Redistribution of NORTIA in response to pollen tube arrival facilitates fertilization 1 in Arabidopsis thaliana 2 3

Redistribution of NORTIA in response to pollen tube arrival facilitates fertilization 1 in Arabidopsis thaliana 2 3 Jing Yuan1,2*, Yan Ju1,2*, Daniel S. Jones4,5, Weiwei Zhang2,3, Noel Lucca1,2, 4 Christopher J. Staiger1,2,3, and Sharon A. Kessler1,2,** 5 6 1Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 7 USA 8 2Purdue Center for Plant Biology, Purdue University, West Lafayette, Indiana USA 9 3Department of Biological Sciences, Purdue University, West Lafayette, Indiana USA 10 4Biology Department, The University of North Carolina at Chapel Hill, Chapel Hill, North 11 Carolina USA (current address) 12 5Department of Microbiology and Plant Biology, University of Oklahoma, Norman, 13 Oklahoma USA 14 *These authors contributed equally 15 16 ** Corresponding author 17 Email: sakessler@purdue.edu 18 19

connected by adjoining cell walls. One exception is pollination, in which pollen (the male 47 gametophyte) is released from an anther, transported to a receptive stigma, and 48 produces a tip-growing pollen tube that grows through the female tissues of the pistil 49 and delivers the two sperm cells to the female gametophyte (also known as the embryo 50 sac, Fig 1A). The pollen tube's journey through the pistil requires cell-to-cell interactions 51  Ngo et al., 2014). In order to follow subcellular changes in NTA-GFP protein 167 localization before, during, and after pollen tube arrival, we used pollen from plants 168 expressing the pollen-specific AUTOINHIBITED Ca 2+-ATPASE9pro::DsRed 169 (ACA9pro::DsRed) reporter and ovules expressing NTApro::NTA-GFP in the semi-in vivo 170 system. Approximately 4 h after pollination, pollen tubes emerged from the style onto 171 the media and were attracted to ovules (Fig 1B). Images in the red and green channels 172 were collected every 5 min from when a pollen tube approached an ovule until after the 173 pollen tube ruptured inside the ovule. In our system, most of the ovules displayed 174 successful pollen tube attraction and reception, while others did not attract a pollen tube 175 during the time course of the imaging experiments (Fig 1B and S1). A second group 176 were imaged under the same conditions and serve as a negative control for 177 environmentally-induced changes in NTA-GFP localization. 83% of the ovules that 178 attracted a pollen tube that successfully burst to deliver the sperm cells (n=93) 179 displayed NTA-GFP redistributed to the micropylar end of the synergid cell (Fig 1C-E). 180 Ovules without NTA-GFP redistribution displayed abnormal pollen tube behavior in 181 which pollen tubes were attracted but stopped growing and never ruptured to release 182 the sperm cells. Neighboring ovules that did not attract a pollen tube (n=103) but were 183 imaged under the same semi-in vivo conditions did not have redistribution of NTA-GFP 184 (Fig 1E), nor did ovules that were incubated on pollen germination media without a 185 pollinated pistil (n=133, Fig 1E). These data suggest that pollen tube arrival is 186 necessary for NTA-GFP redistribution and that the imaging conditions do not trigger 187 redistribution ( Fig 1E). (B, C) Live imaging of pollen tube (PT) reception using NTA-GFP labeled synergids (green signal) and ACA9::DsRed pollen tubes (magenta signal). (B) NTA-GFP redistribution occurred in ovules that attracted a pollen tube (ovules with white stars), while NTA-GFP redistribution did not occur in ovules without pollen tube attraction (ovules with green stars). (C) Timelapse imaging of NTA-GFP movement during pollen tube reception. NTA-GFP before (ovules with yellow stars) and after (ovules with blue stars) the PT resumes growth after initial arrival at the filiform apparatus. (D) Quantification of NTA-GFP signal before (yellow starred ovule) and after (blue starred ovule) pollen tube arrival. Synergid cell from chalazal end to filiform apparatus (FA) end was defined from 0 to 33 µm in length. (E) Quantification of the percentage of ovules with NTA redistribution under different experimental conditions. Bars=50µm (A, C). CC, Central Cell; Syn, Synergid Cells; EC, Egg Cell; An, Antipodal cells; FA, Filiform Apparatus; PT, Pollen Tube.
Our semi-in vivo system also allowed us to determine the timing of NTA-GFP 191 redistribution in relation to the position of the pollen tube as it approached the synergids. We previously determined that NTA is sequestered in a Golgi-associated compartment 215 in synergid cells that have not attracted a pollen tube  . Our live-216 imaging data suggests that NTA-GFP is selectively moved out of the Golgi and 217 trafficked to the region of the filiform apparatus in response to pollen tube arrival; 218 however, it is possible that the observed NTA-GFP movement is a result of massive 219 reorganization of subcellular compartments. To distinguish between these possibilities, 220 we investigated the behavior of Golgi in synergid cells during pollen tube reception. We 221 used the semi-in vivo imaging system described above with a synergid-expressed Golgi 222 marker (Man49-mCherry) co-expressed with NTA-GFP (Jones et al., 2018). In all 223 replicates, the Golgi marker was distributed throughout the synergids, excluded from the 224 filiform apparatus, and co-localized with NTA-GFP as reported previously (Fig 2A). 225 When a pollen tube approached the synergids, NTA-GFP redistributed to the filiform 226 apparatus region of the synergids as observed previously (Fig 1), but the Golgi-mCherry 227 marker remained consistently distributed throughout the synergid cells and did not 228 concentrate near the filiform apparatus ( Fig 2B). In order to examine the behavior of the 229 Golgi during later stages of pollen tube reception, we used the synergid-expressed 230 Golgi marker line (Man49-mCherry) and pollen that was expressing GFP 231 (Lat52pro::GFP). In all cases, the Golgi marker remained randomly distributed 232 throughout the synergid cells, even after pollen tube rupture (   Endosomes also transport molecules from the Golgi and either continue to vacuole or 260 recycle back to the Golgi (Stoorvogel et al., 1991). We previously reported that 261 mCherry-RabA1g is distributed throughout synergid cells and had some overlap with 262 NTA-GFP in synergids of unpollinated ovules (Jones et al., 2018). Using the semi-in 263 vivo system, we confirmed that before pollen tube arrival, mCherry-RabA1g distributed 264 throughout synergid cells ( Fig 3A). Interestingly, as pollen tubes approached, the 265 endosome marker started to accumulate in the filiform apparatus region of the synergid 266 cells (Fig 3A and B). By the time that pollen tube reception was completed, most of the 267 endosome signal was concentrated at or near the filiform apparatus ( Fig 3B-D and S3, 268 Movies S11 and S12). These results indicate that the RabA1g endosome compartments 269 have a distinct response to pollen tube arrival and may play a role in facilitating the 270 intercellular signaling pathway that occurs between the synergids and the pollen tube.

