Genes associated with hypersensitive response (HR) in the citrus EST database (CitEST)

Plants are continuously exposed to pathogen attack, but successful infection is rare because they protect themselves against pathogens using a wide range of response mechanisms. One of them is the hypersensitive response (HR), which is a form of cell death often associated with plant resistance to pathogen infection to prevent the spreadsebpg@cnpq.brsebpg@cnpq.br of the potential pathogen from infected to uninfected tissues. Cell death is activated by recognition of pathogen-derived molecules by the resistance (R) gene products, and is associated with the massive accumulation of reactive oxygen species (ROS), salicylic acid (SA), and other pro-death signals such as nitric oxide (NO). The analysis of the citrus EST (CitEST) database revealed the presence of putative genes likely to be involved in HR through their products, like metacaspases, lipoxygenases, phospholipases, pathogenesis-related proteins, glutathione transferases/peroxidases, enzymes involved in the phenylpropanoid pathway and in the formation and detoxification of ROS, as well as those involved in the formation and regulation of ion channels, SA and NO. By analysis of the EST database of Citrus, it was possible to identify several putative genes that code for key enzymes involved in HR triggering and also in plant defense against biotic and abiotic stress.


Introduction
Plant-pathogen interactions involve complex, specific, and dynamic mechanisms.When a plant is resistant to a pathogen (incompatible interaction), its recognition often results in a hypersensitive reaction (HR), a rapid cell death of the plant tissues at the infection site that contributes to the limitation of growth and spread of the pathogen (Gabriel and Rolfe, 1990;Parker and Coleman, 1997;Lee and Hwang, 2005).It is generally believed that recognition of incompatible pathogens, via specific resistance genes encoded by the plant genome, leads to the activation of a number of different signaling mechanisms that initiate at the plasma membrane (Pontier et al., 2002).
The initial events following the recognition of the pathogen elicitor by the plant are calcium influx, alkalinization of the extracellular space, protein kinase activation, production of reactive oxygen species (ROS) and nitric oxide (NO), and transcriptional reprogramming (Dangl and Jones, 2001).The H 2 O 2 produced by the oxidative burst in the dark does not play an important role in the execution of cell death, but an early and massive production of fatty acid hydroperoxides by lipoxygenases has a prominent role in cell death (Montillet et al., 2005).NO and ROS could also contribute to rapid transcriptional activation of a battery of "defense genes" in and surrounding the infected cell.Some of these genes encode peroxidases, glutathione S-transferases, proteinase inhibitors, and various biosynthetic enzymes, such as phenylalanine ammonia lyase (PAL), which is the first enzyme in the phenylpropanoid pathway and is involved in the synthesis of low molecular weight, antimicrobial compounds known as phytoalexins (Klessig et al., 2000).Other defense mechanisms that are activated include biosynthesis of salicylic acid, induction of ethylene biosynthesis, cell-wall strengthening, and lignification (Scheel, 1998;Lam et al., 2001).The oxidative burst leads to a HR and then to the establishment of systemic acquired resistance (SAR) (Rentel et al., 2004).
HR has been a source of interest and controversy since its recognition in 1915; however, in recent years, the combination of molecular genetics, computer-enhanced microscopy, new cytochemical techniques and a better understanding of plant signal transduction and cell biology have helped to unravel this apparently universal plant response (Heath, 2000).
A functional genomics project has been initiated to study the molecular characterization of the main biological and agronomical traits of citrus (Forment et al., 2005) and the present work also tries to help to uncover the genetic machinery of Citrus species.The objective of this work was to identify expressed sequence tags (ESTs) involved in the codification of key enzymes related to the HR in citrus plants.By searching the citrus EST database (CitEST), we describe putative pathways that may lead to HR in citrus plants.

Materials and Methods
In the Citrus EST Sequencing Project (Millennium Project-CNPq), various libraries of citrus cDNA were constructed with tissues from Citrus sinensis, C. reticulata, C. limonia, C. aurantifolia, C. aurantium, C. limettiodes, C. latifolia, C. sunki and Poncirus trifoliata, in different combinations of limiting factors (biotic and abiotic), physiological conditions (adult x juvenile) or tissue (leaf, bark, fruit, root, flower, and seed).The libraries description can be found in Targon et al. (this issue).The CitEST database contains over 181,000 sequenced ESTs (see CitEST: http:// biotecnologia.centrodecitricultura.br),which were used in this work.
Reads with E value < 10 -10 were individually inspected, excluding sequences unrelated to the target enzyme, whereas the remaining sequences were clustered using the CAP3 (Huang and Madan, 1999).In order to analyze phylogeny among CitEST contigs and coding region of the proteins identified in the NCBI database of known HR genes, cladograms were made using ClustalW program (http:// www.ebi.ac.uk/clustalw/).

