JXB Advance Access originally published online on March 28, 2008
Journal of Experimental Botany 2008 59(6):1305-1313; doi:10.1093/jxb/ern036
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation
1Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA
2Department of Nematology, University of California, Riverside, CA 92521, USA
* To whom correspondence should be addressed. E-mail: philip.roberts{at}ucr.edu
Received 5 November 2007; Revised 22 January 2008 Accepted 23 January 2008
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Abstract |
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Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites with a broad host range which includes economically important crop species. Cowpea (Vigna unguiculata L. Walp) is an important food and fodder legume grown in many regions where root-knot nematodes are a major problem in production fields. Several sources of resistance to root-knot nematode have been identified in cowpea, including the widely used Rk gene. As part of a study to elucidate the mechanism of Rk-mediated resistance, the histological response to avirulent M. incognita feeding of a resistant cowpea cultivar CB46 was compared with a susceptible near-isogenic line (in CB46 background). Most root-knot nematode resistance mechanisms in host plants that have been examined induced a hypersensitive response (HR). However, there was no typical HR in resistant cowpea roots and nematodes were able to develop normal feeding sites similar to those in susceptible roots up to 9–14 d post inoculation (dpi). From 14–21 dpi giant cell deterioration was observed and the female nematodes showed arrested development and deterioration. Nematodes failed to reach maturity and did not initiate egg laying in resistant roots. These results confirmed that the induction of resistance is relatively late in this system. Typically in pathogen resistance HR is closely associated with an oxidative burst (OB) in infected tissue. The level of reactive oxygen species release in both compatible and incompatible reactions during early and late stages of infection was also quantified. Following a basal OB during early infection in both susceptible and resistant roots, which was also observed in mechanically wounded root tissues, no significant OB was detected up to 14 dpi, a profile consistent with the histological observations of a delayed resistance response. These results will be useful to design gene expression experiments to dissect Rk-mediated resistance at the molecular level.
Key words: Cowpea, histology, hypersensitive response, Meloidogyne incognita, reactive oxygen species, root-knot nematode, Vigna unguiculata
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Introduction |
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Cowpea (Vigna unguiculata L. Walp) is a food and fodder legume of significant economic importance worldwide especially in semi-arid regions of Africa. It is also grown in North and South America, southern Europe, and Asia. Cowpea is cultivated in an estimated area of 12.5 million hectares with an annual production of three million tonnes of dry grains worldwide (Singh et al., 1997). In the United States, cowpea is a crop of minor interest grown on an area of about 80 000 hectares (Fery, 1985, 1990).
Root-knot nematodes (RKN) are one of the most important nematode pests of crop plants and have a diverse host range. RKN (Meloidogyne spp.) are sedentary root endoparasites and are involved in the development of specialized feeding structures known as giant cells. The infective stage of the nematode is the second-stage juvenile (J2). The J2 penetrate the roots and go through three successive moults to become adult females or males. Several of the most important root-knot nematode species, including M. incognita, reproduce by obligate mitotic parthenogenesis (Jung and Wyss, 1999).
The mechanism of feeding site development by root-knot nematodes is not well understood as it is a very dynamic and complex process involving genes from both the nematode and the host plant. The secretions from the oesophageal glands of the nematode are important in initiating the development of feeding structures (Davis et al., 2000; Williamson and Kumar, 2006). The first sign of giant cell induction by RKN is the formation of a binucleate cell. Rapid divisions of the nuclei continue in the absence of cytokinesis (acytokinetic mitosis) which gives rise to several large multinucleate cells. The surrounding cells divide to form the characteristic galls often known as ‘root-knots’ (Gheysen and Fenoll, 2002). The xylem parenchyma cells become transfer cells by forming finger-like wall invaginations (Jones and Northcote, 1972). This helps in water transport from the xylem to the feeding sites.
Root-knot nematodes are important pests of cowpea worldwide and host plant resistance is a preferred strategy for managing this problem in infested cowpea fields (Roberts et al., 1995; Ehlers et al., 2002). The Rk locus in cowpea has been used extensively to breed root-knot nematode resistant varieties in the USA and other countries. This gene locus was first designated as Rk by Fery and Dukes (1980) and it confers resistance to many populations of M. incognita, M. arenaria, M. hapla, and M. javanica. Genetic studies have indicated that this locus may have several alleles including rk, Rk, and Rk2. Rk2 confers broad-based resistance to different races of M. incognita and M. javanica (Roberts et al., 1996). It is to be confirmed whether Rk2 is an allele at the Rk locus or is a tightly linked separate locus. A single recessive gene unlinked to Rk, which confers broad-based additive resistance when combined with Rk, was identified and named rk3 (Ehlers et al., 2000). These resistance loci provide a good resource for future studies and cultivar development.