The CaMBD is important for NTA's function in pollen tube reception 274
The timing of the NTA redistribution during pollen tube arrival is similar to the start of (NTA Δ481 ), all fused to GFP, were generated and expressed under the synergid-296 expressed MYB98 promoter in the nta-1 background (Fig 4). Ovule counts in 297 homozygous lines revealed that all three constructs had significant reductions in 298 unfertilized ovules compared to nta-1. However, only NTA Δ481 rescued at similar levels 299 as full-length NTA (Fig 4B), indicating that the C-terminal tail after the CaMBD is 300 dispensable for NTA function. Both NTA Δ450 and NTA W458A partially rescued nta-1, 301 suggesting that either removal or disruption of the CaMBD have a similar impact on 302 NTA's function. disrupted variants could be due to disrupted localization patterns before and/or after 314 pollen tube arrival. In virgin ovules, both NTA Δ481 and NTA W458A were distributed 315 throughout the synergid cell in punctate compartments and were predominantly 316 excluded from the filiform apparatus (Fig 4E and F). NTA Δ450 accumulated in punctate 317 compartments throughout the synergid, but was also detected near the filiform 318 apparatus and in the vacuole (Fig 4D). Although the two variants with disrupted 319 CaMBDs (NTA Δ450 and NTA W458A ) both partially rescued the nta-1 unfertilized ovule 320 phenotype at similar levels, they had different distributions in the synergid cell. This 321 suggests that differences in localization between these two variants may not be 322 functionally relevant to pollen tube reception. Due to this, we focused primarily on 323 NTA W458A for our downstream analyses and comparisons with wildtype NTA so as to not 324 further complicate the interpretation of our results. to pollen tube arrival, the semi-in vivo system described above was used to monitor 343 NTA W458A movement in the nta-1 background. In this background, the partial rescue by 344 NTA W458A leads to some ovules having normal pollen tube reception, while others 345 exhibit pollen tube overgrowth and a failure of pollen tube rupture. In the semi-in vivo 346 system, NTA W458A ovules that did not attract a pollen tube, maintained distributions 347 outside of the filiform apparatus, consistent with unpollinated flowers above ( Fig S5A  348 and C). In ovules with successful pollen tube reception, NTA W458A redistributed to the 349 filiform apparatus region, but in many cases this redistribution was not as complete as 350 with the wild-type NTA-GFP protein, with some GFP signal remaining outside the 351 filiform apparatus region (Fig 5 and Fig S5, B and D). In ovules where pollen tube 352 reception was not successful due to pollen tube overgrowth, NTA W458A did not 353 accumulate at the filiform apparatus region of the synergids (Fig 5C). These data 354 suggest that an active CaMBD enhances NTA's redistribution to the filiform apparatus 355 region during pollen tube reception and that NTA redistribution is correlated with pollen 356 tube rupture. The selective targeting of NTA-GFP from the Golgi apparatus to the filiform apparatus 364 region of the synergid cells during pollen tube arrival (Fig 1-2) and the link between 365 NTA W458A redistribution and pollen tube reception (Fig 5) suggests that NTA 366 accumulation at the pollen tube/synergid interface is important for the intercellular 367 communication process that occurs between the pollen tube and synergids. In nta-1 368 mutants, around 30% of ovules display pollen tube overgrowth and fail to complete  Fig 6B) was 386 sufficient to direct the fusion protein to the filiform apparatus region of the synergids, in 387 a pattern very similar to MLO1-GFP (Fig 6A-C). Quantification of the GFP signal along 388 the length of the synergids from the chalazal end to the filiform apparatus in the NTA-389 GFP, NTA-MLO1 Cterm -GFP, and MLO1-GFP confirmed that the MLO1 tail was sufficient 390 to move the NTA protein to the filiform apparatus end of the cell (Fig 6D). In all MLO1-391 GFP and NTA-MLO1 Cterm -GFP ovules, the majority of GFP signal was detected in the 392 lower 20-40% of the synergids and most of the signal overlapped with the diffuse FM4-393 64 staining in the filiform apparatus ( Fig 6E). In contrast, NTA-GFP is excluded from the 394 filiform apparatus (Fig 6A and (Jones et al., 2017).

