Differentially expressed stress-related genes in the compatible citrus-Citrus leprosis virus interaction

Leprosis, caused by Citrus leprosis virus, cytoplasmic type (CiLV-C), is the main viral disease in the Brazilian citrus industry. This occurs because of the widespread source of inoculum and the year-round presence of the vector, the tenuipalpid mite Brevipalpus phoenicis, in citrus plants. In addition, while some Citrus species are resistant to CiLV-C, C. sinensis, the main cultivated species in the country, is extremely susceptible to the disease. The main objective of this work was to identify genes in C. sinensis cv. Pêra plants that were differentially expressed after the host was challenged with CiLV-C. In order to accomplish that, cDNA libraries were constructed from healthy and CiLV-inoculated sweet orange leaves. Two hundred and fifty-four genes were found to differ significantly in terms of expression, with 193 of them induced and 61 repressed after inoculation. Here we discuss the possible roles of a sub-set of these genes involved in metabolism, energy, signaling and cell rescue, defense and virulence, and indicate which kind of response may take place in the initial steps of the disease. Although the symptoms induced by CiLV-C in its compatible interaction with sweet orange resemble those of hypersensitive response (HR) in incompatible interactions, our data indicate that, apparently, the manifestation of leprosis symptoms should not be considered HR.


Introduction
The citrus industry is one of most important agribusinesses in Brazil, with exports of concentrated juice representing 80% of the world market (Boteon and Neves, 2005).However, Brazilian average productivity is very low due to drought problems, associated with various pathogens that affect the crop.Citrus leprosis virus, cytoplasmic type (CiLV-C) has been considered the main citrus viral pathogen in Brazil for the last several years.Its importance has significantly increased in other countries as well, since it is now present in most South and Central American countries, and has reached the South of Mexico (Bastianel et al., 2006a).
Even though the disease has been known for decades, only recently important progress has been made in understanding its etiology and interactions.Leprosis is a very complex pathosystem involving the causal virus, the Brevipalpus mite vector, the susceptible Citrus plant and, possibly, an endosymbiont (Novelli et al., 2005).
CiLV-C presents morphological -but no sequencesimilarity with rhabdoviruses (Locali-Fabris et al., 2006;Pascon et al., 2006).It is transmitted by a tenuipalpid mite, and induces only local lesions in its susceptible hosts (Bastianel et al., 2006a).Because of its unique properties, it has been proposed as a type member of a new virus genus named Cilevirus (Locali-Fabris et al., 2006).
Typical leprosis symptoms include chlorotic and/ or necrotic local lesions in stems, fruits, and leaves of susceptible hosts.These lesions can coalesce, cause dieback and even the death of young plants, but the virus does not invade the plant systemically (Bastianel et al., 2006a).For years, it has been known that sweet oranges (Citrus sinensis) are more susceptible to leprosis than mandarins (C.reshni, C. reticulata), lemons (C.limon), limes (C.aurantifolia), and citrus hybrids (Bastianel et al., 2006a).However, only recently the genetics of the disease is starting to be understood.Bastianel et al. (2006b) have suggested that one or few major genes are related to leprosis resistance in Murcott tangor (a hybrid between sweet orange and mandarin).
It is known that plants respond in a complex way to biotic and abiotic stresses.However, few studies have tried to elucidate their response to complex pathosystems, especially when viral agents are involved.Compatible responses of sweet oranges to CiLV-C macroscopically resemble those of a hypersensitive response (HR) observed in resistant genotypes to some bacteria and viruses, but with a significant delay in symptom appearance.While HR is often visible after several hours or few days from pathogen challenge, symptoms of leprosis in citrus are evident only weeks after CiLV-C inoculation (Bastianel et al., 2006a).It is not known, however, whether or not the response to CiLV-C resembles that of incompatible interactions at the molecular level.
Information derived from EST (expressed sequence tags) sequencing can help unravel the reason why some citrus species or hybrids are resistant or tolerant to the disease while others are susceptible, through their differential gene expression profiles.Also, through the comparison of differential gene expression between susceptible plants prior and after inoculation, it is possible to draw conclusions regarding how a particular species or variety behaves in response to the virus.
This work is the first attempt to better understand the initial steps that will lead to the appearance of leprosis symptoms in citrus through the comparison of gene expression patterns of C. sinensis inoculated and non-inoculated with CiLV-C.
Several transcripts involved in stress response were up-or down-regulated in CiLV-C challenged plants when in silico compared to the non-inoculated ones, and they provide valuable information for understanding the initial steps of infection in this compatible interaction.However, other categories of genes were found as well.A high number of transcripts involved in plant metabolism was found to be differentially expressed in our libraries.This is not a surprise, since virus infections frequently have dramatic effects on plant metabolism, affecting a variety of essential cellular processes such as synthesis of nucleic acids and proteins, lipids and carbohydrates metabolism, and hormone and enzyme functions (Hull, 2002).Nevertheless, some of the findings differed from those reported either for systemic viral infections or viral-induced HR, and will be discussed below.