Results and Discussion
The clusterization of the reads identified using keywords and BLASTX with known HR genes from other plant species in the CitEST database resulted in 227 contigs and 174 singletons.Table 1 shows 18 proteins involved in the HR process identified in the CitEST, including the total number of contigs and singletons obtained.The presence of these proteins permitted inference of a putative pathway that leads to HR in citric plants.
The NCBI database (http://www.ncbi.nlm.nih.gov/) has 184,881 and 28,822 nucleotides sequences of Citrus and Poncirus species, respectively.Searches in this database revealed the presence of two sequences related to glutathione peroxidase, one peroxidase, two superoxide dismutase, five phenylalanine ammonia-lyase, three cinnamic acid 4-hydroxylase, three chalcone synthase, three lipoxygenase, four chitinase and one osmotin in Citrus sequences, while for the Poncirus species there are just one superoxide dismutase, two nitrate reductase and one glutathione transferase.Some HR genes were not identified in this database, such as metacaspase, calcium and potassium channel related proteins, nitrate reductase, isochorismate synthase, chorismate mutase, phospholipase and glutathione transferase, however they were identified in the CitEST database.According to these data, this work would be con-944 Guidetti-Gonzalez et al. sidered the first attempt to catalog the majority of genes related to HR in Citrus and Poncirus species.The identification of resistance (R) genes that permits plants to perceive the pathogen invasion and trigger the ion flux, which culminates in HR, complements this work, and is reported in Guidetti-Gonzalez and Carrer (this issue).Several distinct MAPK (mitogen-activated protein kinases) cascades have been implicated in the regulation of plant disease resistance through signal transduction between perception of the elicitors and the activation of disease resistance genes.These MAPKs are reported in Mehta et al. (this issue).Besides, several pathogenesis related (PR) proteins are known to be expressed in the context of HR and usually act as antifungal factors at the end of the HR signaling cascade.Herein, the expression of two types of PR proteins typically presenting antifungal activity (chitinases and osmotins/thamatins/permeatins) were analyzed within the CiEST database.Campos et al. (this issue) are presenting a complete analysis of citrus PR proteins under biotic and abiotic stress.
The proteins involved in HR, found in the present work, are detailed below:

Metacaspases
Apoptosis in animals is characterized and commonly defined by the activation of a family of cysteine-dependent aspartate-specific proteases, or caspases (Pennell and Lamb, 1997).Caspases can proteolytically activate downstream caspases or cut various cellular substrates, leading to cell death (Utz and Anderson, 2000).Caspase homologues in plants have been reported and called metacaspase (Hoeberichts et al., 2003;Vercammen et al., 2004).In tomato, a metacaspase LeMCA1, is induced during infection of leaves with Botrytis cinerea (Hoeberichts et al., 2003).Searching within the CitEST, we found 18 reads that match with metacaspases of A. thaliana, L. esculentum and O. sativa, which formed three contigs and four singletons (Table S1).One of these singletons, with similarity of 3e-84 to L. esculentum, came from P. trifoliata Citrus tristeza virus-infected leaf library, which suggests involvement in plant disease resistance, as observed in other species (De Jong et al., 2000;Hoeberichts et al., 2003).

Calcium and Potassium channels
Some of the earliest signaling events detected during the HR include ion fluxes, influx of calcium and H + , and efflux of K + (Atkinson et al., 1990).
Calcium, as the divalent cation (Ca 2+ ), is an essential plant nutrient.It is required for structural roles in the cell wall and membranes, for inorganic and organic anion uptake in the vacuole, and as an intracellular messenger in the cytosol.Polarization or depolarization of the membrane voltage opens the gate subunits and allows calcium flow through the cell membrane.When calcium ions enter plant cells through Ca 2+ -permeable ion channels, tonoplast and/or endoplasmic reticulum, cellular responses to a diverse range of developmental cues and environmental challenges are initiated (White, 2000;Sanders et al., 2002).
The search for calcium channel sequences within the CitEST database revealed the presence of 15 reads (Table S1).The clustering identified as potential calcium channels, two contigs and two singletons, contig 1 (C1) was mainly composed of C. sinensis sequences with only one sequence from P. trifoliata and one occurrence of C. reticulata.Contig 2 (C2) was mostly composed of P. trifoliata sequences (two reads) and one read from C. reticulata.Singletons were found in C. limonia and P. trifoliata.
Contig 1 showed 75% identity of deduced protein sequence with the Two-pore channel 1 (TPC1) from Arabidopsis thaliana (Furuichi et al., 2001).Contig 2 showed 61% amino acid identity with the Two-pore calcium channel TPCB1 from N. tabacum.One singleton is 74% identical to the N. tabacum TPC and the other is 50% identical to the tobacco TPC1B.
The wheat channel homologue to TPC1 from A. thaliana was cloned and expressed under abscisic acid (ABA) and various stress treatments indicating that these channels might be the first gate of control for several cell phenomena related to Ca 2+ influx as cell signaling, homeostasis and stress response (Wang et al., 2005).
Potassium is the predominant inorganic ion of plant cells, where it plays a major role in contributing to cellular hydrostatic (turgor) pressure, growth, and responses to the environment (Michard et al., 2005).
The HLM1 gene encodes a member of the CNGC (cyclic nucleotide -gated channel -CNGC4) ion channel family (Maser et al., 2001).The study of this gene product showed that CNGC4 is permeable to both K + and Na + , is activated by cGMP and cAMP, and its expression is induced in response to pathogen infection and some pathogenrelated signals.So HLM1 may constitute a common downstream component of the signaling pathways leading to HR (Balague et al., 2003).In our analyses, we found only one singleton similar to HLM1 (6e-96), the other 15 singletons and 17 contigs showed similarity to other members of the CNGC family (Table S1), possibly with potassium channel activity.Our comparative analysis of the potassium channel revealed its presence in the C. reticulata, C. sinensis, C. latifolia, C. aurantifolia and P. trifoliata libraries.These results were expected because potassium is involved in wide environmental responses and is possibly involved in the citrus HR as well.

