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Model systems can provide an excellent base for understanding a wide range of biological processes (Nakagami et al., 2010). Rice (Oryza sativa) has proved to be an excellent monocot model plant and the most readily transformable economically important cereal crop (Zhang et al., 1988; Hiei et al., 1994). Annual yield losses as a result of plant-parasitic nematodes on this crop range from 10 to 25% worldwide (Bridge et al., 2005) and the global agricultural losses have been estimated to be c. $157 billion yr−1 (Abad et al., 2008). One of the most damaging plant-parasitic nematodes in all major rice-growing areas is the rice root rot nematode (RRN) Hirschmanniella oryzae (Karakas, 2004). Hirschmanniella spp. infest 58% of the world's rice fields, causing 25% yield losses (Hollis & Keoboonrueng, 1984; Bridge et al., 2005). H. oryzae is a unique nematode in the sense that the nematode is perfectly adapted to constant flooding conditions (Bridge et al., 2005), making them the most serious and damaging nematodes in flooded/irrigated rice regimes. Having a high potential for damage at relatively low population densities, they cover a broad range of hosts in monocotyledonous and dicotyledonous plants (Karakas, 2004). As a migratory endoparasite, it causes necrosis of penetrated epidermal cells, and damages and destroys cortical cells, resulting in cavities in the cortex and necrotic regions. Above-ground symptoms on plants are growth retardation, decreased plant height, delayed tillering, and weight reduction of dry matter. The juveniles and adults enter behind the root tip at some distance from the tip and move freely in the air channels between the radial lamellae of the parenchyma. They may be found anywhere between the root base and the tip. A few days after entry, the female starts laying eggs which hatch in 4–5 d inside the roots. The nematode population increases by sexual reproduction and under suitable conditions the life cycle is completed in about 1 month. The nematode can have two or more generations per growing season. They can survive without food for several weeks and can overwinter in dead roots as eggs, juveniles or adults.
Studies involving plant–nematode interactions provide an opportunity to unravel plant defense signaling in root tissues especially with regard to plant hormones. Plants generally respond to pathogen infection using a sophisticated battery of defense mechanisms, in which the plant hormones salicylate (SA), jasmonate (JA) and ethylene (ET) play central signaling roles (Robert-Seilaniantz et al., 2007; Lopez et al., 2008; Grant & Jones, 2009). Each of these hormones generates and transmits distinct defense signals that are most likely enhancing resistance to a class of invaders. Some defense response genes require intact JA, ET, and SA signaling pathways after pathogen challenge (Campbell et al., 2003). On the other hand, their signaling pathways influence each other through a complex network of synergistic and antagonistic interactions (Koornneef & Pieterse, 2008), allowing the plant to tailor its defense reaction efficiently, depending on the type of attacker.
The complex signaling network involved in the plant's defense against bacterial, viral, and fungal pathogens has been extensively reviewed for dicotyledonous plants (Glazebrook, 2005; Lopez et al., 2008; Bari & Jones, 2009) and, although there are exceptions, it is generally accepted that an SA-dependent defense is active against pathogens with a biotrophic lifestyle, whereas JA/ET-dependent defenses are active against necrotrophic pathogens (Glazebrook, 2005) and herbivorous insects (Kessler & Baldwin, 2002; Rojo et al., 2003; Howe & Jander, 2008). In fact, the nature of the specific plant defense response is largely influenced by the biotrophic or necrotrophic lifestyle of the pathogen. Additional factors, such as the infection stage, age and type of plant tissue, are also important. These different factors interactively determine the nature of the specific defense response (De Vos et al., 2005). Recently, our research has shown that ET-induced systemic defense in rice involves a strong activation of JA biosynthesis and signaling genes, indicating that the JA pathway, which is modulated by ET, is the key defense pathway involved in rice root innate immunity against Meloidogyne graminicola, the sedentary biotrophic root knot nematode (RKN). The SA pathway, on the other hand, has a minor positive effect on root immunity against the RKN (Nahar et al., 2011).
