In order to understand the genetic responses resulting from physiological changes that occur in plants displaying citrus variegated chlorosis (CVC) symptoms, we adopted a strategy of comparing two EST libraries from sweet orange [Citrus sinensis (L.) Osbeck]. One of them was prepared with plants showing typical CVC symptoms caused by Xylella fastidiosa and the other with non-inoculated plants. We obtained 15,944 ESTs by sequencing the two cDNA libraries. Using an in silico hybridization strategy, 37 genes were found to have significant variation at the transcriptional level. Within this subset, 21 were up-regulated and 16 were down-regulated in plants with CVC. The main functional categories of the down-regulated transcripts in plants with CVC were associated with metabolism, protein modification, energy and transport facilitation. The majority of the up-regulated transcripts were associated with metabolism and defense response. Some transcripts associated with adaptation to stress conditions were up-regulated in plants with CVC and could explain why plants remain alive even under severe water and nutritional stress. Others of the up-regulated transcripts are related to defense response suggesting that sweet orange plants activate their defense machinery. The genes associated with stress response might be expressed as part of a secondary response related to physiological alterations caused by the infection.

CitEST project resulted in the construction of cDNA libraries from different Citrus sp. tissues under various physiological conditions. Among them, plantlets of Rangpur lime were exposed to hydroponic conditions with and without water stress using PEG6000. RNA from roots was obtained and generated a total of 4,130 valid cDNA reads, with 2,020 from the non-stressed condition and 2,110 from the stressed set. Bioinformatic analyses measured the frequency of each read in the libraries and yielded an in silico transcriptional profile for each condition. A total of 40 contigs were differentially expressed and allowed to detect up-regulated homologue sequences to well known genes involved in stress response, such as aquaporins, dehydrin, sucrose synthase, and proline-related synthase. Some sequences could not be classified by using FunCat and remained with an unknown function. A large number of sequences presented high similarities to annotated genes involved with cell energy, protein synthesis and cellular transport, suggesting that Rangpur lime may sustain active cell growth under stressed condition. The presence of membrane transporters and cell signaling components could be an indication of a coordinated morphological adaptation and biochemical response during drought, helping to explain the higher tolerance of this rootstock to water stress.

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.

Due to the economic importance of gummosis disease for the citriculture, studies on P. parasitica-Citrus interaction comprise a significant part in the Brazilian Citrus genome data bank (CitEST). Among them, two cDNA libraries constructed from two different growth conditions of the P. parasitica pathogen are included which has generated the PP/CitEST database (CitEST - Center APTA Citros Sylvio Moreira/IAC- Millennium Institute). Through this genomic approach and clustering analyses the following has been observed: out of a total of 13,285 available in the Phytophthora parasitica database, a group of 4,567 clusters was formed, comprising 2,649 singlets and 1,918 contigs. Out of a total of 4,567 possible genes, only 2,651 clusters were categorized; among them, only 4.3% shared sequence similarities with pathogenicity factors and defense. Some of these possible genes (103) corresponding to 421 ESTs, were characterized by phylogenetic analysis and discussed. A comparison made with the COGEME database has shown homology which may be part of an evolutionary pathogenicity pathway present in Phytophthora and also in other fungi. Many of the genes which were identified here, which may encode proteins associated to mechanisms of citrus gummosis pathogenicity, represent only one facet of the pathogen-host Phytophthora - Citrus interaction.

Citrus is the most important fruit crop in Brazil and Citrus tristeza virus (CTV) is considered one of the most important pathogens of citrus. Most citrus species and varieties are susceptible to CTV infection. However, Poncirus trifoliata, a close relative of citrus, is resistant to the virus. In order to better understand the responses of citrus plants to the infection of CTV, we constructed expressed sequence tag (EST) libraries with tissues collected from Poncirus trifoliata plants, inoculated or not with Citrus tristeza virus at 90 days after inoculation, grafted on Rangpur lime rootstocks. We generated 17,867 sequence tags from Poncirus trifoliata inoculated (8,926 reads) and not (8,941 reads) with a severe CTV isolate. A total of 2,782 TCs (Tentative Consensi sequences) were obtained using both cDNA libraries in a single clusterization procedure. By the in silico hybridization approach, 289 TCs were identified as differentially expressed in the two libraries. A total of 121 TCs were found to be overexpressed in plants infected with CTV and were grouped in 12 primary functional categories. The majority of them were associated with metabolism and defense response. Some others were related to lignin, ethylene biosynthesis and PR proteins. In general, the differentially expressed transcripts seem to be somehow involved in secondary plant response to CTV infection.

