JXB Advance Access originally published online on September 5, 2005
Journal of Experimental Botany 2005 56(420):2683-2693; doi:10.1093/jxb/eri261
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RESEARCH PAPER |
Resistance of sunflower to the biotrophic oomycete Plasmopara halstedii is associated with a delayed hypersensitive response within the hypocotyls
UMR 1095 INRA-UBP ‘Amélioration et Santé des Plantes’, Université Blaise Pascal, 24 avenue des Landais, F-63177 Aubière Cedex, France
* To whom correspondence should be addressed. Fax: +33 4 73407914. E-mail: m-fouad.bouzidi{at}univ-bpclermont.fr
Received 30 June 2005; Accepted 7 July 2005
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Abstract |
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The biotrophic oomycete Plasmopara halstedii is the causal agent of downy mildew in sunflower. It penetrates the roots of both susceptible and resistant sunflower lines and grows through the hypocotyls towards the upper part of the seedling. RT-PCR analysis has shown that resistance is associated with the activation of a hsr203J-like gene, which is a molecular marker of the hypersensitive reaction in tobacco. Activation of this gene was specifically observed during the incompatible interaction and coincided with cell collapse in the hypocotyls. This HR was also associated with the early and local activation of the NPR1 gene which is a key component in the establishment of the SAR. No such HR or a significant activation of the hsr203J-like gene were observed during the compatible combination. These results suggest that the resistance of sunflower to P. halstedii is associated with an HR which fails to halt the parasite. By contrast, this HR triggers a SAR which takes places in the upper part of the hypocotyls and eventually leads to the arrest of parasite growth. A model describing the resistance of plants to root-infecting oomycetes is proposed.
Key words: Defence related genes, Helianthus annuus, hypersensitive reaction, Plasmopara halstedii
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Downy mildew caused by the biotrophic Plasmopara halstedii (Farl.) Berl. & de Toni, is one of the principal diseases causing economic losses in the cultivation of sunflowers (Helianthus annuus L.). Resistance to this parasite is controlled by major dominant genes denoted Pl (Vranceanu et al., 1981
). In previous studies, microscopic observations had shown that both susceptible and resistant sunflower lines could be invaded by this soil-borne oomycete (Mouzeyar et al., 1993). However, about 5 d after root infection, a hypersensitive-like reaction develops within the resistant hypocotyls; in most cases, the parasite does not reach the leaves (Mouzeyar et al., 1993, 1994). This so-called Cotyledon Limited Infection (CLI, Gulya et al., 1991) is a general phenomenon found in sunflower–P. halstedii interaction, but little is known about its molecular basis.
During incompatible plant–pathogen interactions, recognition of a potential pathogen often results in a hypersensitive reaction (HR), with the activation of programmed cell death (PCD) at the site of the attack designed to halt the spread of the pathogen. This HR response is characterized by numerous physiological and molecular changes, i.e. depolarization of the plasma membrane, changes in respiration rates, an oxidative burst, the production of nitric oxide (NO), reactive oxygen species (ROS), an oxidative cross-linking of various cell wall compounds, and the production of pathogenesis-related (PR) proteins (Atkinson et al., 1985; Apostol et al., 1989; Lamb et al., 1989; Bradley et al., 1992; Hammond-Kosack and Jones, 1996; Delledonne et al., 1998; Desikan et al., 2001; Concetta de Pinto et al., 2002). During an HR, a small set of cells close to the pathogen undergoes rapid PCD usually within 12–24 h of inoculation (Hermanns et al., 2003).
From challenged HR-developed cells, some particular diffusible molecules known as stress phytohormones, i.e. salicylic acid (SA), jasmonic acid (JA) and ethylene, may play a crucial signalling function in establishing the acquisition of healthy distal tissue resistance, both locally (local-acquired resistance, LAR) and systemically (systemic-acquired resistance, SAR). In addition, sites of the HR are invariably focal points for the transcriptional induction of plant defence genes in neighbouring cells (Dangl et al., 1996). Their appearance is correlated with the expression of a set of pathogenesis-related (PR) proteins, some of which have been shown to exhibit antimicrobial activity in vitro or to confer increased resistance when over-expressed in plants (Broekaert et al., 1995; Smith 1996; Hu and Reddy, 1997; Morrissey and Osbourn, 1999). Such systemic immunity provides the plant with an effective state of acquired resistance throughout its different organs against subsequent attack by a broad spectrum of virulent pathogens (Dempsey et al., 1999).