Besides its signaling roles in diverse processes of plant growth and development, as well as in plant responses to various abiotic stresses (Yamaguchi-Shinozaki & Shinozaki, 2006), the plant hormone abscisic acid (ABA) has also emerged as a key signaling molecule in plant–pathogen interactions (Mauch-Mani & Mauch, 2005; Asselbergh et al., 2008), although its role is less direct than that of other defense regulatory plant hormones. ABA plays a complex role in the plant's defense response while it promotes resistance in some plant–pathogen interactions, whereas it increases susceptibility in others (Asselbergh et al., 2008; Ton et al., 2009). With only a few exceptions, exogenous application of ABA enhances the susceptibility of various dicotyledonous plant species to bacterial and fungal pathogens (Ward et al., 1989; Audenaert et al., 2002; Mohr & Cahill, 2003, 2007; Thaler & Bostock, 2004; Achuo et al., 2006; Asselbergh et al., 2007; de Torres-Zabala et al., 2007; Fan et al., 2009), by counteracting other hormone-dependent basal defenses. For example, according to de Torres-Zabala et al. (2009) bacterial pathogen-modulated ABA signaling antagonized SA-mediated plant basal defenses in Arabidopsis, suggesting an important role of ABA in the pathogenicity. It has also been suggested that in Arabidopsis exogenous application of ABA suppresses the JA/ET-activated transcription of defense genes (Lorenzo et al., 2004; Adie et al., 2007). Moreover, in support of these findings Anderson et al. (2004) also revealed that the ABA signaling pathway interacts with the JA signaling pathway, which, in itself, partially overlaps with the ET signaling pathway in regulating defense gene expression and resistance in Arabidopsis to biotrophic fungal pathogens. By contrast, a positive role for ABA in defense against insects and fungal pathogens has been suggested in Arabidopsis (Ton & Mauch-Mani, 2004; Bodenhausen & Reymond, 2007).
The role of ABA against fungal pathogens also appears ambivalent in the monocot model plant rice. ABA is a susceptibility factor for biotrophic blast (M. grisea) and interacts antagonistically with the SA pathway in the rice–M. grisea interaction (Koga et al., 2004; Jiang et al., 2010). By contrast, ABA induces rice defense against the necrotrophic brown spot pathogen Cochliobolus miyabeanus involving mitogen-activated protein (MAP) kinase-mediated repression of ET signaling (De Vleesschauwer et al., 2010). To date, the role of ABA and its interaction with other hormones have mostly been investigated for defense against leaf pathogens; only very little research has been conducted on root pathogens, such as nematodes. Karimi et al. (1995) compared the reproduction of Meloidogyne incognita on potato roots of ABA-treated plants with the reproduction on control plants. The number of egg masses was 70% lower in ABA-treated plants than in control plants, indicating that exogenous ABA would have a negative effect on plant susceptibility. It has been suggested by Volkmar (1991) that ABA may be involved in growth inhibition of Heterodera avenae-infected roots but appear to play no direct role in determining tolerance.
Unlike the role of hormonal pathways in the infection process between plants and sedentary nematodes, about which there is some knowledge (Glazer et al., 1985; Wubben et al., 2001, 2008; Mazarei et al., 2002, 2007; Nandi et al., 2003; Branch et al., 2004; Chinnasri et al., 2006; Bhattarai et al., 2008; Tucker et al., 2010; Nahar et al., 2011), almost nothing is known about the interaction between plants and migratory nematodes. In this report, we present an in-depth characterization of the roles of SA, JA, ET and ABA in systemic-induced defense in rice to the migratory root rot nematode (RRN) H. oryzae. We show that exogenous benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH), a salicylate analog, methyl jasmonate (MeJA) or 2-chloroethyl phosphonic acid (Ethephon, Eth) application on the shoots is actively inducing systemic defense against this migratory nematode in rice roots. Studies with mutants, transgenics and hormone biosynthesis inhibitors demonstrate that these three hormone pathways are a prerequisite for systemically induced defense and that the ET and JA pathways work independently of each other in root defense against H. oryzae. On the other hand, we also show that foliar ABA treatment suppresses rice basal immunity against this migratory nematode and show the antagonistic interaction between ABA and the SA/JA/ET pathways.