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

In silico expression profiles, of the discovered 3,103 citrus ESTs putatively encoding for PR protein families (PR-1 to PR-17), were evaluated using the Brazil citrus genome EST CitEST/database. Hierarchical clustering was displayed to identify similarities in expression patterns among citrus PR-like gene families (PRlgf) in 33 selected cDNA libraries. In this way, PRlgf preferentially expressed by organ and citrus species, and library conditions were highlighted. Changes in expression profiles of clusters for each of the 17 PRlgf expressed in organs infected by pathogens or drought-stressed citrus species were displayed for relative suppression or induction gene expression in relation to the counterpart control. Overall, few PRlgf showed expression 2-fold higher in pathogen-infected than in uninfected organs, even though the differential expression profiles displayed have been quite diverse among studied species and organs. Furthermore, an insight into some contigs from four PRlgf pointed out putative members of multigene families. They appear to be evolutionarily conserved within citrus species and/or organ- or stress-specifically expressed. Our results represent a starting point regarding the extent of expression pattern differences underlying PRlgf expression and reveal genes that may prove to be useful in studies regarding biotechnological approaches or citrus resistance markers.

Os viróides são os menores fitopatógenos conhecidos, sendo constituídos de uma molécula de RNA fita simples, circular, com forte estrutura secundária. Possuem genomas que variam entre 246 e 401 nucleotídeos e, por não traduzirem proteínas próprias, são totalmente dependentes da célula hospedeira para sua replicação. São classificados em duas famílias e sete gêneros. O Citrus exocortis viroid (CEVd) pertence ao gênero Pospiviroid, enquanto o Hop stunt viroid (HSVd) é o único membro do gênero Hostuviroid, ambos pertencentes à família Pospiviroidae. Estes patógenos apresentam ampla distribuição mundial e infectam um grande número de hospedeiras. No Brasil, isolados do CEVd e do HSVd foram detectados em citros e videiras. Com o objetivo de caracterizar e estudar a variabilidade genética destes viróides, promoveu-se extração de RNA total de folhas de videira Vitis vinifera 'Cabernet Sauvignon' e V. labrusca 'Niagara Rosada' provenientes de Bento Gonçalves, RS, e RT-PCR com oligonucleotídeos específicos para os cinco viróides já descritos em videira [HSVd, CEVd, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2) e Australian grapevine viroid (AGVd)]. Amostras de folhas de Citrus medica infectadas pelo CEVd provenientes de São Paulo também foram analisadas. Os fragmentos de DNA amplificados foram eluídos, clonados e seqüenciados. As análises das seqüências revelaram que as amostras de videira estavam duplamente infectadas com o CEVd e HSVd. As análises filogenéticas mostraram que os clones de HSVd de videira aqui caracterizados agruparam-se com outros variantes de videira, formando um grupo separado de um segundo formado por variantes de citros. Já os clones de CEVd de videira agruparam-se com isolados de citros e videira.

Viroids, non-protein-coding small (246-401 nt) circular single-stranded RNAs with autonomous replication, are currently classified into two families. Within the family Pospiviroidae, Citrus exocortis viroid (CEVd) belongs to the genus Pospiviroid while Hop stunt viroid (HSVd) is the single member of the genus Hostuviroid. These pathogens are distributed worldwide and infect a large number of hosts. In Brazil, isolates of CEVd and HSVd have been detected in both citrus and grapevine. To characterize and study the genetic variability of these viroids, total RNA from leaves of grapevine Vitis vinifera 'Cabernet Sauvignon' and V. labrusca 'Niagara Rosada' from Bento Gonçalves, RS, was used as a template for RT-PCR amplification with specific primers for the five viroids described infecting grapevines [HSVd, CEVd, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2) and Australian grapevine viroid (AGVd)]. Leaf samples of Citrus medica infected with CEVd from São Paulo were also analyzed. The resulting products were separated by agarose gel electrophoresis and DNA fragments of the expected size were eluted, cloned and sequenced. The grapevine samples analyzed were doubly infected by CEVd and HSVd. A phylogenetic analysis showed that the Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants, forming a specific group separated from citrus variants, whereas the Brazilian CEVd variants clustered with other citrus and grapevine variants.