Open Access

Transcriptome dynamics of Arabidopsis thaliana root penetration by the oomycete pathogen Phytophthora parasitica

  • Agnès Attard1,
  • Edouard Evangelisti2,
  • Naïma Kebdani-Minet1,
  • Franck Panabières1,
  • Emeline Deleury1,
  • Cindy Maggio1,
  • Michel Ponchet1 and
  • Mathieu Gourgues1Email author
BMC Genomics201415:538

DOI: 10.1186/1471-2164-15-538

Received: 4 October 2013

Accepted: 3 June 2014

Published: 29 June 2014

Abstract

Background

Oomycetes are a group of filamentous microorganisms that includes both animal and plant pathogens and causes major agricultural losses. Phytophthora species can infect most crops and plants from natural ecosystems. Despite their tremendous economic and ecologic importance, few effective methods exist for limiting the damage caused by these species. New solutions are required, and their development will require improvements in our understanding of the molecular events governing infection by these pathogens. In this study, we characterized the genetic program activated during penetration of the plant by the soil-borne pathogen Phytophthora parasitica.

Results

Using all the P. parasitica sequences available in public databases, we generated a custom oligo-array and performed a transcriptomic analysis of the early events of Arabidopsis thaliana infection. We characterized biological stages, ranging from the appressorium-mediated penetration of the pathogen into the roots to the occurrence of first dead cells in the plant. We identified a series of sequences that were transiently modulated during host penetration. Surprisingly, we observed an overall down regulation of genes encoding proteins involved in lipid and sugar metabolism, and an upregulation of functions controlling the transport of amino acids. We also showed that different groups of genes were expressed by P. parasitica during host penetration and the subsequent necrotrophic phase. Differential expression patterns were particularly marked for cell wall-degrading enzymes and other proteins involved in pathogenicity, including RXLR effectors. By transforming P. parasitica with a transcriptional fusion with GFP, we showed that an RXLR-ecoding gene was expressed in the appressorium and infectious hyphae during infection of the first plant cell.

Conclusion

We have characterized the genetic program activated during the initial invasion of plant cells by P. parasitica. We showed that a specific set of proteins, including effectors, was mobilized for penetration and to facilitate infection. Our detection of the expression of an RXLR encoding gene by the appressorium and infection hyphae highlights a role of this structure in the manipulation of the host cells.

Keywords

Phytophthora Arabidopsis thaliana Appressorium Transcriptome Root infection

Background

Plant pathogenic oomycetes have a particular physiology and are known for their devastating effects on agricultural crops and natural ecosystems. A small number of oomycetes, including downy mildews and Phytophthora and Pythium species, which are pathogenic on virtually all dicots and on some cereals [1], have a major impact on agriculture worldwide. Oomycete control strategies are currently very limited, because very few chemicals are effective against these microorganisms. Indeed, most of the molecules used to reduce the incidence of plant diseases caused by filamentous pathogens target fungal functions that are absent or dispensable in oomycetes, such as melanin, sterol or chitin biosynthesis. These organisms are phylogenetically related to brown algae and diatoms within the Stramenopiles and they have unusual biological, genetic and physiological features [2, 3]. The selection of resistant plant genotypes remains an efficient anti-oomycete strategy, but this approach is costly and time-consuming and is, thus, restricted to crops with a high added value. The development of new methods to combat oomycetes thus requires improvements in our knowledge of the physiology and infection strategies of these pathogens. In particular, early events in infection, including the mechanisms underlying penetration and the modulation of plant responses to ensure successful infection are poorly documented. The modification of these processes through the development of new chemicals or plant engineering would reduce the incidence of diseases caused by oomycetes.

Characterizations of plant penetration by oomycetes at the cellular level have focused mostly on Phytophthora spp. Entry into the host is mediated principally by a specialized cellular structure called the appressorium [48]. Poor nutrient content, surface hydrophobicity and topography have been shown to induce appressorium differentiation in Phytophthora infestans[9]. This process also requires calcium [10]. Little is known about the molecular events governing the differentiation and functioning of appressoria. Gene inactivation strategies have clearly demonstrated a requirement for four proteins for the penetration process. The RNAi-mediated silencing of a family of four cellulose synthase genes from P. infestans revealed that cell wall organization is important for appressorium differentiation and plant infection [11]. The PiHMP1 gene encodes a membrane protein that accumulates in appressoria and haustoria and is required for early infection [12]. The PiBZP1 transcription factor from P. infestans is required for appressorium differentiation [13]. Finally, the silencing of the P. sojae mitogen-activated protein kinase PsSAK1 greatly decreases appressorium differentiation, providing a first clue to the signaling pathways involved in the control of this process [14].

Beyond candidate gene silencing strategies, a few medium- to large-scale analyses have been performed to decipher the molecular mechanisms governing appressorium differentiation in Phytophthora spp. Kramer and coworkers first detected, by 2D-SDS-gel electrophoresis, stage-specific peptides in P. infestans appressorium-like structures differentiated on artificial surfaces [15]. A comparative analysis then showed an accumulation of transcripts and proteins involved in amino-acid synthesis during the formation of appressorium-like structures in vitro[16]. These findings were confirmed by a second analysis of protein extracts from appressorium-like structures [17]. This study highlighted the accumulation of proteins involved in protein synthesis and energy metabolism, together with putative pathogenicity factors, such as the Crinkling and Necrosis protein CRN2 and proteins involved in protection against reactive oxygen species. Grenville-Briggs and coworkers recently made use of a coupled liquid chromatography/MS-MS system to identify membrane and cell wall-associated proteins [18]. These proteins included a transglutaminase, a glycosyl hydrolase and a group of three previously unknown but related proteins containing two repeats that accumulated in P. infestans appressoria. Numerous proteins previously characterized as PAMPs (pathogen-associated molecular patterns), such as CBEL-like proteins or elicitins, and crinkler-like proteins accumulated in the cell walls of germinating cysts, suggesting that all these proteins accumulate before infection. Recent transcriptome studies, based on full genome data for P. infestans, P. sojae and P. capsici, have highlighted the upregulation of transcripts encoding proteins involved in gene expression and translation, primary metabolism, protein kinases, cell wall-degrading enzymes (CWDE) and various proteins used to manipulate plant cells (effectors) during in vitro appressorium differentiation [1921]. Similar observations have been obtained with an expressed sequence tag (EST) approach applied to the broad-host range pathogen P. parasitica. An investigation of the genes expressed during appressorium formation identified sequences encoding numerous cell wall-degrading enzymes and pathogenicity-related proteins [8].

Taken together, these analyses provide interesting clues to the developmental program occurring during appressorium differentiation. However, all the studies investigating early events in plant-oomycete interactions have made use of artificial surfaces to induce appressorium differentiation or were performed on aerial parts of plants [16, 1820]. Artificial hydrophobic surfaces cannot be pierced and only the events from zoospore encystment to the differentiation of appressorium-like structures can be analyzed. It is not possible to characterize the steps from appressorium maturation to early plant penetration with such systems. Furthermore, most pathogenic oomycetes infect plant roots and the molecular events described on aerial tissues may not reflect those governing root infection. Information is therefore required concerning the molecular events occurring during the appressorium-mediated penetration of the host root system by oomycetes.

We addressed this question, by analyzing early events in P. parasitica infection by a transcriptome analysis. This species infects the roots of a wide range of plants and is emerging as a model species [22]. We made use of the P. parasitica/Arabidopsis thaliana pathosystem to analyze the events occurring during the first few hours after the inoculation of roots with motile zoospores [23]. We hybridized a custom oligoarray containing almost one quarter of the P. parasitica genome with samples recovered from a time-course of infection ranging from penetration to the switch to necrotrophy. We identified a subset of sequences that specifically accumulated or were repressed during appressorium-mediated penetration of the host. We then investigated the functions of the proteins encoded by these sequences.

Methods

P. parasiticaand plant culture conditions

Phytophthora parasitica INRA-310 strain, selected as the reference for the P. parasitica genome sequencing project was mainly used for this study. Cultures were performed on V8 Medium and zoospore production was induced as previously described [24]. A P. parasitica race 0 strain was kindly provided by Elodie Gaulin (Toulouse III University) and was used for transformation experiments.