Reactive oxygen species
The generation of reactive oxygen species (ROS), such as singlet oxygen, superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals is one of the earliest responses during HR just after ion fluxes (Atkinson et al., 1990).These ROS not only have direct antimicrobial activ-ity (Lamb and Dixon, 1997), but also act as signaling molecules leading to up-or down-regulation of numerous genes involved in plant defense responses such as the induction of defense-related genes and initiation of programmed cell death (PCD) at the site of the attempted infection (Neill et al., 2002;Apel and Hirt, 2004).This localized PCD generates a physical barrier restraining nutrient availability because of the rapid dehydration caused by tissue death (Parker and Coleman, 1997).
Plants have a number of ways of generating extracellular ROS (Bolwell and Wojtaszek, 1997).During the incompatible pathogen interaction, superoxides are produced enzymatically outside the cell and are rapidly converted to H 2 O 2 , which is able to cross the plasma membrane (Melillo et al., 2006).Cell wall-bound peroxidase (POXs) and a plasma membrane-bound NADPH oxidase are considered the major sources for ROS production as defense mechanisms during biotic stresses (Heath, 2000;Kawano, 2003;Melillo et al., 2006), even though there is evidence supporting a differential ROS generation for different stimuli as well (Bolwell, 1999).
The balance of production and detoxification of ROS is essential for aerobic life (Apel and Hirt, 2004) and the key enzymes or enzyme families involved in the production, as well as scavenging of ROS and induction of HR in plants are nicotinamide-adenine dinucleotide phosphate [NAD(P)H] oxidase, peroxidases and superoxide dismutases.Superoxide radicals generated by the one-electron reduction of molecular oxygen are rapidly converted within the chloroplast to hydrogen peroxide by CuZn-superoxide dismutase (Asada, 1999).The superoxide dismutases (SODs) are extremely effective antioxidant plant enzymes (Skinner and Baek, 2006), since they dismutate two superoxide anions into H 2 O 2 and O 2 (Fridovich, 1991).
Three contigs and three singletons harboring NAD(P)H oxidase gene sequences were obtained from the CitEST database.The three contigs comprised 16, 4 and 5 reads, respectively.They were assembled with ESTs from both C. sinensis and P. trifoliata libraries.However contig 2 (C2) and contig 3 (C3) presented only reads from the leaf libraries, contig 1 (C1), with the largest number of reads, comprised sequences from fruit tissue as well.The three contigs contained partial gene sequences and exhibited similarity with NADPH oxidase protein from either Nicotiana sp. or A. thaliana as best BLASTX hits (Table S1).
Several interesting inferences could be drawn from these sequences.Considering that glutathione S-transferase/peroxidase comprises a large number of reads, which are from all of the seven Citrus and Poncirus species and also the six tissues available at the CitEST, we can infer that the expression of these transcripts are constitutive not only in the genus Citrus but, likely, in other related rutaceae.
However, in a few cases, the specificity of the contigs is evident, with some particular conditions leading to the clustering of similar sequences in well-defined contigs.One of the contigs (C63) was composed of three C. limonia sequences, all of them were from the library constructed from roots undergoing water stress, suggesting that this transcript may code for a peroxidase involved in drought response.Interestingly, three contigs (C22, C64 and C65) were composed by reads from a library constructed from C. sinensis symptomatic for citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, one of the most devastating diseases of the crop, indicating that peroxidases may be involved in the response of citrus to this particular biotic stress.
The multicopy pattern of superoxide dismutase (SOD) gene observed in various hosts was also found within the CitEST database, which yielded 19 contigs and 16 singletons with similarity to translated SOD sequences (Table S1).
As in the peroxidases, at least one contig harbored 43 sequences from most of the species and tissues from where the libraries were constructed.Interestingly, no SOD gene was found to be expressed in the flower tissue, but that might be due to the relatively low number of reads of this library (4,330) compared to the others (Targon et al., this issue).On the other hand, some contigs comprised reads from one or few species.While three contigs (C3, C18 and C19) were formed of only Poncirus sp.reads, the other 11 were formed of only Citrus sp.sequences.Within these latter, some harbored sequences from a particular species (two contigs from C. reticulata alone, five from C. sinensis, and one from C. latifolia), suggest high specificity of these transcripts.
Likewise the peroxidases, but on a lower level, it was possible to determine some tissue-specificity of the SOD transcripts.At least six contigs (C3, C4, C12, C13, C18, and C19) harbored sequences only from the leaf libraries, while the other three (contigs C7, C9, and C16) were formed solely by reads from the fruit libraries.Furthermore, few contigs comprised reads from a particular condition.One of them (contig C12), of great interest, was composed of 39 reads from both the C. sinensis and C. reticulata leaf libraries infected with X. fastidiosa.This transcript was similar to a Cu/Zn superoxide dismutase from C. sinensis (CAA03881.1)and, since the two species are susceptible and resistant to CVC, respectively, it is likely that such transcript is not involved in disease resistance, but in the response to this specific biotic stress.

