Pollen tube-triggered accumulation of NORTIA at the filiform apparatus facilitates fertilization in Arabidopsis thaliana

During gamete delivery in Arabidopsis thaliana, intercellular communication between the attracted pollen tube and the receptive synergid cell leads to subcellular events in both cells culminating in the rupture of the tip-growing pollen tube and release of the sperm cells to achieve double fertilization. Live imaging of pollen tube reception revealed dynamic subcellular changes that occur in the female synergid cells. Pollen tube arrival triggers the trafficking of NORTIA (NTA) MLO protein from Golgi-associated compartments and the accumulation of endosomes at or near the synergid filiform apparatus, a membrane-rich region that acts as the site of communication between the pollen tube and synergids. Domain swaps and site-directed mutagenesis reveal that NTA’s C-terminal cytoplasmic tail with its calmodulin-binding domain influences the subcellular localization and function of NTA in pollen tube reception and that accumulation of NTA at the filiform apparatus is necessary and sufficient for MLO function in pollen tube reception.

through the female tissues of the pistil and delivers the two sperm cells to the female 48 gametophyte (also known as the embryo sac, Figure 1A). The pollen tube's journey through the 49 pistil requires cell-to-cell interactions with the female that allows water and nutrient uptake and 50 enables the detection of cues important for guidance toward the female gametes [4]. tube to induce changes that result in pollen tube rupture and delivery of the sperm cells [4,9].
[21]. At the end of pollen tube reception, NTA protein is only detected at the filiform apparatus, 108 indicating that this protein changes its subcellular localization during pollen tube reception [13].

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This suggests that pollen tube-triggered regulation of the synergid secretory system may be a 110 crucial subcellular response to pollen tube arrival and that NTA function may be related to its 111 subcellular distribution; however, the precise timing and significance of NTA's redistribution 112 remain unclear.

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Here, we use a live-imaging system to further characterize synergid cellular dynamics during  and place so that double fertilization can be completed. Based on static images, we previously 131 reported that NTA-GFP fusion protein localizes to a Golgi-associated compartment in synergids 132 prior to pollen tube attraction [21]. When imaged after pollen tube reception, NTA-GFP is concentrated at the micropylar end of the synergid (in or near the filiform apparatus) [13]. NTA-134 GFP does not accumulate at the filiform apparatus in fer ovules with pollen tube overgrowth, 135 suggesting that FER-mediated signaling during pollen tube reception triggers NTA-GFP 136 redistribution that in turn contributes to the interaction of the synergid with the pollen tube [13].

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An alternative hypothesis is that pollen tube rupture triggers NTA-GFP redistribution and is a 138 symptom of pollen tube reception rather than an important contributor to the signaling pathway.

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To distinguish between these two possibilities, we used a semi-in vivo pollination system 140 combined with time-lapse spinning disk confocal microscopy to determine the timing of NTA-141 GFP redistribution during the pollen tube reception process. In the semi-in vivo system, pollen 142 tubes grow out of a cut style and are attracted to ovules arranged on pollen germination media 143 [28]. This system has previously been used to quantify and track pollen tube attraction to ovules 144 and to image [Ca 2+ ]cyto reporters during pollen tube reception [10,12,[29][30][31][32]. To follow 145 subcellular changes in NTA-GFP protein localization before, during, and after pollen tube arrival, 146 we used pollen from plants expressing the pollen-specific AUTOINHIBITED Ca 2+-147 ATPase9pro::DsRed (ACA9pro::DsRed) reporter and ovules expressing NTApro::NTA-GFP in the 148 semi-in vivo system. Approximately 4 h after pollination, pollen tubes emerged from the style 149 onto the media and were attracted to ovules ( Figure 1B). Fluorescence images in both channels 150 were collected every 5 min from when a pollen tube approached an ovule until after the pollen 151 tube ruptured inside the ovule. In ~83% (n=93) of the ovules that attracted a pollen tube and 152 successfully burst to deliver the sperm cells, NTA-GFP accumulated at the filiform apparatus of 153 the synergids (Figures 1C-E). The rest of the ovules (~17%) attracted pollen tubes that stopped 154 growing in the micropyle and did not rupture. In these ovules, NTA-GFP did not accumulate at 155 the filiform apparatus. To exclude the possibility that prolonged imaging causes stress in 156 synergids which leads to filiform apparatus accumulation of NTA-GFP, we analyzed neighboring ( Figure 1F). Likewise, ovules that were incubated on pollen germination media without a 160 pollinated pistil (n=133, Figures 1F and S1) and imaged over the same time frame did not 161 accumulate NTA-GFP at the filiform apparatus. These data indicate that pollen tube arrival is 162 necessary for NTA-GFP accumulation at the filiform apparatus rather than being retained in the 163 Golgi, and that this accumulation is not an artifact of the semi-in vivo imaging system. (Figure   164 1F).

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Our semi-in vivo system also allowed us to determine the timing of NTA-GFP accumulation at Movies S1 and S2). Even though only one of the synergids receives the pollen tube, NTA-GFP 181 accumulated at the filiform apparatus in both synergid cells in response to pollen tube arrival, 182 similar to the activation of [Ca 2+ ]cyto oscillations in both synergids during pollen tube reception 183 reported in [12].

