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Introduction

Plant diseases are amongst the major limiting factors of agricultural crop production worldwide. Depending upon the time of infection and severity of the disease, they can cause average yield losses of about 10–90 %. Plant disease directly or indirectly affects the life of human, as it may cause famine, mass migration and even death. For example, Irish potato famine of 1845–1846 killed hundreds of thousands of people. This event initiated a large scale migration. Within decade that follows the population of Ireland dropped from 8 million to 4 million (Ristaino 2002). Plant diseases significantly influence world economy, as crop plants make up large proportion of the world’s economy, and in many countries constitute main sustenance for humans. According to one estimate, plant diseases could cost the US alone $33 billion per year (Maor and Shirasu 2005). To meet the ever increasing food demands of the rapidly increasing population, crop production will need to increase by 50 % by 2025 (Khush 2001). Currently, worldwide crop losses due to diseases are estimated to exceed $140 billion (Shani et al. 2006). Although application of fungicides and pesticides has helped in controlling plant diseases, chemical control is economically costly as well as environmentally undesirable. Keeping in view of the global food scarcity, there is, hence, an urgent need to develop crop plants with increased biotic stress tolerance so as to meet the global food demands. A detailed study of the molecular interactions between crops plants and their pathogens would, therefore, be of primary importance for devising new strategies based on plants self defense mechanisms to develop crops with increased disease tolerance for sustainable agricultural production.

When a plant and a pathogen come into contact, close communications occur between the two organisms (Hammond-Kosack and Jones 2000). Pathogen activities focus on colonization of the host and utilization of its resources, while plants are adapted to detect the presence of pathogens and to respond accordingly with antimicrobial defenses and other stress responses. During the long process of host-pathogen coevolution, plants have developed various elaborate mechanisms to ward off pathogen attack. Whereas some of these defense mechanisms are preformed and provide physical and chemical barriers to hinder pathogen infection, others are induced only after pathogen attack (Yang et al. 1997). Intercellular detection of the pathogen activates the first line of defense, termed innate or basal resistance, which involves recognition of evolutionarily conserved and essential features of pathogens such as bacterial lipopolysaccharides (LPS) or fungal chitin. These Extracellular signals are perceived by host cells through plasma membrane (PM) receptors that transduce the signals to an intracellular signal transduction cascade. It ends in the activation of transcription of the appropriate set of genes, which results into alteration or modification of cellular metabolism, accumulation of barrier forming substances (thickening of cell walls) and production of anti microbial compounds. In most cases, the transduction of signal relies on post translational modifications of the signaling proteins and the generation of so called secondary messenger molecules.

Among all post-translational modifications, phosphorylation has been studied most intensively (Pawson and Scott 2005), which can lead to changes in conformation, protein–protein interaction and protein activity. In eukaryotic cells, protein phosphorylation occurs predominantly on serine, threonine and tyrosine residues, but has also been described to occur on aspartate and histidine residues. The regulatory mechanisms of plant–pathogen interaction are extremely complex and dynamic, and the ongoing interactions between the pathogen and the plant are difficult to monitor with more traditional genetic and biochemical methods. The two approaches that are most promising for understanding the full network of the responses are microarray and proteomic analyses. Both permit a global analysis of cellular regulation while the microarray is restricted to the analysis of gene expression. The proteomics follows the accumulation and modification of proteins directly responsible for final cellular responses. Recent advancement in liquid chromatography–tandem mass spectrometry (LC–MS/MS) has greatly improved the throughput and sensitivity of protein measurements. In order to efficiently describe the status of phosphorylated molecules, a variety of enrichment strategies for phosphorylated peptides have been developed. The most commonly used are based on affinity purification of phosphoryl-group containing peptides and include immobilized metal affinity chromatography (IMAC), strong cation exchange chromatography (SCX) and metal oxide affinity chromatography (MOAC) (Mithoe and Menke 2011).

Over the last few years, significant progress has been made in understanding the signaling processes involved in plant-pathogen interactions. In this chapter, we focus on signaling pathways involved in plant defense against pathogens and the role of proteomics technology in understanding the underlying mechanisms.

Plant Signal Transduction in Plant Defense Against Pathogens

Plants have integrated signaling system that mediate the perception and responses to the hormones, nutrients, environmental and stresses that govern plant growth and development. Interactions between plants and pathogens induce a series of plant defense responses (Hammond-Kosack and Jones 1996). The rate at which the plant cell mobilise its defenses often determines whether it survives or succumbs to the attack. Therefore, highly sensitive perception systems for either pathogen derived (exogenous) or plant-derived (endogenous) elicitors are the key to successful plant pathogen defense. The sensing of stress signals and their transduction into appropriate responses is crucial for the adaptation and survival of plants. Plant receptors are instrumental for signal recognition and initiation of an intracellular signal transduction cascade mediating activation of multifaceted defense reactions, both in host and non-host incompatible plant pathogen interactions.