Synergids respond to a signal from the approaching pollen tube 419
Successful pollination and production of seeds requires a series of signaling events 420 between the male gametophyte (pollen tube) and both sporophytic and gametophytic 421 cells of the female. In this study, we used live imaging to characterize dynamic 422 subcellular changes that occur in the synergid cells of the female gametophyte in 423 response to the arrival of the pollen tube. We showed that both the NTA protein and 424 endosomes are actively mobilized to the filiform apparatus region where male-female 425 communication occurs during pollen tube reception (Fig 7). Disruption of NTA's CaMBD 426 partially compromised NTA redistribution and function in pollen tube reception, revealing 427 that Ca 2+ may play a role the synergid response to the signal from the pollen tube.

Signal-mediated protein trafficking 450
Signal-mediated regulation of protein trafficking is an elegant mechanism to control the 451 delivery of molecules to the precise location where they are needed for critical signaling 452 events that occur over relatively short time frames. Selective protein targeting similar to 453 NTA movement in response to pollen tube arrival has also been observed during cell-to- and harmful microbes (reviewed in (Chen et al., 2015). In most cases, the mechanism 486 for decoding Ca 2+ spikes into a cellular response is not known, but Ca 2+ -binding proteins 487 such as calmodulin (CaM) and calmodulin-like proteins could play a role in relaying Ca 2+ 488 signals (Chin and Means, 2000). In nta-1 mutants, the [Ca 2+ ]cyto oscillations still occur, 489 but at a lower magnitude than in wild-type synergids, suggesting that NTA could be 490 involved in modulating Ca 2+ flux (Ngo et al., 2014). The source of Ca 2+ during these 491 oscillations is not known, but it is possible that NTA regulates Ca 2+ channels to regulate 492 the flow of Ca 2+ ions into or out of the apoplast near the filiform apparatus. In this study, we showed that signals from an approaching pollen tube trigger the 543 movement of NTA out of the Golgi and to the region of the filiform apparatus and that 544 this redistribution is correlated with pollen tube reception. However, localization of the 545 NTA-MLO1 CTerm fusion protein was able to complement the nta-1 mutant phenotype, 546 indicating that the final localization of the NTA protein may be more important than the 547 active trafficking from the Golgi compartment. Future work will focus on determining the 548 mechanism through which NTA becomes polarly redistributed and on identifying the 549 signals from the pollen tube that lead to important subcellular changes in the synergids 550 during pollen tube reception.  Table S1). The NTA W458A point mutation was generated using the same NTA template