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Keywords:

  • defence-related genes;
  • phenolics;
  • potato-late blight;
  • real-time PCR;
  • rishitin;
  • Solanum tuberosum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Defence responses were investigated in two potato cultivars with different levels of resistance to late blight, Russet Burbank (susceptible) and Kennebec (moderately resistant), after inoculation with single isolates representing Phytophthora infestans genotypes US-1 (previously predominant, mildly aggressive) and US-8 (currently predominant, highly aggressive). The accumulation of brown lignin-like materials and an increase in the cell wall affinity to trypan blue 24 h after inoculation were observed in cv. Kennebec inoculated with US-1, but not in Kennebec inoculated with US-8, or cv. Russet Burbank inoculated with either US-1 or US-8. The expression of PAL-1, HMG-2, PR-1 and PR-5 was investigated in three leaf strata (local, proximal and distal) and at different times after inoculation, using SYBR real-time RT-PCR. The activation of these defence-related genes was affected not only by P. infestans genotype, but also by the potato cultivar and the proximity to the inoculation site. These genes were up-regulated earlier in Kennebec than in Russet Burbank and in response to US-1 than to US-8. Over all, the earliest and strongest up-regulation of these genes occurred in Kennebec inoculated with US-1. Furthermore, PAL-1 and HMG-2 were down-regulated at the site of infection while such down-regulation was not observed for PR-1 or PR-5. In parallel, the accumulation level and location of phenolics and rishitin matched those of PAL-1 and HMG-2 transcripts, respectively. These results strongly suggest that changes in either the activation or suppression of defence responses by the pathogen shape the level of susceptibility of potato cultivars to late blight.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Both race-specific and race non-specific resistance to Phytophthora infestans, the cause of late blight, have been described in potato (Vleeshouwers et al., 2000). Based on the gene-for-gene concept, race-specific resistance is conferred by a major resistance gene (R) which encodes a specific receptor recognizing the elicitor encoded by the corresponding Avr gene in the pathogen. To date, 11 race-specific R genes have been introgressed from Solanum demissum into potato (Gebhardt & Valkonen, 2001). The R-genes, taken individually, have initially mediated successful resistance but have been defeated by new emerging races of P. infestans (Fry & Goodwin, 1997). Race non-specific resistance is commonly observed in wild Solanum species (Fry & Goodwin, 1997) and is believed to be more durable than race-specific resistance. Race non-specific resistance could result from a plant's intrinsic properties or may be induced by non-specific elicitors produced by different races of P. infestans (Vleeshouwers et al., 2000). Recent evidence has also suggested that R-gene receptors recognized by specific pathogen elicitors might be involved in the race non-specific resistance to late blight (Kamoun et al., 1999). In potato, the hypersensitive response (HR) has been observed in cultivars with either race-specific or race non-specific resistance to P. infestans. In the latter case, HR has been related by certain authors to the presence of weak R-Avr interactions or gene dosage effects (Kamoun et al., 1999; Vleeshouwers et al., 2000).

Many defence-related genes, such as PAL and HMG, control secondary metabolism pathways. PAL encodes phenylalanine ammonia lyase, which converts phenylalanine to an activated hydroxycinnamic acid, an essential step for the synthesis of a wide variety of phenolic compounds such as flavonoids, isoflavonoids, and lignin (Dixon et al., 2003). HMG encodes 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is essential for the biosynthesis of sesquiterpene phytoalexins in potato, i.e. rishitin, solavetivone and lubimin (Choi et al., 1992). Both PAL and HMG have been previously shown to be involved in potato resistance to late blight (Yao et al., 1995; Yoshioka et al., 1999). Other defence-related genes such as those controlling pathogenesis-related (PR) proteins have also been reported in this interaction (Liu et al., 1994; Wang et al., 2005, 2006).

In the last two decades, the population structure of P. infestans in North America has changed dramatically. In Canada, like in other parts of the world, the previously predominant genotype of P. infestans (US-1) has been displaced by new, more aggressive genotypes (i.e. US-8) (Daayf & Platt, 2000; Daayf et al., 2000). As a result, late blight epidemics went from sporadic to frequent and severe, consequently leading to an increase in yield loss and a reduction of the growers’ income, along with a high reliance on costly application of pesticides (Johnson et al., 1997). A better understanding of the mechanisms involved in the race non-specific resistance to different strains of P. infestans will be beneficial for the utilization of host resistance and the development of new resistance management and breeding strategies.