Construction of cDNA libraries
Two cDNA libraries were constructed from mRNA isolated from leaves of Pêra sweet orange (C.sinensis L. Osbeck) grafted on Rangpur lime (C.limonia Osbeck).One of them was constructed from non-inoculated leaves and served as a mock control library.The other one was prepared from leaves collected 48 h after CiLV-C inoculation by viruliferous Brevipalpus phoenicis mites.The presence of the virus in the mites was confirmed in a sub-set of vector population by RT-PCR as described by Locali et al. (2003) with modifications (Freitas-Astúa et al. unpublished data).Details about RNA extraction, cDNA libraries construction and sequencing can be found in Targon et al. (this issue).

Database analyses
Reads from the two libraries were clusterized using the CAP3 tool (Huang and Madan, 1999), with default parameters.For each assembled tentative consensus (TC), the relative abundance of transcripts was calculated, using a correction factor of 10,000 for normalizing, and the significance was given by using the methodology described in Audic and Claverie (1997) for two libraries, with a threshold of p-value ≤ 0.05.The TCs were automatically compared, through BlastX algorithm, to sequences available in the public databases GenBank (http://www.ncbi.nlm.nih.gov/BLAST/), Arabidopsis Genome Initiative dataset (http://www.arabidopsis.org/),and KEGG (Kyoto Encyclopedia of Genes and Genomes -http://www.genome.jp/kegg/).TCs that yielded hits with e-values lower than e -10 were categorized using the Munich Center for Proteins and Sequences Functional Categories (MIPS FunCat) v. 1.3 (http://mips.gsf.de).When the TCs were automatically classified into the 98 (classification not yet clear cut) or 99 (unclassified protein) classes, an additional manual categorization was performed.More details on the bioinformatic analyses can be found in Reis et al. (this issue).

Results and Discussion
cDNA libraries were constructed from healthy and CiLV-C inoculated sweet orange leaves.They yielded 9,536 and 5,664 reads (8,188 and 4,852 valid reads), respectively, that were clusterized in TCs.Using an in silico hybridization strategy, 254 of those putative genes were found to be differentially expressed, with 193 of them induced and 61 repressed by the pathogen, and distributed within 19 categories classified according to the MIPS FunCat (Table 1).This large number of categories observed was expected since, in compatible hosts, viral invasion triggers numerous biochemical and physiological changes in cells, tissues, and even whole plants, causing significant changes in host gene expression (Moller and Chua, 1999;Maule et al., 2002;Huang et al., 2005).A subset of such categories was chosen to be studied in details due to their involvement in stress responses.They included, in addition to genes involved in cell rescue, defense and virulence, those involved in cellular communication/signal transduction mechanisms, metabolism, and energy.