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Most studies on systemic signals mediated by plant hormones in plant–pathogen interaction focus on the classical defense hormones SA, JA and ET (Hammond-Kosack & Parker, 2003; Robert-Seilaniantz et al., 2007; Bari & Jones, 2009), but the involvement of other hormones, such as ABA, has become apparent (Robert-Seilaniantz et al., 2011). Broadly speaking, the SA-dependent pathway triggers resistance against biotrophic and hemibiotrophic pathogens, whereas resistance against necrotrophic pathogens and insects is usually controlled by the JA/ET-dependent pathway (Felton & Korth, 2000). However, crosstalk among them, either synergistically or antagonistically, is crucial in induced defense against diverse types of pathogens (Durrant & Dong, 2004).
The research described here aims to characterize the role of SA-, JA-, ET- and ABA-mediated systemic defense signaling in rice and their importance in root defense against the RRN H. oryzae. Studies involving plant–nematode interactions provide an opportunity to observe defense signaling in root tissues, which is an inadequately represented perspective in plant defense research. The migratory endoparasite H. oryzae destroys the tissue while moving through the root cortex, resulting in large necrotic root lesions. The data presented in this report show that exogenous Eth, MeJA and BTH are potent inducers of systemic root defense against RRN attack in rice, while ABA application induces susceptibility to this nematode.
Although the production of SA is generally linked to systemic acquired resistance (SAR) in other plants (van Loon et al., 2006), SAR in rice has not been completely elucidated, but SA probably plays a different role based on the pathogen encountered. It has been suggested that local endogenous SA protects rice from oxidative damage caused by aging as well as by biotic and abiotic stress (Yang et al., 2004), and BTH application has been shown to induce defense against many fungal and bacterial pathogens (Rohilla et al., 2002; Shimono et al., 2007; De Vleesschauwer et al., 2008). A number of studies have also been conducted on dicotyledonous plants showing that application of BTH/SA to the roots of Arabidopsis (Arabidopsis thaliana) or tomato (Solanum lycopersicum) and shoots of okra (Abelmoschus esculentus), cowpea (Vigna unguiculata) or grapevines (Vitis vinifera) enhanced resistance to two types of sedentary nematodes, namely the cyst nematodes and the RKNs (Owen et al., 2002; Nandi et al., 2003; Branch et al., 2004; Wubben et al., 2008), possibly by elevated expression of defense-related genes. Based on the data provided here from exogenous BTH treatments, SA biosynthesis inhibition, SA-deficient plants and qPCR analyses, we can conclude that the SA-dependent defense pathway in rice induces a strong defense against the migratory nematode H. oryzae. Recently our research group has shown that exogenous foliar application with the SA analog BTH only results in a minor induction of systemic defense pathways against sedentary RKN attack, while Eth and MeJA are more potent defense inducers in the RKN–rice interaction (Nahar et al., 2011). In the rice–RRN interaction, however, SA, JA and ET each appears to play equally important roles. The different degree to which the SA effect induces defense against RKNs and RRNs might be explained by the different infection strategy of both nematodes. Migratory nematode-affected plant cells are either fed upon or left behind as the nematode migrates away, while RKNs induce specialized feeding structures. During their intimate interaction with the plant, RKNs strongly suppress the plant's innate immunity system both locally and in systemic tissues, mainly through interference with the SA and ET pathways (Kyndt et al., 2012a,b). Such a strong interference with the innate immune system has not been observed for RRN-infected plants, where the SA, JA and ET pathways are instead induced early after infection (Kyndt et al., 2012a,b). Our results show that BTH can still induce defense against RRNs in the JA-deficient hebiba plants, signifying that the SA-induced systemic defense does not need any activity in the JA pathway for this rice–nematode interaction. Similarly, MeJA spraying induces defense against RRNs in NahG plants, containing a lower endogenous concentration of SA (Gaffney et al., 1993; Delaney et al., 1994), indicating the mutual independence of the SA and JA pathways in defense against RRNs. SA and JA cross-talk in either negative or positive ways in different plant–pathogen interaction systems (Kunkel & Brooks, 2002; Rojo et al., 2003; Bostock, 2005; Leon-Reyes et al., 2010). In Arabidopsis, it has been suggested that separate JA- and SA-dependent defense pathways are essential for resistance involving the activation of separate sets of genes encoding antimicrobial proteins to distinct microbial pathogens (Thomma et al., 1998).
