Candidatus Liberibacter asiaticus accumulation in the phloem inhibits callose and reactive oxygen species

CLas inhibits callose deposition in the sieve pores and the accumulation of reactive oxygen species to favor its cell-to-cell movement.


Supplemental
Seeds were collected from fruit stored at 4°C until seed vasculature extraction. The testa and the tegmen of the seed were removed with forceps to expose the vascular tissue of the embryo. The vasculatures were carefully removed from the apex at the point of junction with the embryo. The extracted vasculatures were used either immediately (microscopy analysis), stored at 4°C for staining, or stored at -20°C for molecular analysis.

Electron microscopy and imaging analysis
Electron microscopy analysis was performed as previously described (Achor et al., 2020;Folimonova and Achor, 2010), using a standard fixation procedure as follows.

Confirming the accumulation of CLas in the seed vasculature
The accumulation of the bacteria in the seed vasculature was assessed with fluorescent in situ hybridization following a previously reported protocol (Ghanim et al., 2009) and adapted for use with plant tissue (Hilf et al., 2013). Samples were visualized with a Leica SP8 laser-scanning confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) with a 488nm argon excitation laser for Alexa Fluor 488 and a near UV diode 405nm for DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride). Emission signals were respectively detected at 520nm and 480nm with a gain of 600.

H2O2 concentration assay
Samples of leaves were collected from the field and immediately placed in a solution of 1mg/ml 3,3'diaminobenzidine (Sigma-Aldrich) (DAB) in water, pH 3.8, prepared as previously reported (Daudi and O'Brien, 2012;Kumar et al., 2014). Seed vasculatures collected from the fruits, stored at 4°C, were placed in DAB solution. The staining protocols were performed as follows: overnight staining in DAB on a shaker at 50 rpm. The following day destain for 20 minutes (for leaf samples) or 10 minutes (for seed vasculature samples) in a bleaching solution with the composition of ethanol:glycerol:acetic acid in a 3:1:1 ratio at 92°C (Daudi and O'Brien, 2012;Kumar et al., 2014).
After the initial bleaching, the solution was replaced with fresh bleaching solution and the samples were left at room temperature for 30 minutes before the observation. Leaf samples were collected from 10 plants (5 healthy and 5 infected) and at least 6 leaves selected randomly in each plant were observed. For seed vasculature analysis, seed vasculatures were extracted from 3 fruits chosen randomly from each of 10 plants (5 healthy and 5 infected). For each fruit at least 10 seed vasculatures were observed, and pictures were acquired with a stereo microscope Leica KL300 on a white background (Leica Microsystem 2021) and using the LASX software (Leica Application Suite, Leica microsystem 2021). For each picture the same exposure time, light and white balance conditions were applied.
Leaf pictures were analyzed with FIJI software (Schindelin et al., 2012). On each picture, color deconvolution was performed with the H DAB algorithm (Crowe and Yue, 2019). The channel corresponding to the DAB color was separately saved and used for the analysis. For each leaf the mean gray value (intensity of the light) of the whole lamina was analyzed, extracting the lamina from the background.
Vasculature tissue was analyzed with the workflow as described above. For each vasculature, 5 regions of interest (ROIs), each 576 pixels squared in size, were chosen randomly to avoid readings affected by the size of the vasculature. For each ROI, the mean gray value was recorded.
The mean gray value was transformed to optical density value with the following formula: log(max intensity / mean intensity) in which the maximum intensity for 8-bit pictures has a value of 255 (Nguyen and Nguyen, 2013).

Gene expression analysis
Total RNA extraction on Duncan seed vasculatures and leaf midribs were performed using Trizol reagent (Invitrogen). RT-qPCR was performed as previously described , starting from roughly 100 mg of fresh tissue. RNA reverse transcription was carried out using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems), following the manufacturer instructions starting from 500 ng of RNA. To determine difference in modulation of the genes, RT-PCR was performed using SYBR Green FastMix (Quantabio, Beverly, MA, USA) starting from 300 ng of cDNA in a total reaction volume of 15 μL with a concentration of each primer of 400 nM. All gene primers used for RT-qPCR are listed in Table 1. Gene expression was compared to the citrus GAPDH reference gene, and analysis was performed using the 2 -ΔΔCt method (Livak and Schmittgen, 2001). For each analysis, Student's t-test was used to determine significant differences among the treatment group means (healthy or infected) with p < 0.05.