Signaling and cell rescue, defense and virulence
All of the five TCs composed of reads related to cellular communication/signal transduction mechanisms that were differentially expressed in our two conditions (comprising genes that code for glycoproteins, lectin, cyclophillin and calcium ion binding proteins) were induced after the CiLV-C challenge in the citrus plants.This may be due to their role in triggering downstream processes that will result in the compatible interaction (= disease).At least three of these transcripts have been also related to defense against biotic stressors such as insects [lectin (Gatehouse et al., 1999)], pathogens [cyclophillin (Romano et al., 2004) and calcium ion binding protein (Chiasson et al., 2005)].Particularly for calcium ion binding proteins, their role in defense has been strongly associated with the occurrence of HR, since these proteins regulate downstream targets as part of a coordinated cellular response to a given stimulus (Chiasson et al., 2005).It should be noted that the symptoms induced by CiLV-C in its compatible interaction with sweet orange resemble those of HR in incompatible interactions, and at first glance it appeared that similar responses would occur in both situations.However, with few exceptions, such as the induction of calcium ion binding protein genes, other aspects of the interactions are completely distinct and will be discussed throughout this manuscript.
Within the rescue, defense and virulence functional category, our data revealed five and 15 TCs harboring transcripts that were, respectively, down-and up-regulated during virus infection.Amongst the induced genes, those that code for a prolin rich protein, a cytochrome P450, a Lea5, a catalase, a miraculin, a gluthatione reductase, a hsp20, a superoxide dismutase, a metallothionein, and a zing finger protein (Table 2) can be highlighted.
Even though they can be important in biotic and abiotic stress responses, their role in our compatible pathosystem does not seem to be pivotal.The oxidative burst may play the role of an internal emergency signal for induction of the metabolic cascade for active defense (Doke et al., 1996).The activation of antioxidant enzymes as mechanism of cellular protection is commonly observed in plants in response to pathogens, and is one of the first defense responses activated that may be responsible for triggering the beginning of HR (Grant and Loake, 2000).These authors report the isolation of a transcript that presented similarity with the enzyme superoxide dismutase (SOD).This enzyme is responsible for the synthesis of hydrogen peroxide, toxic to microorganisms.Catalase protects cells from hydrogen peroxide which can be generated from an SOD catalyzed reaction.
A putative cytochrome P450 gene from Capsicum sp. was identified using cDNA microarray analysis of gene expression following induction of HR by leaf inoculation with the non-host pathogen Xanthomonas axonopodis (Kim et al., 2006).This indicates that cytochrome P450 in pepper plants may play a role in the defense response pathways (Kim et al., 2006).However, even though we found genes that are often involved in HR to be induced in our infected library, it should be noted that they comprised a small number of the total transcripts related to HR (Guidetti-Gonzalez et al., this issue).

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Genes involved in the sweet orange response to Citrus leprosis virus It remains clear that, even though interesting differential gene expression was observed between the two libraries regarding traditional biotic stress response, it is not yet sufficient to explain the main changes observed in sweet orange infected with CiLV-C.In fact, other genes within the MIPS FunCats of energy and metabolism seem to play more relevant roles in such compatible interaction, and will be discussed below.