Methyl jasmonate application to roots of oat (Avena sativa) and spinach (Spinacia oleracea) and to shoots of tomato enhanced resistance to RKNs, possibly by elevating the concentrations of compounds that are toxic to nematodes, such as phytoectosteroids, flavonoids, and proteinase inhibitors (Soriano et al., 2004a,b; Cooper et al., 2005). Similar to these studies and our previous findings on RKN–rice interaction (Nahar et al., 2011), the current study shows that MeJA is an effective elicitor of systemically induced defense mechanisms against migratory RRNs in rice roots. Foliar application with Eth was shown to activate not only the ET pathway but also the JA pathway and the expression of the pathogenesis-related (PR) genes OsPR1a and OsPR1b in rice roots (Nahar et al., 2011). It has been demonstrated that some defense genes, such as the Arabidopsis genes PDF1.2 (Anderson et al., 2004), need a simultaneous production of both JA and ET. On the other hand, ET alone can also activate JA-independent defense responses in Arabidopsis (Lorenzo et al., 2003). The data provided in the current study show that ET works independently of JA to promote defense against RRNs in rice roots, with ET-induced defense still active in the JA biosynthesis mutant hebiba. However, while Eth also induces defense against RKNs in rice (Nahar et al., 2011), its activity against RKNs was shown to be dependent on activation of the JA pathway, since it was not functional in hebiba. To explain these apparent contradictions in ET dependence on JA, we have to consider the infection strategy of both nematodes and the way they manipulate plant defense pathways to their own benefit. Infection by RRNs involves wounding of the root, and hence the JA and ET pathways will be activated, as shown by Kyndt et al. (2012b) and the qRT-PCR results in this study. Enhancing these pathways by external Eth application leads to higher expression of PR genes (Nahar et al., 2011), indicating stronger defense. However, transcriptional analyses of root galls (Kyndt et al., 2012b) showed a suppression of the ET pathway, but not the JA pathway, in roots infected by RKNs. Also in hebiba plants, ET will be suppressed by the RKNs, which explains why Eth application does not lead to defense in these plants.
The effects of ABA have been shown to be different depending on the pathogen's lifestyle, both in monocots and dicots (Mauch-Mani & Mauch, 2005; Asselbergh et al., 2008; De Vleesschauwer et al., 2010). Most studies have shown that ABA leads to enhanced pathogen susceptibility (Ward et al., 1989; McDonald & Cahill, 1999; Audenaert et al., 2002; Mohr & Cahill, 2003; Koga et al., 2004; Achuo et al., 2006; Fan et al., 2009; Jiang et al., 2010). Pretreatment of potato plants with ABA increased susceptibility to Phytophthora infestans and Cladosporium cucumerinum (Henfling et al., 1980). ABA-deficient tomato plants were more resistant to the necrotrophic pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae pv tomato (Pst), whereas exogenous application of ABA restored the susceptibility to this pathogen in the mutant plants (Audenaert et al., 2002; Thaler & Bostock, 2004). ABA treatment drastically enhanced disease susceptibility in rice not only to compatible but also to incompatible strains of Magnaporthe grisea (Jiang et al., 2010). Our experiments with exogenous ABA and ABA biosynthesis inhibitors reveal that ABA also plays a negative role in rice defense against migratory nematodes in roots. Nevertheless, Karimi et al. (1995) revealed a lower reproduction of the sedentary nematode M. incognita on potato roots of ABA-treated plants, but in that study higher ABA concentrations were used (100 μM), and the effect might vary depending on the lifestyle of the nematode.
