A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by prunus necrotic ringspot virus and peach latent mosaic viroid
© Herranz et al.; licensee BioMed Central Ltd. 2013
Received: 17 December 2012
Accepted: 21 May 2013
Published: 28 May 2013
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.
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.
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.
Plant viruses and viroids are obligate parasites that, consequently, depend on host factors to complete their life cycle. Subversion of host factors for viral replication and spread often disrupts cellular homeostasis and leads to pathogenesis and disease symptoms . Among the most important consequences of viral pathogenesis are changes in the expression of host genes [2–4]. However, the functional significance of transcript changes in specific plant-pathogen interactions is still not well understood [2, 5, 6]. Thus, an improved understanding of these interactions should lead to new and creative methods to control plant virus/viroid diseases.
Stone fruit viruses and viroids can cause significant crop damage and crop losses . Viroids are pathogens of food, industrial and ornamental plants. PLMVd, a member of the family Avsunviroidae, consists of a circular monocatenary RNA of 335-351 nucleotides and is distributed worldwide on peach . Although PLMVd was initially considered as a latent pathogen it has been described that several isolates of PLMVd can cause deformation, discolored spots and cracked sutures in infected peach fruit . PNRSV is a member of the genus Ilarvirus in the family Bromoviridae. PNRSV has been reported to cause a reduction of 12% to 70% in tree growth and 5% to 70% yield losses depending on the cultivar [7, 9].
Microarray technology is a powerful method to simultaneously evaluate changes in the expression of large amounts of genes in response to specific conditions. Currently, an increasing number of studies employ microarray profiling and de novo RNA sequencing to describe gene expression changes in virus/viroid-infected plants . These studies reveal a significant impact of the viral/viroidal infection on host gene expression. Among the cellular processes affected upon infection are defense/stress responses, cell wall structure, chloroplast function, hormone signaling, protein metabolism, silencing and diverse other functions [2, 5, 10–17]. Moreover, infection leads to the modulated expression of various key regulators such as numerous transcription factors, antioxidants, metabolic enzymes and transporters . However, the majority of studies analyzing gene expression changes upon infection are limited to specific host-pathogen interactions involving a given virus and a model host plant, usually Arabidopsis thaliana or Nicotiana benthamiana. Transcriptome analysis upon PNRSV infection, for example has only been performed in N. benthamiana plants . PLMVd infection has not been investigated at the transcript level. Here, we explore and compare the gene expression profiles of peach, a natural host of PNRSV and PLMVd, upon single and double infection with the virus and the viroid in field conditions. Mixed infections of two pathogens occur in natural situations and have biological and epidemiological implications: frequently, simultaneous infections cause symptoms with a higher severity than those induced in single infections by either of the two pathogens. This phenomenon is known as synergism in pathology  and has been mainly studied between two different viruses as, for example, mixed infection of Potato virus X (PVX) and Potato virus Y (PVY) [21–24].
Double infection of peach with PNRSV and PLMVd frequently occurs in the field. Although the absence of symptoms was a common characteristic for all the samples analyzed in this study, we wondered whether the double infection with PNRSV and PLMVd could affect host gene expression differently than single infection. To approach this question, we conducted microarray profiling of peach fruits originating from Chile, singly and doubly infected with PNRSV and PLMVd using microarray slides containing 4,261 unigenes obtained from mesocarp and exocarp tissues of two full-sib progeny contrasting for chilling injury (CI) . Our results clearly demonstrate, for the first time, a synergistic effect at the transcriptomic level between a viroid and a plant virus. The synergistic effect is represented by considerably increased amplitude of differentially expressed genes. The functional identity of those genes with significant alterations upon viral and/or viroidal infection and their putative role in the disease process are discussed.
Sampling and experimental design
Between 2006 and 2008 a number of orchards of different size were visited to assess their sanitary status (Fiore et al., unpublished data) in austral hemisphere spring (September to November) in Chile. Fruits of a total of 2,456 peach trees were sampled during the whole survey period and tested for the presence of viruses and viroids known to affect stone fruit plants in Chile. Among others, samples were tested positive for PNRSV and Prune dwarf virus (PDV), which are the viruses most frequently encountered in southeastern peach, Tomato ringspot virus (TomRSV), Plum pox virus (PPV) and PLMVd. Interestingly, the analysis also revealed that the most recurrent mixed infection was PLMVd-PNRSV (Fiore et al., unpublished data).
Comparison of the PNRSV and PLMVd concentrations in infected peach fruits
Cumulative changes in gene expression
Taken together, transcript analysis revealed a higher number of repressed than induced genes upon infection. In addition, double infection with PNRSV and PLMVd resulted in many more expression changes compared to single infection with either pathogen. Thus, PNRSV and PLMVd appear to exhibit a synergistic effect with respect to the regulation of host gene expression.