The current knowledge of plant signal transduction pathways has come from the identification of the sensors and receptors that perceive the signal, the transcription factors and target genes that coordinate the response (Hammond-Kosack and Jones 1996). The activation of defense responses in plants is initiated by host recognition of pathogen-encoded molecules called elicitors (e.g., microbial proteins, small peptides, and oligosaccharides, etc.). The term ‘elicitor’, originally coined for compounds that induce accumulation of antimicrobial phytoalexins in plants, is now commonly applied to agents stimulating any type of defense response (Ebel and Scheel 1997). Elicitors of diverse chemical nature and from a variety of different plant pathogenic microbes have been characterized and shown to trigger defense responses in intact plants or cultured plant cells. These elicitors include (poly) peptides, glycoproteins, lipids and oligosaccharides. Binding of the elicitor ligand to its receptor initiates a signal transduction cascade that may involve protein phosphorylation, ion fluxes, reactive oxygen species (ROS), nitric oxide (NO) and other signaling events.

Ion Fluxes and Reactive Oxygen Species

The earliest reactions of plant cells include changes in plasma membrane permeability leading to calcium and proton influx and potassium and chloride efflux (McDowell and Dangl 2000). Various fungal and bacterial elicitors have been reported to trigger fluxes of H+, K+, Cl-, and Ca2+ across the plasma membrane (Atkinson et al. 1996; Mathieu et al. 1991; Bach et al. 1993; Kuchitsu et al. 1993; Popham et al. 1995). In suspension cells of parsley, a transient influx of Ca2+ and H+ and an efflux of K+ and C1- are initiated within two to five minutes after the addition of a fungal oligopeptide elicitor (Hahlbrock et al. 1995). Ion fluxes subsequently induce extracellular production of reactive oxygen intermediates, such as superoxide (O2–), hydrogen peroxide (H2O2) and hydroxyl free radical (OH-), known as oxidative burst, catalyzed by a plasma membrane-located NADPH oxidase and/or apoplastic-localized peroxidases (Somssich and Hahlbrock 1998). Oxidative burst is a central component of plants defense machinery (Alvarez et al. 1998). The generation of ROS is likely dependent on the activation of a plasma membrane NADPH oxidase similar to that present in mammalian phagocytes. Using the mammalian system as a model, homologues of the large gp91-phox protein of NADPH oxidase have been cloned from several plant species including Arabidopsis and rice (Desikan et al. 1998; Torres et al. 1998). Eight such sequences have been identified in the Arabidopsis genome (The Arabidopsis Genome Initiative 2000).

Interestingly, plant homologues contain calcium-binding EF-hand regions, suggesting that calcium may be important in the regulation of their activity (Desikan et al. 1997). Immunological evidence points to the presence of the NADPH oxidase cytoplasmic peptides too, in several species of plants including Arabidopsis (Desikan et al. 1996), tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), soybean (Glycine max) and cotton (Gossypium hirsutum) (Dwyer et al. 1996; Tenhaken et al. 1995; Xing et al. 1997).

Cell wall peroxidases have also been reported to be involved in ROS generation following pathogen challenge (Bolwell et al. 1995). For example, in French bean (Phaseolus vulgaris) ROS production in response to a cell wall elicitor from Colletotrichum lindemuthianum was dependent on an exocellular peroxidase, and Arabidopsis plants expressing an antisense peroxidase from French bean exhibit enhanced disease susceptibility (Bolwell 1999). Therefore, it can be seen that there are several potential sources of ROS in plant tissues, and future research should aim to elucidate the role of distinct sources of ROS not only in plant defense, but also in response to a variety of abiotic stresses.

Superoxide anion and H2O2 generated during the oxidative burst play multiple roles in plant defense responses. During a hypersensitive response (HR), a highly localized accumulation of H2O2 was found in the lettuce cell walls adjacent to invading bacteria (Bestwick et al. 1997). In addition, constitutive expression of an H2O2 generating glucose oxidase in the transgenic potato was shown to confer enhanced resistance to the bacterial pathogen, Erwinia carotovora pv. carotovora and the fungal pathogen, Phytophthora infestans (Wu et al. 1995). H2O2 was also demonstrated to have direct antimicrobial activity (Peng and Kuc 1992) and to contribute to cell wall reinforcement by stimulating lignification and crosslinking of cell wall hydroxyproline-rich glycoproteins (Bradley et al. 1992; Brisson et al. 1994). Furthermore, superoxide anion and H2O2 may also act as secondary messengers to induce plant defense-related genes (Levine et al. 1994; Green and Fluhr 1995) and hypersensitive host cell death (Doke 1983a; Doke 1983b; Doke and Ohashi 1988; Levine et al. 1994). Elicitor-stimulated superoxide anion from the oxidative burst was also shown to be essential in triggering defense gene activation and phytoalexin synthesis in parsley (Jabs et al. 1997).