Prior to the present study, the differential expression of PAL-1, HMG-2, PR-1, PR-2, PR-3, PR-5 and PR-9 has been described, using Northern blot analysis in cvs Russet Burbank and Kennebec inoculated with isolates from the US-1 and US-8 genotypes of P. infestans (Wang et al., 2004, 2005, 2006). The differential expression of these genes was studied both locally at the infection site and remotely in proximal and distal sites. The analysis suggested that the success of P. infestans US-8 genotype may be related to its greater ability to locally suppress the expression of defence-related genes PAL and HMG, whereas no sign of suppression of PR-genes PR-1 and PR-5 was observed.

The objectives of the present study were to (i) assess the accumulation of PAL-1, HMG-2, PR-1 and PR-5 transcripts using quantitative real-time RT-PCR as a more accurate tool to confirm their differential accumulation patterns in response to the two P. infestans isolates; (ii) describe related histological changes at the local site of inoculation in response to the two isolates; and (iii) assess the accumulation of metabolites deriving from the PAL and HMGR pathways, as a result of either their induction or suppression after inoculation with each of the two isolates.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plants

Russet Burbank (RB, highly susceptible) and Kennebec (KB, moderately resistant) were used in this study. KB carries the R1 resistance gene, whereas RB does not have any of the known R genes. Potato plants were grown in clay pots containing a mix composed of soil, sand, peat and perlite at the ratio of 4:4:4:1. Potted plants were kept in a growth chamber maintained at 20 ± 2°C with 16 h photoperiod.

Isolates

Phytophthora infestans isolates (FA1, US-1 genotype and D1901, US-8 genotype) were grown on V8-PDA medium consisting of 10 g of agar, 10 g of PDA, 150 mL V8 juice, 850 mL of H2O and 3 g of CaCO3 at 20 ± 2°C. Their physiological races were previously determined (Wang et al., 2005), based on their performance on a differential set of potato cultivars harbouring late blight single resistance genes R1 to R11. This was based on the presence or absence of the hypersensitive reaction, and the extension of the infection lesion with or without sporulation on leaves of each differential cultivar (Daayf et al., 2001). Isolate FA1 (US-1) was race 1, 2, 4, 7, 9 and isolate B1901 (US-8) was race 1, 2, 3, 4, 6, 7, 8, 9, 10.

Inoculation

Inoculations were carried out using whole plants. One hundred microlitres of the sporangial suspension (5 × 106 sporangia mL−1), freshly collected from 10–14-day-old P. infestans cultures (Wang et al., 2005), were applied to the primary leaflet of the fourth full grown potato leaf. The inoculum was deposited on the upper surface of the potato leaflet as multiple tiny droplets (15–20 droplets of about 5 µL each), using a micropipette to prevent the inoculum from running off. Potato plants were then kept under saturated atmosphere for 48 h.

Microscopy

Histology of the interaction was examined on detached leaves, after P. infestans inoculum was deposited on the abaxial surface of detached leaflets from the fourth fully grown leaf. Leaf discs (6 mm in diameter) each containing one inoculum droplet, were excised 24 and 48 h after inoculation (h.a.i.). The excised leaf discs were first discoloured and fixed in Farmer's solution consisting of 95% ethanol, chloroform and acetic acid (6:3:1, v/v/v) until it had been fully cleared (approximately 1 h). Leaf discs were then stained with trypan blue (Colon et al., 1993) for the observation of mycelial growth and phloroglucinol-HCl (O’Brien & McCully, 1981) for the detection of lignin-like materials. Fifty leaf discs per treatment and time point were cut and subjected to the trypan blue (25 discs) or phloroglucinol-HCl (25 discs) staining.

Genomic DNA extraction and Southern blot hybridization

Genomic DNA was extracted from healthy control plants (fourth fully grown leaves) of cvs Kennebec and Russet Burbank using the method described by Taylor & Powell (1982). Five micrograms of DNA were digested with AluI or MseI overnight then size-separated on 1% agarose gel. Following electrophoresis, DNA was blotted onto Hybond-N+ membrane (La Roche-Hofmann) and hybridized with probes for PAL-1, HMG-2, PR-1 and PR-5 (Table 1). Probe labelling and the hybridization were performed as previously described (Wang et al., 2006) except that the hybridization temperature was decreased to 42°C.

Table 1.  Primers used for the synthesis of Southern blot probes and for quantitative real-time RT-PCR assessment of four defence-related genes (PAL-1, HMG-2, PR-1 and PR-5)
DesignationGenBank Acc. No.Primers for probe synthesis (F and R)/quantitative real-time PCR (RTF and RTR)(°C)aSize (bp)
  • a

    Annealing temperature.

