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A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by prunus necrotic ringspot virus and peach latent mosaic viroid.

Herranz MC, Niehl A, Rosales M, Fiore N, Zamorano A, Granell A, Pallas V - Virol. J. (2013)

Bottom Line: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits.We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections.These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia 46022, Spain. carhergor@gmail.com

ABSTRACT

Background: Microarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees.

Results: Upon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection.

Conclusions: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

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Validation of microarray data using quantitative RT-PCR. The Microarray (upper graph) and quantitative RT-PCR (lower graph) data are shown for nine randomly selected genes with statistically significant expression changes in the microarray experiments: Glutamate dehydrogenase 2 (GDH2) (ppa006458m.g), Cysteine proteinase (RD21A) (ppa005328m.g), Invertase/pectin methylesterase inhibitor family protein (ppa011831m.g), Universal stress protein (USP) family protein (ppa012560m.g), CBL-interacting protein kinase 6 (CIPK6) (ppa005365m.g), Phytoene synthase (ppa005962m.g), Auxine response protein (IAA9) (ppa007194m.g), Glutamate descarboxilase (ppa004796m.g) and Expansin (EXP8) (ppa010260m.g). Clathrin adapter complex (ppa005912m.g) was used as a control gene with unchanged expression upon infection. Values represent the log2ratio.
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Figure 5: Validation of microarray data using quantitative RT-PCR. The Microarray (upper graph) and quantitative RT-PCR (lower graph) data are shown for nine randomly selected genes with statistically significant expression changes in the microarray experiments: Glutamate dehydrogenase 2 (GDH2) (ppa006458m.g), Cysteine proteinase (RD21A) (ppa005328m.g), Invertase/pectin methylesterase inhibitor family protein (ppa011831m.g), Universal stress protein (USP) family protein (ppa012560m.g), CBL-interacting protein kinase 6 (CIPK6) (ppa005365m.g), Phytoene synthase (ppa005962m.g), Auxine response protein (IAA9) (ppa007194m.g), Glutamate descarboxilase (ppa004796m.g) and Expansin (EXP8) (ppa010260m.g). Clathrin adapter complex (ppa005912m.g) was used as a control gene with unchanged expression upon infection. Values represent the log2ratio.

Mentions: As reference genes for quantitative RT-PCR we chose peach orthologs of Arabidopsis F-box family protein (F-BOX) and elongation factor 1-α (EF1-α), which were previously reported to exhibit stable expression upon virus infection in Arabidopsis [29] and the clathrin adaptor complex (CAC) as internal control of the microarray. All reference genes exhibited similar expression levels in virus, viroid infected or healthy peach fruits, demonstrating their suitability as reference genes in peach. To validate the results obtained by microarray analysis we performed quantitative RT-PCR with nine randomly selected genes displaying induced or reduced expression upon infection plus the CAC. The nine selected genes were Phytoene synthase, Auxin response protein (IAA9), Expansin (EXP8), Glutamate descarboxilase, Glutamate dehydrogenase 2 (GDH2), Cysteine proteinase (RD21A), Invertase/pectin methylesterase inhibitor family protein, Universal stress protein (USP) family protein, CBL-interacting protein kinase 6 (CIPK6) and Phosphate-responsive protein, putative (E). Out of these nine genes analyzed, seven displayed consistent gene expression changes with both methods (Figure 5). Phytoene synthase, Auxin response protein, Glutamate descarboxilase, Glutamate dehydrogenase 2 (GDH2), Cysteine proteinase (RD21A), Invertase/pectin methylesterase inhibitor family protein, CBL-interacting protein kinase 6 (CIPK6) exhibited reduced expression in PNRSV, PLMVd and PNRSV-PLMVd infected samples compared to the healthy control plants, and Glutamate decarboxylase was induced upon infection. As already seen by microarray analysis, all expression changes were more pronounced upon double infection with PNRSV and PLMVd. Expression levels for Expansin, however, differed in Microarray and quantitative RT-PCR experiments. While the gene was upregulated in microarray analyses, it was downregulated by qRT-PCR in PNRSV infected samples. Expansins exist in a multigene family in Arabidopsis [30]. It is likely, that also peach has several expansin isoforms. Thus, the differences in expression of Expansin upon infection may result from several isoforms recognized by the cDNA array and the specific detection of only one isoform in our quantitative RT-PCR experiments. In a similar way, expression levels for Universal stress protein (USP) family protein are distinct in the two different analysis. A similar situation to that observed for Expansins occurs with this gene and thus, the same explanation could be valid for this protein family. Taken together, the results of qRT-PCR analyses confirmed the gene expression changes seen by microarrays analysis.