Cellular Generation of Nitric Oxide

Nitric Oxide (NO) like ROS, is an important signaling molecule that is rapidly generated after recognition of pathogens (Perchepied et al. 2010). It is a small gaseous radical with broad spectrum of regulatory functions in lateral root development, germination, leaf expansion, stomatal closure, flowering, hormonal signaling, defense against biotic and abiotic stresses and cell death (Leitner et al. 2009). The sources of NO synthesis in plants include nitrate reductase (NR) dependent NO formation, oxidation of arginine to citrulline by NO Synthase (NOS) like activity, and a non-enzymatic NO generation system in the apoplast (Leitner et al. 2009). Although a number of studies had demonstrated NOS-like activity in plants, no gene or protein that has a sequence similar to known mammalian-type NOS has been establish in plants (Leitner et al. 2009). Guo et al. (2003) reported a NOS-like enzyme activity from Arabidopsis thaliana (At NOS1) with a sequence similar to a protein that has been implicated in NO synthesis in the snail Helix pomatia. Recently, the only postulated plant NOS (AtNOA1/RIF1) has been shown to have no NOS activity (Moreau et al. 2008). Instead, it is the chloroplast-targeted GTPase essential for proper ribosome assembly (Moreau et al. 2008). Mutation in this gene leads to reduced NO accumulation, probably because of its rapid reaction with the elevated amounts of ROS observed in the Atnoa1 mutant (Moreau et al. 2008). Arabidopsis mutant noa1, however, is still useful for its phenotype, which shows reduced levels of NO in plant growth, fertility, hormonal signaling, salt tolerance, and plant-pathogen responses. Knocking out or down NOA1 expression provides a powerful tool to analyze NO function (Asai et al. 2008). So, the identification and characterization of NO-producing enzymes in plants, other than NR, still remains a challenging tasks for plant biologists.

Recently, Perchepied et al. (2010) reported that NO production in Arabidopsis leaf was significantly reduced by the mammalian NO synthase (NOS) inhibitor L-NAME (37% inhibition). They further demonstrated that like ROS, NO is an early-induced signal during the interaction between Arabidopsis and S. sclerotiorum. In order to genetically determine the role of the signaling molecules during the interaction, mutants altered in their production were tested for their response to S. sclerotiorum and found that NO synthesis was strongly reduced in noa1 mutant (83% inhibition) and also in nia1 nia2 mutant (62% inhibition). Therefore, these results demonstrate that NO might have an important role in disease resistance to S. sclerotiorum. To further analyze the role of ROS and NO in the activation of defenses to S. sclerotiorum, Perchepied et al. (2010) also demonstrated that expression of PR1, PDF1-2, and ABI1 was found to be abolished or strongly reduced compared with the wild type in double mutant nia1 nia2, whereas VSP1 expression was delayed by 24 h. Similarly, in the rboh-D rboh-F double mutant, PR1 expression was abolished, PDF1-2 and VSP1 expression was delayed, and ABI1 expression was upregulated. These results suggest a differential regulatory effect of NO and ROS on the different defense pathways (Perchepied et al. 2010).

G Proteins

G Proteins are one of the most important components of signaling system. There are two types of G proteins, monomeric and heterotrimeric. The heterotrimeric G proteins contain α, β and γ subunits, and the monomeric small G proteins appear to be similar to free α subunits, operating without the βγ heterodimer. The Gα subunit has both GTP-binding and GTPase activity and acts as a molecular switch for signal transduction. G proteins have been implicated in plant defense, however much remains to be explored especially in case of Arabidopsis and rice. The genome of the model plant Arabidopsis thaliana contains one prototypical Gα (GPA1), one Gβ (AGB1), and two identified Gγ (AGG1 and AGG2) subunits (Jones and Assmann 2004) and one RGS protein, AtRGS1 (Chen et al. 2003). Two Gα subunits (PGA1 and PGA2) are reported from pea (Marsh and Kaufman 1999).

A variety of evidence suggest that heterotrimeric GTP-binding proteins are involved in transferring elicitor signals from the receptor to calcium channels that activate downstream reactions, such as the oxidative burst and phytoalexin accumulation (Gelli et al. 1997; Xing et al. 1997). Llorente et al. (2005) reported that the ERECTA receptor-like kinase and G proteins are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis. In cultured soybean cells, mastoparan, a G protein-activating peptide, was found to stimulate calcium influx, increases in cytosolic calcium levels and production of reactive oxygen species in the absence of elicitor (Chandra and Low 1997). Ectopic expression of the cholera toxin A1 subunit inhibiting GTPase activity of G proteins in tobacco plants resulted in high salicylate levels, constitutive expression of PR proteins and enhanced pathogen resistance (Beffa et al. 1995). Recently, Trusov et al. (2006) found in Arabidopsis that Gβ deficient mutants are more susceptible to infection with A. brassicicola and F. oxysporum when compared to wild type (Col-0), while Gα-deficient mutants are less susceptible to the disease than wild type. They also reported that the Gβγ subunit is an integral component and a positive regulator of the JA-signaling cascade.