PAL-1cDNA, potato X63103F: 5′-GCGATTTTCGCTGAAGTG-3′60596
R: 5′-TGTTGCTCGGCACTCTGA-3′
RTF: 5′-TTGCACAAGTTGCATCCATT-3′62 84
RTR: 5′-CACCAGCTCTTGCACTTTCA-3′
HMG-2cDNA, potato AB041031F: 5′-TGACGCAATGGGAATGAA-3′60506
R: 5′-CCTCCAGGAACGGTAGTA-3′
RTF: 5′-ACAAGAAGCCAGCAGCAGTT-3′62120
RTR: 5′-CCACAAGAGCAGCAACTTCA-3′
PR-1cDNA, potato AJ250136F: 5′-TCACTCTTGTGATGCCCAAA-3′60580
R: 5′-AGTGGAAACAAGAAGATGCA-3′
RTF: 5′-GCATCCCGAGCACAAAATTA-3′60 99
RTR: 5′-GAAATCACCACTTCCCTTGG-3′
PR-5cDNA, potato X67244F: 5′-TAATGCTTCCGGCGTATTTG-3′60509
R: 5′-AATCGGTAGGACCACATGGA-3′
RTF: 5′-ATGGGGTAAACCACCAAACA-3′60121
RTR: 5′-GTTAGTTGGGCCGAAAGACA-3′
β-tubulinS. tuberosum (TUBST1) mRNA for β-tubulin Z33382F: 5′-TCTGCAACCATGAGTGGTGT-3′60557
R: 5′-ATGTTGCTCTCGGCTTCAGT-3′
RTF: 5′-CAAGGCTTTCTTGCATTGGT-3′60 72
RTR: 5′-ATGTTGCTCTCGGCTTCAGT-3′

Leaf sampling procedures for real-time PCR analysis

Potato leaflets were collected from healthy and inoculated plants at various time points after inoculation. The sampling included terminal leaflets from individual healthy potato plants (healthy controls); inoculated terminal leaflets from the fourth fully grown leaf (local, L), non-inoculated leaflets within the same leaf where P. infestans was inoculated (proximal, P) and non-inoculated leaflets from the leaf adjacent to the inoculated leaf (distal, D; Fig. 1). Potato leaflets were collected at 0, 6, 12, 24, 48, 72, 96 and 120 h.a.i. The harvested leaf samples were reduced to a fine power immediately after sampling and stored at –80°C until used for total RNA extraction. For every single time point, leaflets from three separate pots were collected and RNA was extracted separately and used for real-time PCR analysis.

image

Figure 1. A simplified diagram representing a potato plant and highlighting the positions of the leaflets inoculated with Phytophthora infestans and included in the analysis of defence-related genes.

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Total RNA extraction and cDNA synthesis

Total RNA was extracted with the method described by Verwoerd et al. (1989). The absorbance at 260 nm and A260/A280 ratio was used to determine the concentration and purity of RNA samples. Leaf samples collected from three replicates were used separately for RNA extractions. Prior to cDNA synthesis, RNA samples were treated with RNAase-free DNAase (Invitrogen Inc.), following the manufacturer's protocol. Reverse transcription was performed using 2 µg of total RNA, random primers and Superscript II RT (Invitrogen Inc.) in a total volume of 20 µL. The reaction was incubated at 25°C for 10 min followed by incubation at 42°C for 50 min. The reaction was terminated by incubation at 65°C for 10 min followed by RNAase treatment (Invitrogen Inc.).

SYBR green real-time PCR assays

Real-time PCR was performed using SYBR@Green I technology on a Smart Cycle thermocycler (Cepheid) in the presence of 2·5 mm MgCl2. For each PCR sample, a 20 µL reaction solution was prepared in a LightCycler capillary (Cepheid) containing 2 µg cDNA preparation, 0·5 µm of each primer and 10 µL of qPCR Mastermix (Invitrogen Inc.). Samples were run for 40 cycles under the following thermal cycling protocol: samples were preheated at 95°C for 15 s. Then, 40 amplification cycles were performed consisting of 15 s at 95°C, 5 s at the annealing temperature of each specific primer as indicated in Table 1, and 15 s at 72°C. The primers were designed using OligoPerfectTM Designer software (InvitrogenTM Life Science Software, Invitrogen Inc.) and the sequences of primers used for PAL-1, HMG-2, PR-1, PR-5 and tubulin are listed in Table 1. The amplification efficiency of primer pairs for genes of interest was tested. Standard curves were developed for each gene by plotting the logarithm of known concentration (10-fold dilution series from 10 ng to 1 pg per 25 µL reaction volume) of the gene of interest against Cycle of threshold (Ct) value and used to calculate the amount of expression in samples (µg per 25 µL). Ct value is the threshold cycle number at which the fluorescence emission of the PCR product becomes significantly distinguishable from the background. This value is inversely related to the logarithm of the initial template concentration. Potato tubulin gene was included in the real-time PCR assay and used as a constitutively expressed internal control. To minimize the possible variation from reverse transcription, the ratio of gene of interest/tubulin was used to establish the relative expression curves.