Energy and metabolism
Nineteen differentially expressed TCs were classified within the MIPS energy category, with clear evidence of reduction in aerobic respiration in challenged sweet orange plants when compared to the non-challenged ones.In addition to a TC that codes for rubisco, other transcripts involved in aerobic respiration were mostly repressed by the pathogen, most of them coding for chlorophyll a/b binding proteins (Table 2).Several studies have been conducted on the influence of viral diseases in host respiration rate.Hull (2002) summarizes these data stating that, for host-virus combinations where necrosis does not occur, the respiration rate starts to increase, often even before the appearance of symptoms, and continues rising as disease develops.In chronically infected plants, respiration is normally lower than normal, and sometimes unchanged.In host-virus inter-actions characterized by the appearance of necrotic local lesions, the respiration rate increases as necrosis develops.
These results are contrary to our data, which suggest a reduction in respiration rate during citrus-CiLV-C interaction.However, it is important to take into consideration that, even though the virus induces necrotic local lesions in sweet orange, the samples for the library construction were collected 48 h after inoculation, several weeks prior to the appearance of symptoms.It cannot be discarded that the respiration rate may increase as necrosis develops, as often observed in other combinations.It should also be noted that there was no evidence to support the hypothesis that citrus response to leprosis would be similar to that of HR.If the viral particle is not recognized by the host plant, a compatible interaction -which favors the virus -is established (Hammond-Kosack and Jones, 2000;Stange, 2006).In an incompatible reaction, development of the receptor-ligand complex triggers a cascade of transduction signals that ultimately leads to the HR response.This response is a local reaction characterized by programmed cell death (PCD) at the infection site (Heath, 2000).Importantly, during HR, reactive oxygen species are produced (Lamb and Dixon, 1997), callose (Shimomura and Dijkstra, 1975) and lignin are synthesized, the levels of salicylic acid increase (Naylor et al., 1998) (Yalpani et al., 1991).As a result, plants limit the short and long-distance movement of the pathogen (Stange, 2006;Guidetti-Gonzalez et al., this issue).
As will become clearer throughout this paper, we found several major differences between the molecular responses normally associated with typical HR induced by viruses in an incompatible interaction and the necrotic local lesion caused by CiLV-C in this compatible combination.
Interestingly, out of the 54 differentially expressed TCs automatically classified into the MIPS metabolism category (28 induced and 26 repressed; Table 2), several of their putative products are involved in the generation of precursor metabolites and energy and are related to photosynthesis and respiration.It was possible to observe a clear repression in genes that code for key enzymes involved in photorespiration, such as glycine hydroxymethyltransferase/serine hydroxymethyltransferase (SHMT), glycine decarboxylase, and alanine: glyoxylate aminotransferase (Taylor et al., 2002;Vol et al., 2006).These data suggest that even before the appearance of the typical chlorotic and necrotic symptoms of leprosis, genes involved in respiration are repressed.It strengthens the hypothesis that the response of citrus to CiLV-C is different from that observed in other hosts that develop necrotic local lesion when challenged with a viral agent.Altogether, this suggests that host-leprosis interaction presents more similarities with the initial steps of infection of other compatible plant-virus interactions (Hull, 2002).
Repression in gene expression was also observed for some transcripts that code for the key chlorophyll synthesis enzymes protoporphyrin IX magnesium chelatase and glutamyl-tRNA reductase (GluTR) suggesting that, as expected, the presence of the virus has influence on photosynthesis as well, even before the appearance of macroscopic symptoms (Van Kooten et al., 1990).
In fact, in a compatible host-pathogen interaction, studying the up-regulated genes is very important, but a careful review of repressed ones can be relevant as well.The repressed genes can lead to important cues to understanding viral interference in plant metabolism in order to establish an adequate environment for the development of the disease.Repression of genes involved in chlorophyll synthesis has been found not only in plants undergoing biotic, but also abiotic -such as cold -stress (Mohanty et al., 2006).Similarly, it has been shown that enzymes involved in the photorespiratory pathway, such as SHMT, may play an important role in the response not only to biotic, but also to abiotic stress (Moreno et al., 2005).The authors found that a recessive mutation in the Arabidopsis shmt1 gene (shmt1-1) causes aberrant regulation of cell death resulting in chlorotic and necrotic lesion formation under a variety of environmental conditions.Hence, the repression of the shmt gene in the compatible citrus-CiLV interaction studied may be involved in the intensification of the typical leprosis symptoms observed.