Arabidopsis thaliana plantlets were grown, inoculated and observed as previously described [23]. A. thaliana Col-O ecotype was used. An A. thaliana transgenic line expressing the mCherry plasma membrane marker PM-RB was kindly supplied by Professor Torii from Washington University and was used for the cytological analysis of the expression profile of a P. parasitica appressorium specific gene [25].

Appressorium differentiation was induced on onion epidermis as previously described [8].

Sample preparation and RNA extractions

For hybridization and subsequent validations of expression patterns by RT-qPCR, a series of two biological replicates each corresponding to RNA extractions of the following biological conditions were used: 1-Vegetative mycelium (recovered from two samples of 4 day-old cultures in liquid V8 medium at 24°C), 2- Motile zoospores (recovered from 8 independent cultures), 3-Appressoria differentiated on onion epidermis (epidermis from 20 onion bulbs inoculated with zoospores collected from 8 independent Petri dishes); appressoria collected 3 hours after inoculation (24°C), 4- Infection of A. thaliana roots by P. parasitica zoospores (samples recovered at 2.5, 6, 10.5, 30 and 96 hours post inoculation; 5 inoculated plants for each sample) as already reported [23].

As a rule, independent samples were used for Array hybridizations and RT-qPCR analyzes with an exception for purified appressoria RNA that are hardly obtained. RNA extraction from inoculated plant tissues was performed as described by [26]. P. parasitica RNA was extracted using Trizol reagent (Invitrogen, France).

P. parasiticaoligoarray design and hybridizations

An unisequence set was obtained by clustering all P. parasitica EST sequences available in dbEST (Additional file 1: Figure S1). They originated from vegetative mycelium [27], zoospores and germinating cysts [28, 29], appressoria [8] and P. parasitica-infected tomato plantlets, displaying symptoms of the necrotic step of the invasion [30]. Clustering was performed using TGICL package (http://compbio.dfci.harvard.edu/tgi/software/) and default parameters (Additional file 1: Figure S1). Unisequence composition is detailed in the supporting information (Additional file 2: Table S1). Ascribing sequences to plant or Phytophthora was initially done as already described [8]. This was supported by a subsequent BLASTN search against the recently released P. parasitica genome V2.0 (http://www.broadinstitute.org). Functional annotation was performed using blastx against the NCBI non redundant protein database (E value < 1E-05), searches against the Interpro database [31] and using Blast2GO annotation tool [32]. The P. parasitica oligoarray manufactured by NimbleGen systems (NimbleGen Systems, Reykjavik, Iceland) contained 11 independent 60-mer probes per unisequence with 4 technical replicates (Additional file 1: Figure S1). This array is fully described in the platform GPL17781 stored in the Gene Expression Omnibus (GEO) at NCBI (http://www.ncbi.nlm.nih.gov/geo).

CDNA synthesis, sample labeling, hybridization procedures, data acquisition and normalization were performed at the NimbleGen facilities (NimbleGen Systems, Reykjavik, Iceland). The complete expression dataset is available as series accession number GSE51252 in the GEO at NCBI. Average expression levels were calculated for each gene from the independent probes on array and were used for further analysis. Genes were considered as not expressed in a sample (background level) if the normalized value was less that the 95th percentile of random probes found on the array. Data were subjected to the Anaïs statistical framework to identify differentially expressed sequences (Pvalue <0.05) [33]. Data were mean-centered and log-2 transformed using Epclust (http://www.bioinf.ebc.ee/EP/EP/EPCLUST/). Hierarchical clustering (Pierson correlations, average linkage) and K-mean clustering (default parameters) were performed using Genesis program [34].

Real time RT-PCR analyses

Total RNA was treated with DNAse I (Ambion, Austin, USA) and reverse transcribed (1 μg) to cDNA using I-Script cDNA synthesis (Biorad, Hercules, USA). Real Time PCR experiments were performed using 5 μl of 1:50 dilution of first strand cDNA and SYBRGreen (Eurogentec SA, Seraing, Belgium) using the Opticon 3 (Biorad, Hercules, USA). All assays were carried out in triplicates. Gene-specific oligonucleotides were designed using primer3 (http://frodo.wi.mit.edu) and their specificity was validated by the analysis of dissociation curves using genomic DNA as a template. The genes encoding ubiquitin conjugating enzyme (Ubc, CK859493), the 40S ribosomal protein S3A (WS21, CF891675), and the P. parasitica homolog (PpGAM26e01: BlastN, 91% identity) of the P. infestans Mago-Nashi protein (Pi000681) described to be stably expressed were selected as constitutive internal controls [19, 35]. Quantification of gene expression was performed using delta CT method [36].

GO enrichment analysis

Full Gene Ontology files were downloaded at http://www.geneontology.org/GO.downloads.ontology.shtml. After reconstitution of GO pathways, occurrences of each GO term and their parent terms were numbered for each cluster and for the P. parasitica array dataset. Proportion of GO terms in each cluster was then compared to the proportion observed on the P. parasitica array. Terms with significant enrichment were identified using Fisher Exacts test with a p-value cut off at 0.05. To facilitate the analysis, only GO parent terms with level higher than 4 were considered.

P. parasiticatransformation

A Gateway vector was obtained to accelerate gene expression analyses in Phytophthora spp. using transcriptional and translational fusions. The reporter system, corresponding to a fusion between Green Fluorescent protein (GFP) and Escherichia coli β-glucuronidase (GUS), was derived from the pKGWSF7 vector dedicated to plant transformation (from University of Gent, http://gateway.psb.ugent.be/; [37]). It was modified to be suitable for Phytophthora transformation. A geneticin resistance cassette was obtained as a ClaI (blunt ends with DNA pol I)/ SacI fragment from the pTefGHNH vector [30]. It contains the P. parasitica translation elongation factor 1 (Tef1) promoter, the NPTII coding sequence conferring geneticin resistance and the Ham34 terminator from Bremia lactucae. This fragment was cloned into a vector fragment corresponding to an AflII (blunt)/SacI fragment from the pKGWFS7 Gateway cloning vector, replacing the kanamycin resistance cassette used for plant selection. The resulting plasmid was named pIPO-1.

To obtain a translational fusion between the promoter sequence of a RXLR encoding gene (CL380) with the GFP-GUS reporter system (pCL380::GFP-GUS), a 1144 bp fragment upstream of CL380 sequence start codon was amplified on genomic DNA from P. parasitica PP-INRA310 and cloned into the pIPO-1 vector. P. parasitica race 0 protoplast transformation was performed as previously described [30].

Confocal microscopy

A Zeiss LSM 510 META confocal microscope was used (Carl Zeiss GmbH, Jena, Germany). GFP excitation was obtained at 488 nm. Penetration structures were observed two to thirty hours after inoculation of 5×105 zoospores on onion epidermis. Infection of A. thaliana roots was observed eight and thirty hours after inoculation of 5×105 zoospores. For imaging of penetration and invasion on onion epidermis, acquisition parameters were calibrated at two hours post inoculation and unchanged at the following time points to enable comparison.

Results

Design of the P. parasiticaoligoarray

We retrieved all the P. parasitica EST sequences available from public databases. These sequences were obtained at various developmental stages and from cell types involved in plant infection: mycelium, zoospores, appressoria differentiated on onion and tomato necrotrophic stage of infection [8, 2730]. In total, 12632 ESTs were assembled into 1572 contigs and 3879 singletons, giving a final 5451-unisequence set (Additional file 2: Table S1). A NimbleGen custom oligo-array was designed (Additional file 1: Figure S1). Approximately 97% of the unisequences were represented by more than five 60-bp probes. Only 24 sequences did not fit the NimbleGen criteria for probe design and were discarded from the analysis. Following this design step, 56524 probes corresponding to 5427 sequences were spotted onto the array. We attributed 4398 sequences (81%) to P. parasitica and 729 sequences (13%) to the plant (Additional file 1: Figure S1). The origin of 324 sequences (6%) remained unknown (Additional file 3: Table S2). In total, 4700 probe sets corresponded to sequences from P. parasitica or of unknown origin. The remaining probe sets matched sequences of plant origin. Finally, 163000 random probes were added to complete the array, as controls.