Nitric oxide
Nitric oxide (NO) has well-known biological functions in mammals, but it was only recently recognized as a signal compound in plants (Zhao et al., 2005).In fact, it is now known that NO plays an important role in diverse physiological processes in plants (Grun et al., 2006).The Avr factors from pathogens stimulate NO production, which promotes disease resistance in plants, collaborating with ROS in the oxidative burst (Delledonne et al., 1998).The relative level of NO and H 2 O 2 appears to be very important to HR-associated cell death in soybean cells (Delledonne et al., 2001) and it was observed that only the simultaneous increase of NO and H 2 O 2 in tobacco cells induced typical programmed cell death (de Pinto et al., 2002).
Two potential enzymatic sources of NO in plants are NO synthase (NOS) and nitrate reductase (NR).NOS is a family of well characterized enzymes in mammalian cells (Desikan et al., 2002) that was found in several plants (Neill et al., 2002), and serves as a signal for plant growth, development, and defense (Delledonne et al., 1998;Klessig et al., 2000;Neill et al., 2002).NR is a central enzyme of nitrogen assimilation in plants (Lea, 1999;Kaiser et al., 2002), and is induced in potato tubers treated with either Phytophthora infestans or an elicitor derived from this oomycete pathogen (Yamamoto et al., 2003).
Despite the large number of citrus ESTs sequenced in the CitEST Project, we did not find reads similar to NOS, which suggests that the NO synthesis in these plants can be preferentially performed by NR.Within the CitEST database, three contigs and one singleton similar to plant nitrate reductase were assembled (Table S1).One of these contigs (C10), similar to Petunia x hybrida and also Tilia platyphyllos (e-values = 0.0) nitrate reductase, seems to be expressed especially within non-infected leaf tissue from C. reticulata and P. trifoliata, while the unique read from bark P. trifoliata is from Phytophthora-infected library.These results suggest that this contig has species-and tissue-specific expression, showing that the bark tissue of P. trifoliata may be involved in the production of NO for plant defense, as with potato tubers infected with P. infestans (Yamamoto et al., 2003).
Another contig (C11), similar to NR from Tilia platyphyllos (2e-152) presents reads solely from C. reticulata and a tendency of expression in Xylella fastidiosa-infected leaves (Figure 1a).This specific expression pattern suggests that this protein is important to plant defense specifically in C. reticulata.The third contig (C7) did not exhibit a species-specific expression, but comprised mainly sequences from the C. reticulata and C. sinensis leaf libraries infected with X. fastidiosa, indicating that this transcript may be involved in the response to this biotic stress.Figure 2 shows the estimate of a phylogeny among contigs C7, C10 and C11, with nitrate reductase from Petunia x hybrida (gi|294113), Tilia platyphyllos (gi|24474445) and Ricinus communis (gi|11119240).As expected, it was observed that Contigs C10 and C11 are more closely related to each other and are related with Tilia platyphyllos and Petunia x hybrida, and C7 is more closely related to Ricinus communis.

Phenylpropanoid pathway
In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, and flavonoids (Hwang et al., 2003).
In the plant phenylpropanoid pathway, chorismate mutase catalyses the reaction that forms phenylalanine, and the enzyme phenylalanine ammonia-lyase (PAL) deaminates phenylalanine to yield cinnamic acid (Gozzo, 2003;Kaneko et al., 2003).Several other enzymes are present in the phenylpropanoid pathway, with cinnamate 4-hydroxylase (C4H)-the first cytochrome P450 upstream in this pathway -leading to the synthesis of a variety of compounds including lignin monomers, flavonoids, and furocoumarins (Morant et al., 2003;Gravot et al., 2004).Chalcone synthase (CHS) is an enzyme that catalyses the reaction that leads to the production of flavonoids and phytoalexins (Yang et al., 2002).The final substance produced by this pathway (4,2', 4', 6'-tetrahydroxychalcone) serves as the backbone for the production of a wide array of flavonoid compounds that function in such diverse roles as flower pigmentation, UV protection, signaling, male fertility, and defense against microbial pathogens (Winkel-Shirley, 2001).
Within the CitEST database, we found five contigs and four singletons similar to chorismate mutase (Table S1), some of them showed specific patterns of expression.The contig 4 (C4) that is similar to A. thaliana chorismate mutase (accession number AAC73018) showed specific expression in the C. sinensis fruit, whereas the contig 6 (C6) (1e-67) (similar to accession number AAM61395) showed expression in C. reticulata fruits and in C. sinensis X. fastidiosa-infected leaves (Figure 1b).The other three contigs did not show an expression pattern, presenting reads from different libraries.Figure 3 shows a phylogeny analysis among contigs C4, C5, C6 and C7, and other chorismate mutase sequences from NCBI, showing coherence between contigs and the best hit in BLAST search (Table S1).
Four contigs and seven singletons were found in the search for phenylalanine ammonia-lyase (PAL) within the CitEST database (Table S1).The contig 10 presents high similarity (2e-107) with PAL from Nerium oleander and was expressed solely in P. trifoliata Phytophthora-infected bark (two reads), suggesting its importance in specific defense of this species.Another contig (C8), comprising sequences with similarity to that from C. limon, comprised 20 reads from different libraries.In C. sinensis, as in C. reticulata, reads showed a specific pattern of expression within X. fastidiosa-infected leaf libraries.However, PAL expression was also found in C. reticulata within the healthy fruit library.The contigs C3 (65 reads) and C9 (four reads) were similar to PAL from Citrus clementina x Citrus reticulata and Jatropha curcas, respectively, although they were highly similar to PAL (e-values = 0.0), they did not show any specific expression pattern, since they were formed by reads from a variety of libraries.
In a study with Valencia sweet orange, two cinnamic acid 4-hydroxylase (C4H) cDNAs were cloned and showed differential expression regulation (Betz et al., 2001).One of the cDNAs, C4H2, appeared to be expressed constitutively and seemed to play the role of a `housekeeping' gene in the phenylpropanoid pathway.Conversely, expression studies showed C4H1 to be strongly wound-inducible, but even in wounded tissue it reached much lower levels of mRNA than C4H2 (Betz et al., 2001).Within the CitEST, we found three contigs and two singletons related to C4H2 (Table S1).One of these contigs (C3) was assembled from 46 reads and showed 100% of identity with C4H2, for which the majority reads originated from C. sinensis and C. reticulata in different stages of fruit development.Contig 1 (C1) showed 91% of identity with C4H2 and was formed by 11 reads, mostly from C. sinensis (10 reads) in the early stages of fruit development and one read from the P. trifoliata Citrus tristeza virus-infected library.These results suggest a possible pattern of expression of this protein in the fruits of C. sinensis and C. reticulata.Moreover, a possible involvement in plant defense against pathogen in P. trifoliata cannot be excluded.
In contig 2 (C2), we observed 81% of amino acid identity with Medicago sativa C4H protein (Table S1), in agreement with its pattern of expression during fruit ripening (Fahrendorf and Dixon, 1993), since the two reads of this contig were originated from the C. sinensis fruit libraries.Figure 4 shows the phylogeny pattern among contigs C1, C2 and C3, and cinnamic acid 4-hydroxylase from Citrus sinensis (AAF66066) and Medicago sativa (gi|166372); coherence can be observed with the best hit in BLAST search and the phylogenetic relationship.
Two cDNA clones encoding chalcone synthase (CHS) were isolated (CitCHS1 and CitCHS2) from citrus (Moriguchi et al., 1999).The accumulation of CitCHS2 mRNA was notably induced by embryogenesis but CitCHS1 mRNA was not.There was no detectable accumulation of flavonoid in the undifferentiated calli, but flavonoid accumulated after the morphological changes to embryoids.These results indicate that two CHS genes differentially expressed during citrus somatic embryogenesis and CitCHS2 may regulate the accumulation of flavonoids in citrus cell cultures (Moriguchi et al., 1999).Within CitEST, we found four contigs and 12 singletons related to chalcone synthase; among them, three contigs and eight singletons related to CitCHS2.Contig 3 (C3) is formed by 75 reads that mostly came from C. sinensis fruits in the early stages of development and is 100% identical to the most homologous BLAST hit (C.sinensis).Contig 4 (C4) was composed of 54 reads mostly from C. reticulata in the early stages of the fruit development and shares 92% identity with C. sinensis CHS.Finally, another contig (C1) was formed by nine C. sinensis reads and presented a tendency of expression in early stages of X. fastidiosa-infected leaves (Figure 1c), and was 79% identical to C. sinensis CHS from NCBI.The singletons related to CHS came especially from C. sinensis and C. reticulata fruit libraries.Figure 5 shows the expected phylogeny relationship between these contigs and chalcone synthase from Citrus sinensis (BAA81664) and Betula pendula (CAA71904).
These results indicate a possible presence of at least two CHS genes in C. sinensis that are expressed during fruit development as shown in other species (Aharoni and O'Connell, 2002;Kumar and Ellis, 2003).In addition, other CHSs may be involved in citrus response to pathogens.For more information of flavonoid biosynthesis, see Lucheta et al. (this issue).

