deviation is included within the graph line width. Note the log10 scale.
Mentions: The P. ostreatus genome sequencing project has revealed the existence of at least nine non‐allelic genes coding for MnP gene family members (Table 1; http://genome.jgi‐psf.org/PleosPC15‐1). To date, only four of these genes (mnp1–4) have been studied (Cohen et al., 2001; 2002b). Of these known genes, only mnp3 encodes a Mn2+‐dependent peroxidase, whereas the others encode VPs (Mn2+‐independent peroxidases). We designated the additional five genes mnp5–9 (Table 1). The deduced protein sequences of mnp5–9 indicate that MnP6, 7, 8 and 9 are Mn2+‐dependent peroxidases, whereas MnP5 is most likely a VP (Asada et al., 1995; Ruiz‐Dueñas et al., 1999; Giardina et al., 2000; Irie et al., 2000; Cohen et al., 2001). In order to study the effect of Mn2+ on the transcription profile of P. ostreatus MnP gene family members we used relative real‐time PCR quantification analysis. The fungus was grown for 7 days in either a liquid medium amended with 27 µM Mn2+ (+Mn treatment) or a non‐amended medium (−Mn treatment). The results presented in Fig. 2 show the relative expression of the nine different MnP gene family members. The primers used for real‐time PCR analyses (Table 1) were verified to be gene‐specific, and examination of melting curves indicated highly specific amplification of the respective cDNAs (data not shown). The endogenous control gene used was β‐tubulin, and the calibrator was the −Mn treatment. Transcripts of all the nine mnp genes were detected in both Mn2+‐amended and non‐amended cultures. However, Mn2+ in the medium affected the transcript abundance level of the mnp genes analysed in different manners. The transcripts levels of mnp3 and mnp9 were about 200‐fold higher when Mn2+ was present in the medium; conversely, mnp4 transcript abundance was about 70‐fold higher in the non‐amended medium. The transcript levels of all the other genes were increased by only 1.4‐ to 3‐fold in the presence of Mn2+, and were therefore not considered to be substantially induced. In addition, absolute quantification of the basal expression levels of the MnP gene family members, based on evaluation deduced from the real‐time PCR amplification plots, indicate that, relative to the treatment, the transcript levels of mnp3, 4 and 9 were at least eightfold higher than those of the other mnp genes. Similar results were observed on the basis of semi‐quantitative RT‐PCR. The abundance of the β‐tubulin transcript was not affected by the presence of Mn2+. We therefore concluded that the presence of Mn2+ can affect different mnp genes in opposing ways.

Bottom Line: Relative real-time PCR quantification analysis confirmed that all the nine genes are transcribed, and that Mn(2+) amendment results in a drastic increase in the transcript levels of the predominantly expressed MnP genes (mnp 3 and mnp 9), while decreasing versatile peroxidase gene transcription (mnp 4).Knock-down of mnp 3 resulted in the reduction of fungal OII decolourization capacity, which was co-linear with marked silencing of the Mn(2+)-dependent peroxidase genes mnp 3 and mnp 9.This is the first direct genetic proof of an association between MnP gene expression levels and azo dye decolourization capacity in P. ostreatus, which may have significant implication on understanding the mechanisms governing lignin biodegradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

Show MeSH
Related in: MedlinePlus