Phospholipid Derived Molecules

Phospholipids are emerging as important secondary messengers in plant defense signaling. Recent research has begun to reveal the signals produced by the enzymes phospholipase C (PLC), phospholipase D (PLD) and phospholipase A2 (PLA2), and their putative downstream targets (Laxalt and Munnik 2002). Upon perception of the invading pathogen, several phospholipid hydrolyzing enzymes are activated that contribute to the establishment of an appropriate defense response. These include activation of G proteins based signaling leading to the production of oxylipins and jasmonates, as well as the potent second messenger, phosphatidic acid (PA) (Canonne et al. 2011).

Three PAT-PLA genes (AAF98368, AAF98369, AAF98370) found in tobacco, were strongly induced in response to microbial infections or elicitors before the accumulation of jasmonic acid in the infiltrated zone, but poorly induced in response to wounding (Dhondt et al. 2002), indicating that further studies are required. Additionally, PLA2 activity was found to be involved in the wound-activated cascade leading to the production of aldehydes by generating C20 fatty acids in diatoms (Pohnert 2002). PLA activation has been reported in elicitor-treated cultured parsley cells. Virus infection also activates PLA (Ryu 2004). The role of PLA in cell elongation, auxin signaling, shoot gravitropism, pollen maturation, anther dehiscence and flower opening are well established, but its role in plant defense is still need to be revealed.

The involvement of PLC in stress signaling has been indicated in a number of studies. The genes encoding PI‑PLC were found to be induced to a significant extent under environmental stresses (Tuteja and Sopory 2008). Van der Luit et al. (2000) reported that tomato cells responded to general elicitors, such as xylanase, flagellin or chitotetraose, by rapidly (i.e. within minutes) and transiently producing phosphatidic acid (PA). Role of PLC in Avr‑induced disease resistance has also been implicated. In this case PLC/diacylglycerol kinase (PLC/DGK) mediated production of PA was found to be involved in disease resistance signaling (Andersson et al. 2006).

PLD activity has been associated to a variety of stress responses in plants. Activation of PLD activity during plant defense was first described in rice, after infection by Xanthomonas oryzae (Young et al. 1996). In Arabidopsis, expression of the α, β and γ class of PLD genes is induced after infiltration by both virulent and avirulent strains of P. syringae (de Torres Zabela et al. 2002). The effects of PLD activation during plant-pathogen interactions are varied. Indeed, PA has been shown to induce ROS production1 and activate defense-related or ethylene-responsive genes PLDs also participate in salicylic acid-dependent signaling (Canonne et al. 2011). Five different tomato PLDs have been cloned and only one of them, PLDβ1, is specifically upregulated in response to xylanase. Silencing of this gene in tomato cell suspensions resulted in the loss of the xylanase-PLD response, indicating that PLDβ1 generates PA in response to xylanase treatments (Laxalt and Munnik 2002).

Protein Kinase

Protein phosphorylation plays an important role in plant responses to pathogen attack. Signaling systems which involve phosphorylation, and can lead directly to altered gene expression pattern in cells, are the MAPK (Miotgen Activated Protein Kinase) pathways (Hancock et al. 2002). The MAPK cascades are highly conserved modules in all eukaryotes (Pitzschke et al. 2009). In plants, MAPK pathways are involved in the regulation of development, growth and programmed cell death in responses to a diversity of environmental stimuli including cold, heat, reactive oxygen species, UV, drought and pathogen attack (Colcombet and Hirt 2008). These cascades are minimally composed of a MAPKKK (MAPK kinase kinase), a MAPKK (MAPK kinase) and a MAPK. The Arabidopsis genome contains about 110 genes coding for putative MAPK pathway components: 20 MAPKs, 10 MAPKKs and more than 80 MAPKKKs (MAPK Group 2002). In plants, pathogen challenge along with cold, drought and phytohormones may lead to the activation of MAPK cascades, resulting in the modulation of nuclear gene expression (Hirt 1997). Exogenous hydrogen peroxide can lead to the activation of MAP kinases (Kovtun et al. 2000; Samuel et al. 2000). In Arabidopsis suspension cultures and leaves, hydrogen peroxide treatment activates AtMPK6 (Desikan et al. 2001). One of the potential targets of NO in cells is also the MAPK cascade. MAPK activation by NO has been reported in Arabidopsis (Clarke et al. 2000) and tobacco (Durner and Klessig 2000). Activation of such MAPK cascades is likely to lead to alteration in gene expression profiles. In Arabidopsis, MPK3, MPK4 and MPK6 are all activated by bacterial and fungal elicitors (Desikan et al. 2001; Nuhse et al. 2000). The flagellin derived peptide, flg22 triggers a rapid and strong activation of MPK3, MPK4 and MPK6 (Droillard et al. 2004). MPK4 andMPK6 are also activated by harpin proteins, which are encoded by hrp (hypersensitive response and pathogenicity) genes in many plant pathogenic bacteria. This activation is followed by the induction of pathogenesis-related (PR) genes (Desikan et al. 2001), encoding for proteins with antimicrobial activities. Similarly, various NLPs (necrosis and ethylene-inducing peptide1-like proteins) trigger MAPK activation and induce defence responses (Qutob et al. 2006).