Salicylic acid
The involvement of salicylic acid (SA) as a signal molecule for plant defense has been extensively studied (Shah and Klessig, 1999;Shah, 2003;Stout et al., 2006).SA quickly accumulates at the site of infection during pathogen attack and plant HR, and it spreads to other parts of the plant to induce a wide range of defense responses (Zhao et al., 2005).Besides inducing PR protein genes, SA is assumed to act before the divergence of the two metabolic branches leading to the phenylpropanoid pathway and the activation of the oxidative burst, respectively.In this way, SA amplifies its own synthesis, which, in addition, is stimulated by H 2 O 2 (Gozzo, 2003;Zhao et al., 2005).
Previous work has suggested that plants synthesize SA from phenylalanine utilizing chorismate mutase (Gozzo, 2003).However, evidence showed that, in the chloroplast of Arabidopsis, SA is synthesized from isochorismate catalysed by isochorismate synthase.This route, typical of bacteria, seems to be required even in other plants to produce SA for defense against pathogens (Métraux, 2002;Gozzo, 2003).
Although evidence from tobacco and potato would indicate that salicylic acid in plants is derived from phenylalanine via benzoic acid, catalysed by benzoic acid 2-hydroxylase (Yalpani et al., 1993;Coquoz et al., 1998) in citrus SA, formation seems to occur via the isochorismate route since the benzoic acid 2-hydroxylase was not found within the CitEST database.
Analysis within CitEST resulted in two contigs and one singleton similar to isochorismate synthases from M.  truncatula, Capsicum annuum and Catharanthus roseus (Table S1).Only one contig (C2), similar to M. truncatula isochorismate synthase, showed a specific pattern of expression, presenting a tendency to be expressed in C. sinensis leaves, in the early stage of X. fastidiosa infection (Figure 1d).This result may suggest that this contig was formed by a transcript that codes for a protein involved in the production of SA upon plant infection by a pathogen.