Validation of expression changes by quantitative RT-PCR
Functional categorization of genes with induced and repressed expression upon double infection with PNRSV and PLMVd
Gene distribution of the three functional categories with the highest number of statistically significant genes according to MIPS Functional Catalogue Database
plasma membrane/membrane attached
intracellular transport vesicles
protein folding and stabilization
protein targeting sortening and translocation
assembly of protein complexes
nitrogen, selenium and sulfur metabolism
c-compound and carbohydrate metabolism
lipid, fatty acid and isoprenoid metabolism
metabolism of vitamins, cofactors and prosthetic groups
Statistical analysis of functional categories
To identify overrepresented functional categories among induced or repressed genes, we subjected genes to analysis by Babelomics 4.2 FatiGO and single enrichment analysis (SEA). FatiGO uses Fisher’s exact test for 2×2 contingency tables to check for significant over-representation of GO terms. Singular enrichment analysis identifies enriched GO terms in a list of microarray probe sets or gene identifiers. We used this test to identify over- or underrepresented GO terms in the set of genes with significant changes upon infection with both pathogens compared to the whole set of genes represented on the microarray. This analysis should enable us to predict a role of a certain biological processes in response to infection rather than merely calculate a number of up-regulated and down-regulated genes.
Non-reduntant GO categories identified as enriched in down-regulated genes in double infected plants
352 diferential genes, >1.5 fold
Alcohol metabolic process
Response to Stress
Protein catabolic process
Response to cadmiun ion
Metal ion binding
oxidoreductase activity, acting on NADH or NADPH
150 genes diferenciales, >2 fold
Cellular amino acid metabolic process
Amine metabolic process
Response to water deprivation
Response to abiotic stimulus
Response to cadmium ion
To gain a more precise idea about functional classes of proteins with altered expression upon double infection with PNRSV and PLMVd, we increased the threshold of genes subjected to over-representation analysis to at least 2-fold changes in expression level. With these settings, the majority of the functional groups over- represented within GO Biological Process was “related with cellular amino acid metabolic process” and “response to abiotic stresses”. Interestingly, “response to water deprivation” was overrepresented five times compared to the normal distribution of this functional class on the array (Table 2). No functional category was over- or under- represented in GO Molecular Function and only one category with 19 genes, “vacuole”, was overrepresented in GO Cellular Component.
Viroids and viruses depend on host components to complete their life cycle and thus interfere with host processes, such as host gene expression during infection. The identification of genes with altered expression in response to a certain virus/viroid infection can provide clues about the viral life cycle and requirements which need to be met by the host cell. Moreover, a better understanding of host responses to infection facilitates the development of methods to control pathogen infection. To date, the effects of viroid infection on the host transcriptome have been reported for four viroids. These studies used PCR-based cDNA library subtraction ; differential display RT-PCR and quantitative real-time RT-PCR [31, 32] or cDNA microarray [17, 33] to detect host gene expression changes. Currently, a number of studies explore gene expression changes in virus-infected plants using DNA microarrays. All these studies involve a given virus and a model host plant, usually A. thaliana or N. benthamiana. In the present work, we collected peach fruits, a natural host of both PNRSV and PLMVd, at development stage (S4) and studied changes in their transcriptome upon infection with the two pathogens. Today, no array including the whole peach fruit genome is available. Thus, we used a cDNA microarray containing probes for 4,261 genes expressed during chilling injury (CI) development .