Blastx searches of sequences attributed to P. parasitica against a protein set based on the recently released genome annotation gave 3806 hits with identity levels of more than 95%. Blastx searches (e-value = 1E-20) using the 4398 P. parasitica sequences as queries also revealed that 320 sequences (7%) had no ortholog in the genomes of P. infestans, P. ramorum and P. sojae and therefore constituted probable species-specific sequences. Functional annotation of the 4398 and 324 sequences attributed to P. parasitica and of unknown origin, respectively, was performed following blastx searches (E-value cut off: 1E-05) against the NCBI non-redundant (NR) protein database, with the blast2GO tool [32]. A GO term was associated with 3516 sequences (65%), whereas the function of 1935 sequences remained unknown (Additional file 3: Table S2).

Analysis of gene expression during early steps of the P. parasitica/A. thalianainteraction

The custom oligo-array was used to identify the modulations of the P. parasitica transcriptome during the onset of a compatible interaction. The samples analyzed corresponded to vegetative mycelium, motile zoospores, purified appressoria and a time course of early infection in A. thaliana. The A. thaliana samples were collected 2.5, 6, 10.5 and 30 hours after inoculation (hai), from roots inoculated with P. parasitica motile zoospores, thus reflecting the natural mode of infection. We selected stages ranging from appressorium-mediated penetration (2.5 hai) to the first occurrence of dead cells, indicative of the switch to necrotrophy (30 hai), as previously described [23]. There were two biological replicates. Each replicate corresponded to RNA extracts from pooled independent biological samples for each condition, to ensure the robustness of our results.

Only hybridization data relevant to P. parasitica or sequences of unknown origin were retained for analysis. In total, 4194 sequences (89% of the 4700 probes corresponding to P. parasitica sequences or sequences of unknown origin) gave a hybridization signal stronger than the background in at least one condition and were considered to correspond to expressed genes. The oligo-array contained 314 of the 320 putative P. parasitica-specific sequences. Significant expression in at least one biological sample was detected for 266 of these sequences, confirming that they corresponded to P. parasitica-specific expressed genes. Interestingly, 2864 sequences (61%) and 2987 sequences (64%) were expressed in purified appressoria and during the appressorium-mediated penetration of A. thaliana roots (2.5 hai), respectively. Thus, with the inoculation method used for this analysis, a high proportion of transcripts can be detected even at the earliest stages of the interaction.

The Anaïs statistical framework was used to identify differentially expressed sequences [33]. Using a P-value threshold of 0.05, 3783 sequences were considered to display differential patterns of accumulation between at least two sets of conditions. Maximum fold-change, defined as the ratio of the maximum and minimum hybridization signal values, was calculated for each sequence. In total, 3471 sequences had a maximum fold-change of more than 2 and 1806 sequences had a maximum fold-change of at least 4.

Cluster analysis of gene expression patterns

The expression patterns of the 1806 sequences displaying at least a four-fold modulation of expression were analyzed. We performed a hierarchical clustering of the expression patterns (Figure 1A) and genes with coordinated expression were grouped into 12 major clusters (K-mean clustering, Figure 1B, Additional file 4: Table S3).
Figure 1

Hierarchical and K-mean clustering of four-fold expressed genes. (A) Hierarchical clustering of the 1806 genes modulated by 4-fold. Pearson correlation method with average linkage on conditions was used. Gene expression level is indicated as a Log2 transformation of relative value calculated on gene average signal value. Green and red were used for down-regulated and up-regulated conditions respectively. Lines mark major K-mean groups. (B) Expression profiles were K-mean clustered based on euclidean distance. The twelve main K-mean clusters are represented. The center line represents average expression with standard deviation. Number of genes within each cluster is indicated. The six clusters used for subsequent characterization of the molecular events occurring during early infection are colored in red. Myc, mycelium; Zoo, zoospores, App, Appressorium; 2.5 h, Arabidopsis thaliana infected roots recovered 2.5 hours after inoculation (appressorium-mediated penetration); 6 h, A. thaliana infected roots recovered 6 hours after inoculation (biotrophic growth, two to three cells invaded); 10.5 h, A. thaliana infected roots recovered 10.5 hours after inoculation (invasive growth along the stele); 30 h, A. thaliana infected roots recovered 30 hours after inoculation (switch to necrotrophy).

Clusters I (124 sequences), II (154 sequences), III (144 sequences), IV (388 sequences), V (94 sequences), VI (101 sequences) and XX (92 sequences) contained sequences for which expression was modulated during the onset of infection (Figure 1B). Clusters I, II, V, VI and XX contained sequences with a specific modulation of expression (induced or repressed) during early contact with plant cells, as they were detected in purified appressoria and during the first few hours of A. thaliana infection (2.5, 6 and 10.5 hai). By contrast, clusters III and IV contained sequences whose accumulation or repression begins in motile zoospores, corresponding to the pre-infection stage.

Sequences from cluster VII (366 sequences) were specifically modulated in zoospores. Interestingly, clusters VIII (70 sequences) and IX (130 sequences) contained sequences with similar patterns of expression in zoospores and in A. thaliana roots collected 2.5 hours after inoculation (Figure 1B). This may be due to the contamination of A. thaliana root tissues by zoospores, either motile or initiating cyst formation, in the first sample collected for the interaction. This finding is consistent with previous reports indicating that plant infection by motile P. parasitica zoospores is an asynchronous process. It thus highlights the advantages of the simplified system previously used to obtain purified appressoria [8].

Cluster XI (74 sequences) grouped together sequences that accumulated preferentially in mycelium (K-mean clustering, Figure 1B). However, most of the sequences found in this cluster also accumulated 2.5 hours after A. thaliana inoculation. This may be due to cysts failing to penetrate and the plant and instead initiating mycelial growth around A. thaliana roots, as reported during the initial steps of tomato root infection by P. parasitica[30].Finally, transcripts from cluster XII (69 sequences) accumulated throughout the development of plant infection (Figure 1B). This accumulation increased during infection, reaching a maximum at the last stage studied (30 hai).

Validation of the transcriptome data by RT-qPCR

The expression patterns observed on hybridization of the P. parasitica oligo array were validated by quantitative RT-PCR. We selected 58 sequences from the six clusters containing sequences with expression modulated during the appressorium-mediated penetration of the host (clusters I, II, III, IV, V and VI; red on Figure 1). A biological sample corresponding to the A. thaliana/ P. parasitica interaction was added, to monitor expression of the selected sequences during the necrotrophic stage. This sample corresponded to inoculated A. thaliana root tissues collected four days after inoculation with zoospores. Forty-four of the 58 genes assessed (75%) displayed a modulation of expression during the onset of the interaction, consistent with the results of array hybridization (Figure 2 and Additional file 5: Table S4). Nine of the 14 sequences for which expression profiling results were not validated by RT-qPCR were from cluster V (Additional file 5: Table S4).
Figure 2

Quantification of mRNA corresponding to on gene of each of the clusters I to VI. Relative mRNA levels were quantified by quantitative RT-PCR in biological replicates of samples used in Figure 1. A condition corresponding to A. thaliana roots 4 days after inoculation (4d), corresponding to necrotrophic stage of the interaction was added. Data are presented as expression ratios relative to UBC, WS21 and Mago Nashi reference genes (2−ΔCT). Grey bars represent mean signal obtained following oligoarray hybridizations (black left axis). Red point and crosses represent relative expression values obtained for two independent biological replicates analyzed using qRT-PCR (red right axis).

This cluster probably included sequences with artifactual hybridization signals. After we had discarded data for the members of this cluster, the expression patterns for 43 of 48 (90%) sequences were validated by RT-qPCR, demonstrating the robustness of our transcriptome data.