Lipoxygenases
Lipoxygenases (LOXs) constitute a family of enzymes that deoxygenate polyunsaturated fatty acids containing cis, cis-1,4-pentadiene structure.The hydroperoxides produced by the LOX reaction are converted into a series of compounds involved in several physiological processes including growth, fruit ripening and response to biotic and abiotic stresses (Hildebrand et al., 1989;Gardner, 1991;Siedow, 1991;Brash, 1999).The most common substrates for plant LOXs are linoleic and linolenic acids, which are important constituents of plant membrane phospholipids.Plant LOXs are classified according to their positional specificity of linoleic or linolenic acid oxygenation, which occurs either at the C-9 or at the C-13 of the hydrocarbon backbone of the fatty acid.Therefore, these LOXs are often referred to as 9-LOXs or 13-LOXs.
Regarding sequence similarity, plant LOXs can be classified into two gene subfamilies.Enzymes with no transit peptide share a similarity higher than 75% with one another and are designated type 1-LOXs.The other subfamily, called type 2-LOX, contains a putative chloroplast transit peptide sequence and shares a sequence similarity of approximately 35% with each other (Feussner and Wasternack, 2002).In C. jambhiri, a LOX containing typical features of a transit peptide for chloroplastic targeting has been identified and expressed upon wounding and inoculation with nonpathogenic isolates of Alternaria alternata (Gomi et al., 2002b).
The analysis of the CitEST database revealed 323 sequences related to lipoxygenases, which formed 16 contigs and 18 singletons.The best hit in a BLASTX search as well as the homologous organism are shown in Table S1.The data available from the CitEST database does not allow us to classify the LOXs found and further studies are necessary to characterize the different citrus LOXs.Moreover, most contigs and singletons were incomplete and therefore a classification of the LOXs encountered, based solely on partial sequence comparison, may be premature.The three largest contigs obtained (C3, C7 and C11) revealed deduced amino acid sequences of 921, 945 and 900 amino acids, respectively.The comparison of these sequences with those of the 8 other LOXs showed high similarity to the citrus LOXs (Table S1) revealed that the sequence of contig 7 presented an exclusive amino acid insertion sequence at po-sitions 574 to 606.Contig 7 was formed by reads from healthy and X. fastidiosa-infected leaves of C. reticulata as well as X. fastidiosa-infected leaves of C. sinensis and showed 73% identity with lox1 of Sesbania rostrata.Contig 3 showed 71% identity with a lox2 of Zantedeschia aethiopica and contig 11 showed 78% identity with a lox of C. jambhiri.Contigs 3 and 11 represent complete sequences of the lox gene.Some 9-LOXs have been associated to plant resistance and lipid peroxidation, which is involved in membrane damage and HR (Rance et al., 1998, Kolomiets et al., 2000;Jalloul et al., 2002).The biosynthesis of oxygenated unsaturated fatty acids via LOX pathway may be important for plant defense against pathogen attack (Peng et al., 1994).Most of the reads showing similarity to LOXs were obtained from pathogen-infected or fruit libraries.LOXs were highly expressed in the early stages of X. fastidiosainfected libraries of sweet orange (C.sinensis) and mandarin (C.reticulata) (up to 30 days after inoculation-DAI).Some ESTs associated with lipoxygenase were also observed in C. reticulata up to 60 DAI.Several studies have reported that LOX activity in plants is higher and more rapid upon infection of avirulent pathogens as compared to virulent pathogens (Ocampo et al., 1986;Yamamoto and Tani, 1986;Siedow, 1991;Melan et al., 1993).C. reticulata is resistant to X. fastidiosa and, as expected, a higher expression of LOX was observed in X. fastidiosa-infected mandarin plants.LOX was also expressed in C. sinensis and P. trifoliata libraries infected with Citrus leprosis virus (CiLV) and Citrus tristeza virus (CTV), respectively.P. trifoliata is resistant to CTV and therefore the expression of LOX in this interaction was expected.
Several fruit ESTs associated with LOX were also identified within the CitEST database.The role of LOX in fruit development has been well documented (Griffiths et al., 1999;Chen et al., 2004a).LOXs play an important role in the production of volatile molecules that can influence flavor and aroma of many plant products (Robinson et al., 1995).LOXs can also alter shelf life and organoleptical and nutritional characteristics of plant products (Robinson et al., 1995).These are important features for the citrus industry, since fruit quality is essential for juice production as well as for the commercialization of fresh fruits.
Three C. limonia EST sequences related to LOX were also expressed during water stress.Membranes are main targets of degradative processes induced by drought and lipid peroxidation is one of the most important causes of cell deterioration, resulting in changes in the composition of fatty acids that affect the structural and functional properties of cell membranes (Smirnoff, 1993;Asada, 1999).The expression of LOXs during drought has been reported (Gigon et al., 2004;Sofo et al., 2004); however, little is known about the relationship between drought stress and LOX activity.Further studies are necessary to better understand the function of citrus LOXs in drought response.Guidetti-Gonzalez et al.

