The citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xac), is a serious disease that is adversely affecting citrus groves worldwide. The major goal of citrus breeding programs is to develop commercial citrus varieties with resistance to citrus canker. However, this strategy has not produced results so far, because the genus Citrus presents limitations for improvement via conventional breeding, and this are directly associated with the reproductive biology of Citrus, such as nucellar polyembryony and a long juvenile period. The identification of genes involved in Xac pathogenicity and virulence and the identification of plant defense systems is important to understand the pathosystem and to develop strategies to control the citrus canker. Thus, this project aims to investigate, through the technology of RNA-Seq (Whole Transcriptome Shotgun Sequencing), the temporal expression of Xac genes and plant genes of mandarin and sweet orange varieties resistant and susceptible to citrus canker. Cell suspensions of Xac 306 strain will be inoculated into leaves of the resistant variety Satsuma mandarin (Citrus unshiu) and the susceptible variety Hamlin sweet orange (Citrus sinensis) and these leaves will be collected at 24, 48 and 72 hours after inoculation. These leaves will be frozen and will be macerated in liquid nitrogen and total RNA will be extracted using Trizol reagent. The purification of plant mRNAs will be made by affinity chromatography on oligo(dT). The bacterial total RNA will be extracted by Illustra RNAspin Mini Isolation kit (GE Heathcare Life Sciences) from the bacterial cells recovered from Xac cells exudates of inoculated leaves cut into strip in distilled water. High throughput sequencing of expressed genes will be used to identify gene by RNA-Seq technology using the HiScanSQ System (Illumina). After sequencing, the BLAST tool will be used to identify expressed genes of Xac and genes of citrus in each time. For Xac sequences, the genome sequence of Xac strain 306 will be used for comparison and for citrus sequences, the comparison will be made to GenBank sequences and the genome sequence of Citrus sp. that is already available.
News published in Agência FAPESP Newsletter about the scholarship: