Phloem

Raw EPG data recorded by EPG Systems Stylet+d was manually annotated using EPG Systems Stylet+a software v01.30 (13-04-2016)/B27. Annotated waveforms were non-probing (np), pathway (C), phloem salivation (E1), phloem ingestion (E2), derailed stylet mechanics (F), xylem feeding (G) and intracellular puncture—potential drop (pd). Waveforms were identified based on the waveform pattern, amplitude, relative voltage level, R/emf origin, frequency, and the context of the waveform as described in the previous EPG studies of B. tabaci (Jiang et al., 1999 (link); Johnson and Walker, 1999 (link); Liu et al., 2012 (link); Civolani et al., 2014 (link); Zhou, 2014 ; Prado Maluta et al., 2017 (link)).
Annotation files were then directly passed to a modified version of the Ebert 3.0 program in SAS Enterprise Guide 7.1, SAS 9.4 statistical software (SAS Institute, Cary, NC, USA) for further analysis which produces the same parameters as the popular Sarria excel workbook (Sarria et al., 2009 (link); Ebert et al., 2015 (link)). The modified version is provided in the Supplementary Material. The modified version utilizes the series of BoxCox power transformation to determine the best possible transformation, as implemented in the PROC TRANSREG statement (Osborne, 2010 ). The results of this power transformation were inspected visually using histogram and Q-Q plots. For certain parameters, power transformations are unsuitable as they cannot approximate the necessary S-curve. Therefore, the Arcsine transformation was applied before the BoxCox transformation if this was necessary. The modification of the original Ebert 3.0 program also utilizes a macro script developed by Piepho (2012 ) that mitigates the case when varying standard error of a difference causes the traditional algorithm to fail to represent all significant differences of the means using the letter grouping. The Piepho algorithm solves the problem as it is able to generate a discontinous line display (Piepho, 2012 ). As a consequence, seeing discontinous assigned letters such as “ac” or “acd” is not uncommon (Piepho, 2014 (link); Poosapati et al., 2014 (link); Santos et al., 2015 (link); McCaghey et al., 2017 (link)).

The collection of phloem and xylem saps from wheat seedlings was performed according to the method of Hazama et al. (2015) (link). Treated and untreated seedlings of 27 days cut on surfaces of the stems near the petioles of mature leaves with a razor blade and exuded drops (excluding the first drop) collected as phloem sap using micropipettes. Samples of phloem sap were stored in eppendorf previously washed with 0.1 M HNO₃ for 2 days and then with double distilled water three times to eliminate metals. The samples were stored at -20°C until analysis.
After the collection of phloem sap, stems were cut at 2 cm above the interface of the shoot and root, and xylem sap exudates were collected for 30 min using micropipettes. The measured pH of the xylem sap was 6.0–6.4. Samples of xylem sap were also stored at -20°C until analysis.
For the estimation of Zn in phloem and xylem saps above, the described procedure (for Zn) was followed.

Phloem, mature leaves, flowers, and fruits at different stages (expanding fruit, half-red, and full-red) of the ‘Junzao’ cultivar and the wild jujube ‘Qingjiansuanzao’ (8 years old) were collected in 2013 and 2014, respectively. All the samples were immediately frozen in liquid nitrogen. Total RNAs were isolated using a modified CTAB method and then treated with RNase-free DNase I (Promega, USA). First-strand cDNAs were synthesized using a Clontech kit. RNA-Seq libraries were constructed using the NEB Next UltraTM RNA Library Prep Kit (NEB, USA) and sequenced on a HiSeq 2000/2500 system. RNA-Seq reads were mapped to the ‘Junzao’ genome using TopHat [38 (link)]. The total numbers of aligned reads (read counts) for each gene were normalized to the reads per kilobase exon model per million mapped reads (RPKM) [66 (link)]. DESeq [67 (link)] was used to identify differentially expressed genes.

Transcriptome data of 9 samples comprising phloem, root and leaf tissues as well as, developmental periods and growth condition (hydroponics or terrestrial) obtained from Genome Sequence Archive database were used. Transcript abundance of the BnGR2R3-MYB genes was calculated as fragments per kilobase of exon model per million mapped reads (FPKM). Log2(FPKM +1) values were displayed according to the color code. Detailed FPKM values were listed in Supplementary Table S7.

Phloem and xylem tissue were homogenised in liquid nitrogen with mortar and pestle and then stored at at -80 °C for further RNA or protein extraction. 100 mg of the homogenized tissue was placed in a frozen 2 mL tube containing a ceramic bead and ground for 60 s at a frequency of 26 1/S with a TissueLyser II (Qiagen, Cat. 85,300). Total RNA was isolated using RNeasy Plant Mini kit (Qiagen, Cat. 74,903) with DNase treatments (RNase-Free DNase Set cat. No. 79254) according manufacturer’s instructions. The RNA was quantified using the Qubit 4 fluorometer (ThermoFisher) with the Qubit RNA BR Assay Kit (ThermoFisher, Cat. Q10211). RNA integrity (RIN) was tested with the Agilent RNA 6000 Nano Kit (Agilent, Cat. 5067–1511) on a 2100 Bioanalyzer instrument (Agilent, Cat. G2939BA) and all samples used for RNA-Seq had a RIN greater than 7.
The Illumina NeoPrep Library Prep System was used to prepare samples from 50 ng of total RNA extraction (Illumina, Documents: 15049720v01, 15049725v03, 15059581v02). TruSeq Standard mRNA Library Prep (Illumina, NP-202–1001) was used with the default indexes adapters A to P. At the last step, each processed sample collected from the library card was analysed for library quality check using a DNA 1000 chip on the 2100 Bioanalyzer (Agilent, Cat. 5067–1504). Finally, each sample was normalized manually at 10 nM and then pooled (5 μL × 16 samples) in one library for the Illumina sequencing platform.
The libraries composed of the 16 samples were sequenced into two lines in Rapid-Run Mode (16 samples/line) in a single flow cell for paired-end 100 bp with an Illumina HiSeq 2500 sequencing system. The samples were sequenced at the Centre Hospitalier de l'Université Laval sequencing platform (Quebec City, Canada).

Proteins were extracted from phloem and xylem samples and prepared for proteomic analysis using the following protocol. 200 mg of the homogenized phloem and tissue samples were added to 500 µL of extraction buffer (0.5% sodium deoxycholate, 50 mM 1,4 dithiotreitol, 1 µM Pepstatin (Thermo Fisher Scientific), 1X Complete Mini Roche (Sigma Aldrich) in 50 mM ammonium bicarbonate) was added to each sample. Mechanical extraction was then performed using a Mixer mill MM400 (Retsh) with three inox beads of two cycles of 2 min at 30 Hz, turning the tube racks 180° between the cycles. Samples were centrifugated at 10 000 × g for 15 min at 4 °C to remove pellet debris. The supernatant was then filtered using a 0.45 µm Centrifugal Filter Membrane (Millipore) at 12 000 × g. Five volumes of acetone at -20 °C was added to the filtered sample and incubated at -20 °C overnight. After centrifugation at 16 000 × g for 15 min at 4 °C, the major part of the supernatant was discarded and the remaining acetone was left evaporated under the fume hood. The pellet was then resuspended with 50µL of 50 mM ammonium bicarbonate and protein concentration was measured using Bradford assay.
For each sample, a volume corresponding to 20 µg of proteins was used for subsequent analysis. Volumes were adjusted to 30 µL using 50 mM ammonium bicarbonate and sodium deoxycholate was added to a final concentration of 1%. The samples were heated at 95 °C for 5 min for protein denaturation. Cysteine disulfide bridges were reduced and alkylated using the following procedure. 1,4 dithiothreitol was added to a final concentration of 0.2 mM and incubated at 37 °C for 30 min. This was followed by the addition of iodoacetamide to a final concentration of 0.8 mM and incubated at 37 °C for 30 min in the dark.
Enzymatic digestion of the protein samples was initiated using 400 ng of trypsin enzyme (Promega), corresponding to an enzyme:protein ratio of 1:50, followed by an incubation at 37 °C overnight. Enzymatic digestion was stopped by acidification using 30µL of 3% acetonitrile, 1% trifluoroacetic acid, 0.5% acetic acid. This step also allowed the precipitation of sodium deoxycholate. Samples were finally centrifugated at 16 000 × g for 5 min and the supernatants were collected. The peptides resulting from trypsin digestion contained in these supernatants were purified on StageTips according to [33 (link)] using C18 Empore reverse phase. The samples were finally vacuum dried and stored at -20 °C prior to mass spectrometry analysis.