We also used the RT-PCR results to improve our clustering analysis. The grouping of sequences in cluster I, which contained transcripts transiently accumulating during the onset of infection was validated, because eight of the nine sequences had expression patterns consistent with that deduced from oligoarray hybridization (Figure 2, Additional file 5: Table S4). The transcripts of these genes accumulated in smaller amounts during the necrotrophic phase of the P. parasitica/A. thaliana interaction (4 days after inoculation). This cluster included sequences that accumulated during early infection of the host. The content of clusters III and IV was also validated with all and 8 of the 10 sequences, respectively, displaying the predicted expression pattern (Figure 2, Additional file 5: Table S4). The use of the sample corresponding to necrotrophy also confirmed that the sequences grouped in these clusters accumulated or were repressed transiently in zoospores and during the penetration process. Sequences from cluster II had lower accumulation during early infection than during vegetative mycelial growth and the necrotrophic phase (4 days after inoculation). Nevertheless, seven of these nine sequences also accumulated to low levels in zoospores (Figure 2, Additional file 5: Table S4). This suggests that cluster II could be grouped with cluster IV for a single analysis. Cluster VI contained sequences specifically induced during the penetration of onion epidermis (purified appressoria, Figure 1). This cluster was poorly validated because five of the 10 sequences accumulated during the penetration of both A. thaliana roots and onion (Additional file 5: Table S4). This confirms that the results obtained with our simplified inoculation protocol reflect the situation occurring in natural conditions of plant penetration [8]. On the basis of this RT-qPCR analysis, clusters I and VI were grouped together for subsequent analyses.

Functions of the sequences accumulating during appressorium-mediated plant penetration

We focused our analysis on transcripts modulated during the onset of a compatible interaction (clusters I-VI). A functional annotation of the 20 genes displaying the highest fold-change in each cluster is presented in Table 1. We performed a GO enrichment analysis of the clusters, for annotation purposes. Sequences specifically accumulating during the appressorium-mediated penetration of the host plant (purified appressoria, A. thaliana 2.5 hai, 6 hai and 10.5 hai, clusters I and VI) were highly enriched in cell wall-degrading enzymes (CWDEs, Additional file 6: Table S5_I and Additional file 7: Table S5_VI). There were 64 sequences encoding CWDE on the array: 14 and 8 had expression patterns characteristic of clusters I (grouping 124 sequences) and VI (grouping 101 sequences), respectively (Additional file 4: Table S3). Figure 3A shows the expression patterns of all the sequences associated with GO terms relating to cell wall degradation. Thirty CWDEs were found to be preferentially expressed during the first few hours of infection. The other 34 enzymes were expressed in zoospores, mycelium or both mycelium and at the necrotrophic stage of interaction with A. thaliana. GO terms associated with the modulation of plant defenses were also overrepresented during the early stages of infection (Additional file 6: Table S5_I). Hence, six of the 14 sequences encoding RXLR effectors present on the oligoarray were present in cluster I. One of these six sequences corresponded to the PSE1 effector, which has recently been demonstrated to contribute to P. parasitica pathogenicity [38]. The expression patterns of sequences encoding cytoplasmic (RXLR and CRN) effectors are presented in Figure 3B. Twelve of the RXLR effectors are induced during the penetration process, whereas only a few CRN effector transcripts accumulated at this stage. Other functions relevant to pathogenicity or the elicitation of plant defense responses were also identified in clusters I (2 elicitin-like, 2 NPP1-like and 1 M81 elicitor-like) and VI (a putative protease inhibitor) (Table 1 and Additional file 4: Table S3). GO annotations relating to ribosomes were also enriched in cluster VI, providing evidence for the activation of the translation machinery during early infection (Additional file 7: Table S5_VI).
Table 1

Functional annotation of the sequences from clusters I-VI

Normalized hydridization signal

 

Blastx best hit

CL

Sequence

Myc

Zoo

App

2,5h

6h

10,5h

30h

FC

ID

Description

E value

I

CL1533Contig1

102

749

95

10813

1022

712

139

114

 

no hit

 

I

ppgam36a11r.1

127

131

336

1972

3482

13284

713

105

XP_002895059.1

elicitin-like protein INF4 [P. infestans]

3E-10

I

ppo3h07a24t.1

97

104

9105

1711

4473

1226

450

94

XP_002998010.1

secreted RxLR effector peptide protein, [P. infestans]

2E-13

I

CL1408Contig1

120

178

10915

1767

3707

5999

7470

91

XP_002896645.1

Amino Acid/Auxin Permease (AAAP) Family [P. infestans]

1E-134

I

ppo3h11p12t.1

296

334

4984

26369

10531

11172

2378

89

 

no hit

 

I

CL262Contig1

196

391

5444

14553

9894

3628

1081

74

 

no hit

 

I

CL380Contig1

108

120

7702

1192

3335

848

336

72

XP_002998010.1

secreted RxLR effector peptide protein, [P. infestans]

5E-19

I

CL1335Contig1

333

1011

13535

15140

18621

22234

10825

67

 

no hit

 

I

ppgam18a12r.1

112

116

119

6434

1077

814

167

57

 

no hit

 

I

ppo3h08i21t.1

122

128

6892

5236

3381

818

288

57

XP_002901148.1

carbohydrate-binding protein, [P. infestans]

2E-13

I

ppgam09e02r.1

100

110

116

5457

539

400

126

55

XP_001339148.2

PREDICTED: polymerase polyprotein-like [Danio rerio]

3E-53

I

CL42Contig1

132

134

7202

5692

3174

681

233

55

XP_002901145.1

hypothetical protein PITG_11596 [P. infestans]

1E-09

I

ppt4j31g10r.1

126

120

135

5163

639

381

163

43

 

no hit

 

I

ppo3h12o13t.1

118

154

4926

1188

4607

2779

1464

42

XP_002900991.1

conserved hypothetical protein [P. infestans]

2E-32

I

ppo3h06d21t.1

190

699

990

3799

7709

4416

2482

41

XP_002904652.1

ATP-binding Cassette (ABC) superfamily [P. infestans]

2E-17

I

ppo3h05p06t.1

96

96

2003

1513

3273

884

212

34

Q56TU4.1

S-adenosylmethionine synthase 1; Daucus carota

1E-13

I

CL382Contig1

180

190

5393

3807

3160

987

403

30

XP_002901148.1

carbohydrate-binding protein, [P. infestans]

6E-11

I

CL1026Contig1

125

144

900

1436

3606

2879

845

29

XP_002901054.1

secreted RxLR effector peptide protein, [P. infestans]

2E-15

I

CL366Contig1

122

228

274

3032

2717

1029

146

25

 

no hit

 

I

CL1109Contig1

135

138

3025

1229

3268

2982

2582

24

XP_002898673.1

ribonuclease, [P. infestans]

1E-89

II

CL1024Contig1

5212

1169

195

341

160

154

481

34

XP_500020.1

YALI0A12705p [Yarrowia lipolytica] emb

1E-13

II

ppgam23f03r.1

3620

1975

358

224

136

126

158

29

XP_002895853.1

sporangia induced phosphatidyl inositol kinase [P. infestans]

1E-142

II

CL1233Contig1

3966

6902

240

430

353

738

1651

29

XP_002899328.1

Ca2+−transporting ATPase endoplasmic reticulum type, [P. infestans]

0

II

CL1149Contig1

903

2978

308

207

297

436

462

14

XP_002905211.1

protein kinase, [P. infestans]

0

II

CD051670.1.1

1313

2611

2387

248

185

207

625

14

 

no hit

 

II

CL1407Contig1

10595

4542

757

1385

2211

3020

2338

14

XP_002908946.1

dihydroflavonol-4-reductase, [P. infestans]

1E-159

II

ppt4j11b07r.1

636

2408

175

259

352

341

343

14

XP_002903886.1

conserved hypothetical protein [P. infestans]

3E-99

II

CL772Contig1

4458

2341

605

336

991

969

1198

13

XP_002997937.1

conserved hypothetical protein [P. infestans]

0

II

CL864Contig1

9998

4700

787

1220

1070

1225

2643

13

XP_002898044.1

conserved hypothetical protein [P. infestans]