Tentative consensi comprising transcripts involved in saturated fatty acid synthesis, methionine metabolism, and biosynthesis of phenylpropanoids and terpenes were also found to be repressed in our analyses.Four genes belonging to the latter groups stand out: phenylalanine-ammonia lyase (pal), isoflavone reductase (ifr) and hydroxycinnamoyl transferase (hct), related to phenylpropanoid biosynthesis, and terpene synthase (tps), which encodes the primary enzyme in the formation of low-molecular-weight terpene metabolites.The pal gene codes for the PAL protein, the first enzyme of the phenylpropanoid biosynthesis, involved in an array of functions, from synthesis of flavonoids to phytoalexins, lignin, and other important secondary metabolites associated with plant development, structure and defense (Klessig et al., 2000).One of the major roles of the isoflavones is to provide the disease resistance response by inducing the formation of the defense compounds against phytopathogenic microorganisms.The enzyme IFR catalyzes a NADPH-dependent reduction and acts in the biosynthesis of important and related phenylpropanoid-derived plant defense proteins (Dixon and Steele, 1999;Verica et al., 2004).Lignins are extremely resistant to microbial degradation and are often induced at sites of pathogen infection, playing important roles in cell wall reinforcement and, consequently, increased defense response against infection (Lange et al., 1995;Kawasaki et al., 2006).The HCT enzyme is an acyltransferase that is potentially implicated in the pathway both upstream and downstream of the 3-hydroxylation step in lignin biosynthesis.The induction of lignin synthesis or lignin-related genes after virus challenge has been reported in incompatible interactions in herbaceous plants (Jaeck et al., 1992) and, recently, in citrus (Cristofani-Yaly et al., this issue).Since in both cases lignification seems to help prevent viral infection, it is not surprising to observe that in the compatible citrus-CiLV-C combination, we found evidence for repression of lignin production.
Terpene metabolites are important compounds in direct defense against microbes and insects (Loreto et al., 2004), and several tps genes are induced in leaves by herbivore attack (Bohlmann et al., 1997;Steele et al., 1998).Similarly, the repression of tps found in our analyses suggests that these compounds did not play a relevant role in increasing defense against leprosis.However, our data do not explain why tps was repressed, and therefore should be addressed in further studies.
Three other TCs that had their expression repressed after challenge by CiLV-C harbored genes involved in abiotic stress response.One of them codes for an inositol-3-phosphate synthase (Myo-inositol-1-phosphate synthase, MIPS), for which expression has been reported as either reduced (Chun et al., 2003) or increased (Buchanan et al., 2005) under salt and osmotic stress, for sesame and sorghum, respectively.The second transcript codes for phosphoethanolamine N-methyltransferase (PEAMT), an 986Genes involved in the sweet orange response to Citrus leprosis virus important enzyme for the synthesis of glycinebetaine (betaine).Betaine is a compatible solute that accumulates in large amounts in certain plants and which seems to play a role in the protection from salt stress (Tabuchi et al., 2005).Finally, a third repressed transcript codes for S-adenosylmethionine decarboxylase (SAMDC), which is involved in increased resistance or tolerance to numerous abiotic stresses, since it is a key enzyme in polyamine (PA) biosynthesis.In a recent paper, Wi et al. (2006) showed that the overexpression of the carnation SAMDC gene generated a broad-spectrum tolerance to abiotic stressors in transgenic tobacco plants.The authors found that the transgenic plants had an increased net photosynthetic rate and showed attenuated stress-induced damage and the appearance of yellowing and chlorophyll degradation symptom, suggesting that PAs may have pivotal importance in stress tolerance in plants.Interestingly, Hao et al. (2005) isolated and cloned two SADMC cDNAs from apple plants and showed that only one of them was actually involved in stress responses, while the other was likely to be involved in fruit development and cell growth.It is interesting that at least three transcripts associated with abiotic stress responses were repressed in the compatible interaction studies; however, whether or not they play any role in the susceptible response of sweet orange to leprosis remains to be tested.
Another repressed TC was composed of reads that code for a putative acyl CoA synthetase (LCAS2), which has roles in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments.It also seems to be involved in normal cuticle development in Arabidopsis and lacs2 mutant phenotype causes, among other things, reduced leaf size and plant growth (Schnurr et al., 2004).Interestingly, two TCs harboring genes that code for proteins related to lipid metabolism were found to be induced in the same sweet orange library.One of them, an amino-alcohol phosphotransferase, is involved in the biosynthesis of phospholipids, while the other, a thiolase, is involved in the degradation of fatty acids.Lipids are connected to the plant defense response against biotrophic fungi, bacteria, and viruses (Thomma et al., 2001;Murphy and Carr, 2002) through their potential action as signaling molecules (Laxalt and Munnik, 2002;Maldonado et al., 2002).In fact, production of fatty acids is an early step in cell death caused by oxidative burst (Montillet et al., 2005) that will result in HR.Our data demonstrated not only a repression in the synthesis of fatty acids, but also an increase in its degradation, suggesting that fatty acid metabolism is important to the susceptibility of Pêra sweet orange to leprosis.Also, this result clearly points out that the host responses to CiLV-C significantly differ from those observed during HR.