High pressure liquid chromatography (HPLC) analysis of secondary metabolites

Soluble phenolics of potato leaves were extracted from local, proximal and distal leaflets according to the protocol described by Daayf et al. (1997). Briefly, 200 mg of potato leaf tissue were reduced to a fine powder in liquid nitrogen and extracted with 10 mL of 80% methanol overnight in the dark at room temperature. After centrifugation, the pellet was re-suspended twice in 2·5 mL of 80% methanol. Pooled methanolic fractions were incubated under a nitrogen stream until the methanol had evaporated. The remaining aqueous phase was then mixed with petroleum ether to remove chlorophylls, waxes and lipids from the leaf extracts. The cleared aqueous phase was thereafter extracted three times with ethyl acetate. After a final evaporation of the ethyl acetate fraction, the residue was suspended in 200 µL of pure methanol and immediately subjected to analysis or stored at –20°C until used.

For HPLC analysis, a Waters 2690 separation module was used. This module is equipped with an autosampler and a Waters 996 photodiode array detector, and fitted with a 5 µm LiChrospher® 100 RP-18 guard column (LiChroCART® 4-4) and a reverse-phase 5 µm 250-4 LiChrospher® 100 RP-18 column (LiChroCART® 4-4). Results were analyzed using Millenium Software, version 3·2. The column was eluted at a flow of 1 mL min−1 with a gradient using a solvent system composed of A: 0·1% H3PO4-acidified water and B: HPLC grade acetonitrile. The gradient used to carry the analysis was as follows: (time [min]/A [%]/B [%]) = 0/100/0, 5/95/5, 10/95/5, 14/90/10, 20/80/20, 23/80/20, 30/65/35, 35/65/35, 43/50/50, 48/25/75, 55/0/100, 60/0/100. Injected volumes were 15 µL per sample and each injection was repeated three times.

Thin layer chromatography (TLC) analysis of rishitin accumulation

Extraction of sequiterpene phytoalexins including rishitin, the main sesquiterpene phytoalexin resulting from the activity of HMG genes, was carried out using local, proximal and distal leaflet material (200 mg) in 70% methanol followed by a purification step using ethyl acetate (Lyon, 1972). The final residue was dissolved in pure methanol and stored at –20°C until used. Detection of the phytoalexin in the plant extracts was performed on TLC plates (Silica gel plate, SHGF254). A total of 50 µL of final methanol extract was loaded on TLC plates. The plates were then developed in 4% methanol in chloroform as described by Lyon (1972). After a complete migration of the mobile phase through the plate, a spray of 5% vanillin in 5% sulphuric acid in ethanol was performed and the plates were heated using a hot air gun. Under these conditions rishitin appears as a blue spot at an Rf of 0·25.

Data analysis

All the experiments were conducted following a series of trials in a randomized complete block design. For each trial, 25 replicates were used per treatment ‘cultivar × isolate’ and each trial was repeated independently three times. For real-time RT-PCR data, the relative expression of the studied defence-related genes was reported by reference to the expression of the constitutively expressed β-tubulin. These ratios were arcsine-transformed and subjected to anova analysis (SAS Institute Inc.). When F-test was significant (P < 0·05), mean values of three independent replicates were compared among treatments according to Tukey's test. For HPLC analysis, three independent extractions were performed per treatment isolate cultivar and time series. The extracts were then analyzed three times using HPLC. Peaks representing soluble phenolics were integrated using the Millenium Software, version 3·2. Area under the peak was then converted to µg equivalents of chlorogenic acid per g of FW, by reference to a pre-established standard curve using commercial chlorogenic acid. The collected data were then analyzed by anova and the averages were compared among treatments according to Tukey's test (P < 0·05).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Histological observations

The first observations were carried out 24 h.a.i., at which time the pathogen has already penetrated the host cells in all treatments. In cv. Kennebec tissues inoculated with US-1 and stained with phloroglucinol-HCl, brown lignified-like materials commonly accumulated (Fig. 2a vs. Fig. 2b–d) along with an increase in the cell wall affinity to trypan blue, especially in cells nearby the penetration sites (Fig. 2e vs. Fig. 2f–g). The growth of hyphae was also limited to the inoculation site surroundings. Conversely, in leaf discs from cv. Kennebec inoculated with US-8, or from cultivar Russet Burbank inoculated with either US-1 (Fig. 2c) or US-8 (Fig. 2d), mesophyll cells were indistinguishable from those of the healthy controls. Neither an accumulation of brown lignin-like materials nor an increase in cell wall affinity were observed with phloroglucinol-HCl or trypan blue staining, respectively.