Compatible and incompatible reactions lead to differential plant responses to nematode infection. A complex cascade of plant genes is activated upon nematode invasion and there are some visible reactions observed in the plant cells (Williamson, 1999). Based on the limited number of reported studies, a common response to root-knot nematode attack in host plants carrying a resistance gene is an early hypersensitive reaction (HR)-mediated cell death around the nematode feeding site, which prevents the nematode from further feeding resulting in nematode death. For example, strong early HR responses have been observed in Mi-1-mediated resistance in tomato (Williamson, 1999), Mex-1-mediated resistance in coffee (Anthony et al., 2005), Me3-mediated resistance in pepper (Pegard et al., 2005), and incompatible interactions in soybean (Kaplan et al., 1979). Accumulation of phenolic compounds, especially chlorogenic acid, at the site of infection was also reported in resistant pepper roots by Pegard et al. (2005).
Non-hypersensitive reactions have been observed in Hsp1pr°–1-mediated resistance in sugarbeet against the cyst nematode Heterodera schachtii, where the J2 died due to degradation of the feeding structure (Holtmann et al., 2000). A delayed hypersensitive cell death was observed in the case of Hero-mediated gene responses in tomato against the cyst nematodes Globodera pallida and G. rostochiensis (Sobczak et al., 2005), in which the nematodes became sedentary and died at a late juvenile stage due to HR-mediated cell death in the developed syncytium. Gene H1 confers resistance to G. rostochiensis pathotype Ro1 in potato. Studies of root ultrastructure in resistant potato plants harbouring gene H1 showed an early HR around the J2 (Rice et al., 1985; Williamson, 1999). Here, syncytial development was restricted by HR leading to the restriction in the development of the nematode and increased numbers of males and reduced numbers of females.
In cowpea there is no detailed histological documentation of RKN-induced changes during compatible and incompatible reactions. This study was done to provide a detailed histological characterization of Rk-mediated resistance in cowpea. It is known that an oxidative burst is typically associated with HR in incompatible host–pathogen interactions including nematode–plant interactions (De Gara et al., 2003). A significant oxidative burst has been recorded in incompatible tomato (Mi-1)–RKN interactions (Melillo et al., 2006). Therefore, the accumulation of reactive oxygen species in the Rk-mediated incompatible cowpea–RKN interaction was also investigated.
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Materials and methods |
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Plant material
Two near-isogenic lines (NIL) differing in the presence or absence of the gene Rk were used. The two parents used to develop the NIL were M. incognita race 3 resistant cowpea genotype ‘CB46’ (homozygous resistant, RkRk) and a highly susceptible genotype ‘Chinese Red’ (homozygous susceptible, rkrk). The F1 was backcrossed to recurrent parent CB46 (BC1) and homozygous Rk plants were discarded in BC1F2 and non-segregating rkrk plants were advanced to the next back-cross (BC2). Repeated backcrossing and selection was used to recover the rkrk line in the CB46 background. BC4F4 progenies were used for all the experiments described here. The rkrk line is referred to as the null-Rk line from here on.
Nematode inoculum
Eggs of M. incognita race 3 (isolate Beltran) cultured on susceptible tomato host plants were extracted from roots using 10% bleach solution (Hussey and Barker, 1973). This isolate is avirulent to gene Rk in CB46. Eggs were hatched in an incubator at 28 °C and J2 were collected in fresh deionized water. The J2 inoculum was prepared according to the experimental requirements.
Histological experiments
Seeds of CB46 and null-Rk cowpea genotypes were grown in growth pouches under controlled environmental conditions of 26.7±0.5 °C constant temperature and daily light/dark cycles of 16/8 h. This temperature was used because it lies within the optimum temperature range of 26–28 °C for development and reproduction of M. incognita on cowpea in growth pouches (Ehlers et al., 2000). Each pouch was inoculated with 3000 J2 in 5 ml of deionized water, 12 d after planting (dap). Three pouches from each genotype were mock inoculated with 5 ml of deionized water as negative controls. The presence of nematodes in the roots was confirmed by acid fuchsin staining (Byrd et al., 1983) 24 h post-inoculation (hpi).