Two TCs that had their expression induced after CiLV-C challenge were composed of reads that encode the enzymes pectinesterase and cellulase, related to the degra-dation of pectin and cellulose, respectively.Since they are the main components of the plant cell wall, it seems evident that, even prior to symptom appearance, there is a disruption of the cell physical structure, which will ultimately lead to cell collapse and death.Interestingly, not only cellulose gene expression was induced, but also a cellulose synthase gene was found to be repressed in the library constructed from inoculated leaves, suggesting a role in the symptoms observed.
As expected, several other TCs harbored transcripts involved in carbohydrate metabolism were found to be differentially expressed between the two libraries.Two of them, that code for a GDP-mannose 3',5'-epimerase and a triosephosphate isomerase, were repressed.GDP-man 3',5'-epimerase is a late methyl jasmonate-responsive enzyme that helps control the carbon flux into the vitamin C pathway in response to the redox state of the cell, stress conditions, and GDP-sugar demand for cell wall/glycoprotein biosynthesis (Wolucka and Van Montagu, 2002;Wolucka et al., 2005).Triosephosphate isomerase, an enzyme in the glycolysis pathway, is also involved in stress response, and reduction in its expression is reported in plants after abiotic or biotic stress conditions (Riccardi et al., 1998;Morris and Djordjevic, 2001).
Three TCs composed of transcripts involved in carbohydrate metabolism were found to be induced in the CiLV-C-inoculated library, one encoding a sucrose synthase, another one, a glucose-6-phosphate isomerase, and a third one, a mannose pyrophosphorilase.Numerous reports have indicated that carbohydrate metabolism in the source leaf is influenced by viral infection (Tecsi et al., 1994a(Tecsi et al., , 1994b(Tecsi et al., , 1996)).Infected source leaves are usually characterized by a decrease in the concentration of soluble sugars, and often starch accumulation (Goodman et al., 1986;Fraser, 1987).Changes in the capacities of enzymes in various metabolic pathways have been measured during infection of cotyledons of Cucurbita pepo L. with Cucumber mosaic virus (CMV).CMV infection significantly altered carbohydrate metabolism, with a sharp increase in the concentrations of soluble sugars observed in the infected leaves.These changes were associated with a decrease in leaf starch content.An earlier study indicated an increase in reducing sugars and a reduction in starch content due to CMV-induced higher starch hydrolase and lower ADP-Glc pyrophosphorylase activities (Tecsi et al., 1994b).It has been proposed that the inhibition of starch accumulation and/or starch degradation is probably due to the increased demand for soluble sugars required to maintain the high respiration rate (Tecsi et al., 1996;Shalitin and Wolf, 2000).
In our data analysis we not only found an increase in the expression of transcripts that code for enzymes involved in synthesis of soluble sugars, but also in the expression of transcripts related to starch degradation.Interestingly, it has recently been shown, through histological studies using light microscopy, that there is a clear reduc- Freitas-Astúa et al. tion in starch accumulation in the epidermis of sweet orange leaves and stems symptomatic for leprosis (Marques et al., 2005), corroborating our gene expression data and suggesting a common response to viruses that invade systemically or cause only local lesions in their hosts.

Final Considerations
Our study showed that CiLV-C infection induced immediate and subsequent changes in host gene expression and that the infection can potentially give advance signaling of an imminent infection.Even though there was clear variation in the expression of genes involved in cell rescue, defense and virulence, the most relevant molecular changes observed seem to have occurred in the MIPS energy and metabolism categories.It can be understood as a new data, since it is a compatible interaction, different than most of the other studies done on plant-pathogen combinations.
In addition, the fact that CiLV-C causes only chlorotic and necrotic local lesions in susceptible hosts, and never invades them systemically, has raised questions regarding whether or not the symptoms could be a variation of the HR observed in viral incompatible interactions.However, our data strongly indicate that the two responses are very different at the molecular level and hence, the manifestation of leprosis symptoms should not be considered HR.

Table 1 -
Categorization of differentially expressed transcripts in CiLV-C inoculated Citrus sinensis leaves according to Munich Center for Proteins and Sequences Functional Categories (MIPS).

Table 2 -
Result of the BlastX search of induced and repressed C. sinensis genes after CiLV-C inoculation according to Arabidopsis thaliana GenBank data.

Table 2 (cont.)
and pathogenesis related proteins are produced