Salicylic Acid, Jasmonic Acid and Ethylene

Most of the inducible, defense-related genes are regulated by signal pathways involving one or more of the three regulators jasmonate, ethylene and salicylic acid (Sticher et al. 1997; Reymond and Farmer 1998; Ananieva and Ananiev 1999).

SA levels increase in plant tissue following pathogen infection, and exogenous application of SA results in enhanced resistance to a broad range of pathogens (Kunkel and Brooks 2002). Genetic studies have shown that SA is required for the rapid activation of defense responses that are mediated by several resistance genes, for the induction of local defenses that contain the growth of virulent pathogens, and for the establishment of systemic acquired resistance (SAR) (Ryals et al. 1996). Several studies have also demonstrated that when SA accumulation is prevented, resistance is compromised. Transgenic tobacco and Arabidopsis plants unable to accumulate SA because of the expression of the Pseudomonas putida nahG gene encoding salicylate hydroxylase, exhibit poor induction of PR genes after pathogen infection and fail to develop SAR (Gaffney et al. 1993; Delaney et al. 1994). The signal transduction pathway downstream of SA leads to the expression of a number of PR genes, such as PR-1 and β-1,3-glucanase (Ryals et al. 1996). Activation of R-gene-mediated defense signaling induces SA synthesis and downstream defense responses. Significantly, the application of SA activates the expression of R genes of the toll-interleukin-2 receptor (TIR)–nucleotide-binding site (NBS)–leucine-rich repeat (LRR) type (Shirano et al. 2002). Similarly, SA activates expression of RPW8, which confers resistance to the powdery mildew pathogen (Xiao et al. 2003).

SA also activates expression of the EDS1 gene, which is required for SA accumulation and resistance conferred by these R-gene-activated pathways (Feys et al. 2001). Arabidopsis mutants that are impaired in SA responsiveness, such as npr1 (nonexpressor of PR) or are defective in pathogen-induced SA accumulation, such as eds1 (enhanced disease susceptibility 1), eds5 (enhanced disease susceptibility 5), sid2 (isochorishmate synthase) and pad4 (phytoalexin deficient 4), exhibit enhanced susceptibility to pathogen infection and show impaired PR gene expression (Venugopal et al. 2009).

Jasmonates are produced from the major plant plasma membrane lipid, linolenic acid via the octadecanoid biosynthetic pathway (Yang et al. 1997). First indications for a role of jasmonates in the regulation of gene expression were obtained by Parthier and co-workers who observed the accumulation of jasmonate inducible proteins (JIPs) in senescing barley leaves (Weidhase et al. 1987; Mueller-Uri et al. 1988). The rapid accumulation of jasmonate has been observed in many cultured plant cells in response to various elicitor treatments (Ebel and Scheel 1997; Gundlach et al. 1992). In suspension-cultured rice cells, an N-acetylchitohepatose elicitor induces the synthesis of the phytoalexin, momilactone A, which is preceded by transient accumulation of jasmonate (Nojiri et al. 1996). A. thaliana mutants that are impaired in JA production (e.g. fatty acid desaturase fad3/fad7/fad8 triple mutants) or perception (e.g. coronatine insensitive1 [coi1] and jasmonic acid resistant1 [jar1]) exhibit enhanced susceptibility to a variety of pathogens, including the fungal pathogens Alternaria brassicicola, Botrytis cinerea, and Pythium sp., and the bacterial pathogen Erwinia carotovora (Thomma et al. 1998; Stintzi et al. 2001; Vijayan et al. 1998; Staswick et al. 1998; Norman-Setterblad et al. 2000). Perchepied et al. (2010) demonstrated that the JA-insensitive coi1-1 arabidopsis mutant was highly susceptible to S. sclerotiorum, thus indicating that JA is a major signal for activation of defenses against this fungus. In the same study, however, jar1-1, a jasmonate-resistant mutant shown to exhibit enhanced sensitivity to the fungal necrotroph Pythium irregulare, was not affected for responsiveness to S. sclerotiorum. Wounded tissue rapidly activates JA biosynthesis, and increased JA triggers the SKP1/Cullin/F-box E3 ubiquitin ligase complex containing the F-box subunit CORONATINE INSENSITIVE1 (SCFCOI1) to degrade the repressors of JA signaling – the JASMONATE-ZIM (JAZ) family proteins – by the ubiquitin/26S-proteasome pathway (Chini et al. 2007; Yoshida et al. 2009). In addition to their local synthesis and action, JAs also move systemically via vascular strands to transmit wound signals to distal tissues (Thorpe et al. 2007).