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Golgi do not concentrate at the filiform apparatus during pollen tube reception 186 We previously determined that NTA is sequestered in a Golgi-associated compartment in 187 synergid cells that have not attracted a pollen tube [21]. Our live-imaging data suggest that 188 NTA-GFP is selectively moved out of the Golgi and transported to the filiform apparatus in 189 response to pollen tube arrival. However, it is possible that the observed NTA-GFP 190 accumulation at the filiform apparatus is a result of massive reorganization of subcellular 191 compartments. To distinguish between these possibilities, we investigated the behavior of the 192 Golgi in synergid cells during pollen tube reception. We used the semi-in vivo imaging system

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Movies S11 and S12). These results indicate that the RabA1g endosome compartments have a 234 distinct response to pollen tube arrival and may play a role in facilitating the intercellular 235 signaling pathway that occurs between the synergids and the pollen tube.

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Pollen tube-independent targeting of NTA to the filiform apparatus is not toxic to the 239 synergids 240 The selective targeting of NTA-GFP from the Golgi to the filiform apparatus during pollen tube 241 arrival (Figures 1 and 2) suggests that NTA trafficking to the pollen tube/synergid interface is 242 important for the intercellular communication process that occurs between the pollen tube and 243 synergids. In nta-1 mutants, around 30% of ovules display pollen tube overgrowth and fail to 244 complete double fertilization, but the other 70% are fertilized normally [13]. This indicates that 245 NTA is not absolutely required for pollen tube reception, but may function as a modifier of the 246 signaling pathway. Since FER signaling in the synergids leads to cell death as pollen tube 247 reception is completed [17,23,24], NTA trafficking to the filiform apparatus could be a 248 mechanism to regulate this death and would thus require sequestration of "toxic" NTA in the 249 Golgi before pollen tube arrival. To test this hypothesis, we took advantage of sequence Early targeting of NTA-MLO1 CTerm -GFP to the filiform apparatus was sufficient to complement 281 the nta-1 pollen tube reception phenotype, but did not give an indication of whether filiform 282 apparatus targeting is necessary for NTA function. Since NTA accumulation is triggered by the 283 arrival of a growing pollen tube, drugs that block membrane protein trafficking are not 284 appropriate to inhibit NTA accumulation at the filiform apparatus because they would also inhibit 285 pollen tube growth. Therefore, we used a genetic approach to inhibit NTA trafficking to the 286 filiform apparatus by adding a Golgi retention signal (RNIKCD) to the C-terminus of our MLO-287 GFP variants. This Golgi retention signal has been reported to cause Golgi-retention when 288 added to proteins that would normally be targeted to the trans-Golgi network and tonoplast [36].
To test if RNIKCD works on MLO proteins, we added RNIKCD to the C-terminus of MLO1 290 (MLO1-RNIKCD) and NTA (NTA-RNIKCD) and co-infiltrated tobacco leaves with these 291 constructs and the Golgi-mCherry marker (35S::Man49-mCherry). Both NTA and NTA-RNIKCD  Figure 5A) and transformed into the nta-1 background. In 328 contrast to wild-type NTA and NTA Δ481 , which result in an even higher fertility in nta-1 mutants 329 than in the Ws controls, NTA W458A only partially complemented the nta-1 fertility phenotype 330 ( Figure 5E). Before pollen tube arrival, NTA W458A co-localizes with a Golgi marker in synergids 331 similar to wild-type NTA ( Figure 5D and Figure S6). However, during pollen tube reception, 332 NTA W458A displays different accumulation patterns that correlate with the ability of the pollen 333 tube to rupture. In our semi-in vivo system, ovules with normal pollen tube rupture had at least 334 partial NTA W458A accumulation at the filiform apparatus, while ovules with pollen tube overgrowth 335 did not accumulate NTA W458A at the filiform apparatus ( Figure 6). These data reveal that an 336 intact CaMBD enhances NTA's redistribution to the filiform apparatus during pollen tube 337 reception and that NTA accumulation at the filiform apparatus promotes pollen tube rupture, 338 while the C-terminal tail after the CaMBD is dispensable for NTA function.

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Disrupting the ability of NTA to pass through the secretory system by adding a Golgi retention 362 signal compromised its ability to participate in pollen tube reception, revealing that NTA

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In this study, we showed that signals from an approaching pollen tube trigger the movement of 470 NTA out of the Golgi and to the filiform apparatus and that this redistribution is necessary for 471 pollen tube reception. Future work will focus on determining the mechanism through which NTA 472 polarly accumulates at the filiform apparatus and on identifying the signals from the pollen tube 473 that lead to important subcellular changes in the synergids during pollen tube reception.

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Genes were amplified with primers that had attB1 and attB2 sites for recombination via BP 537 reaction into the Gateway-compatible entry vector pDONR207. Full-length NTA entry vectors 538 used in this study was generated as described previously [21] . NTA truncation was amplified 539 using NTA full-length entry vector as a template with forward primer NTA-FattB1 and the 540 reverse primer NTA481-RattB2 (See all primer sequences in Table S1). The NTA W458A point 541 mutation was generated using the same NTA template and amplifying two fragments of NTA 542 with desired point mutations introduced into the primers: NTA-FattB1 + NTAW458A-R and 543 NTAW458A-F + NTA-RattB2. The two fragments were purified and pasted together with 544 overlaps using a PCR-pasting protocol. The NTA-MLO1 CTerm construct was generated using the 545 full-length entry vectors of NTA and MLO1 used in previous study [21] as templates and 546 amplifying two fragments of NTA and MLO1 using the two pairs of primers: NTA-FattB1 +NTA-547 R19 and MLO1-F + MLO1-RattB2. The two fragments were purified and pasted together with 548 overlaps using a PCR-pasting protocol. The NTA-GFP-RNIKCD construct was generated using