Alternatively, a HR may be the consequence of a mechanism that is actually killing both host and pathogen cells. In fact, other authors have suggested that, in at least some cases, HR cell death is not required to halt pathogen growth (Century et al., 1995; Hammond-Kosack et al., 1996; Yu et al., 1998). For example, Rx-mediated resistance against the PVX virus occurs without an HR in the potato (Bendahmane et al., 1999). Similarly, the induction of defence responses in bean after infiltration with a Pseudomonas syringae pv. tabaci hrp– mutant did not correlate with the induction of a hypersensitive reaction, suggesting that this reaction might be associated with, but is not essential to, establishing resistance (Jakobek and Lindgren, 1993). However, many of the models presented deal with foliar infecting parasites and little is known about those parasites which infect plant roots.
In a recent work, Sesma and Osbourn (2004), who studied the rice and blast fungus Magnaporthe grisea interaction, reported that this fungus can undergo a different and previously uncharacterized set of programmed developmental events that are typical of root-infecting pathogens. They also showed that when the resistant rice cultivar CO39 carrying the resistant gene Pi-CO39(t) is infected by an avirulent strain of M. grisea, the development of the fungus is restricted to the outer part of the roots, indicating that gene-for-gene type specific disease resistance that is effective against rice blast in leaves also operates in roots. Similarly, Holub and Beynon (1997) collected Hyaloperonospora parasitica isolates from oospore infections of Arabidopsis roots, and found that the isolates were often avirulent when sporangia were used to inoculate cotyledons of the same host genotype. This finding raises an important question as to whether the initial signal for this response occurs in the roots, following root infection.
When Arabidopsis leaves are infected by a genetically incompatible strain of Hyaloperonospora parastica, a hypersensitive reaction develops within 24 h of infection. In contrast, when the infection occurred in the roots, the parasite formed intercellular hyphae and haustoria which were similar to those observed in the compatible combination and no hypersensitive reaction was observed, even 72 h post-infection (Hermanns et al., 2003). Interestingly, the resistance gene and many of the components of the R-mediated signalling pathways leading to resistance were expressed in the roots in both combinations, indicating that resistance in this pathosystem is specified by the organ (Hermanns et al., 2003).
In this paper, the oomycete Plasmopara halstedii was used to inoculate the roots of sunflower seedlings and the microscopic and molecular reactions in both compatible and incompatible combinations were then analysed. It was shown that resistance was associated with a hypersensitive reaction, which took place in the hypocotyls without halting the parasite, and with SAR, which seemed to be involved in the late stages of the resistance process. A model for plant resistance to biotrophic root-infecting oomycetes is then proposed.
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Materials and methods |
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Sunflower genotypes and infection procedure
Sunflower lines QIR8 (resistant) and CAY (susceptible) were used during this study. Resistance in the QIR8 line is controlled by the Pl8 locus containing CC-NBS-LRR sequences (Radwan et al., 2003
, 2004). Infection procedures with Plasmopara halstedii race 300 and the conditions prevailing in the growth chamber were as described by Mouzeyar et al. (1993). Briefly, sunflower seeds were surface-sterilized with hypochlorite and allowed to germinate for 48 h at 24 °C. Two-day-old seedlings are then infected by immersing them in 10 ml of a suspension containing 50 000 zoosporangia ml–1. Five hours later, the zoosporangia suspension was removed and the seedlings were transferred to a tray containing sterile soil-less compost in a growth chamber. The growth conditions were 18±1 °C, 16/8 h light/dark and about 200 µE m–2 s–1 light intensity. Control plants underwent the same procedures, except that sterile water replaced the zoosporangia suspension.
Histochemical procedures
For microscopic observations, the sunflower seedlings were fixed in FAA (formalin–acetic acid–ethanol, 5:5:90 by vol.). Light microscopy observations were carried out on free-hand sections as described by Mouzeyar et al. (1993). Lactophenol trypan blue was used to detect the parasite in some cases. The accumulation of hydrogen peroxide (H2O2) was studied histochemically using H2O2-specific 3.3-diaminobenzidine (DAB) dye (Thordal-Christensen et al., 1997). Photographs were taken using an Axio cam HRC, Zeiss digital camera.