0

II

CL112Contig2

5791

4284

1116

2745

2166

1535

463

13

XP_002895909.1

ATP-binding Cassette (ABC) Superfamily [P. infestans]

0

II

ppgam04e11r.1

649

2816

231

262

328

349

557

12

 

no hit

 

II

CL1478Contig1

771

2708

235

250

347

341

514

12

XP_002907456.1

conserved hypothetical protein [P. infestans]

7E-76

II

ppgam07d02r.1

634

2263

196

293

225

275

345

12

XP_002998017.1

transmembrane protein, [P. infestans]

7E-71

II

CL471Contig1

5487

4101

582

848

477

756

1567

12

XP_002896830.1

conserved hypothetical protein [P. infestans]

1E-100

II

CL1485Contig1

1608

838

158

143

186

180

347

11

XP_002897750.1

short/branched chain specific acyl-CoA dehydrogenase, [P. infestans]

1E-125

II

CL349Contig1

1340

4691

549

502

543

431

729

11

XP_002909307.1

conserved hypothetical protein [P. infestans]

0

II

ppgam33a03r.1

2179

1553

202

508

579

938

701

11

XP_002905400.1

L-aminoadipate-semialdehyde dehydrogenase, [P. infestans]

1E-128

II

CL874Contig1

1039

2304

216

452

351

573

601

11

XP_002997338.1

HECT E3 ubiquitin ligase, [P. infestans]

7E-89

II

CL176Contig1

2239

5389

710

506

573

549

834

11

XP_002897342.1

conserved hypothetical protein [P. infestans]

8E-61

II

ppt4j38b11r.1

681

2149

332

246

230

202

432

11

XP_002896848.1

conserved hypothetical protein [P. infestans]

1E-108

III

ppo3h14g02t.1

142

11120

10795

18486

7479

2452

503

130

XP_002997786.1

glycoside hydrolase, [P. infestans]

1E-43

III

ppo3h14f01t.1

126

5446

7895

14463

5042

1485

437

115

 

no hit

 

III

CD051442.1.1

218

15744

14847

23012

17903

9763

5659

105

 

no hit

 

III

CL369Contig1

122

11151

4457

12379

4521

1277

268

102

XP_002997786.1

glycoside hydrolase, [P. infestans]

1E-138

III

CD051500.1.1

157

15890

5422

7601

5856

5131

1149

101

 

no hit

 

III

CL306Contig1

148

14591

3542

5108

3343

3089

710

99

XP_002898718.1

conserved hypothetical protein [P. infestans]

1E-146

III

CL804Contig1

130

9313

6270

2748

2823

2240

1429

72

XP_002904112.1

sulfatase-like protein [P. infestans]

1E-132

III

CL515Contig1

131

7782

2639

1063

1153

1103

519

59

XP_002904421.1

conserved hypothetical protein [P. infestans]

0

III

CD051686.1.1

135

6845

2453

1660

868

844

290

51

 

no hit

 

III

CL1187Contig1

181

9106

1049

3912

5197

6878

5043

50

XP_002896478.1

poly [ADP-ribose] polymerase, [P. infestans]

1E-174

III

CF891673.1.1

157

5465

5871

2105

4405

2699

1995

37

 

no hit

 

III

CL232Contig1

200

7317

3970

3044

4343

2281

3612

37

XP_002904949.1

conserved hypothetical protein [P. infestans]

1E-168

III

CL1195Contig1

135

4941

2491

3037

2412

1683

724

37

XP_002895053.1

conserved hypothetical protein [P. infestans]

1E-129

III

CL181Contig1

276

7495

9615

1909

1423

1212

1047

35

XP_002900508.1

conserved hypothetical protein [P. infestans]

1E-119

III

ppo3h09b05t.1

172

5371

703

1742

819

937

463

31

ABG80552.1

cell 5A endo-1,4-betaglucanase [P. ramorum]

2E-97

III

ppo3h07h24t.1

112

3238

1398

543

840

680

469

29

XP_002906516.1

transmembrane protein, [P. infestans]

1E-117

III

ppo3h13d10t.1

115

3128

1867

885

647

518

171

27

XP_002998505.1

GPI-anchored serine-rich elicitin INL3b-like protein [P. infestans]

1E-38

III

CL449Contig1

125

3223

2186

1589

1510

843

410

26

XP_002898095.1

conserved hypothetical protein [P. infestans]

7E-96

III

ppt4j29b11r.1

158

3765

2184

1048

557

849

399

24

XP_002902649.1

Drug/Metabolite Transporter (DMT) Superfamily [P. infestans]

1E-108

III

CL330Contig1

1867

23600

43890

40265

40618

35214

14295

24

XP_002904340.1

conserved hypothetical protein [P. infestans]

1E-102

IV

CL8Contig1

44525

163

325

452

825

9662

7144

274

XP_002899844.1

conserved hypothetical protein [P. infestans]

1E-152

IV

CL639Contig1

22961

606

806

221

192

166

413

139

ABG23233.1

unknown [Hyaloperonospora parasitica]

1E-36

IV

ppo3h02e04t.1

19582

157

3139

1151

2639

7487

7743

124

XP_002900352.1

Proton-dependent Oligopeptide Transporter (POT) Family [P. infestans]

8E-98

IV

ppgam01h01r.1

17201

173

339

204

164

142

881

121

XP_002901432.1

conserved hypothetical protein [P. infestans]

6E-33

IV

ppo3h10m18t.1

25224

224

6090

1667

2610

4991

19398

113

 

no hit

 

IV

CL90Contig1

13934

167

1552

489

7712

8560

18029

108

XP_002999240.1

pyrophosphate vacuolar membrane proton pump, [P. infestans]

0

IV

CL32Contig1

14467

189

275

148

258

429

4440

97

AAM18483.1

AF494014_1 exo-1,3-beta-glucanase [P. infestans]

0

IV

ppgam24d04r.1

13873

149

200

667

295

697

973

93

XP_002907089.1

protein kinase, [P. infestans]

1E-127

IV

CL210Contig1

12224

133

178

647

362

905

11665

92

XP_002909387.1

conserved hypothetical protein [P. infestans]

7E-69

IV

ppgam36c10r.1

16676

187

4612

1365

566

1463

8770

89

XP_002904661.1

Major Facilitator Superfamily (MFS) [P. infestans]

1E-126

IV

CL119Contig1

10489

126

163

144

246

461

494

84

XP_002900778.1

Annexin (Annexin) Family [P. infestans]

1E-166

IV

ppgam37e05r.1

15664

342

205

390

190

188

2589

83

XP_002907653.1

oxidoreductase, [P. infestans]

4E-85

IV

CL71Contig1

10898

137

1568

339

1504

4481

9186

79

ABH11757.1

elicitin-like protein 6 precursor [P. nicotianae]

4E-44

IV

ppo3h07a23t.1

9263

118

124

161

162

175

1752

78

XP_002907297.1

P-type ATPase (P-ATPase) Superfamily [P. infestans]

1E-120

IV

CL92Contig1

12589

213

871

357

5799

12282

16142

76

XP_002998388.1

carbohydrate-binding protein, [P. infestans]

1E-127

IV

ppgam07a10r.1

8342

249

671

275

1663

9237

18817

76

XP_002903354.1

mucin-like protein [P. infestans]

8E-38

IV

ppgam02h08r.1

4632

136

625

454

3462

10250

8890

75

XP_002896569.1

glucosylceramidase, [P. infestans]

1E-109

IV

ppt4j09a01r.1

8222

115

199

259

220

399

8676

75

XP_002909387.1

conserved hypothetical protein [P. infestans]

2E-46

IV

ppgam18e03r.1

15537

715

353

538

227

207

2704

75

XP_002902980.1

acyl-CoA synthetase short-chain family member, [P. infestans]

1E-124

IV

CL758Contig1

12392

169

311

676

908

2489

3671

73

XP_002904197.1

glucosylceramidase, [P. infestans]

1E-156

V

ppo3h06f02t.1

409

777

214

8582

1544

1290

509

40

XP_002997773.1

conserved hypothetical protein [P. infestans]

7E-78

V

ppgam12h06r.1

781

868

171

5385

359

274

138

39

 

no hit

 