During infection, the concentration of PNRSV and PLMVd in fruits is significantly higher (100:1) than in leaves [35, 36]. In addition, the occurrence of peach trees doubly infected with both, PNRSV and PLMVd in the field is considerable (e.g. Fiore et al., unpublished data); . Here, we compared transcript changes in asymptomatic singly and doubly PNRSV or PLMVd infected peach fruits. Overall, our gene expression analysis revealed a relatively low number of differentially expressed genes in single PNRSV infections compared to the healthy control plants (82 genes). This is consistent with the results obtained by Dardick  in which only 89 genes were significantly differentially expressed in PNRSV-infected N. benthamiana plants compared to mock controls. Peach fruits infected with PLMVd also mounted only a moderate response at transcript level (16 genes). The PLMVd variants detected in our study are all considered as latent. In future studies it would be interesting to compare the host transcripts changes caused by variants causing the “calico”  syndrome with those caused by milder variants. PNRSV infected samples were also asymptomatic and all the sequence variants recovered belonged to the PV32 group . Remarkably, in the doubly infected peach fruits a significant synergistic effect on the host transcriptome was observed. Considering the three different infections, the total number of genes with significantly altered expression (at least 1.5 fold change in expression level) was 783, which represents 18% of the whole array. From these 783 genes and eliminating those with significant expression changes occurring in common upon the three scenarios, 627 have orthologs in Arabidopsis (Additional file 3: Table S2). Among those genes 211 exhibited induced and 416 reduced expression. Thus, the total number of genes with reduced expression exceeded that of genes with induced expression by two-fold. This is surprising as comparative analysis of the Arabidopsis transcriptome during compatible interaction with plant viruses  revealed that there was a greater number of up-regulated than repressed genes in the course of viral pathogenesis. However, the authors demonstrated that each virus-host interaction is unique in terms of the genes with altered expression levels and to find a common pattern among different viruses is difficult . In addition, in one of the few studies in which a temporal analysis after virus/viroid infection was carried out, Rizza et al.,  showed that the pattern of up-regulated vs down regulated genes can change in pre-symptomatic when compared to post-symptomatic stages of Etrog citron infected with Citrus exocortis viroid (CEVd).
The overlap in significantly altered gene expression among the two single and the double infections was low. Common genes that were differentially expressed during each infection scenario are shown in Additional file 4: Table S3. This is not surprising considering the small number of genes with significant changes in both single infections. Consistently, Postnikova and Nemchinov  have recently shown that the number of host genes commonly affected during infection of Arabidopsis with either of twelve different viruses studied is very limited. Due to the nature of this specialized database it is difficult to establish a relationship between genes involved in peach ripening and those affected by the pathogens. Nevertheless in our study we observed some genes previously described as related with ripening with altered expression. Most of them did it in the, a priori, unexpected wayside slowing down the ripening (see Additional file 5: Table S4 and references [38–41]). Although we did not follow with great detail whether or not mixed infections had modified the ripening it is noteworthy to emphasize that alterations in ripening date have been described in several virus-host interactions regarding fruit trees (see  for review).
The higher number of genes significantly altered exclusively upon double infection with PLMVd and PNRSV led us to investigate whether some specific functional categories were over- or under- represented among those genes. Interestingly, among the 198 significantly induced genes unique to double infection (Figure 4) none of the functional categories appeared to be enriched in comparison with the normal distribution of genes in the whole microarray. As the array used in this study is enriched with sequences of genes that are implicated in chilling injury development, the lack of overrepresentation of functional categories among the induced genes upon infection compared to the genes represented on the array may indicate a similarity between the functional classes induced upon the two types of stresses, chilling injury and virus/viroid infection. By contrast, we identified over-represented functional categories and subcategories among the set of repressed genes belonging to each of the main GO domains (Table 2, threshold > 1.5 fold). We found genes involved in “response to external stimulus”, “defense response”, “catabolic processes” and “binding function or cofactor requirement”. Downregulation of these functions is presumably due to the virus counterattack against host defense-related pathways. Moreover, these host functions are frequently found in gene expression studies in response to virus infection .
By increasing the threshold to at least 2 fold changes we identified enriched functional categories only belonging to two of the main GO domains: Biological Process and Cellular Component (Table 2). Using this more stringent analysis we found groups of genes related with “response to stress”, to “external stimulus”, “amino acid metabolism” and “vacuole”. Importantly, the most overrepresented functional category was “response to water deprivation”. Among the genes assigned to this functional group genes related with “plant hormones” were found. Plant hormones play important roles in regulating developmental processes and signalling networks involved in plant responses to a wide range of biotic and abiotic stresses. For instance we identified a tonoplast resident H+-pump (At1g15690 H+-PPase AVP1) that contributes to vacuolar acidification, regulation of apoplastic pH and auxin transport . Auxin acts as an important component of hormone signaling network involved in the regulation of defense responses against various biotrophic and necrotrophic pathogens. Additionally, this hormone regulates the expression of genes associated with the biosynthesis, catabolism and signaling pathways of other hormones . Viral pathogens manipulate auxin signaling components to promote virulence and cause disease. One example is the interaction of Tobacco mosaic virus (TMV) replicase with Aux/IAA proteins. Development of symptoms promoted by this interaction has been described in Arabidopsis and tomato [45–47] and in addition disrupts the nuclear localization of several Arabidopsis Aux/IAA proteins. A second example is the protein phosphatase AtPP2CA (At3g11410) which acts as strong negative regulator of ABA signal transduction during seed germination and the regulation of stomatal closure . Tobacco plants infected with TMV showed increased ABA levels and treatment with ABA enhanced TMV resistance in tobacco . Some pathogens might have evolved the ability to produce ABA or ABA-mimicking substances to interfere with host defence. In any case, the role of ABA during plant-pathogen interactions depends on the individual plant-pathogen combination .