Amplification, cloning and sequencing of defence genes
Sunflower defence-related genes were cloned using degenerate primers designed for conserved regions detected after the alignment (CLUSTAL X, Thompson et al., 1997) of defence gene sequences from various species listed in GenBank databases. Degenerate primers were designed for hsr203J [For: 5'-CGC(TC)TAGCGCG(ATGC)GTGGC(ATCG)AACGC-3', Rev: 5'-(TG)A(ATGC)GTT(ATGC)CCGCCGGAGC(ATGC)GTCTCC-3'], NPR1 [For: 5'-GC(AGCT)(AGCT)T(AGCT)GA(CT)TC(AGCT)GA(CT)GATGT(AGCT)GA-3', Rev: 5'-GCAA(AGCT)ATG(AGCT)AG(AGCT)(AG)C(GT)GT(AG)TA(AT)CC-3'], and glutathione-S-transferase (GST) [For: 5'-(CA)C(GCA)(AT)T(AC)(TC)T(CAGT)CCT(TC)(CA)(TGA)GATCCTTA-3', Rev: 5'-T(CG)GCTA(TA)C(CTA)GCAGC(CTA)ACATCGAC-3']. The resulting PCR products were purified using the GFX PCR purification system kit (Amersham-Pharmacia-Biotech, France), cloned in pGEMT-easy vector (Promega), and entirely sequenced using Genome express (Grenoble, France). After sequence verification, specific primers (Table 1) were designed for each sunflower defence-related gene described above for RT-PCR purposes. For PR-5 and defensin (PDF) accession no. AF364865 and AF364864, respectively (Hu et al., 2003), were used to design the primers. The sunflower elongation factor EF-1
sequence (accession no. AAM19764) was used as an internal standard. To check for the presence of the oomycete within sunflower tissues during the time-course of RT-PCR analysis, specific primers targeting the P. halstedii elongation factor TEF1 (accession no. CB174619) were used.
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RNA manipulations and RT-PCR procedures
Total RNAs were extracted using the method described by Bogorad et al. (1983)
, then treated with DNase I to remove genomic DNA contaminations. Two µg of DNase-treated RNA were reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (InVitrogen, France). A ‘minus’ reverse transcriptase PCR reaction was used to test each mRNA sample for genomic DNA contaminations where no reverse transcriptase enzyme was added during the cDNA synthesis reaction.
Defence gene expression
The transcriptional expression of each gene was analysed using RT-PCR. cDNA was diluted 1/6, and then 2 µl was used in 40 µl of the PCR mix containing 0.3 U (0.3 µl) of Taq DNA polymerase (Adventage 2, Clontech, France), 1x Taq polymerase buffer, 0.1 mM of each dNTP, and 0.75 mM of each primer. PCR was carried out in a Bio-Rad thermocycler under the following conditions: an initial denaturation at 94 °C for 3 min followed by 25–32 cycles (depending on the experiments, see Table 1) of 15 s at 94 °C, 15 s at Tm °C (Table 1) and 20 s at 72 °C. PCR amplification products were separated on TAE-agarose gels (3%). Gels were stained with ethidium bromide (0.5 mg ml–1). All PCR products were cloned and sequenced once to ensure the specificity of amplifications.
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Results |
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Hypersensitive-like reaction
In both genetically compatible and incompatible combinations in roots, intercellular hyphae and haustoria were observed within 3 d of infection (3 dpi) (Fig. 1A, B).
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By 6 dpi in the genetically compatible combination, the oomycete had extended half-way along the hypocotyl and cells containing haustoria had a normal appearance with no evidence of hypersensitive cell death (Fig. 1C). In the genetically incompatible combination, sets of host cells were undergoing a hypersensitive-like reaction (Fig. 1D).
By 9 dpi, in the genetically compatible reaction, intercellular hyphal growth with abundant haustoria and an absence of necrotic parenchyma cells was apparent in the hypocotyls (Fig. 1E), whereas in the genetically incompatible combination, the parenchyma cells penetrated by haustoria had undergone cell death (Fig. 1F).