image

Figure 2. Phloroglucinol-HCl (a–d) and trypan blue (e–k) staining of potato leaf tissues inoculated with Phytophthora infestans 24 and 48 h.a.i. An abundant presence of brown lignin-like materials is observed in mesophyll cells of KB leaves inoculated with P. infestans US-1 isolate and ongoing HR 24 h.a.i (a). A reduced presence of this material is observed 24 h.a.i. in KB × US-8 (b), RB × US-1 (c) or RB × US-8 (d). Trypan blue staining of leaf tissues inoculated with P. infestans 24 h.a.i. shows an increased affinity to trypan blue in mesophyll cells of KB leaves inoculated with P. infestans US-1 and ongoing HR 24 h.a.i. (e). No such an affinity was observed 24 h.a.i. in KB × US-8 (f), RB × US-1 (g) or RB × US-8 (h). (i–k): reveal three types of symptoms that were observed around the infection sites (arrows) 48 h.a.i. (i) represents hypersensitive-like necrotic lesion, in which the development of mycelia was limited KB × US-1; (j) shows expanding necrotic lesions, in which the cells surrounding the infection site became necrotic and intercellular mycelium developed extensively KB × US-8; and (k) highlights cells found near the penetration site with no necrotic lesions and an extensive development of intercellular mycelium RB × US-1, RB × US-8. BM, brown lignin-like materials; H, hyphae; TA, trypan blue aggregation. KB and RB are cvs Kennebec and Russett Burbank respectively.

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At 48 h.a.i., expanding necrotic lesions (Fig. 2j) and biotrophic growth of P. infestans mycelia (Fig. 2k) were frequently noticeable in all sampled tissues. Observations made in areas surrounding the penetration sites showed symptoms ranging from very limited necrotic lesions with a restricted mycelium growth that can be qualified as a hypersensitive-like type of symptom (KB × US-1 and occasionally KB × US-8; Fig. 2i), to expanding necrotic lesions, in which the cells surrounding the infection site become necrotic with an extensive intercellular mycelium development (KB × US8, Fig. 2j; RB × US8, Fig. 2k). Another type of symptom was characterized by the absence of visible necrotic cells nearby the penetration site and an extensive intercellular growth of the pathogen mycelium (RB × US8; RB × US1; Fig. 2k). Hypersensitive-like necrotic lesions were commonly observed in cv. Kennebec leaf tissues in response to inoculation with the US-1 and occasionally with the US-8 isolate. These symptoms were not, at any time, observed in cv. Russet Burbank leaf tissues inoculated with either US-1 or US-8 isolate. However, the growing hyphae remained ahead of the cells ongoing the hypersensitive-like reaction. These reactions were ineffective in preventing further development of the hyphae since they converted into necrotic sporulating lesions 72 h.a.i. (data not shown).

Southern blot analysis

Southern blot analyses were used to determine whether there were similarities in the distribution of PAL-1, HMG-2, PR-1 and PR-5 genes across Kennebec and Russet Burbank genomes. Emphasis was given to the isogeny and copy number of these defence-related genes among the two cultivars. Using the specific probes developed in this work, the patterns of hybridization of all four investigated genes were similar (Fig. 3), indicating that these genes are fairly conserved among the two tested cultivars. For each cultivar, multiple hybridization bands were observed, suggesting a presence of multi-gene families for PAL-1, HMG-2, PR-1 and PR-5, due either to regular duplication or to the tetraploidy of the species S. tuberosum. There were also differences in band intensity, which could result from either sequence diversity or variation among families of genes controlling various isoforms that still share high homology (Fig. 3).

image

Figure 3. Southern blot analysis showing the presence of multi-copies of defence-related genes coding for PAL (phenylalanine ammonia lyase), HMGR (3-hydroxy, 3-methylglutaryl CoA reductase), PR-1 and PR-5 (pathogenesis-related proteins) in potato cvs Russet Burbank (RB) and Kennebec (KB). Genomic DNA from both cultivars was digested with restriction enzymes RsaI and AluI, and hybridized against probes designed for potato PAL-1, HMG-2, PR-1 and PR-5 genes. Arrows on the right indicate the size of products of interest.