Three root tips, each 
50 mm in length, were harvested randomly from two plants of each genotype at 3, 4, 5, 9, 14, 15, 16, 17, 18, 19, 20, and 21 d post-inoculation (dpi) and immersed in half-strength Karnovsky's fixative (2.5% glutaraldehyde and 4% formaldehyde in 50 mM phospahte buffer, pH 7.2). The roots were left overnight in the fixative at 4 °C. The roots were dehydrated by passing through a graded ethanol series (10–100%). Infiltration and embedding was done with a JB-4 methacrylate embedding kit (Polysciences Inc., Pennsylvania, USA). Semi-thin sections 4 µm thick were cut using a DuPont-Sorval JB-4 microtome using triangular glass knives. The sections were stained in 0.5% toluidine blue O in borate buffer (pH 4.4). Digital micrographs were taken using a Spot CCD camera (Spot RT colour system, model no. 2.2.1, Diagnostics Instruments Inc.) attached to a Leica DM LB2 compound bright-field microscope. Giant cell diameters were measured at 5, 9, 14, 19, and 21 dpi. Three well-developed giant cells were selected for each time point in both resistant and susceptible cowpea genotypes, being chosen from sections in a sequential series that optimized the giant cell size. The diameter was measured at three positions for each giant cell using a stage micrometer and the mean of the three measurements was used as the diameter for that giant cell. Giant cell measurements from root sections provide a relative measure of cellular changes in infected resistant and susceptible cowpea roots and do not represent an absolute measurement.
Root penetration studies
Penetration of avirulent nematodes in root tissue was studied on susceptible null-Rk and resistant CB46 cowpea genotypes. Plants were grown in growth pouches at 26.7 ± 0.5 °C constant temperature and daily dark/light cycles of 16/8 h. The inoculum level used was the same as for the histological experiments. Each pouch was inoculated with 3000 J2 in 5 ml of deionized water, 12 dap. The inoculated roots were harvested at 24, 48, and 72 hpi and immersed in 1.5% NaOCl solution for 15 min followed by rinsing with tap water to remove excess NaOCl. The roots were then stained with 1 ml of 3.5% acid fuchsin stain (Byrd et al., 1983), the solution was heated to boiling, followed by cooling to room temperature, and excess stain was removed by rinsing in running water. The root material was placed in acidified glycerin. The stained roots were pressed between glass slides and observed under the microscope. Three plants were selected for each sampling time point and three root tips from each plant were selected randomly and numbers of J2 inside the root tissue were counted.
An analysis of variance (ANOVA) was used to compare the penetration rate between resistant and susceptible roots. Data from all three root systems for a given genotypextime point were pooled together for analysis.
Egg mass production
Numbers of egg masses per root system were counted using an egg mass specific stain erioglaucine (Omwega et al., 1988; Ehlers et al., 2000) at 30 dpi to confirm the susceptibility of the null-Rk line used in the experiments. An inoculum of 3000 J2 per root system was used for the egg mass production assays and 20 plants each from CB46 and null-Rk were screened.
Quantitative detection of ROS release
A fluorometric assay was designed to detect ROS accumulation using a membrane permeable probe, dichlorofluoroscein (DCFH). DCFH alone does not have fluorescence but when it reacts with ROS it oxidizes to DCF which is fluorescent. Root pieces were collected from infected root systems of the susceptible and resistant genotypes at 24, 48, and 72 hpi. Also, to detect the presence of a late oxidative burst, root samples were collected at 5, 9, and 14 dpi. The root samples (250 mg) were processed as described by Melillo et al. (2006). Phosphate-buffered saline (20 mM, pH 7.2) was used in place of potassium phosphate buffer. A 1 ml aliquot of the processed sample was collected and used to detect the increase in fluorescence (excitation 488 nm, emission 521 nm) caused by oxidation of DCFH using a fluorescence spectrophotometer (SPEX FluoroLog-3, Horiba Jovin Yvon). Mock inoculated roots (5 ml of deionized water per root system) were used as negative controls and mechanically injured roots were used as a positive control. Mechanical wounding was achieved by puncturing plant roots with a hypodermic needle. Five biological replicates were taken and the entire experiment was repeated once. The duplicate experiments did not differ according to ANOVA tests, therefore data from the independent duplicate experiments were combined for analysis. Blank samples without plant material were processed in parallel to eliminate any spontaneous change in fluorescence.