In situ localization of H2O2
In situ localization of H2O2 requires the presence of endogenous peroxide activity in order to ensure the formation of a visible polymer, so H2O2 production was monitored using 3.3-diaminobenzidine (DAB), which forms an insoluble reddish-brown polymer. In the case of the P. halstedii–sunflower model, differences were observed between the genetically incompatible and compatible combinations. In the compatible interaction, a slight DAB coloration around the hyphae was observed. Cells that had been penetrated by haustoria did not produce H2O2 (Fig. 2A). In the incompatible combination, local H2O2 production in cells that had been in contact with the pathogen or close to it, was observed by 6 dpi (Fig. 2B). No H2O2 was observed in unstained resistant and susceptible infected seedlings (Fig. 2C, D).
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Expression of the sunflower hsr203J homologue as a marker of HR
hsr203J is a tobacco gene that is rapidly induced in cells activated to undergo a hypersensitive reaction (Pontier et al., 1994
, 1998, 1999; Tronchet et al., 2001). In the sunflower, degenerate primers were used to amplify the hsr203J homologue (accession no. AY645206) and then a specific primer pair was used to detect the corresponding transcript. In the incompatible reaction, the accumulation of gene transcripts was strongly induced in the hypocotyls 36 h (1.5 dpi) after infection and then decreased after 9 d post-infection (dpi) (Fig. 3A). In contrast, no significant transcriptional induction of this gene was observed in control plants or during the compatible reaction (Fig. 3A). In the cotyledons, no increase in the Ha-hsr203J transcript was observed during the time-course of the experiment (Fig. 4). Interestingly, the high level of transcriptional accumulation of this gene by 6 dpi coincided with the collapse of some cells close to the oomycete.
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The sunflower NPR1 homologue is rapidly and locally induced
The NPR1 gene encodes a protein which is involved in establishing SAR in uninfected tissues (Cao et al., 1994
). The transcript accumulation of a sunflower NPR1 homologue was examined in both the hypocotyls and the cotyledons. In the hypocotyls, transcript accumulation of the Ha-NPR1 had significantly increased 3 d after infection of the resistant line (QIR8), when compared with the control (Fig. 3A). This activation reached its highest level by 6 dpi and then declined back to the control level by 12 dpi. In the susceptible line (CAY), the transcript accumulation of Ha-NPR1 had not significantly increased after infection, compared with the control (Fig. 3A). In the cotyledons, no increase in transcript accumulation was observed during the time-course of the experiment (Fig. 4). Interestingly, transcript accumulation of the sunflower Ha-NPR1 started before the hypersensitive reaction was observed under the microscope.
Rapid and systemic induction of Ha-GST in the incompatible interaction
During the incompatible combination, the accumulation of Ha-GST was rapid and systemic when compared with the compatible combination. About 36 h after infection (1.5 dpi), Ha-GST transcript accumulation was significantly higher in resistant, infected hypocotyls than in susceptible hypocotyls or uninfected plants. Ha-GST activation lasted until 9 dpi and then declined to control levels (Fig. 3). Ha-GST activation in resistant cotyledons was observed before the oomycete reached this tissue, as demonstrated by the amplification failure of the P. halstedii TEF1 gene (Fig. 4). Thus, the activation of Ha-GST in distant cotyledons reflected a systemic response and transmission of a signal from the lower parts of the plant.
Expression of the defence-related Ha-PR5 and Ha-PDF genes
In order to investigate whether the cellular reactions observed at the microscopic level were associated with the induction of defence-related genes, a set of genes were cloned and sequenced which were monitored by RT-PCR analysis using specific primers. Ha-PR5 and Ha-PDF transcripts accumulated in the same way. The increase in transcript accumulation of these genes was observed in resistant hypocotyls at 6 dpi and reached its maximum at 9 dpi (Fig. 3). By contrast, in the compatible combination, activation of these genes was delayed and observed at 12 dpi (Fig. 3). Similarly, the induction of Ha-PR5 and Ha-PDF genes was observed in resistant cotyledons as early as 6 dpi, whereas only a weak induction was observed at 15 dpi in susceptible cotyledons (Fig. 5).