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Expression analysis and detection of the final products of defence-related genes

Expression of PAL-1

No variation in the accumulation of PAL-1 transcripts was observed in the healthy control over the time of the experiment. In local leaflets, a weak synthesis/accumulation of PAL-1 transcripts was observed in Kennebec in response to both P. infestans US-1 and US-8 genotypes at 96 and 120 h.a.i. (Fig. 4). No significant synthesis/accumulation of PAL-1 transcripts was found in Russet Burbank local leaflets infected with US-1 or US-8 during the whole period of the experiment (120 h.a.i.). In proximal leaflets, PAL-1 transcripts were first detected at 12 h.a.i. in Kennebec in response to US-1 and the same level of transcripts accumulation was maintained throughout the sampling period. A peak of PAL-1 transcripts synthesis/accumulation was also detectable in leaflets of cv. Russet Burbank in response to US-1 at 72 h.a.i. No significant transcripts synthesis/accumulation was observed in Russet Burbank proximal leaflets in response to US-8 while in cv. Kennebec leaflets a weak accumulation was noticeable at 120 h.a.i. in response to the same isolate. In distal leaflets, PAL-1 transcripts were only weakly synthesized/accumulated in Kennebec leaflets (48 h.a.i.) and in Russet Burbank leaflets (120 h.a.i) in response US-1. The synthesis/accumulation of PAL-1 transcripts in distal leaflets was much weaker and delayed than in proximal leaflets.

image

Figure 4. Quantitative real-time RT-PCR analysis of the relative expression of PAL-1, HMG-2, PR-1 and PR-5 in three potato leaf strata (see Fig. 1): local (L, infected terminal leaflet of the 4th fully grown leaf), proximal (P, non-infected lateral leaflet from the infected leaf) and distal (D, non-infected leaflet from an adjacent non-infected leaf), of cvs Russet Burbank (susceptible) and Kennebec (moderately resistant), in response to infection with Phytophthora infestans isolates representing US-1 and US-8 genotypes. The relative expression of each defence-related gene represents the ratio of the accumulated transcripts to β-tubulin transcripts used as internal control. Results are expressed as means of three independent replicates ± SE. Asterisks show significant accumulation of transcripts at P < 0·05 according to Tukey's test. (inline image, US-1; inline image, US-8; and inline image, Control). Data points from 6 and 96 h.a.i. were analyzed but not included in the figure.

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Over all, the expression of PAL-1 was recorded earlier in both Kennebec and Russet Burbank in response to US-1 compared to US-8. This expression was stronger and earlier in cv. Kennebec and occurred initially in proximal, then in local, and later on in distal leaflets.

Analysis of the soluble phenolics pool in infected potato leaves

HPLC analyses of the soluble phenolics resulting from the activation of expression of PAL genes in infected potato leaves in response to different genotypes of P. infestans showed that the accumulation of these compounds started at 24 h.a.i. with a maximum reached at 120 h.a.i., especially in cv. Kennebec infected with genotype US-1 (Fig. 5). Comparison of the phenolics pool content over time showed an earlier and strong accumulation first in proximal followed by local leaflets especially in Kennebec in response to genotype US-1 (Fig. 5).

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Figure 5. Variation over time (0–120 h.a.i.) of the total content of soluble phenolics in potato leaflets of cvs Russet Burbank (RB, susceptible) and Kennebec (KB, moderately resistant) in response to two Phytophthora infestans isolates representing US-1 and US-8 genotypes. Three types of leaflets were included in the analysis (inline image, local; inline image, proximal; and inline image, distal; see Fig. 1). Asterisks show significant accumulation of soluble phenolics among all treatments (P < 0·05).

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Expression of HMG-2

No changes in the level of HMG-2 transcripts synthesis/accumulation were recorded over time in the healthy control samples. No variation in the synthesis/accumulation of HMG-2 transcripts was found in local leaflets from both cultivars infected with either US-1 or US-8 genotype (Fig. 4). In proximal leaflets, HMG-2 transcripts were transiently detectable at a significant level in Kennebec leaflets in response to US-1 as early as 12 h.a.i. and until 96 h.a.i. No variation in the synthesis/accumulation of HMG-2 transcripts was found in distal leaflets collected from all four analyzed treatments.

Over all, the expression of HMG-2 was recorded earlier in cv. Kennebec in response to US-1 compared to US-8, and occurred first in proximal leaflets.

Accumulation of the phytoalexin rishitin in infected potato leaves

Extracts from local or distal leaflets of cultivar Kennebec infected with genotype US-1 did not display any accumulation of rishitin over time (Fig. 6). However, extracts from proximal leaflets exhibited an accumulation of rishitin over time as early as 24 h.a.i. The rest of analyzed treatments including different strata of leaflets from either Kennebec infected with genotype US-8 or Russet Burbank infected with either US-1 or US-8 genotype did not reveal any rishitin accumulation (data not shown).

