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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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Experimental design for microarray hybridizations. Samples tested positive for either PNRSV, PLMVd, both pathogens simultaneously, or healthy controls were analysed in four independent biological replicates and two technical replicates (dye swaps: Cy3-healthy/Cy5-infected or Cy3-infected/Cy5-healthy). Each biological replicate (numbered 1 to 4) for each infection and healthy plants consisted of a pool of four infected peach fruits from four different trees. The different sets of probes were hybridized to a cDNA microarray representing all the unigenes in the ChillPeach database. In detail, microarray hybridizations were performed by hybridizing each virus infected biological replicate and the healthy control replicate to an array resulting in a total of 16 hybridizations. Dye swaps were performed for two of the four replicates of each infection.
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Figure 1: Experimental design for microarray hybridizations. Samples tested positive for either PNRSV, PLMVd, both pathogens simultaneously, or healthy controls were analysed in four independent biological replicates and two technical replicates (dye swaps: Cy3-healthy/Cy5-infected or Cy3-infected/Cy5-healthy). Each biological replicate (numbered 1 to 4) for each infection and healthy plants consisted of a pool of four infected peach fruits from four different trees. The different sets of probes were hybridized to a cDNA microarray representing all the unigenes in the ChillPeach database. In detail, microarray hybridizations were performed by hybridizing each virus infected biological replicate and the healthy control replicate to an array resulting in a total of 16 hybridizations. Dye swaps were performed for two of the four replicates of each infection.

Mentions: For microarray analysis, peach fruits infected either with PNRSV, PLMVd or simultaneously with both pathogens were collected at developmental stage (S4), at approximately 100 days after boom, period where the fruit reaches the final full size and enters the fruit ripening or climacteric stage according with the description provided by Zanchin et al. [26]. The tissue used for RNA extraction was exocarp and mesocarp. The analysis of the PLMVd sequences recovered from the infected trees revealed a mix of quasispecies (e.g. PLMVd-4tun, ZZ46, lib P7, PC-C29 etc.) none of them associated to symptomatic strains. For the PNRSV all the sequence variants recovered belong to the PV32 group for which no specific symptomatology has been observed [9]. To obtain robust and statistically accurate data, four biological replicates and two technical, dye swapped replicates of healthy, virus infected, viroid infected, and doubly infected samples were analyzed. Each biological replicate consisted of a pool of four peach fruits from four different trees previously tested positive (for the infected samples) or negative (for the healthy controls) (Figure 1). RNA of the different sets of samples was hybridized to cDNA microarrays representing all the unigenes in the ChillPeach (http://bioinfo.ibmcp.upv.es/genomics/ChillPeachDB) database.


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)

Experimental design for microarray hybridizations. Samples tested positive for either PNRSV, PLMVd, both pathogens simultaneously, or healthy controls were analysed in four independent biological replicates and two technical replicates (dye swaps: Cy3-healthy/Cy5-infected or Cy3-infected/Cy5-healthy). Each biological replicate (numbered 1 to 4) for each infection and healthy plants consisted of a pool of four infected peach fruits from four different trees. The different sets of probes were hybridized to a cDNA microarray representing all the unigenes in the ChillPeach database. In detail, microarray hybridizations were performed by hybridizing each virus infected biological replicate and the healthy control replicate to an array resulting in a total of 16 hybridizations. Dye swaps were performed for two of the four replicates of each infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672095&req=5

Figure 1: Experimental design for microarray hybridizations. Samples tested positive for either PNRSV, PLMVd, both pathogens simultaneously, or healthy controls were analysed in four independent biological replicates and two technical replicates (dye swaps: Cy3-healthy/Cy5-infected or Cy3-infected/Cy5-healthy). Each biological replicate (numbered 1 to 4) for each infection and healthy plants consisted of a pool of four infected peach fruits from four different trees. The different sets of probes were hybridized to a cDNA microarray representing all the unigenes in the ChillPeach database. In detail, microarray hybridizations were performed by hybridizing each virus infected biological replicate and the healthy control replicate to an array resulting in a total of 16 hybridizations. Dye swaps were performed for two of the four replicates of each infection.
Mentions: For microarray analysis, peach fruits infected either with PNRSV, PLMVd or simultaneously with both pathogens were collected at developmental stage (S4), at approximately 100 days after boom, period where the fruit reaches the final full size and enters the fruit ripening or climacteric stage according with the description provided by Zanchin et al. [26]. The tissue used for RNA extraction was exocarp and mesocarp. The analysis of the PLMVd sequences recovered from the infected trees revealed a mix of quasispecies (e.g. PLMVd-4tun, ZZ46, lib P7, PC-C29 etc.) none of them associated to symptomatic strains. For the PNRSV all the sequence variants recovered belong to the PV32 group for which no specific symptomatology has been observed [9]. To obtain robust and statistically accurate data, four biological replicates and two technical, dye swapped replicates of healthy, virus infected, viroid infected, and doubly infected samples were analyzed. Each biological replicate consisted of a pool of four peach fruits from four different trees previously tested positive (for the infected samples) or negative (for the healthy controls) (Figure 1). RNA of the different sets of samples was hybridized to cDNA microarrays representing all the unigenes in the ChillPeach (http://bioinfo.ibmcp.upv.es/genomics/ChillPeachDB) database.

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