Ethylene (ET) plays a critical role in the activation of plant defenses against different biotic stresses through its participation in a complex signaling network that includes jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA) (Adie et al. 2007). ET has been implicated in both local and systemic defense responses to A. brassicicola through its regulation of GLIP1. This secreted lipase has antifungal properties and is induced by ET but not by SA or JA (Oh et al. 2005). It is believed that crosstalk between ethylene and JA pathways enables plants to optimize their defense strategies more efficiently and economically (Baldwin 1998). It has also been reported that ethylene and jasmonate pathways converge in the transcriptional activation of ETHYLENE RESPONSE FACTOR1 (ERF1), which encodes a transcription factor that regulates the expression of pathogen response genes that prevent disease progression. The expression of ERF1 can be activated rapidly by ethylene or jasmonate and can be activated synergistically by both hormones (Lorenzo et al. 2003). In another study with ET-insensitive (Tetr) tobacco plants, it was reported that ET is essential for the onset of SA-dependent SAR that is triggered upon infection by tobacco mosaic virus (Verberne et al. 2003). Moreover, ET was shown to enhance the response of Arabidopsis to SA, resulting in a potentiated expression of the SA-responsive marker gene PR-1 (Lawton et al. 1994; De Vos et al. 2006). This synergistic effect of ET on SA-induced PR-1 expression was blocked in the ET-insensitive mutant ein2 (De Vos et al. 2006), which indicates that the modulation of the SA pathway by ET is EIN2 dependent and thus functions through the ET signaling pathway. Therefore, the SA, JA and ET response pathways serve as the backbone of the induced defense signaling network in plants.

Plant-Pathogen Interactions and Proteomics

The common approach utilized for proteomics based experiments comprises two-dimensional gel electrophoresis protein profile followed by MS analysis of differential expressed spots (MALDI-TOF or MS-MS) and identification by DNA, EST or protein database searching using specific algorithms (i.e. MASCOT, phenyx and OMSAA). In brief, the workflow of a standard proteomics experiment includes all or most of the following steps: experimental design, sampling, sample preparation, protein extraction/fractionation/purification, labeling/modification, separation, MS analysis, protein identification, and statistical analysis of data and validation. The most appropriate protocol to be used depends on and must be optimized for the biological system and type of tissue/cells, as well as the objectives of the research (descriptive, comparative, Post Translational Modifications, interactions, targeted Proteomics) (Jorrin-Novo et al. 2009). There are number of technical advances available and constantly evolving particularly for sample preparation, gel free proteomics, protein identification and data analysis, but this will not be the focus of this chapter. Those interested in further reading can refer to the reviews (Ong et al. 2003; Chen and Harmon 2006; Domon and Aebersold 2006; Rossignol et al. 2006; Everberg et al. 2008; Carpentier et al. 2008; Chen 2008; Jorrin-Novo et al. 2009). Herein, we address the challenges in proteomics and phosphoproteomics studies of plant pathogen interactions.

Comparative Proteomics

The aim of most of the proteomics studies was to compare the plant response to infection by the pathogen and to identify and characterize common and specific changes in protein expression patterns. Geddes et al. (2008), using two-dimensional Electrophoresis (2-DE) coupled with LC-MS/MS, identified differentially expressed proteins in Fusarium head blight-resistant and Fusarium head blight-susceptible barley genotypes under infected and uninfected conditions. In this study, approximately 600 protein spots were resolved in the pH range of 4–7 in the 2-DE gels. A total of 16 different acidic proteins associated with resistance mechanisms against Fusarium head blight were identified, out of which 12 proteins were associated with oxidative burst response and 4 proteins were associated with PR-Proteins. Takemoto et al. (1997) reported that chitinase (PR3) and osmotin (PR5) were associated with the actin cytoskeleton that is involved in cytoplasmic aggregation in the early stages of the hypersensitive response (HR) between Phytophthora infestans and potato. Fusarium head blight, caused mainly by F. graminearum, is one of the most destructive diseases of wheat. The interaction between F. graminearum and wheat has been investigated by Zhou and his coworkers in 2006. They found that 33 plant proteins were expressed in response to F. graminearum in wheat spikes. These proteins were divided into two groups, each related to defense response or metabolism. The authors suggested that several of these proteins were directly involved in mounting the plant defense against infection by protecting against the oxidative burst inside the plant cell. Such a burst can be caused in plant cells by invading fungus.