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Effect of salicylic acid on Ha-hsr203J and defence genes
In hypocotyls of the resistant line (QIR8), no significant transcription of the Ha-hsr203J gene was induced by treatment with SA (2 mM) (Fig. 5). These results suggest that the activation of Ha-hsr203J is related to the HR occurring in hypocotyls infected by P. halstedii. The transcript accumulation of Ha-GST and Ha-NPR1 had increased within 3 h post-treatment (hpt) with salicylic acid, compared with the controls (Fig. 5). A significant activation of Ha-PR5 and Ha-PDF transcription was observed in response to SA by 12 hpt (Fig. 5).
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Discussion |
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During this study, a sunflower resistant line (QIR8) and a susceptible line (CAY) were used to analyse modulation of the transcriptional expression of a set of genes during infection with the biotrophic oomycete Plasmopara halstedii, which is the causal agent of sunflower downy mildew. At the same time, microscopic observations and RT-PCR were carried out to describe resistance events at both the molecular and tissue levels.
Our microscopic observations confirmed the findings of previous studies which had shown that both resistant and susceptible sunflower lines are penetrated by Plasmopara halstedii (Viranyi and Mohamed, 1985; Gray and Sackston, 1985; Sackston, 1992; Mouzeyar et al., 1993). Parasite penetration takes place in the roots and at the developing transition area between the roots and the hypocotyl of germinated seeds. In both susceptible and resistant seedlings, the parasite initiates a systemic invasion towards the cotyledons, after which a hypersensitive-like reaction develops within the hypocotyls of the resistant line without, seemingly, halting parasite development.
H2O2 accumulates differentially in resistant and susceptible infected sunflower lines
The oxidative burst, which includes hydrogen peroxide (H2O2), is a characteristic early feature of the hypersensitive response (HR) during incompatible interactions between resistant plants and avirulent pathogens or non-pathogens (Hammond-Kosack and Jones, 1996; Lamb and Dixon, 1997). The results obtained by Delledone et al. (2001) suggest that an HR requires the correct relative levels of both NO and H2O2 to be induced in a plant. The results using diaminobenzidine (DAB) staining (Fig. 2A, B) showed that H2O2 accumulates differentially between susceptible and resistant sunflower lines. While DAB staining was slight around intercellular hyphae in the susceptible line, a more intense staining indicative of a higher level of H2O2 was observed within cells undergoing a HR in the resistant line. These results show that the resistance of sunflower to P. halstedii correlates with a differential accumulation of H2O2. Detectable levels of H2O2 were observed around the mycelia in the intercellular spaces of susceptible hypocotyls. This H2O2 production is R gene-independent and may correspond to the non-specific phase I of ROS production in plants. These results suggest that either H2O2 alone cannot trigger a hypersensitive reaction, or that a critical dose of H2O2 is required to initiate such a reaction. Alternatively, H2O2 may require the presence of other compounds that are not produced during the compatible combination, such as NO (Delledone et al., 2001). This latter hypothesis is supported by data from other plant parasite interactions. For example, the Arabidopsis AtbohF/AtbohD double mutant in the NADPH oxidase complex, which is thought to generate an oxidative burst, failed to accumulate detectable H2O2 but still activated some early cell death (Torres et al., 2002). These authors suggested that the first cell deaths were H2O2-independent, but that the presence of this compound was required for subsequent cell deaths. Thus, the question still remains as to whether H2O2 accumulation in the sunflower resistant line is the primary signal in the HR observed during infection by P. halstedii.
Resistance of sunflower to P. halstedii is associated with activation of a HR marker
hsr203J is a tobacco gene that is specifically activated during the hypersensitive reaction (Pontier et al., 1994). Its activation is rapid and takes place in various incompatible combinations involving tobacco, suggesting that activation of this gene tends to be associated with cell death rather than with a particular HR accompanying a specific resistance (Pontier et al., 1998). Peptide inhibitors of caspase-like activity suppressed the activation of both the hsr203J gene and HR cell death (Del Pozo and Lam, 1998, 2003). These results suggest that the activation of the hsr203J gene is specific to the HR cell death pathway after the caspase-like proteolytic switch (Pontier et al., 1999). Recent findings have supported the hypothesis that cell death is associated with caspase-like activity (Coffeen and Wolpert, 2004). Transient activation of the hsr203J homologue in the resistant sunflower hypocotyls was observed early after infection, whereas no such activation was observed during compatible infection (Fig. 3A) or in distant uninfected tissues (Fig. 4). These results suggest that the resistance of sunflower to P. halstedii is associated with a hypersensitive reaction which is activated within the hypocotyls during the days after the parasite has penetrated the host.