V

ppgam31f01r.1

119

197

159

2339

520

353

164

20

 

no hit

 

V

ppt4j25b05r.1

97

176

126

1823

424

369

127

19

AAR21576.1

heat shock protein 70 [P. nicotianae]

2E-20

V

CL1505Contig1

480

596

349

5757

3617

1585

1274

17

XP_002895485.1

ATP-binding Cassette (ABC) superfamily [P. infestans]

1E-136

V

ppt4j21a06r.1

445

118

199

1866

303

357

311

16

XP_002895988.1

maltose O-acetyltransferase, [P. infestans]

5E-86

V

ppgam36f04r.1

108

116

171

1707

275

240

140

16

 

no hit

 

V

ppgam17d07r.1

159

458

362

2083

608

514

333

13

XP_002906389.1

conserved hypothetical protein [P. infestans]

2E-65

V

CL344Contig1

138

179

483

1684

534

446

257

12

 

no hit

 

V

ppgam04f05r.1

128

167

462

1554

521

297

166

12

 

no hit

 

V

ppo3h02b24t.1

112

407

356

1344

495

796

671

12

XP_002895175.1

conserved hypothetical protein [P. infestans]

5E-51

V

ppo3h06n03t.1

129

135

526

1544

284

204

175

12

NP_001063268.1

Os09g0438100 [Oryza sativa Japonica Group] dbj

0,000009

V

ppt4j01f03r.1

106

122

165

1257

236

187

145

12

XP_002903318.1

conserved hypothetical protein [P. infestans]

1E-15

V

ppo3h03i13t.1

161

608

253

1692

729

454

216

11

XP_002904470.1

conserved hypothetical protein [P. infestans]

5E-97

V

ppo3h01k13t.1

126

418

348

1275

506

395

230

10

XP_002903425.1

conserved hypothetical protein [P. infestans]

3E-45

V

ppt4j36c04r.1

291

127

168

1285

233

258

241

10

XP_002182839.1

predicted protein [Phaeodactylum tricornutum CCAP 1055/1]

4E-51

V

ppo3h12p16t.1

564

1450

1037

5589

1138

1127

581

10

XP_002898514.1

conserved hypothetical protein [P. infestans]

1E-36

V

ppo3h03o24t.1

240

119

230

1158

360

368

185

10

 

no hit

 

V

ppt4j38e02r.1

102

102

100

943

200

196

117

9

 

no hit

 

V

ppgam22f11r.1

122

120

153

1082

249

223

145

9

XP_002896344.1

conserved hypothetical protein [P. infestans]

2E-14

VI

ppo3h12n08t.1

104

106

37783

103

124

120

116

365

 

no hit

 

VI

ppo3h10g01t.1

113

131

9703

121

207

111

112

87

 

no hit

 

VI

ppo3h12n20t.1

104

128

8715

707

225

478

128

84

XP_002909505.1

mannitol dehydrogenase, [P. infestans]

5E-41

VI

ppo3h13j21t.1

92

94

4826

93

122

112

96

52

 

no hit

 

VI

CL613Contig1

117

129

5082

224

1021

1359

1803

43

XP_002904550.1

folate-Biopterin Transporter (FBT) family [P. infestans]

0

VI

ppo3h03f02t.1

132

996

5251

350

169

144

142

40

XP_002904736.1

conserved hypothetical protein [P. infestans]

2E-69

VI

ppo3h05l14t.1

95

173

3749

261

179

118

114

39

XP_002905279.1

cleavage induced hypothetical protein [P. infestans]

5E-86

VI

CL419Contig1

139

323

5306

737

1879

924

595

38

ABB22029.1

cell 12A endoglucanase [P. sojae]

1E-117

VI

ppo3h06d03t.1

112

124

4159

443

816

580

173

37

XP_002904437.1

conserved hypothetical protein [P. infestans]

1E-112

VI

ppt4j22c09r.1

118

593

3355

2610

422

405

321

28

XP_002909523.1

Amino Acid-Polyamine-Organocation (APC) Family [P. infestans]

1E-115

VI

ppo3h04p13t.1

113

118

2658

565

195

151

129

24

XP_002901038.1

carbohydrate-binding protein, [P. infestans]

1E-58

VI

CL118Contig1

105

106

2438

176

140

160

118

23

XP_002902444.1

conserved hypothetical protein [P. infestans]

1E-107

VI

CL999Contig1

117

131

2593

402

881

527

270

22

XP_002901373.1

conserved hypothetical protein [P. infestans]

1E-86

VI

CL1422Contig1

138

172

3038

797

222

254

913

22

XP_002904844.1

ATP-binding Cassette (ABC) Superfamily [P. infestans]

1E-165

VI

CL317Contig1

160

124

2693

276

585

516

953

22

XP_002901401.1

conserved hypothetical protein [P. infestans]

7E-26

VI

CL1406Contig1

105

112

1979

156

169

164

117

19

ABG23232.1

N-acetyltransferase-like protein [Hyaloperonospora parasitica]

3E-49

VI

CL448Contig1

237

228

4124

1663

1472

745

545

18

XP_002900313.1

cutinase, [P. infestans]

1E-103

VI

CL1513Contig1

124

124

2132

264

650

571

264

17

XP_002903927.1

pectin lyase, [P. infestans]

2E-61

VI

CL1176Contig1

106

116

1784

152

122

124

108

17

XP_002999045.1

conserved hypothetical protein [P. infestans]

1E-142

VI

ppo3h13a15t.1

97

100

1585

101

119

110

102

16

XP_002518625.1

conserved hypothetical protein [Ricinus communis]

1E-51

Annotation proposed for the 20 genes with the highest fold change in each cluster (CL) is presented. For all sequences, normalized hybridization signal in each condition (mean of the two biological replicates) is indicated. Maximum fold change (max FC) observed between the different biological samples and BlastX best hit result (E value < 1E-05) against NCBI non-redundant (NR) protein database are presented.

Figure 3

Hierarchical clustering of the genes encoding cell wall degrading enzymes and cytoplasmic effectors. Based on GO annotations, sequences corresponding to cell wall degrading enzymes (A) and sequences corresponding to RXLR and Crinkler effectors (B) are presented. Pearson correlation method with average linkage on conditions was used. Gene expression level is indicated as a Log2 transformation of relative value calculated on gene average signal value. Green and red were used for down-regulated and up-regulated conditions respectively. Myc, mycelium; Zoo, zoospores, App, Appressorium; 2.5 h, A. thaliana infected roots recovered 2.5 hours after inoculation; 6 h, A. thaliana infected roots recovered 6 hours after inoculation; 10.5 h, A. thaliana infected roots recovered 10.5 hours after inoculation; 30 h, A. thaliana infected roots recovered 30 hours after inoculation.

An analysis of clusters I and VI showed these clusters to contain sequences involved in protein and amino-acid metabolism, including four proteases and four putative amino-acid transporters. Similarly, eight and three sequences encoding functions involved in the detoxification of plant toxic metabolites (ABC transporters and major facilitator superfamily members) were identified in clusters I and VI, respectively (Additional file 4: Table S3). Interestingly, sequences relating to sugar metabolism were poorly represented in these clusters. Cluster I included a sequence corresponding to mannose 6P isomerase, a pyruvate kinase and a NAD-dependent malic enzyme. A single sequence encoding a malate dehydrogenase was identified in cluster VI (Additional file 4: Table S3). A similar situation was observed for sequences relating to fatty-acid catabolism. Only one sequence encoding a long-chain fatty acid CoA ligase and one sequence encoding an acyl-CoA dehydrogenase were identified in clusters I and VI, respectively (Table 1 and Additional file 4: Table S3).

