Regulation of plasmodesmata at specific cell-cell interfaces

Precise exchange of information and resources among cells is essential for multicellular organisms. Intercellular communication among diverse cell types requires differential mechanisms to achieve the specific regulation. Despite the significance of intercellular communication, it is largely unknown how the communication between different cells is regulated. Here, we report that two members of plasmodesmata-located proteins modulate plasmodesmata at two distinct cell-cell interfaces.

Recent findings also highlighted the crucial role of PD in plant immunity against filamentous and 23 bacterial pathogens (Lee et al., 2011;Faulkner et al., 2013;Cao et al., 2015;Aung et al., 2020;24 Tomczynska et al., 2020). The function of PD is controlled by the homeostasis of a plant 25 polysaccharide, callose. Callose synthases (CalSs) and β-1,3 glucanases mediate the biosynthesis 26 and degradation of callose, respectively, at the plasma membrane of PD (De Storme and Geelen, 27 2014). Callose deposition at PD suppresses the PD-dependent movement of molecules between 28 adjoining cells, presumably by narrowing the PD aperture. Other than callose, plasmodesmata-29 located proteins (PDLPs) also play important roles in regulating the plasmodesmal function, but 30 how PDLPs regulate PD function is unclear (Lee et al., 2011;Wang et al., 2020). 31 was detected in epidermis and cortex (Figure 2a). Transgenic plants co-expressing PDLP-YFP and 125 cell type-specific markers tagged with a compatible fluorescent protein (e.g., red fluorescent 126 protein) for simultaneous imaging will further confirm the cell type-specific expression of PDLP6 127 in phloem. In parallel, we determined the promoter activity of PDLP5 and PDLP6 using β-128 Glucuronidase (GUS) reporter gene assay. Two transgenic lines, pPDLP5::GUS and 129 pPDLP6::GUS, were subjected to GUS activity staining as previously described (Li et al., 2016). in the vasculature, likely phloem (Supplemental Figure 3). Together, our findings demonstrate that 137 PDLP5 and PDLP6 are expressed in distinct and non-overlapping cell types. Interestingly, PDLP5 138 was reported to express in lateral root primordium-overlaying cells during lateral root emergence 139 process (Sager et al., 2020). Further investigation will reveal the cell type-specific expression and 140 function of PDLPs at different developmental stages and tissues. 141

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While the molecular mechanism underlying PDLP function is unclear, the expression level of 143 PDLP5 is highly associated with callose accumulation at PD in plants ( Lee et al., 2011;Li et al., 144 2020). Similarly, the expression of PDLP1 is required for callose accumulation at haustoria during 145 downy mildew infection in Arabidopsis (Caillaud et al., 2014). To further investigate the cell type-146 specific roles of PDLP5 and PDLP6, we examined whether the overexpression of PDLP5 or 147 Fluorescence intensity profiles were consistent among data collected from 25 individual transgenic 159 plants for each genotype (Supplemental Figure 4). These findings support that the overexpression 160 of PDLP5 and PDLP6 leads to the higher accumulation of callose at specific cell-cell interfaces.

Tissue Sectioning and Starch Staining 232
Leaf punch samples were collected and fixed in FAA fixative (5% formaldehyde, 5% glacial acetic 233 acid, 50% ethyl alcohol). Samples were dehydrated through graded ethanol series (70, 85, 95, 234 100%) for 3-6 hours each concentration. Samples were infiltrated into LR White hard grade resin 235 (Electron Microscopy Sciences) and polymerized at 55°C for 48 hours. Sections were made using 236 a Leica UC6 ultramicrotome at 1.5 µm thickness. Sections were stained for non-soluble 237 polysaccharides as the following: slides with sections were immersed in periodic acid for 5 238 minutes, rinsed in distilled water for 5 minutes, stained in Schiff's reagent (Electron Microscopy 239 Sciences) for 10 minutes, rinsed in running tap water for 5 minutes, and air dried. Dry slides were 240 coverslipped using Permount mounting media (Fisher Scientific). 241 242

Aniline Blue Staining 243
Mature leaves from 4-week-old Arabidopsis leaves were infiltrated with 0.1 mg/ml aniline blue in 244 1x PBS buffer (pH 7.4). Samples were imaged at 5 minutes after the dye infiltration using confocal 245 microscopy. Ten-day-old seedlings were vacuum-infiltrated with 0.1 mg/ml of aniline blue in 1x 246 PBS buffer (pH 7.4). Stained root tissues were imaged using confocal microscopy. Callose in 247 mature leaves was quantified using FIJI. For epidermis, images were converted from lsm to tiff.

8-bit images were used for analysis. Black and white images highlighting callose were created by 249
Auto Threshold which was set by RenyiEntropy white method. Particle Analysis tool was used to 250 outline callose with size from 0.10 to 20 µm 2 and circularity from 0.15 to 1.00. Quantitative 251 numerical values in µm 2 were then exported. For mesophyll cells, aniline blue-stained callose area 252 was manually selected and the signal intensity was determined by measuring integrated density. 253 Semi-quantitative evaluation of the relative level of aniline blue-stained callose in the root tissues 254 of the transgenic plants were performed using FIJI. Images were converted from lsm to tiff. 16 bit 255 images were used for analysis. Horizontal lines were drawn near the bottom of the images crossing 256 all root cell types as shown in Figure 2d. Plot profile was used to generate a two-dimensional 257 graph. Value on y-axis represents the relative signal intensity as arbitrary unit (AU). and 1 mM X-Gluc). Samples were vacuumed for 10-40 min, followed by incubation in darkness 264 at 37°C for 2-16 h. After staining, samples were de-stained in 75% ethanol. Images were taken 265 using ZEISS Axio Observer. 266 267

Confocal Imaging 268
All confocal images were captured with a confocal laser-scanning microscope (Zeiss LSM 700). 269 A small piece of tissue was mounted with water on a glass slide. For leaf tissues, the abaxial side 270 was imaged. YFP was excited at 514 nm and emission was collected over the range of 510-550 271 nm using SP555. Aniline blue-stained callose was excited at 405 nm and emission was collected 272 over the range of 420-480 nm using SP555. 273 274

Immunoblot Analyses 275
Arabidopsis leaves were frozen with liquid nitrogen and homogenized with 1600 miniG (SPEX). We thank the ABRC for providing the T-DNA insertion mutants and pMDC163 vector. We thank 293 Tracey Stewart from Roy J. Carver high resolution microscopy facility at Iowa State University 294 (ISU) for helping with histological sectioning and starch staining. We thank the Aung lab members 295 Dr. Yani Chen, Dr. Su-Ling Liu, and Haris Variz. We also thank Dr. Yanhai Yin from ISU, Dr.