SAR is activated in the sunflower resistant plants
The onset of HR cell death in the basal part of the hypocotyl is associated with transcriptional activation of a sunflower homologue of the NPR1 gene, which is a key component of systemic acquired resistance in plants (Cao et al., 1994, 1997, 1998; Pieterse et al., 1998; Mou et al., 2003; Fitzgerald et al., 2004). Activation of the sunflower NPR1 homologue occurred as early as 3 d after infection during resistance (Fig. 3A). No such activation was observed in the cotyledons (Fig. 4), suggesting that when the HR took place in the hypocotyl, a SAR was initiated throughout the remaining parts of the plant, including the cotyledons. The transcript profiles of Ha-GST, Ha-PDF, and Ha-PR5 genes confirmed this conclusion, because no activation or a much delayed activation of these genes was observed in the case of susceptibility when compared with resistance (Fig. 3).
Sunflower resistance to P. halstedii is probably accomplished through both HR and SAR
The molecular and microscopic data presented here, and those described elsewhere in the literature, suggest that resistance to P. halstedii in the sunflower involves the sequential action of both HR cell death and the SAR that is associated with it. The HR initiated in the hypocotyls may merely reduce parasite growth without apparently killing it. The HR also initiates a SAR in the upper parts of the infected seedling. Thus, the parasite may encounter gradually stronger defence reactions as it progresses upwards. The systemic response may involve secondary ROS generation beyond the HR site which initiates a series of micro-HR in the upper parts of the hypocotyl that are difficult to observe microscopically (Alvarez et al., 1998). The combination of both phenomena, i.e. reduced growth due to the hypersensitive reaction in the lower part of the hypocotyl and establishment of the SAR in the upper part of this hypcotyl, eventually lead to the arrest of parasite growth.
The hypersensitive reaction also seems to be developmentally controlled, since it occurs when the parasite reaches the hypocotyl but is absent from the roots. A similar organ-specific initiation of resistance was also described by Hermanns et al. (2003) in A. thaliana infected by the causal agent of downy mildew Hyaloperonospora parasitica. In this pathosystem, no difference was observed between the infected roots of susceptible and resistant plants 3 d after infection, even though the corresponding resistance gene RPP1 and components of the signalling pathways were expressed (Hermanns et al., 2003). These authors suggested that the HR could not take place in the roots which are continually in contact with pathogenic microorganisms in the soil, so that HR-associated cell death in the roots would endanger the whole plant. However, due to experimental constraints, the same authors were not able to follow the growth of Hyaloperonospora isolates for more than 72 h after infection. Thus longer observation periods of parasite growth within the hypocotyl (particularly in the case of incompatibility) were not possible (Hermanns et al., 2003). Interestingly, Sesma and Osbourn (2004) used rice and the blast rice fungus Magnaporthe grisea and demonstrated that resistance is controlled in a gene-for-gene manner after root infection and that the fungus growth is restricted to the outer area of the root. However, the same authors did not report if this resistance involves a hypersensitive reaction. Although the host and the pathogen described by Hermanns et al. (2003) and those used during this study were different, there are numerous similarities. For example, both parasites are biotrophic oomycetes that initially infect their host roots in the soil. If these results are combined, they give rise to the hypothesis that resistance to root-infecting parasites could be associated with a delayed and weak hypersensitive reaction which takes several days to become visible, when compared to the leaves where a HR can occur within hours. Moreover, initiation of this HR is displaced from the infection site (the roots) to more distal tissues (hypocotyl) where cell death would be less deleterious (Fig. 6). It would be very interesting to see whether this working hypothesis could be extended to other plant–parasite systems involving root infecting oomycetes.
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Acknowledgements |
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We would like to thank the Egyptian Ministry of Higher Education for a Doctoral Scholarship concerning the first author and PROMOSOL for its financial support.
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References |
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