In order to test the specificity of the reaction, an experiment was done using the ROS scavenging reagent diphenylene-iodonium (DPI, an NADPH oxidase inhibitor). Root pieces (250 mg) from non-infected and 24 h-infected CB46 plants were harvested and pre-incubated for 30 min with 100 µM DPI followed by 30 min in DCFH reaction medium. The decrease in fluorescence was measured as described earlier and reagent blanks were used as reference.
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Results |
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Root penetration
The presence of the gene Rk did not affect juvenile penetration into cowpea roots. The avirulent J2 were able to penetrate the roots of both cowpea genotypes and there was no effect of genotype (P = 0.05) on the number of J2 in roots up to 72 hpi (Fig. 1). In both genotypes, up to 24 hpi the number of J2 that had penetrated into roots was low, but the penetration rate was higher at 48 hpi and a gradual increase in penetration was observed up to 72 hpi.
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Histological response to infection
The resistant line CB46 did not show a HR response to nematode infection. HR, in which a programmed cell death around the area of infection occurs and the development of the pathogen is arrested, is a common plant reaction against different pathogens in resistant genotypes. For example, in root-knot nematode–host plant interactions such as the Mi-1-mediated resistance in tomato an obvious HR occurs within 24 h of infection (Dropkin, 1969; Williamson, 1999). In this study, when 12-d-old seedlings were inoculated with 3000 J2 and longitudinal sections of roots were examined, there was no visible evidence of a HR response in resistant roots up to 21 dpi. The nematodes were able to establish healthy feeding sites in resistant roots in which the giant cells looked similar to those in susceptible roots up to 5 dpi (Fig. 2A, B). Lack of HR response was also confirmed by staining the roots with acid fuchsin at various time points (not shown). The first evident differences between the two genotypes were observed at 9 dpi when the giant cells adjacent to the nematode in resistant roots had some larger vacuoles (Fig. 2C, D), whereas the giant cells in the susceptible roots had uniformly dense cytoplasm with less vacuolation. The giant cells farthest from the nematode appeared to be metabolically more active than giant cells closer to the nematode in resistant roots (Fig. 2D). The nematodes at this time point were developing normally in the genotypes based on observations of their size, shape, and condition of internal contents. This trend in the giant cell conditions continued up to 14 dpi (Fig. 2E, F) when there was still no sign of visible feeding site deterioration in resistant roots. However, the nematodes associated with resistant roots at 14 dpi were arrested in development as they were slightly shrivelled and narrower than the nematodes in susceptible roots. This confirmed that although giant cell deterioration was not visible under bright field microscopy at this stage, giant cells were not metabolically active enough to provide optimum nutrients for nematode development. At 19 dpi (Fig. 2G, H) the differences in feeding sites between the two genotypes were clearly visible. At this stage most of the giant cells in the resistant roots appeared to be on the verge of collapse as they were devoid of any cytoplasm and the common cell walls between the giant cells were also thin, whereas in susceptible roots, healthy giant cell complexes with dense cytoplasm and thick cell walls were present. At 21 dpi the giant cell complexes in resistant roots had collapsed completely and nematode development was severely disrupted; they had a shrivelled appearance and had not advanced to a mature female stage based on lack of gonad development (Fig. 2J). In susceptible roots at 21 dpi most of the nematodes had developed to mature females and their well-developed ovaries could be seen in the sections (Fig. 2I).
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Giant cell dimensions
Giant cell diameter did not differ between infection sites in resistant CB46 and susceptible null-Rk roots until 19 dpi. Giant cell measurements revealed that, by 5 dpi, the giant cells were fully developed and overall there was no significant difference in giant cell diameter between the two genotypes up to 19 dpi (Fig. 3). Although at 14 dpi the giant cells in susceptible roots were found to be larger (P = 0.05) than in resistant roots, it was probably because the giant cells selected for the measurement were not fully representative for this time point. However, at 21 dpi, the mean diameter of the giant cells in resistant roots was much smaller than in susceptible roots due to the collapse of giant cells (Fig. 3).