It has been revealed by Xiong & Yang (2003) that dsRNAi transgenic OsMPK5 plants show constitutive expression of PR genes such as PR1 and PR10 and significantly enhance resistance to fungal and bacterial pathogens in rice. Our data provide evidence that these plants are also more resistant to RRNs. It has been suggested that ABA interacts antagonistically with the SA pathway in the rice–M. grisea interaction (Jiang et al., 2010). In this study, we have also shown that SA and ABA mutually antagonize each other's signaling pathways in the rice–H. oryzae interaction. Foliar ABA application suppresses SA biosynthesis (OsICS1) and SA signaling (OsWRKY45) in rice roots, accompanied by an enhanced susceptibility to H. oryzae infection. Conversely, SA (BTH) suppressed ABA-related gene expression during nematode infection. Interestingly, the compatible rice–H. oryzae interaction induced a strong up-regulation of an ABA biosynthesis gene soon after infection, which results in induction of the ABA-responsive gene OsLip9 at later stages of infection. These data suggest that during a compatible interaction the ABA pathway is activated, resulting in enhanced rice susceptibility. Although it is hard to distinguish whether these observations are the result of nematode effects or systemic hormone signaling effects, we can speculate that the RRN is interfering with the normal hormone homeostasis, resulting in the suppression of the SA pathway and induction of the ABA pathway, to promote root susceptibility. These data, taken together, strongly suggest that the balance of interaction between SA and ABA signaling is an important determinant in the outcome of the rice–H. oryzae interaction.
The JA and ET pathways are known to play synergistic roles in plant innate immunity (Pieterse et al., 2009). In the case of migratory nematode-infected roots, a trend of coregulation of genes involved in these pathways was also observed, with them generally being unchanged or up-regulated compared with noninfected roots, similar to their systemic induction upon RRN infection (Kyndt et al., 2012a). Besides the negative effect of ABA on SA-dependent defense, our data suggest a crucial role of ABA–JA/ET signal interactions in determining rice–H. oryzae outcomes. qRT-PCR analysis shows that JA and ET pathways are suppressed in ABA-treated infected plants compared with untreated uninfected plants, suggesting antagonistic effects of ABA on the JA/ET pathway. It has also been suggested that, in Arabidopsis, ABA negatively regulates defense against the soilborne fungus F. oxysporum, by antagonizing JA and ET signaling networks (Anderson et al., 2004). Recently, Bailey et al. (2009) reported that the exogenous application of ABA drastically reduces ET concentrations in rice and enhances rice blast susceptibility. In addition, RNAi-mediated suppression of OsEIN2b, a central component of ET signal transduction, resulted in ABA hypersensitivity, reduced defense gene expression, and enhanced blast susceptibility. Bailey et al. (2009) suggested that ABA antagonistically interacts with the ET signaling pathway and, consequently, enhances susceptibility of rice to M. grisea.
The data provided in this study suggest that exogenous BTH, MeJA or Eth supplied to the rice shoot induce systemic SA/JA/ET pathways in the infected root tissues, hence protecting the plant from infection with migratory nematodes. When taken together, our results support the idea that ABA opposes the SA/ET/JA signaling pathways to suppress the defense response against H. oryzae in rice.
In closing, we present an in-depth characterization of the roles of SA, JA, ET and ABA in mediating effective systemic rice response to the root nematode H. oryzae. H. oryzae is a migratory endoparasite which can easily escape from the plant defense system by moving through and out of the root system. Yet, to our knowledge, no study has addressed the role of hormones in the plant–migratory nematode interaction. Our results show that application of BTH, MeJA or Eth to the shoots induces systemic defense pathways in rice roots, which are active against the migratory nematode H. oryzae. On the other hand, ABA induces susceptibility to this nematode. Our data also demonstrate that SA and ET act independently of the JA pathway in switching on the induced defense mechanisms in rice–H. oryzae interaction. Moreover, the results presented here and those published previously (Xiong & Yang, 2003; Yasuda et al., 2008; Bailey et al., 2009; de Torres-Zabala et al., 2009; Jiang et al., 2010) emphasize that ABA-induced susceptibility interacts antagonistically with other hormonal pathways (SA/JA/ET).