The prominent functions identified for sequences from cluster III (sequences accumulating in zoospores and during host penetration, 144 sequences) were mostly similar to those identified in clusters I and VI. GO enrichment analysis highlighted the accumulation of sequences with functions relating to ribosome biogenesis (Additional file 8: Table S5_III). However, a detailed analysis of this cluster revealed the occurrence of three additional cell wall-degrading enzymes and numerous infection-associated sequences, including five M81-like sequences, two OPEL-like sequences, one EPIC-like sequence and one elicitin (Table 1 and Additional file 4: Table S3). Three sequences encoding functions involved in the evasion of toxic molecules (ABC transporters and major facilitator superfamily members) were observed. Finally, this cluster also contained four proteases and one amino acid/auxin permease, confirming the importance of amino-acid uptake during early infection. Only two glycolytic enzymes (glucokinase and a glyceraldehyde 3P dehydrogenase) and a fatty-acid metabolism-related short-chain dehydrogenase were identified in this cluster.

Cluster V, which contained 94 sequences specifically expressed during the penetration of A. thaliana roots, but not onion epidermis, was not supported by RT-qPCR experiments, suggesting that this cluster should be considered with caution. Moreover, most of the sequences in this cluster lacked robust annotation. Nevertheless, like clusters I and VI, this group contained various pathogenicity-related sequences, including a range of apoplastic and cytoplasmic effectors: four RXLR effectors, two CRNs, one elicitin-like sequence, one IPI-like sequence, one NPP1-like sequence and one protease (Table 1, Additional file 9: Table S5_V and Additional file 4: Table S3).

Functions of sequences repressed during appressorium mediated penetration of the host

An analysis of the sequences of cluster II (154 sequences specifically repressed during appressorium-mediated penetration) revealed enrichment for functions involved in signaling (Additional file 10: Table S5_B). A more detailed analysis identified 15 potential protein kinases, including three phosphatidyl-inositol kinases and two MAP-kinases (Table 1 and Additional file 4: Table S3). Sequences relating to lipid metabolism (2 acyl-CoA dehydrogenases, 2 acyl-CoA ligases) and ubiquitin-mediated protein degradation (2 E3 ubiquitin ligases and one ubiquitin-like protein) were also identified in this cluster.

For sequences repressed both in zoospores and during the penetration process (cluster IV, 388 sequences), most of the associated GO terms identified related to metabolism (Additional file 11: Table S5_IV). Primary metabolism was globally downregulated, as indicated by the numbers of sequences relating to glycolysis/neoglucogenesis, the pentose phosphate pathway, lipid metabolism, and their overall repression level (Table 1 and Additional file 4: Table S3). Similarly, the ubiquitin-proteasome pathway (12 proteasome components and 9 sequences associated with the proteolytic process) was repressed during early infection (Table 1 and Additional file 4: Table S3).

Surprisingly, an analysis of cluster IV led to the identification of functions otherwise overrepresented during the penetration process. The sequences of this cluster included sequences encoding enzymes involved in cell wall degradation (15 sequences), proteases (13 sequences), elicitin-like proteins (4 sequences) and potential ABC transporters (8 sequences, Table 1 and Additional file 4: Table S3). These findings suggest that multiple functions are achieved by specific proteins during the penetration process, with these proteins being downregulated during the other steps of the P. parasitica life cycle.

An RXLR effector is expressed in the appressorium and infectious hyphae

A significant number of transcripts encoding RXLR effectors were found to accumulate during host penetration. These proteins are generally thought to be delivered into the plant cytoplasm via haustoria [39]. We analyzed the expression pattern of an RXLR sequence from cluster I. This gene, represented by the unisequence CL380, was chosen for study because it was strongly expressed during the penetration process (Table 1). P. parasitica transformants expressing a pCL380::GFP-GUS transcriptional fusion were obtained and used in the onion epidermis-based simplified penetration assay, for a precise analysis of CL380 expression during penetration. Two independent transformants gave similar GFP expression patterns. Expression was detected in germinated cysts (2 hai) and a faint GFP signal was observed in the cyst and germ tube (Figure 4A, 2 h). Fluorescence remained weak in germinated cysts differentiating into appressoria (Figure 4A, 2 h). The GFP signal increased 3 hours after inoculation, at the onset of penetration, and peaked six hours after inoculation (Figure 4A, 3 h and 6 h). The GFP signal was localized in infectious hyphae, and the cysts and appressoria appeared to have no cytoplasm at this stage (Figure 4A, 6 h). The GFP signal decreased 10 hours after inoculation, when the infectious hyphae grew into the cells and became weakly detectable, 30 hours after inoculation, in heavily colonized tissues. As GFP is stable for almost 24 h, the decrease in fluorescence intensity is accounted for by both transcription arrest and the dilution of the existing GFP in the developing infectious hyphae. This result confirmed that the CL380 effector accumulated transiently during the penetration process. Zoospores from transformants expressing the pCL380::GFP-GUS transcriptional fusion were then used to inoculate A. thaliana plantlets. A transgenic line accumulating a plasma membrane-targeted mCherry fluorescent protein was used to visualize the boundaries of the plant cell cytoplasm. We observed mCherry fluorescence around the infectious hyphae, suggesting that P. parasitica invades plant cells by growing between the plasma membrane and the cell wall (Figure 4B). As in onion epidermis, GFP fluorescence was observed just after penetration, in the infectious hyphae of the transgenic P. parasitica strain, whereas no fluorescence was detected with the wild-type strain (8 hours post inoculation for the image presented, Figure 4B). This result confirms that this RXLR-encoding gene is expressed during penetration of the first plant cell. The fluorescence subsequently decreased but continued to be observed in some areas of the infected roots at the late biotrophy stage (24 hours post inoculation for the image presented, Figure 4B). This may be due, in part, to the stability of GFP, which delays the decrease in fluorescence. Moreover, as P. parasitica infection is an asynchronous process, the infection events observed 30 hours after the inoculation of A. thaliana may correspond to different biological stages. Some parts of the roots may undergo early biotrophic P. parasitica colonization, even 30 hours after inoculation. It was more difficult to detect mCherry fluorescence at this stage, which suggests that plant cells are already affected by P. parasitica colonization. Taken together, these results confirm that P. parasitica transiently expresses RXLR effectors during the penetration process.
Figure 4

P. Parasitica appressorium transiently express a RXLR encoding effector during the penetration process. (A and B) Expression pattern of the CL380 RXLR encoding gene using a pCL380::GFP-GUS fusion. (A) Kinetic of early infection steps using a simplified penetration assay based on onion epidermis. GFP fluorescence from a P. parasitica strain carrying the pCL380::GFP-GUS construct was monitored 2 to 30 hours after inoculation of zoospores on onion epidermis. (B) Analysis of the CL380 expression pattern during early A. thaliana infection. A. thaliana plantlets where inoculated with zoospores from a P. parasitica strain carrying the pCL380::GFP-GUS construct and fluorescence was monitored during infection of the first cell (6 hours after inoculation for the presented image) and late biotrophic phase (24 hours after inoculation for the proposed image). Plant cell membranes (red) are visualized with the mCherry plasma membrane marker pm-RB. A representative image for each stage is presented. GFP and mCherry fluorescence was visualized using a confocal laser scanning microscope. Bars: 10 μM; Arrows: appressoria; Stars: Infectious hyphae.