Proteomics analysis was carried out to study the compatible and incompatible interactions between rice and bacteria, Xanthomonas oryzae pv. oryzae (Xoo) (Mahmood et al. 2006). In this study, four different defense-related proteins were identified, namely thaumatin-like protein (PR5), PBZ, SOD, and peroxiredoxin. Overexpression of PR5 in transgenic rice plants enhanced the resistance of rice to Rhizoctonia solani, the causal organism of sheath blight of rice (Datta et al. 1999). Wei et al. (2009) used isobaric tag- based methodology for relative peptide quantification (iTRAQ) coupled with multidimensional liquid chromatography and tandem mass spectrometry to study the response of rice to brown plant hopper (BPH) attack. In this study, three proteins involved in JA biosynthesis were induced in rice in response to infestation by the BPH: cytochrome P450, AOC 4 and alpha-DOX2. Alpha-DOX2 is a dioxygenase that catalyzes the synthesis of 13-hydroperoxylinolenic acid from linolenic acid in JA biosynthesis. Koeduka et al. (2005) reported that alpha-DOX can be induced by blight bacteria infection, and both oxidative and heavy metal stresses, through the jasmonate signaling pathway in the leaves of rice seedling. AOC4 catalyzes the stereospecific cyclization of an unstable allene oxide to (9S, 13S)-12-oxo- (10, 15Z)-phytodienoic acid, and experiments with the JA deficient Arabidopsis mutant opr3 indicate that AOC is the preferential target in the regulation of JA biosynthetic capacity (Stenzel et al. 2003). Since the BPH is a phloem-feeding insect, AOC may have a role in systemic defense signaling. Liang et al. (2008), using 2-DE identified 9 proteins related with defense responses. Out of these two proteins were identifies as methionine adenosyltransferase (MAT) involved in ethylene biosynthesis, and JA-responsive proteins (JR1) MAT catalyzes the synthesis of the ethylene precursor, S-adenosylmethionine (AdoMet) and plays an important role in mediating the cross talk between ethylene and NO signaling pathways (Lindermayr et al. 2006). JA has crucial role in regulating many plant processes including mediation of resistance to pathogens (Creelman and Mullet 1997). JA-responsive (JR) genes, including JR1, have been demonstrated to be induced by wounding (Leon et al. 1998). Oh et al. (2005) started with a proteomic comparison of the proteins secreted by Arabidopsis cultured cells in the presence of salicylic acid (SA). Thirteen different proteins that responded to the SA treatment were identified by MALDI-ToF MS. One of them was GDSL LIPASE 1, or GLIP 1, a SA-induced protein. Upon further characterization, it was found to play a role in the defense against the necrotrophic fungus Alternaria brassicola. In another study, a proteomics analysis was carried out to understand the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. About 1,380 protein spots were resolved on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected which are differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection (Zhou et al. 2006).

Beet necrotic yellow vein virus (BNYVV) is a devastating sugar beet pathogen. Resistance is limited and resistance-breaking isolates are becoming problematic. Larson et al. (2008) studied the differential sugar beet protein expression induced by BNYVV- with multidimensional liquid chromatography. Of more than 1,000 protein peaks detected in root extracts, 7.4 and 11% were affected by BNYVV in the resistant and susceptible genotypes, respectively. Using tandem MALDI-TOF-MS, 65 proteins were identified in this study. Proteomic data suggest involvement of systemic resistance components in Rz1-mediated resistance and phytohormones in symptom development. Several proteins affected by BNYVV are classically associated with plant defense, suggesting inducible resistance may contribute to viral disease suppression. These include pathogenesis-related proteins, such as chitinase, protease, glucanase, peroxidase and defensin. Interestingly, induction of these proteins was not always limited to the resistant genotypes. Some oxidative enzymes, which are also known to contribute to plant defense, appear to have similar timing dependent expression. Polyphenol oxidase, a protein responsible for physical barrier development, and toxic compound and ROS production, is more highly and rapidly expressed in the resistant genotype when compared with expression patterns from the susceptible genotype.

A study on rice proteomics was performed to analyse the protein profile after Magnaporthe grisea infection, and was conducted using infected leaf blades fertilized with various levels of nitrogen (Konishi et al. 2001). Rice plants grown with high levels of nitrogen nutrient are more susceptible to infection by the blast fungus (Long et al. 2000). Though, leaf proteins revealed some minor changes when plants grown under different levels of nitrogen were compared, this study failed to establish any direct correlation between nitrogen application and disease resistance (Rakwal and Agrawal 2003). Twelve proteins, including the rice thaumatin-like protein (TLP) (PR-5), were identified with accumulation changes at different levels of nitrogen. Another study of the same interaction was performed by Kim et al. (2003) using rice suspension cultured cells. In this study, twelve proteins were identified, including the rice pathogenesis-related protein class 10 (OsPR-10), isoflavone reductase-like protein (PBZ1), glucosidase and putative receptor-like protein kinase (RLK), which had not been reported previously in suspension-cultured rice cells. The authors, followed with another proteome study using rice leaves, identified eight proteins newly induced or with increased expression (Kim et al. 2004). The identified proteins belonged to several groups of PR proteins, and included two RLKs, two b-1,3-glucanases (Glu1, Glu2), TLP, peroxidase (POX 22.3), PBZ1 and OsPR-10. Lee et al. (2006) investigated rice sheath leaves after infection with this fungus, Rhizoctonia solani and the results revealed six proteins whose relative abundance varied significantly in the resistant and susceptible lines, and 11 additional proteins which were identified in abundance in response of the resistant line only. These proteins have been reported previously to be involved in antifungal activity, signal transduction, energy metabolism, photosynthesis, protein folding and degradation, and antioxidation, indicating a common pathway for both stress and non-stress plant functions.

