Materiales de herramientas de corte que se utilizan para maquinado de aleaciones de aeromotor

Confocal microscopy of Nicotiana benthamiana leaves infiltrated with both an N-terminal YFP fusion protein-encoding plasmid, and C-terminal YFP fusion protein-encoding plasmid was used to determine the interaction between CPR5 and the proteins identified in the Y2H screening. Full length coding regions of each protein were used for cloning.

To confirm that the BiFC assay works efficiently and properly, EDS1 from Arabidopsis thaliana was used as a control gene to optimize the BiFC assay. The interaction of the positive control (EDS1+EDS1) in this experiment has been confirmed by many studies

A B C No Interaction X cYFP nYFP Z X nYFP cYFP Z No Signal Interaction No Signal X nYFP cYFP Y X cYFP nYFP Y X nYFP cYFP Y OR X Y nYFP cYFP _Interaction Reconstituted signal X Y cYFP nYFP X Y nYFP cYFP OR 496nm Light

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(Feys, Moisan, Newman, & Parker, 2001). EDS1 interaction with itself is known to occur within the nucleus as well as the cytoplasm. Prior to the use of the YFP nonfluorescent halves in BiFC analysis, visible YFP fluorescence was confirmed as well as EDS1 localization. EDS1 fused to full length YFP was transiently transformed to Nicotiana and fluorescence was observed confirming YFP fluorescence can be observed in the cytoplasm and the nucleus (Figure 3.9). Additionally, as a positive control for BiFC interaction, nYFP- EDS1 and cYFP-EDS1 (Figure 3.10B) or EDS1-cYFP (Figure 3.11B) were transiently transformed into Nicotiana and fluorescence was observed, however little interaction was observed within the nucleus as compared to full length YFP-EDS1 which exhibits bright fluorescence in the nucleus (Figure 3.9, 10B). The nuclear fluorescence caused by the interaction of EDS1 within the nucleus may not have been within an observable threshold (Figure 3.9) (Feys, et al., 2001).

To further confirm the system, the following negative controls were tested. Agrobacterium containing plasmids coding for nYFP-EDS1+cYFP-CPR5 (Figure 3.10A), nYFP-EDS1+CPR5-cYFP (Figure 3.11A), cYFP-CPR5+nYFP, cYFP+nYFP-EDS1, cYFP- EDS1+nYFP, and EDS1-cYFP+nYFP (data not shown) was transiently transformed into Nicotiana benthamiana and no detectable YFP fluorescence was detected indicating that the YFP protein was not reconstructed because no interactions were occurring between any of the proteins and protein fragments.

To study protein-protein interactions of CPR5, Agrobacterium containing plasmids coding for CPR5 and one of the 10 genes of interest, or one of the published control genes (SIM, SMR1 and KRP2) were infiltrated into tobacco N. benthamiana. Nine of ten of the proteins of interest, SIM, SMR1 and KRP2 were tested with both cYFP-CPR5 and CPR5-cYFP. SAC9 was not assayed due to the inability to obtain the SAC9 coding sequence without mutation. As no confirmed positive control has been extensively tested and proven for CPR5, no known CPR5 true positive control could be used, however any interaction with SIM, SMR1 and KRP2 will be considered confirmation of their published interaction with CPR5. YFP signal was observed between N-terminal tagged, C-terminal YFP-CPR5 (cYFP-CPR5) and PATL3, PATL5, and FSD1 in the cytoplasm (Figure 3.10C, D, and E). The fluorescence suggests that CPR5 interacts with PATL3, PATL5, and FSD1 outside of the nuclear envelope. YFP signal was also observed between cYFP-CPR5 and KRP2 in the nucleus (Figure 3.10F), suggesting that interaction of the two proteins occurs in the nucleus as observed by Wang et al, (2014).

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YFP signal was also observed between C-terminal tagged, C-terminal YFP-CPR5 (CPR5- cYFP) and N-terminally tagged, N-terminal YFP-CRK4 (nYFP-CRK4) (Figure 3.11B) and PATL6 (nYFP-PATL6) (Figure 3.11C) respectively, in the cell periphery or cytoplasm.

Figure 3.9: EDS1-YFP Fluorescence in the cytoplasm and nucleus.

EDS1-YFP was transiently transformed into Nicotiana leaves, and was visualized using a confocal microscope 3 days after transformation. YFP is positively visualized at 501-600nm. EDS1-YFP fluorescence is positively observed in the nucleus and cytoplasm. EYFP was visualized at 501- 600nm. Bright field was visualized was taken simultaneously using 496nm. Nuclei are noted in red.

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Figure 3.10: BiFC Signal form N. benthamiana Expressing N-terminal tagged, C-terminal-YFP- CPR5 and N-terminal tagged, N-terminal-YFP-GOI.

BiFC assay was carried out in Nicotiana benthamiana leaves. N terminal tagged C-terminal YFP-Full Length CPR5 (cYFP-CPR5) was used for all assays. The N and C terminus of YFP were fused to A) EDS1(nYFP-EDS1) and full length CPR5(cYFP-CPR5) as a negative control, B) EDS1(nYFP-EDS1) and EDS1(cYFP-EDS1), C) PATL3(nYFP-PATL3) and CPR5(cYFP-CPR5), D) PATL5(nYFP-PATL5) and CPR5(cYFP-CPR5), E) FSD1(nYFP-FSD1) and CPR5(cYFP-CPR5), F) KRP2(nYFP-KRP2) and CPR5(cYFP-CPR5). EYFP was visualized at 501-600nm. DAPI served as a nuclear marker and was visualized at 415-491nm. Chloroplast were visualized at 643-746nm. Brightfield was visualized was taken simultaneously using 496nm.

Merged A nYFP-EDS1 + cYFP-CPR5 B nYFP-EDS1 + cYFP-EDS1 C nYFP-PATL3 + cYFP-CPR5 D nYFP-PATL5 + cYFP-CPR5 E nYFP-FSD1 + cYFP-CPR5 F nYFP-KRP2 + cYFP-CPR5

Chloroplast DAPI Brightfield YFP

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Figure 3.11: BiFC Assay was carried out in N. benthamiana Expressing C-terminal tagged, C- terminal-YFP-CPR5 and N-terminal tagged, N-terminal-YFP-GOI

C terminal tagged C-terminal YFP-Full Length CPR5 (CPR5-cYFP) was used for all assays. The N and C terminus of YFP were fused to A) EDS1 (nYFP-EDS1) and full length CPR5 (CPR5-cYFP) as a negative control, B) PATL6 (nYFP-PATL6) and CPR5 (CPR5-cYFP), C) CRK4 (nYFP-CRK4) and CPR5 (CPR5-cYFP). EYFP was visualized at 501-600nm. DAPI served as a nuclear marker and was visualized at 415-491nm. Chloroplast were visualized at 643-746nm. Brightfield was visualized was taken simultaneously using 496nm.