A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by prunus necrotic ringspot virus and peach latent mosaic viroid.

Herranz MC, Niehl A, Rosales M, Fiore N, Zamorano A, Granell A, Pallas V - Virol. J. (2013)

Validation of microarray data using quantitative RT-PCR. The Microarray (upper graph) and quantitative RT-PCR (lower graph) data are shown for nine randomly selected genes with statistically significant expression changes in the microarray experiments: Glutamate dehydrogenase 2 (GDH2) (ppa006458m.g), Cysteine proteinase (RD21A) (ppa005328m.g), Invertase/pectin methylesterase inhibitor family protein (ppa011831m.g), Universal stress protein (USP) family protein (ppa012560m.g), CBL-interacting protein kinase 6 (CIPK6) (ppa005365m.g), Phytoene synthase (ppa005962m.g), Auxine response protein (IAA9) (ppa007194m.g), Glutamate descarboxilase (ppa004796m.g) and Expansin (EXP8) (ppa010260m.g). Clathrin adapter complex (ppa005912m.g) was used as a control gene with unchanged expression upon infection. Values represent the log2ratio.
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Figure 5: Validation of microarray data using quantitative RT-PCR. The Microarray (upper graph) and quantitative RT-PCR (lower graph) data are shown for nine randomly selected genes with statistically significant expression changes in the microarray experiments: Glutamate dehydrogenase 2 (GDH2) (ppa006458m.g), Cysteine proteinase (RD21A) (ppa005328m.g), Invertase/pectin methylesterase inhibitor family protein (ppa011831m.g), Universal stress protein (USP) family protein (ppa012560m.g), CBL-interacting protein kinase 6 (CIPK6) (ppa005365m.g), Phytoene synthase (ppa005962m.g), Auxine response protein (IAA9) (ppa007194m.g), Glutamate descarboxilase (ppa004796m.g) and Expansin (EXP8) (ppa010260m.g). Clathrin adapter complex (ppa005912m.g) was used as a control gene with unchanged expression upon infection. Values represent the log2ratio.
Mentions: As reference genes for quantitative RT-PCR we chose peach orthologs of Arabidopsis F-box family protein (F-BOX) and elongation factor 1-α (EF1-α), which were previously reported to exhibit stable expression upon virus infection in Arabidopsis [29] and the clathrin adaptor complex (CAC) as internal control of the microarray. All reference genes exhibited similar expression levels in virus, viroid infected or healthy peach fruits, demonstrating their suitability as reference genes in peach. To validate the results obtained by microarray analysis we performed quantitative RT-PCR with nine randomly selected genes displaying induced or reduced expression upon infection plus the CAC. The nine selected genes were Phytoene synthase, Auxin response protein (IAA9), Expansin (EXP8), Glutamate descarboxilase, Glutamate dehydrogenase 2 (GDH2), Cysteine proteinase (RD21A), Invertase/pectin methylesterase inhibitor family protein, Universal stress protein (USP) family protein, CBL-interacting protein kinase 6 (CIPK6) and Phosphate-responsive protein, putative (E). Out of these nine genes analyzed, seven displayed consistent gene expression changes with both methods (Figure 5). Phytoene synthase, Auxin response protein, Glutamate descarboxilase, Glutamate dehydrogenase 2 (GDH2), Cysteine proteinase (RD21A), Invertase/pectin methylesterase inhibitor family protein, CBL-interacting protein kinase 6 (CIPK6) exhibited reduced expression in PNRSV, PLMVd and PNRSV-PLMVd infected samples compared to the healthy control plants, and Glutamate decarboxylase was induced upon infection. As already seen by microarray analysis, all expression changes were more pronounced upon double infection with PNRSV and PLMVd. Expression levels for Expansin, however, differed in Microarray and quantitative RT-PCR experiments. While the gene was upregulated in microarray analyses, it was downregulated by qRT-PCR in PNRSV infected samples. Expansins exist in a multigene family in Arabidopsis [30]. It is likely, that also peach has several expansin isoforms. Thus, the differences in expression of Expansin upon infection may result from several isoforms recognized by the cDNA array and the specific detection of only one isoform in our quantitative RT-PCR experiments. In a similar way, expression levels for Universal stress protein (USP) family protein are distinct in the two different analysis. A similar situation to that observed for Expansins occurs with this gene and thus, the same explanation could be valid for this protein family. Taken together, the results of qRT-PCR analyses confirmed the gene expression changes seen by microarrays analysis.

Bottom Line: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits.We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections.These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia 46022, Spain. carhergor@gmail.com

ABSTRACT

Background: Microarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees.

Results: Upon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection.

Conclusions: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

Show MeSH
Related in: MedlinePlus