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Fig. 4 | Genome Biology

Fig. 4

From: Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

Fig. 4

Interference of the N-terminal signal peptide compromises apoplastic localization of the EIG-E80 pollen tube-secreted protein. Secretion of pollen tube-secreted proteins to the proximity of the plasma membrane and the apoplast of tobacco leaf epidermal cells is signal peptide-dependent. a, b Chimeric construct of pollen tube-secreted cysteine-rich polypeptide protein 2 (NtPsCRP2) and its predicted topology showing extracellular localization. c–e Verification of NtPsCRP2 localization near the plasma membrane (PM) by plasmolysis of tobacco epidermal cells co-infiltrated with apoplastic viral AVR2-mCherry. The arrow shows plasmolyzed regions with detached plasma membrane and apoplastic localized PR1a-AVR2-mCherry. f Two-channel confocal laser scan profile during co-localization of NtPsCRP2 with apoplastic marker AVR2-mCherry following plasmolysis. AU arbitrary units. g, h Chimeric construct of elicitor-induced protein E80 (EIG-E80) and its predicted topology showing apoplastic/extracellular localization. i, j Reconstituted GFP:EIG-E80 chimeric construct with blocked signal peptide resulted in partial apoplastic localization and predominant nuclear localization instead. k Subcellular quantification of the modified GFP:EIG-E80 expression. Error bars represent ± standard deviation. l–o Verification of EIG-E80:GFP apoplastic localization by plasmolysis with viral AVR2 apoplastic marker. Arrows indicate plasmolysed regions. p Localization of the unconventionally secreted tobacco pollen tube protein Translationally controlled tumor protein (TCTP) showing nucleoplasm, cytosol, and apoplastic localization. q–s Verification of TCTP apoplastic localization. t Immunodetection verification of ectopically expressed GFP chimeric pollen tube-secreted proteins with rat anti-GFP monoclonal antibodies in tobacco epidermal cells. Commassie G250 stain of total protein extract (left) and immunoblot (right) 48 h post-infiltration. Scale bars = 10 μM. N.b untransformed Nicotiana benthamiana

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