title: Illumina Genome Analyzer IIx sequencing; Grapevine comparative early transcriptome profiling reveals that Flavescence dorée phytoplasma represses plant responses induced by vector feeding in susceptible varieties
design: Grapevine comparative early transcriptome profiling reveals that Flavescence dorée phytoplasma represses plant responses induced by vector feeding in susceptible varieties
library_construction_protocol: Insect rearing and infection Egg-hatched S. titanus individuals, reared in cages in controlled conditions at 24 ± 1°C, 70-75% RH and 16:8 photoperiod, were used for the transmission experiments [20]. The hatched larvae (first instar) were divided into two groups: the first was fed with fresh grapevine leaves collected from FD-healthy grapevine plants cv Pinot gris maintained in screenhouse, while the second was fed with fresh FD-infected leaves (isolate FD-D) from field-grown grapevine plants from the same cultivar, growing in a vineyard monitored since several years (Figure 1). All the source grapevines had been previously tested by PCR to identify the FD-infected and healthy plants, as described later. Moreover, all the FD isolates present in the used plants were previously characterized by RFLP (Random Fragment Length Polymorphism) in the 16-23S region and by further sequencing of the ribosomal and secY genes, as described previously (Filippin et al 2009), in order to confirm their identification as FD-D phytoplasma isolate. From the infected grapevines, only leaves with clear GY symptoms were collected and used for FD acquisition. The detached leaves in the cages, maintained with the petioles immersed in a small tube with water (20), were checked daily and changed every 2-3 days, or earlier if they showed signals of senescence. The insects were reared in the conditions described until their use for the experimental trials.   Transmission trials  The transmission trials were carried out with fifth instars or adults, both FD-free (fed on FD-healthy leaves) and infective (fed on FD-D-infected leaves), after two weeks of acquisition in infected leaves and 4 weeks of latency period, based on data reported in literature (Figure 1) [5]. The micropropagated plantlets (weight 65 to 350 mg each) were placed in transparent boxes with a transpiring lid and wet substrate and separated two by two by means of a transparent wall, so that the insect movements could be monitored every day. Two S. titanus specimens were put on every micropropagated plantlet pair and were allowed to feed for three days. Their movements were observed daily to check whether, and where, they were feeding, and to collect the dead specimens. When both the insects preferred one of the two micropropagated plantlets, one specimen was moved to the other plant to increase the homogeneity of the treatment and the probability of FD transmission. When one or both the S. titanus specimens died within 24 hours, one or two new specimens were added into the box. The dead specimens were frozen at -20°C for PCR analysis to test the presence of the FD phytoplasma. Half of the micropropagated plantlets were collected after three days (3 dpi treatment), while the remaining were kept until six days (6 dpi treatment) (Figure 1). All the plantlets were weighed without roots and frozen at -80°C.  Grapevine plantlets In vitro micropropagated plantlets from T. friulano and Chardonnay, maintained at 25 ± 1°C under a 16:8 h photoperiod at 60 μmol m-2 s-1 cool-white light, were previously tested with ELISA and PCR diagnostic assays to exclude the presence of the most common viral grapevine pathogens, according to well-established protocols  . After multiplication, they were transferred to pots with sterile soil (20 minutes at 120 °C) and moved to a climatic chamber at 24 ± 1°C under a 16:8 h photoperiod and 70-75% RH, until their use for the experimental transmission, approximately after one month. The total RNA was extracted from 50 mg of vegetable tissue from the micropropagated plantlets employed in the trial using the plant/fungi total RNA purification kit (Norgen), then it was added to 1 μl of the RNase inhibitor RNasin Plus (Promega) to avoid it degrading, and treated with DNAse (DNA-free kit Ambion). The quantity and quality of RNA isolated from the micropropagated plantlets were evaluated with spectrophotometer and capillary electrophoresis (Agilent, 2100 BioAnalyzer). Only samples with a RNA Integrity Number (RIN) value higher than 7 were kept for the following analyses. Subsequently, 1.5 μg of the total RNA of each sample was used for library preparation using the Illumina TruSeq RNA library preparation kit (FC-122-1001) according to the manufacturer's instructions. The libraries were amplified with 15 cycles of PCR and then purified and size-selected for an average size of 300 bp by a 2% low-range ultra-agarose gel (Bio-Rad). The libraries were quantified through quantitative RT-PCR (qRT-PCR), as recommended by the TruSeq RNA library preparation protocol.