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Root galling and egg production
Although the Rk-mediated resistance reaction was delayed, the development of female nematodes was arrested in resistant roots such that they did not reach reproductive maturity. The cortical cells surrounding the giant cells in resistant roots started to shrink rapidly at 12–14 dpi and at 19–21 dpi the cortical cells were almost normal in size. Therefore, at 19 dpi, only residual galling was visible on resistant roots even though the giant cells were not collapsed.
External observations of the roots at 21 dpi revealed large well-developed galls in susceptible roots whereas the resistant roots supported only some small residual swelling around the feeding sites (Fig. 4). Acid fuchsin staining at 21 dpi revealed that, in susceptible roots, the females had reached reproductive maturity and started to lay eggs, whereas in resistant roots, the under-developed female nematodes (approximately 90% J4 and 10% immature adults) showed no sign of egg production. This confirmed our observations from the histological root sections. At 30 dpi the number of egg masses per root system ranged from 43 to 99 (mean ±SD = 65.5±15.9) in susceptible roots, whereas nematodes failed to produce any egg masses in resistant roots.
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Quantitative detection of ROS release
In a time-course experiment, ROS activity was studied, starting at the early stages of infection (24 hpi) until the later stages of infection at 14 dpi. Due to nematode infection, an early rise in ROS activity at 24 hpi was observed in root tissue of both resistant CB46 (157% compared with non-infected control) and susceptible null-Rk (153% compared with non-infected control) plants as shown in Fig. 5. This early oxidative burst continued up to 48 hpi in both CB46 (159%) and null-Rk (151%). ROS activity decreased after that with readings for ROS in infected roots compared to non-infected control at 72 hpi being 92% in CB46 and 85% in null-Rk. There was no differential ROS activity between the resistant and susceptible genotypes during these time points. During later time points (5, 9, and 14 dpi) very low levels of ROS activity were detected in both genotypes in infected and non-infected roots. Compared to non-wounded control plants, mechanically wounded roots (positive control) of both CB46 and null-Rk produced a significant early oxidative burst up to 48 hpi, which diminished at 72 hpi (Fig. 6), similar to the response in nematode-infected resistant and susceptible plants. Oxidation of DCFH in CB46 roots was inhibited by the superoxide O2– scavenger DPI (Table 1). DPI is an NADPH-oxidase inhibitor and was the most efficient inhibitor of ROS in Mi-1-mediated resistance in tomato (Melillo et al., 2006). Upon DPI treatment ROS activity was reduced to 53% in RKN infected CB46 roots at 24 hpi. This confirmed that the enzymatic origin of superoxide contributed significantly to the early oxidative burst detected in infected cowpea roots.
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Discussion |
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The gene Rk was identified almost three decades ago (Fery and Dukes, 1980) as a highly effective RKN resistance gene in cowpea. Although the Rk-based resistance has been studied genetically and has been used extensively in cowpea breeding, little was known about the mechanism of Rk-mediated resistance. Two types of mechanisms for RKN resistance in plants have been reported, including pre-infection resistance, where the nematodes cannot enter the plant roots due to the presence of toxic or antagonistic chemicals in root tissue (Haynes and Jones, 1976; Bendezu and Starr, 2003), and post-infection resistance in which nematodes are able to penetrate roots but fail to develop. Post-infection resistance is often associated with an early Hypersensitive Reaction (HR)-mediated cell death, in which rapid localized cell death in root tissue around the nematode prevents the formation of a developed feeding site, leading to resistance. Tomato (Dropkin, 1969; Williamson, 1999), pepper (Pegard et al., 2005), soybean (Kaplan et al., 1979), and coffee (Anthony et al., 2005) host plants that are resistant show typical HR upon avirulent RKN infection. In tomato, HR was observed as early as 24 hpi whereas in pepper, soybean, and coffee the HR was visible at 1–3 dpi, 2–3 dpi, and 4–6 dpi, respectively.
Interestingly in the current study, it was found that the presence of the gene Rk did not affect J2 penetration into cowpea roots and there was no evidence of early HR. In fact, the nematodes were able to initiate and maintain apparently healthy giant cells in resistant roots for about two weeks before visible signs of deterioration occurred, especially vacuolation and cell wall thinning, leading to giant cell collapse. This mechanism appears to be novel for RKN resistance. The only published report for a delayed resistance response against RKN was in tobacco (Powell, 1962) where a late HR was seen in developed giant cells. In cowpea there was no HR even during the later stages of infection. During this time the nematodes were able to feed and develop into late stage juveniles.
A common feature of pathogen-related HR is that it is preceded by loading of vacuoles with hydrolases and toxins and a calcium flux in the cytoplasm (Jones, 2001). A significant difference in vacuolation between the resistant and susceptible cowpea genotypes starting at 9 dpi was observed, and it is possible that the large vacuoles in resistant cowpea roots were filled with hydrolases and toxins that deprived the nematodes of nutrients and led to giant cell collapse, whereas in susceptible roots nematode feeding did not cause the formation of large vacuoles. In Arabidopsis a mutant called dnd1-1 failed to produce an HR response against an avirulent strain of the bacterial pathogen Pseudomonus syringae, but an effective gene-for-gene resistance was still operative (Clough et al., 2000). DND1 codes for a cyclic gated ion channel which facilitates passage of Ca2+, K+ and various other cations. Thus host defence can be effective in the absence of HR and it might be dependent upon subtle changes in ion fluxes.
Reactive oxygen species (ROS) play an important role in plant defence, and during pathogen attack levels of ROS detoxifying enzymes like ascorbate peroxidase (APX) and catalase (CAT) are often suppressed in resistant plants (Klessig et al., 2000). As a result plants produce more ROS and accumulation of these components leads to HR in plant cells. For example, H2O2 plays a major role in triggering HR in incompatible interactions (Dangl and Jones, 2001). However, in the cowpea–RKN incompatible interaction mediated by the gene Rk, a classic HR, which is characteristic for most gene-for-gene resistance pathways, was not seen.
Our results of ROS quantification in RKN-infected cowpea roots showed that although there was an early oxidative burst in both the compatible and incompatible interactions in susceptible and resistant roots, respectively, there was no significant difference between the resistant and susceptible genotypes in level of ROS activity. Typically in incompatible interactions ROS is produced in a biphasic manner (Apel and Hirt, 2004), in which the initial rapid accumulation of ROS is followed by a more stable second burst. However, in cowpea, a biphasic pattern of ROS production was not seen in the incompatible reaction. These results indicated that the initial burst that was detected in cowpea roots upon RKN infection is a part of basal host defence reaction and is independent of gene Rk-mediated resistance. This response, which develops a few days after elicitation, seems to be related to an innate immunity in plants (Iriti and Faoro, 2007) and is triggered by changes in membrane potential, ion fluxes, and production of ROS. It is well known now that perception of parasite and/or wounding and modulation of ROS contribute toward the activation of the plant defence response (Klessig et al., 2000; Gechev et al., 2006). This pattern of ROS production correlated well with the results of the histological profiles of the feeding site and the giant cell development that were found in the resistant and susceptible cowpea roots. The magnitude of H2O2 production apparently was not sufficient enough to trigger HR cell death in cowpea roots. It is also possible that the ROS scavenging mechanism was not suppressed to a level at which enough ROS could be diffused into the cells to trigger HR.
In conclusion, it is reported that Rk-mediated resistance in cowpea is a unique resistance mechanism involving the lack of a HR and based on a delayed defence response. The current study provides a strong platform for designing gene expression studies to identify candidate genes which play an active role in this defence pathway. An evaluation of the expression levels of genes coding for enzymes involved in ROS production and ROS scavenging will be useful for understanding the intricacies of redox changes occurring upon RKN infection in resistant cowpea.
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Acknowledgements |
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The authors would like to thank William Matthews and Teresa Mullens for technical assistance, Dan Borchardt for help with the fluorescence spectrophotometry and Dr Thomas Eulgem for assistance with digital imaging and stimulating discussions. The work was funded in part by Bean/Cowpea Collaborative Research Support Program, USAID Grant no. GDG-G-00-02-00012-00. The opinions and recommendations herein are those of the authors and not necessarily those of USAID.
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Abbreviations |
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CPS, counts per second; DCFH, dichlorofluoroscein; DPI, diphenylene-iodonium; HR, hypersensitive reaction; RKN, root-knot nematode; ROS, reactive oxygen species.
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References |
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