Discussion

We developed a custom oligoarray, using all the P. parasitica sequences available at the start of this project. These sequences were obtained in a range of biological conditions, including plant infection. A set of 5451 unisequences was generated by EST clustering and assembly and oligonucleotides were designed for all but 24 of these sequences. Overall, 87% of these sequences were attributed to P. parasitica or were of unknown origin. Based on the recently released complete P. parasitica genome sequence, we determined that the array made it possible to study the expression of about 20% of the predicted genes of this species. The oligoarray was used to monitor gene expression during the onset of infection. The objective was to characterize the genetic program activated during appressorium differentiation and the penetration of the first host cells. RNA recovered from vegetative cultures, pre-infection structures (motile zoospores and cysts differentiating into appressoria on onion) and samples collected from the plant (corresponding to A. thaliana tissues collected from 2.5 to 30 hours after infection) was used to characterize the initial steps of infection. We detected 89% of the sequences in at least one set of conditions. This high rate of detection is slightly higher than the 69%, 79% and 75% reported during analyses of the early infection process in P. infestans, P. sojae and P. capsici, respectively [1921]. This result is unsurprising, because EST sequences were used for the design of the array, rather than genome-based predicted open reading frames. In particular, 64% of the sequences were detected 2.5 hours after the inoculation of A. thaliana roots, which is not surprising because 20% of the ESTs used for the assembly of sequences analyzed with the array were obtained from germinated cysts with appressoria [8]. Thus, our methods were effective for detecting sequences expressed at early stages of infection. A large proportion of the sequences were differentially expressed, with 74% and 38% displaying modulations of at least two-fold and at least four-fold, respectively, between at least two sets of conditions. This proportion is higher than the proportion of genes displaying a two-fold modulation of expression (46%) described by Judelson and coworkers, who analyzed the events from sporangium cleavage to the in vitro differentiation of appressoria [19]. This difference may reflect the large proportion of genes with expression modulated during plant penetration, a biological condition not tested in the transcriptome analysis for P. infestans. Other studies on fungi have generated findings consistent with this conclusion. Only 29% of Magnaporthe grisea genes have been shown to display a two-fold modulation of expression during in vitro appressorium differentiation, whereas up to 44% of Colletotrichum higginsianum genes were found to display a four-fold modulation of expression in a transcriptome analysis of samples corresponding to plant infection [40, 41].

A clustering analysis identified five highly validated clusters containing sequences modulated during appressorium-mediated penetration of the host. Sequences accumulating transiently during the penetration of the first plant cells, some of which were already accumulating in zoospores before infection, were identified. Similarly, sequences displaying transient downregulation upon host penetration were also identified. Such sequences, displaying specific downregulation in appressoria, were not identified in P. infestans germinated cysts with appressoria obtained in vitro[19]. The perception of environmental cues at the plant surface may contribute to this downregulation.

An analysis of the predicted function of the genes transiently expressed or repressed provided insight into the genetic program activated during early infection. The cluster grouping together sequences specifically repressed during the first few hours of infection (cluster II) contained a large number of genes relating to lipid and sugar metabolism. Consistent with this observation, only a few sequences relating to sugar or lipid degradation were identified in cluster I, which contained sequences transiently accumulating during the penetration process. By contrast, many sequences relating to protein degradation and amino-acid uptake were identified among the sequences accumulating at this stage. Taken together, these results suggest that P. parastica may use amino-acid uptake from the plant as a carbon source, as soon as penetration occurs. By contrast, sugar and lipid metabolism may be repressed at early stages of infection. Jupe and coworkers recently reported an enrichment in functions relating to gene expression and metabolism during the biotrophic phase of tomato infection by P. capsici[21]. They suggested that amino-acid uptake from the plant would not be favored by P. capsici at early stages of infection. This study provided no information about sugar and lipid metabolism but, as we obtained conflicting results, additional studies are required to determine which carbon sources are used by Phytophthora species during early stages of infection.

A large number of sequences relating to signaling were identified in the cluster grouping together sequences transiently repressed during penetration, within which protein kinases were particularly abundant. In addition, few, if any, signaling-related sequences were identified among the cellular functions for sequences accumulating during penetration of the host plant by P. parasitica. As calcium is known to be required for appressorium differentiation in P. infestans, related signaling pathways should have been detected during penetration [10]. The absence of these functions among the penetration-specific genes may be due to the corresponding transcripts and/or proteins accumulating in zoospores before infection, in avoid the need for de novo expression in appressoria and young infectious hyphae. Indeed, the penetration process occurs very rapidly after zoospore germination in P. parasitica[8, 23]. Consistent with this hypothesis, several protein kinases, including a calcium-dependent kinase, and transcription factors were observed among the cluster II sequences transiently expressed in zoospores. Thus, the zoospore, in addition to its role in dissemination, may be considered to be a pre-infection stage expressing important functions required for the penetration process.

An analysis of the sequences transiently accumulating during penetration highlighted functions that have been reported to influence the behavior of the interaction. The proteins concerned included a set of proteins triggering the necrosis of plant tissues and including elicitins, Nep-like proteins and proteins homologous to M81 elicitors. Sequences encoding protease inhibitors and transporters putatively involved in the efflux of toxic plant molecules were also observed. Such sequences have been reported to accumulate in P. infestans germinated cysts and probably constitute general weapons in the arsenal of Phytophthora spp. [18, 19]. Cell wall-degrading enzymes were particularly abundant among the transcripts transiently accumulating during the first few hours of infection. Judelson and coworkers also detected numerous CWDEs in P. infestans germinated cysts [19]. We also noticed that CWDEs displayed very unusual expression patterns. Some enzymes were specific to penetration, whereas others were expressed in the mycelium or during the necrotrophic phase of the interaction. This result confirms our previous hypothesis that a specific set of P. parasitica CWDEs may be mobilized to soften the plant cell wall and facilitate penetration [8]. The finding that other CDWE genes are expressed in vegetative cultures or during the necrotrophic phase of the interaction suggests that necrotrophy is related to saprophytic growth and that this second set of enzymes is used to obtain sugars from the walls of dead cells.

Finally, most of the RXLR effectors represented on the array displayed transient expression during penetration. This finding was not unexpected, because most of these sequences originated from appressorium-derived cDNAs [8]. Nevertheless, this result indicates that, as observed for CWDEs, a specific set of effectors is activated during penetration. No such expression pattern was not observed for transcripts encoding CRN proteins, suggesting different roles for these two classes of cytoplasmic effectors. Previous studies reported the detection of transcripts encoding secreted effectors in appressoria from Phytophthora species such as P. infestans, P. sojae, and P. capsici and in appressoria from ascomycetes, such as Magnaporthe grisea and Colletotrichum higginsianum[19, 21, 40, 41]. However, the secretion of Phytophthora cytoplasmic effectors has been documented only in haustoria to date. By using a transcriptional fusion with the GFP reporter gene, we were able to detect the expression of an effector in the appressorium and infectious hyphae during the penetration process. Our work thus suggests that appressoria and infectious hyphae are not only involved in plant penetration, they may also act as secretory organs for the transfer of cytoplasmic effectors into the host. These penetration-specific effectors may be involved in manipulating plant cells to facilitate establishment of the pathogen. Wang and coworkers described successive waves of effector expression during plant infection by P. sojae[42]. These authors suggested a relay between these successive waves, interfering with plant immunity at various levels. We have shown that the PSE1 effector, which is transiently expressed during penetration, can interfere with auxin physiology to facilitate plant infection [38]. Additional functional analyses of effectors transiently expressed during the onset of infection are required to determine whether this specific set of proteins plays a particular role in modulating plant development.

This study provides new insight into the process by which Phytophthora species penetrate their hosts. Based on our results for about one fifth of the gene content of this species, we propose several hypotheses concerning the biology of the infection process of P. parasitica. Future projects, making use of the full genome sequence, which is now available, will complete this analysis and should make it possible to test our hypotheses.

Conclusion

By using precisely calibrated interaction systems, we characterized the genetic program activated by P. parasitica during the initial infection of A. thaliana cells. Expression of a series of genes is transiently modulated during the penetration of the host. These modulations account for a specific developmental program occurring at the penetration stage. During penetration, genes encoding proteins involved in lipid and sugar metabolism are down-regulated whereas genes encoding functions controlling the transport of amino acids seemed favored. Similarly, cell wall degrading enzymes and other proteins involved in pathogenicity including RXLR effectors are highly modulated. Interestingly, P. parasitica uses distinct genes from these families during penetration and subsequent necrotrophic phase. We reconsidered the respective roles of the different developmental stages occurring during the infection cycle in this study. We suggest that the appressorium is involved in manipulating host cells to facilitate infection.

Declarations

Acknowledgements

This work was funded by grants from INRA, Direction scientifique ‘Plante et Produits du végétal’ and Department ‘Santé des Plantes et Environnement’. We thank Prof. Torii (Washington USA) for providing the A. thaliana transgenic line expressing the PM-RB marker.

Authors’ Affiliations

(1)
UMR Institut Sophia Agrobiotech, INRA1355-CNRS7254-UNSA, Université de Nice Sophia-Antipolis
(2)
Sainsbury Laboratory (SLCU), University of Cambridge

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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