As members of the functional group “response to water deprivation” we also found proteins directly related with protection against oxidative stress like the Aldehyde dehydrogenase (At1g54100) and the Late embryogenesis abundant (LEA)-like protein (AtLEA5) (At4g02380). Aldehyde dehydrogenases (ALDHs) regulate the level of aldehydes by metabolizing excessive amounts of aldehyde molecules, which accumulate as a result of perturbed environmental conditions. Aldehydes are involved in different cellular metabolic processes but in excess they can have toxic effects on the cells. It is important then to maintain an appropriate balance of these molecules to avoid cellular damages. It has been described that over-expression of different ALDHs in A. thaliana confers tolerance to abiotic stress and protects plants against lipid peroxidation and oxidative stress . (LEA)-like proteins protect other proteins from aggregation from desiccation. AtLEA5 is the unique which is induced specifically by reactive oxygen species (ROS) as well as by ABA but it is unlikely to act as an antioxidant enzyme. It has been suggested that LEA5 may cooperate with other factors to protect cellular components against ROS-induced damage or indeed to enhance the turnover of specific proteins during stress to enable rapid acclimation to the prevailing conditions .
In the few cases in which changes in the host transcriptome have been investigated upon double virus infections (e.g. the PVX-PVY interaction in N. benthamiana) a severe oxidative stress was inferred to be induced in infected plant leaves, as increased transcript levels of genes encoding proteins important for lipid peroxidation for the generation of ROS were observed. We did not observe any significant over- or underrepresentation of genes involved in the antioxidative system in the doubly infected peach fruits. This difference between PLMVd-PNRSV-infected peach fruits and PVX-PVY infected N. benthamiana plants may be explained by the nature of the interaction between the two pathogens and the pathogens and the host. While co-infection of N. benthamiana with PVX-PVY can be considered as a true biological synergistic interaction leading to enhanced disease phenotypes compared to single infection with either pathogen, the doubly-infected PNRSV-PLMVd peach fruits did not exhibit an enhanced disease phenotype compared to single infection with either PNRSV or PLMVd. Interestingly, PNRSV was able to induce significant oxidative stress and an imbalance in the antioxidant systems in infected apricot-seeds  resulting in a decrease in seed germination. This observation indicates that host responses are not only specific for the host plant and the pathogen, but also tissue and/or organ-specific.
In the functional category “response to dehydration” which was strongly overrepresented upon double infection with PNRSV and PLMVd, we also found other important players with known functions in plant immunity. Among those was Cysteine protease RD21 (At1g47128), a Papain-like cysteine protease (PLCP). These proteins participate in immune responses and are targeted by pathogen- derived inhibitors. PLCPs are also required to trigger the hypersensitive response (HR) [54, 55]. Consistently, PLCP RD19 is required in Arabidopsis for RRS1-R- mediated resistance against the bacterial pathogen Ralstonia solanacearum producing effector PopP2 . A crucial role of PLCPs in disease immunity is also indicated by the observation that many pathogens produce effectors that manipulate these proteases [56–61].
Our data reveal for the first time a clear synergistic effect between a viroid and plant virus at the transcriptome level. We demonstrate that mixed infections, which occur frequently in the field, can result in a more complex transcriptional response than that observed in single infections under the same experimental conditions. Thus, our analysis takes first steps to illuminate the mechanistic basis of synergistic mixed infections in peach trees and reveals candidate genes which can be tried as targets for crop protection. Future additional studies will further elucidate facilitative or antagonistic interactions between plant viruses/viroids in mixed infections.
A total of 2,456 stone fruit trees were sampled during the whole survey period. Three trees were randomly sampled within each orchard. Twenty leaves per plant were collected from several points of the canopy and pooled leaves of each plant were tested for the presence of viruses and viroids previously reported from Chile, i.e. (PPV), (PDV), (PNRSV), (ACLSV), (ToRSV), (PLMVd) [62–65] and for viruses and viroids until now unrecorded in the country, i.e. Apple mosaic virus (ApMV), American plum line pattern virus (APLPV) and Hop stunt viroid (HSVd). Detection was carried out by nonradioactive molecular hybridization (MH) using the corresponding probes for the different viral/viroidal sequences [27, 66]. Reverse transcription-polymerase chain reaction (RT-PCR) was used on selected positive and negative samples, and in all the samples which showed questionable and weak reactions using MH. Infected and healthy fruits were collected from the selected positive and negative samples and analysed using the same detection methods described above.