Phospholipases
Phospholipases (PLs) in plants are known to be involved in several cellular responses, including plant growth, development, and responses to stress and defense.PLs play a pivotal role in the plant response via lipids pathway.The superfamily is involved in processes such as cytoskeletal rearrangement, membrane trafficking and degradation (Meijer and Munnik, 2003;Wang, 2004).Several studies have associated PL activity to stress response and defense, and the involvement of PLs and other enzymes in cell death has also been reported (Spiteller, 2003;Wang, 2005).
Plant PLs are classified according to their site of hydrolysis on phospholipids as phospholipase A 1 (PLA 1 ), phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD).
The analysis within the CitEST database revealed sequences related to PLs.The assembling of the reads (E-value < -10) provided 9 contigs and 12 singletons with E-value < -10.The best hit in a BLASTX search as well as the homologous organism are shown in Table S1.
The phospholipase A (PLA) activity increased rapidly and systemically in response to wounding by attack of herbivores in leaves of tomato plants (Narvaez-Vasquez et al., 1999).Multiple secretory-phospholipase A 2 (sPLA 2 ) genes have been also identified in plants (Lee et al., 2005) and within the CitEST database, four reads were identified with high similarity to PLA 2 .The clusterization provided only one contig that was confirmed to be 75% identical to a rice PLA 2 form, and this contig was formed by reads from healthy C. sinensis and C. aurantifolia libraries.This PLA 2 contig contains twelve conserved cysteine residues and sequences that are likely to represent the Ca(2+)-binding loop and active-site motif, which are characteristic of animal PLA 2 s.In the CitEST project, the PLA 2 identified was sequenced from bark tissue, probably indicating a secretory possibility and a defense response in plant to a variety of stimuli including biotic and abiotic stresses (Lee et al., 2005).
Under the control of cell surface receptors, the phosphoinositide-specific PLC isozymes hydrolyze the highly phosphorylated lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], generating two intracellular products: inositol 1,4,5-trisphosphate (InsP 3 ), which is an universal calcium-mobilizing second messenger; and diacylglycerol, which is an activator of protein kinase C (Rebecchi and Pentyala, 2000).Evidence suggests that InsP 3 may be involved in the signal transduction pathway for the development of the HR of C. lemon against A. alternata (Ortega and Perez, 2001).A search within the CitEST identified reads homologous to PLC genes.Clustering of these reads resulted in five contigs and nine singletons.The similarity among the deduced PLC proteins found ranged from 59% to 73% identity to PLC from A. thaliana, O. sativa, Nicotiana rustica, and Glycine max.The most representa-tive group for PLC was contig 1, which was formed by reads from the libraries, bark and leaves of healthy C. sinensis and from C. sinensis X. fastidiosa-infected leaves.
PLD mediates the hydrolysis of membrane lipids and diacylglycerol-kinase-mediated phosphorylation of diacylglycerol (Wang, 2002).The expression of several PLD genes has been extensively reported in plant defense induced by the elicitor treatment as well as pathogen infection (Laxalt et al., 2001;De Torres et al., 2002).The PLD group was present in 14 reads clustered into three contigs within CitEST.The PLD genes identified were classified as different isoforms related to the A. thaliana (AAP42742.1)PLD protein.The CitEST PLDs were 80 to 83% identical to their orthologues.The largest PLD was also formed by reads from healthy C. sinensis.
Analysis of the CitEST database revealed the presence of several citrus PLs; however, since most contigs and singletons are incomplete, it was not possible to unequivocally classify them within the family.Further studies are necessary to better understand the genetic and biochemical heterogeneity of PLs and to relate the function of citrus PLs in HR.

Glutathione transferases and Glutathione peroxidases
Plant tissues contain several ROS scavenging enzymes (like glutathione peroxidase) and detoxifying lipid peroxidation products (like glutathione S-transferases and phospholipid-hydroperoxide glutathione peroxidase) (Chen et al., 2004b), which protect cells from ROS and damage from stress conditions.
By using an in-silico analysis of the CitEST database, we have identified 36 contigs and four singletons related to glutathione transferase (GST) genes (Table S1).Taking into consideration that the CitEST database was constructed based on a number of ESTs produced during different stages of the development and challenges, it is obvious that, as seen in other plant species (Sappl et al., 2004), several mechanisms are acting to induce citrus GSTs.
By combining keyword and BLASTX searching within the CitEST database with the sequence from public database that showed the best match with citrus sequences, we found full-length genes with high homology to GST 22 from soybean (McGonigle et al., 2000).Whether this putative gene plays a role in stress tolerance remains to be experimentally demonstrated in plant species, including citrus.In addition, we found full-length copies of LeGST-T3, a GST protein from tomato that was found to protect yeast cells from H 2 O 2 -induced oxidative stress (Kilili et al., 2004).This indicates that the putative citrus genes could play a role in cell protection from pro-oxidant-induced cell death.
Glutathione peroxidases (GPX) are enzymes that reduce organic hydroperoxides produced during oxidative stress.The over-expression of a GPX inhibited oxidative HR in citrus stress -induced PCD in tomato (Chen et al., 2004b).It is strongly suggested that specific GPXs, such as Phospholipid Hydroperoxide GPX (PHGPX), play a specific role in ROS; however, their specific role in plants remains to be elucidated (Milla et al., 2003;Chen et al., 2004b).PHGPX functions, in the removal of phospholipid hydroperoxides, are generated as products of lipoxygenase catalysed oxygenation of fatty acids (Ursini et al., 1985).
The analysis of the CitEST database also revealed sequences related to GPXs, which formed 10 contigs and 2 singletons (Table S1).The analysis of these sequences shows GPX and PHGPX similar to the ones found in orange, pea, Momordica charantia, chickpea, and Arabidopsis.
At least two copies of a gene found in this study code for the PHGPX that was experimentally found to be responsible for salt-stress signaling in citrus (Holland et al., 1993;Avsian-Kretchmer et al., 2004).
As compared to other stress-related genes found in this broad-scale search, an observation that emerged was the proportion of genes that code for GPX in citrus.On the basis of these results, it is suggested that GPX is an enzyme that shows a low number of isoforms and/or copies in the genome of citrus and, probably, plays a role in a condition not strongly induced in this study.