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Figure 6. Thin layer chromatography analysis of rishitin accumulation (0–120 h.a.i.) in local, proximal and distal potato leaflets of cv. Kennebec inoculated with the Phytophthora infestans US-1 isolate. The accumulation of rishitin was detected as a blue band (arrows, Rf = 0·25) using the vanillin/sulphuric acid spray (Lyon, 1972). Purified rishitin was used as a standard (S).

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Expression of PR-1

While no recordable synthesis/accumulation of PR-1 transcripts was observed in non-inoculated healthy leaflets, a differential expression of this PR gene was detected in both cultivars inoculated with US-1 and US-8 genotypes (Fig. 4). In local leaflets, PR-1 transcripts accumulated earlier in response to US-1 rather than to US-8 and more in cv. Kennebec than Russet Burbank. The synthesis/accumulation of PR-1 transcripts was found in Kennebec leaflets infected with US-1 genotype as early as 6 h.a.i. After that, synthesis/accumulation of PR-1 transcripts was detectable from 24 until 120 h.a.i. with a maximum at 96 h.a.i.. Local leaflets of cv. Kennebec infected with the US-8 isolate did not display any accumulation of PR-1 transcripts before 48 h.a.i. Those of Russet Burbank were even later, with an accumulation of PR-1 transcripts at 72 and 120 h.a.i., in response to US-1 and US-8 genotypes, respectively. In proximal leaflets, PR-1 transcripts strongly accumulated at 72 to 120 h.a.i. in Kennebec leaflets in response to US-1, while no such accumulation was found in other treatments. In distal leaflets, PR-1 transcripts were detected only in Kennebec leaflets in response to infection with US-1 (96 to 120 h.a.i.).

Over all, the expression of PR-1 was recorded in both cultivars response to US-1 rather than US-8. This expression was stronger and earlier in cv. Kennebec and occurred locally then remotely from the infection site.

Expression of PR-5

As seen for PR-1, no substantial synthesis/accumulation of PR-5 transcripts was recorded in non-inoculated healthy leaflets of both cultivars. Among all analyzed treatments, PR-5 transcripts were detected in local and proximal leaflets of cv. Kennebec inoculated with the US-1 isolate (Fig. 4). The expression of PR-5 gene appeared first in local leaflets then in proximal and lastly in distal leaflets. No significant variations were recorded in terms of PR-5 transcripts synthesis/accumulation in cv. Kennebec leaflets in response to the US-8 isolate or in Russet Burbank leaflets in response to either US-1 or US-8 at the local, proximal and distal position.

Over all, the expression of PR-5 was recorded earlier in cv. Kennebec in response to US-1, and occurred locally then remotely from the infection site.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In recent studies (Wang et al., 2004, 2005, 2006), Northern blot analyses suggested differential suppression by P. infestans isolates of potato genes controlling the early steps in the phenylpropanoid and isoprenoid pathways (PAL-1 and HMG-2, respectively). Earlier studies suggested the suppression of PAL and HMG genes by P. infestans in potato tubers (Choi et al., 1992; Yoshioka et al., 1996), but as far as is known, this is the first evidence of potato defence gene suppression by P. infestans that is based on (i) both gene expression, using real-time RT-PCR, and their metabolic products, using chromatographic analyses, (ii) comparison of local and systemic host reactions, and (iii) analysis of leaf, not tuber, reactions. This study has shown that gene down-regulation of PAL-1 and HMG-2 resulted in a reduced accumulation of phenolic compounds and inhibition of the phytoalexin rishitin production at the inoculation site, respectively. Further evidence of gene suppression at the local inoculation site was provided by both their up-regulation, and the resulting accumulation of products in their respective pathways, phenolics and rishitin, distant from the inoculation site. Interestingly, PR-1 and PR-5 were both up-regulated at the local, proximal and distal sites. Such a systemic up-regulation of all of the tested defence genes concurs with the systemic acquired resistance (SAR) previously described in potatoes (Cohen et al., 1993).