Due to the absence of a unique peach tree variety infected with all the suggested combinations of virus/viroid for this study, we used three different peach tree varieties. Kawea: for healthy and PLMVd infected samples, White Lady: for PNRSV infected samples and Rosario Red for doubly infected samples. Before proceeding with the microarray analysis, we checked whether the distinct varieties could influence in the transcriptome analysis. For this purpose, we used Real Time qRT-PCR and amplified several genes in different infection scenarios against the same healthy variety. No influence of the different varieties used on the transcriptome profile was observed.
Total RNA from peaches for subsequent functional analyses was extracted as described by Meisel et al. . RNA quality and quantity was analyzed spectrophotometrically by evaluating the absorption ratios: A260/230 and A260/280 and confirmed with an EtBr stained 1.5% agarose gel containing 3% formaldehyde. Presence or absence of the different virus and viroids detected in above leaves was confirmed by MH. Positive tested samples infected either with PNRSV, PLMVd, or both pathogens, as well as healthy controls were selected for microarray profiling.
Microarray hybridization, scanning and data analyses
For each biological replicate equal amounts of samples from four peach fruits belonging to four different trees were pooled and RNA was extracted. To obtain robust and statistically accurate data, four biological replicates and two technical replicates (dye swaps: Cy3-healthy/Cy5-infected or Cy3-infected/Cy5-healthy) were used for all the different samples.
Amplification of RNA samples for microarray hybridization was carried out using the method of Van Gelder et al., 1990 . 1.25 μg of total RNA of each sample were amplified and aminoallyl-labelled using the MessageAmp® II aRNA kit (Ambion, http://www.ambion.com) and 5-(3-aminoallyl)-2´-deoxyuridine-5´-thiphosphate (aa-Dutp, Ambion), following manufacturer’s instructions. A rough amount of 20 μg of amplified RNA (aRNA) was obtained and 7.5 μg from each sample of aminoallyl-labelled aRNA were re-suspended in coupling buffer labelled with either Cy3 or Cy5 Mono NHS Ester (CyTM Dye Postlabelling Reactive Dye Pack, Amersham). Sample purification was carried out with MegaclearTM (Ambion) following manufacturer instructions. Incorporation of Cy3 and Cy5 was measured using 1 μl of the probe in a Nanodrop spectrophotometer (Nanodrop Technologies Inc.; http://www.nanodrop.com/).
Hybridization of samples and reference pool (RP) to the microarray slides was performed manually using Telechem Hybridization Chambers (Corning), according with the protocol described by Ogundiwin et al.  and manufacturer’s instructions. To detect differentially expressed genes in PNRSV, PLMVd or doubly infected samples in comparison to healthy samples, the data were analyzed with the SAM package (Significance Analysis of Microarray, . Statistical significance was assessed using SAM analyses with a false discovery rate of 5% with no fold change cut-off. To study the functional category distribution of genes with expression changes we used the Munich Information Center for Protein Sequences (MIPS FunCatDB, http://mips.helmholtz-muenchen.de/proj/funcatDB/). Gene lists were further analyzed with FatiGO to find differential distributions of gene ontology (GO) terms between statistically differential expressed genes and the rest of genes present on the microarray (Fisher’s exact test in 2 x 2 contingency tables), with P values adjusted after correcting for multiple testing.
Real-time qRT-PCR analysis
Real-time polymerase chain reaction primers, gene targets and the corresponding Arabidopsis thaliana homologs
Locus name (Peach)
Arabidopsis thaliana homolog
Forward primer (5´-3´)
Reverse primer (5´-3´)
Elongation factor 1-alpha (EF1-a)
Clathrin adapter complex (CAC)
Auxine response protein (IAA9)
Invertase/pectin methylesterase inhibitor family
Universal Stress Protein
CBL-interacting protein Kinase 6
Total nucleic acids were first denatured with formaldehyde, serially diluted  and then applied to nylon membranes (positive charged, ROCHE), air dried and covalently UV cross-linked to the membrane (700 × 100 μJ/cm2). Prehybridizations and hybridizations with specific probes for the specific viruses and viroids were conducted as described previously [28, 70]. Chemiluminiscent detection with CSPD reagent as the substrate was performed as recommended by the manufacturer (ROCHE).
We thank Drs M.A. Pérez-Amador y J. Gadea for helping in the result analysis. This work was supported by grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica for the transcriptomic analyses and from the grant 2009CL0020 from the bilateral project INIA-Chile/CSIC-Spain for the phytosanitary evaluation. MC Herranz was the recipient of a contract from the Juan de la Cierva program of the Ministerio de Educación y Ciencia of Spain.
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