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.

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.

Out of the 18,942 flavedo expressed sequences (clusters plus singletons) in Citrus sinensis from the Citrus EST Project (CitEST), 25 were statistically supported to be differentially expressed in this tissue after a double in silico hybridization strategy against leaf-, flower-, and bark-derived ESTs. Five of them, two terpene synthases and three O-methyltransferases, are absent in the other citrus tissues with concomitant 2x2 statistics, supporting the hypothesis that they are putative flavedo-specific expressed sequences. The pattern of these differentially expressed sequences during fruit development suggests that most of them are developmentally regulated. Some expressed gene products, including a putative germin-like protein highly expressed in flavedo, are shown to be promising candidates for further characterization. In addition to promoter seeking, this kind of analysis can lead to gene discovery, tissue-specific and tissue-enriched expression pattern predictions (as shown herein) and can also be adopted as an in silico first, and probably reliable approach, for detecting expression profiles from EST sequencing efforts before experimental validation is available or for heuristically guiding that validation.

In this work we describe all the computational environments, pipelines, and web services developed for the CitEST transcriptome project, on which all the annotation researchers relied. We also present a complete list of CitEST libraries and, for each of them, the general features after the in silico processing, showing some quantitative information.