Using 2-DE, the root protein profiles of M. truncatula were analysed after Aphanomyces euteiches pathogen infection (Colditz et al. 2004). The majority of the induced proteins belonged to the PR-10 family, whereas others corresponded to putative cell wall proteins and enzymes of the phenylpropanoid–isoflavonoid pathway. Another study focused on Zea mays embryos in response to the fungus, Fusarium verticillioides (Campo et al. 2004). The proteins identified included PR proteins, antioxidant enzymes and proteins involved in protein biosynthesis, folding and stabilization.

Phosphoproteomics

Protein phosphorylation plays a key role in signal transduction in plant during defense responses. The importance of phosphorylation in plant basal defense responses is exemplified by the FLS22activated MAP kinase cascade. The phosphorylated proteins are primarily involved in the early steps of signal transduction pathways as demonstrated by Lecourieux-Ouaked et al. (2000) in tobacco by using cryptogein, an elicitor of defense reactions. Kinases are implicated in direct interactions with R protein signaling complexes (RPS5/PBS1) and also in the modification of the key effector protein RIN4. Additional kinase activities are necessary for downstream signaling events. Proteomics not only monitors the steady state level of proteins but also co- and post-translational modifications of proteins. These include not only kinases and phosphatases but also their substrates (Xing et al. 2002).

Lecourieux-Ouaked et al. (2000), using 2-DE, tested the in vivo phosphorylation status of proteins after cryptogein, staurosporine (a kinase inhibitor), and calyculin A (a phosphatase inhibitor) treatments in tobacco cells. Out of about 100 phospholabelled polypeptides, 19 showed increased 32P incorporation after cryptogein treatment and 12 of these depended upon calcium influx. Staurosporine inhibited the phosphorylation induced by cryptogein whereas calyculin A activated the phosphorylation of 18 polypeptides indicating that the phosphorylation of these proteins were activated by certain protein kinases and inhibited by certain phosphatases. These results demonstrate the power of phosphoproteomics to identify key proteins.

In suspension-cultured cells of Arabidopsis, Peck et al. (2000) used 32P pulse-label method in conjunction with 2-DE and MS to identify proteins that are rapidly phosphorylated in response to bacterial and fungal elicitors. One of these proteins, AtPhos43 was found to be phosphorylated within minutes after treatment with flagellin. They also found that phosphorylation of AtPhos43 after flagellin treatment was dependent on FLS2, a receptor-like kinase involved in flagellin perception (Gomez-Gomez and Boller 2000). It has also been found that this protein was phosphorylated in response to both bacterial and fungal elicitors, and related proteins are phosphorylated in other monocot and dicot species (Peck 2003; Peck et al. (2000)).

Jones et al. (2006) describe the application of differential mass tags (iTRAQ) to provide relative quantification of phosphorylated peptides during the early stages in plant pathogen interaction the model plant Arabidopsis thaliana after challenging with three strains of Pseudomonas syringae pv. tomato DC3000 (DC3000). The HR induced by the avrRpm1/RPM1 interaction was compared with basal resistance through examination of responses to the hrpA mutant of DC3000. They identified five proteins which showed reproducible differences between a mock-inoculated control and different bacterial challenges 3 hours post inoculation (hpi), thus identifying proteins. Four of the five proteins, a dehydrin, a putative p23 co-chaperone, heat shock protein 81 and a plastidassociated protein (PAP)/fibrillin, are known to be phosphorylated or have potential phosphorylation sites. One another protein, the large subunit of Rubisco, showed a significant difference between tissue undergoing the hypersensitive response and a basal defence response. This novel study shows the application of iTRAQ to plant–pathogen interactions and the challenge of examining phosphoproteins from intact green leaf tissue, rather than the more commonly used cell culture system.

Conclusion and Future Perspectives

In the post genomic era, proteomics has emerged as an indispensable tool for understanding signaling mechanisms of plant against pathogen, its potential impact in plant pathology, and the study of plant-pathogen interaction. Previously, a limited number of genes involved in infection process had been identified using conventional molecular genetics and biochemical methods. With the advent of proteomics technology, a number of proteins involved in plant defenses have been identified. Phosphoproteomics will continue to play a major role in identifying post-translational modifications and, therefore, have an additional benefit of identifying signaling components that may not be revealed by transcriptome analysis alone. Currently, the major challenges for the plant phosphoproteomics are to identify the relevant phosphorylation sites from the vast majority of phosphopeptides. A high throughput technical advancement, therefore, will be an important development that will help to identify these relevant phosphorylation sites in proteins of interest. Another much needed improvement, highly desired in phosphoproteomics study, is the development of improved and novel enrichment strategies for phosphorylated peptides. In future, the integration of proteomics with genomics, transcriptomics, and metabolomics will play a major role in understanding the plant biology and will uncover many unexpected links within the signaling networks in plants. The continued proteomics advances in unrevealing the molecular mechanisms will lead to a better understanding of plant-pathogen interactions, which may ultimately contribute to the development of novel disease tolerant varieties of agriculturally and economically important crops.