Regarding the mechanism of suppression, previous studies have suggested that P. infestans suppressors inhibit the interactions between pathogen elicitors and their corresponding receptors in the host plant by blocking the binding sites (Doke et al., 1979; Garas et al., 1979). If this was true in the present study, it would mean that P. infestans elicitors that induce PR genes are different from the ones that induce PAL and HMG. Others have suggested that suppressors could act by blocking the transduction of signal(s) during the elicitor-mediated activation of defence responses (Yoshioka et al., 1990; Shiraishi et al., 1991). In the present study, the systemic induction of locally-suppressed genes suggests that the signal transduction and translocation were not affected in this case. Other authors have suggested that suppressors could act by directly affecting the formation of binding complexes in the promoter region, hence leading to the suppression of expression of specific defence-related genes at the transcription level (Wada et al., 1995). This last suggestion is the most compatible with the current findings, and studies will be conducted to assess possible effects on defence gene promoters.

The initial interaction between hosts and their fungal/oomycete pathogens often occurs via substances that are secreted by the spore into its germination fluids. Cystospores of P. infestans secrete small water-soluble glucans into their germination fluid. These glucans suppress both cell death, during the hypersensitive reaction, and the production of the phytoalexin rishitin in potato (Doke et al., 1980; Andreu et al., 1999; Ozeretskovskaya et al., 2001). Other studies have shown that P. infestans is capable of producing molecules other than β-glucans with an ability to suppress potato defence responses. Extra-cellular protease inhibitors, i.e. kazal-like extracellular serine proteases, have been recently identified in P. infestans and are thought to directly interact with, or inhibit, host proteases (Tian et al., 2004). Whether this suppression is occurring upon a mechanism involving soluble glucans, extra-cellular proteases, or other molecules is still unknown. However, it is clear from the present findings that the localized suppression of PAL-1 and HMG-2 does not inhibit the translocation of signal(s) that lead to the induction of these defence-related genes remotely from the inoculation site.

Previously, Garas et al. (1979) reported that suppressors from the virulent races of P. infestans were more active in inhibiting the hypersensitive reaction and the accumulation of rishitin and lubimin than those from the avirulent race. The weaker hypersensitive reaction induced in cv. Kennebec by the US-1, as compared to the US-8 isolate, concur with these observations and suggest that the latter has more effective defence suppressors than the US-1 isolate. However, in the present study, both isolates were virulent on both cultivars, and were only different in their level of aggressiveness. Therefore, despite being under control of race non-specific resistance a hypersensitive-like reaction occurred in both cultivars at early stages of infection, but earlier and longer in Kennebec (moderately resistant, R1) than in Russet Burbank (susceptible, no known R gene). However, it was ineffective in stopping the progress of the lesions on both cultivars, which is similar to the trailing hypersensitive reaction, described by Vleeshouwers et al. (2000), where hyphae developed ahead of hypersensitive reaction-responding cells. This type of reaction is apparently associated with all forms of resistance to P. infestans, including both race-specific and race non-specific resistance. The latter form of resistance is often related to the presence of quantitative trait loci (QTL) (Léonards-Schippers et al., 1994). Several studies of these QTL revealed a linkage with different known late blight R genes (Léonards-Schippers et al., 1994; Gebhardt & Valkonen, 2001) while others have not yet shown any association of these QTL with known R genes (Collins et al., 1999; Oberhagemann et al., 1999; Ewing et al., 2000; Ghislain et al., 2001). Knowing that some dominant R genes can also confer quantitative resistance (Lauge et al., 1998) combined with observations about the ‘trailing’ hypersensitive reaction (Vleeshouwers et al., 2000), it was suggested that dominant R genes could act as components of a QTL and become indirectly involved in quantitative resistance to late blight.

Besides the difference in the reaction level of the two cultivars, the hypersensitive-like reaction was induced earlier and more frequently in response to the US-1 than to the US-8 isolates. This might be related to the weak interaction between potato R genes and P. infestans ligands, proposed by Kamoun et al. (1999), where there is low affinity and poor interaction between the R and the Avr gene products.

The current findings, along with recent studies (Wang et al., 2004, 2005, 2006), are the first to show that down-regulation of potato defence genes (i) occurs locally and not systemically, (ii) takes place not only in tubers but also in the foliage, (iii) is specific to some defence-related genes (i.e. PAL-1 and HMG-2) and not to others (i.e. PR-1 and PR-5); and (iv) occurs differentially in response to isolates of P. infestans from different genotypes. Furthermore, PR-1 and PR-5 were found to be induced both locally and systemically. This suggests that the expression of the two PR genes may be regulated through a signalling system that is independent from the one regulating the expression of PAL and HMG.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported by a grant from the Natural Sciences and Engineering Research Council (NSERC-Canada) to Dr Fouad Daayf. Additional funds were provided by the Department of Plant Science, University of Manitoba. US-1 isolate of P. infestans used in these studies was a courtesy of Dr Patrice Audy, AAFC New Brunswick, Canada.

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  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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