Transcriptome of grapefruit during citrus canker development using RNA-Seq

DAURELIO, L.D.; KRAISELBURD, I.; ESTEBAN, L.; GONZALEZ, M.; TALÓN, M.; TADEO, F.; ORELLANO, E.G.

Rosario

Congreso; 4to. Congreso Argentino de Bioinformática y Biología Computacional (4CAB2C) y 4ta. Conferencia Internacional de la Sociedad Iberoamericana de Bioinformática (SolBio); 2013

Sociedad Argentina de Bioinformática y Biología Computacional (4CAB2C), Sociedad Iberoamericana de Bioinformática (SolBio)

Background: Citrus trees, one of the most important fruit crops in the world, are currently exposed to diverse pathogens. Xanthomonas citri subsp. citri (Xcc) produces citrus canker, one of the most damaging citrus disease worldwide. This disease produces damage in every tissue of all citrus trees species, producing losses in productivity and commercialization in Argentine. Several studies have been made regarding transcriptome response of citrus leaves to Xcc using microarrays. Here we analyzed the citrus transcriptome in fruit peel tissue using next generation sequencing or RNA-Seq.         Materials and methods: Citrus paradise fruits were treated with 107 UFC/ml of Xcc. Copper-mancozeb, a chemical used to prevent citrus canker, was used as a negative control (Ctr). Total RNA was extracted using Trizol® reagent from manually dissected grapefruits peels at 1, 3 and 5 day post treatment (dpt), using three fruits from three different plants. The RNAs integrity was checked using the Agilent 2100 Bioanalyzer kit. Equimolar pools were obtained by mixing RNAs of the three Xcc treated samples at each time and by mixing one sample of each time for Ctr. cDNA libraries were subjected to lllumina sequencing in Lifesequencing service (www.lifesequencing.com). Quality control, alignment to reference genome of Citrus sinensis (www.phytozome.net), count of transcripts and statistical analysis to detect DEG were performed with RobiNA software (http://mapman.gabipd.org/web/guest/robin). The functional analysis was performed with MapMan software (http://mapman.gabipd.org/web/guest/mapman).         Results: Over 360 million of 50-base pair reads were generated from pooled grapefruit samples and over 210 million of these reads presented at least one reported alignment. Mapping reads onto the reference genome of Citrus sinensis detected approximately the 92% of these genes (23,568). RNA-Seq analysis uncovered 787 DEG in at least one Xcc treatment in comparison to Ctr or between different times post treatments. No DEG were found between Xcc 1 dpt and Ctr while a high number of DEG were observed between Xcc 5 dpt and Ctr, in correlation with disease symptoms progress on the tissue. We decided to analyze 326 DEG which presented differential expression between Xcc5 dpt - Ctr, in the intersections with the Xcc3 dpt - Ctr, Xcc3 dpt - Xcc1 dpt and Xcc5 dpt - Xcc3c dpt treatments. The most represented categories in this functional analysis were "regulation of expression", "cell wall related proteins", "hormone metabolism", and "protein metabolism", among others.           Conclusions: This study presents a novel transcriptome RNA-Seq analysis during citrus canker disease, using fruits from trees grown under field conditions similar to those used for citriculture. Genes involved in key steps during citrus canker development in fruits are revealed, that could be used in future strategies of molecular breeding for citrus disease resistance.