Pathogenesis-related proteins
The induction of PR proteins is triggered stereotypically in plants by infection with fungal, bacterial, and viral pathogens, and is part of a more general response to biotic and abiotic stress (Datta and Muthukrishnan, 1999).PR proteins comprise a group of 17 protein classes (Christensen et al., 2002), most of which present antifungal activity, such as the PR-3, PR-4, PR-8 and PR-11 proteins (also known as chitinases) and the PR-5 proteins (also known as osmotins, permatins or thaumatins).
The chitinases, which are vacuolar PR proteins, act in concert to defend against a specific class of pathogens, fungi (Wirdnam et al., 2004).Moreover, there is also an indication that chitinases may act as lysozymes, by degrading bacterial cell walls (Tiffin, 2004).This class of proteins can be induced by ethylene treatment and in association with HR (Leubner-Metzger and Meins, 1999).
The analysis of the CitEST database revealed several reads related to chitinases, which formed 29 contigs and 24 singletons (Table S1).The analysis of these sequences shows various acidic classes I and II chitinases that match with citrus libraries from elsewhere.In addition, we found a number of sequences that relate to chitinase, in general, from other plant species such as A. thaliana, Tulipa bakeri, Hevea brasiliensis, Gossypium hirsutum, and N. tabacum.In rough lemon (C.jambhiri), the expression of the two acidic chitinases (I and II) was not constitutive and accumulated in leaves two hours after induction by wounding or inoculation with non-pathogenic isolates of A. alternata (Gomi et al., 2002a).Our results reveal that a number of acidic class I and II chitinases are expressed in citrus.However, it remains unclear whether the citrus chitinases found in our study refer to isoforms of the same gene.
The PR-5 proteins are expressed in response to abscisic acid, ethylene, auxin, salinity, lack of water, cold, UV light, wounding, viral, fungal and oomycetal infection (Nelson et al., 1992;Zhu et al., 1995;Sato et al., 1996).Within the CiEST database, we found 63 unique reads related to PR-5-like proteins, clustered into 10 contigs and four singletons (Table S1).These singletons encode a 219amino acid ORF homologous to a sunflower (H.annuus) PR-5-like protein, a 190-amino acid ORF homologous to a turnip (B.rapa) PR-5-like protein, as well as a 182-and a 280-amino acid ORF, both homologous to a tobacco (N.tabacum) PR-5-like protein (Table S1).These singletons were originated from fruit libraries, from CTV-infected leaf libraries or from X. fastidiosa-infected leaf libraries, either from C. reticulata, C. sinensis or from P. trifoliata origin.The 10 PR-5-like contigs were assembled from two up to 13 reads each, mainly from the fruit libraries, but also from seed, bark, and flower libraries.Despite the fact that PR-5 proteins are expressed also in response to biotic stress, there were only five reads composing contigs which were originated from leaves infected either with X. fastidiosa or with CTV.Moreover, the majority of the PR-5-like reads forming contigs were from C. sinensis origin, secondarily from C. reticulata or P. trifoliata and seldomly from C. aurantifolia or C. aurantium.Two contigs homologous to tomato PR-5-like proteins, one homologous to an Arabidopsis PR-5-like protein (accession NP_173261), one homologous to a cultivated apple PR-5like protein and one homologous to a grape PR-5-like protein presented complete coding sequences when aligned with the best hit in BLASTX search.As expected, a CitEST contig homologous to a C. sinensis PR-5-like protein (accession AAC02549; Table S1) presented high amino acid identity (97%).The 14 PR-5-like clusters found within the CitEST database, i.e., 10 contigs and the four singletons, were considerably similar (from 73% to 97% similarity) to their corresponding most homologous amino acid sequences (Table S1).
To conclude, the CitEST database is considerably representative of citrus PR-5-like sequences, expressed in various biological situations, tissues and citrus species.Unexpectedly, within the CitEST database, no PR-5-like sequences associated with drought stress or with infection by Phytophthora parasitica were found.Surprisingly, there were only seven reads found, out of 63 reads in total, which originated either from X. fastidiosa infected-leaves or from CTV infected-leaves.Although most of the PR-5-like reads found within the CitEST database are associated with abiotic stress and plant development processes, these sequences potentially encode PR-5 proteins involved also in citrus HR.Therefore, the possible role of the CitEST PR-5-like clusters in HR must be demonstrated experimentally.Further investigation is also necessary to clone complete citrus PR-5-like sequences, to validate their biological function and to better characterize their expression patterns under biotic and abiotic stress conditions.

Concluding Remarks
Despite a large number of potential pathogens that attempt to infect cultivated plants, disease is not the rule.
Plants have an arsenal of mechanisms to defend themselves against pathogen invasion.One of these strategies is the expression of HR genes, elicited when resistant hosts are infected by incompatible races of a pathogen.It is characterized by localized cell death at the site of attempted infection, generating a physical barrier that restricts nutrient availability to the pathogen and prevents the dispersion of the pathogen to uninfected tissues.HR has been investigated extensively, and the increase of genomic sequences available has made it possible to indicate the existence of homologous genes in all plant species by in silico analysis.Moreover, the use of tools to search for sequence similarities at the amino acid level can reveal the overall patterns of gene network related to HR responses.In the present work, we tried to uncover the pathways that may lead to HR in citrus, based on the CitEST database.We found putative deduced proteins that can be important for HR and thus we have shown the putative pathways involved in this process.However, further studies are necessary to confirm the functions of these proteins and to better understand the genetic and biochemical network in citrus HR.To our knowledge, this is the first genome study that attempts to identify genes involved in HR in citrus plants.Functional characterization of them may contribute for the development of molecular tools to overcome economically important citrus diseases.

Table 1 -
Classes of HR proteins identified in CitEST database.

Table S1 -
Description of citrus ESTs enconding proteins involved in HR.Between parentheses are represented the contigs (C) commented in the Results and Discussion.As search for different proteins had been made in separate archives, different contigs (representing different proteins) can have the same number. *