The full length SDE1 gene is not transcribed during Agrobacterium-mediated transient expression in N. benthamiana
The levels of expression of SDE1 were measured in N. benthamiana leaves subjected to Agrobacterium-mediated transformation to determine the reason for the lack of induction of cell death during transient expression experiments of the full length SDE1 [31]. The coding sequences of full length SDE1 and mature SDE1 (SDE1mp) were separately cloned into the binary vector pHB (Fig. 1a). The transient expression of SDE1mp induced chlorosis in N. benthamiana at 3 days post infiltration (dpi), while no chlorosis was observed in the infiltration area that transiently expressed the full-length SDE1 (Fig. 1b). Using EF1a as an internal control, quantitative RT-PCR (qRT-PCR) was conducted to evaluate the transcript levels of SDE1 and SDE1mp. The results showed that a high transcript level of SDE1mp was detected. In contrast, the transcript level of SDE1 was extremely low (Fig. 1c). No transcript level of SDE1 was detected from uninoculated leaves or the leaves infiltrated with Agrobacterium that harbored empty vector pHB in all the qRT-PCR analyses (Fig. 1c). To determine the expression of SDE1 at protein level, SDE1 and SDE1mp were fused to the C-terminus of red fluorescent protein (RFP) in pGDR vector. At 2 dpi, a strong fluorescence signal was observed from N. benthamiana cells that expressed the RFP-SDE1mp fusion, and the signal was distributed in the cell membrane, cytoplasm, and nucleus (Fig. 1d). In contrast, no fluorescence was observed from the cells that expressed RFP-SDE1 fusion (Fig. 1d). These results demonstrated that the full length SDE1 did not induce cell death in N. benthamiana merely because it could not be transcribed in Agrobacterium-mediated transient transformation. The mature protein SDE1mp was used to test phenotypes in N. benthamiana in the following research.
Four SDE1-interacting proteins are screened from N. benthamiana
Using SDE1 as bait, a Y2H experiment was conducted to identify SDE1-interacting proteins from a N. benthamiana cDNA library. We obtained five positive clones, which corresponded to four N. benthamiana genes. The duplicate clone was DEAD-box RNA helicase DDX3, while the other three included a 26S proteasome non-ATPase regulatory subunit PSMD14, an ARM repeat protein Niben101Scf05290g02006.1, and unknown protein Niben101Scf04231g02014.1 (Table 1). The amino acids of four interactors shared more than 58% identity with their homologs in citrus species, including Citrus sinensis and C. clementine (Table 1). The pGADT7 plasmids recovered from yeast were co-transformed separately into AH109 cells using pGBKT7-SDE1. Each transformant was subjected to growth analysis on selective dextrose (SD) media SD/-Ade/-Leu/-Trp/-His/ and assayed for β-galactosidase to confirm the interaction (Fig. 2).
Table 1
The SDE1-interacting proteins of Nicotiana benthamiana and homologs in citrus plants
No. | Gene ID | Gene products | Homologs in citrus plants |
Citrus sinensis (Identity%) | Citrus clementine (Identity%) |
1 | Niben101Scf02762g01005.1 | DEAD-box ATP-dependent RNA helicase 3 | orange1.1g011124m (70) | clementine0.9_002897 m (70) |
2 | Niben101Scf05290g02006.1 | ARM repeat protein | orange1.1g005044m (81) | clementine0.9_003622 m (81) |
3 | Niben101Scf04231g02014.1 | Uncharacterized | orange1.1g033279m (58) | clementine0.9_025591 m (58) |
4 | Niben101Scf02762g01005.1 | DEAD-box ATP-dependent RNA helicase 3 | orange1.1g011124m (70) | clementine0.9_002897 m (70) |
5 | Niben101Scf07364g00017.1 | 26S proteasome non-ATPase regulatory subunit 14 | orange1.1g032384m (99) | clementine0.9_016406 m (99) |
Silencing of NbDDX3 results in mottled leaves
To investigate the functions of four screened proteins, we created plants in which the respective transcripts were knocked down by virus-induced gene silencing (VIGS) using a tobacco rattle virus (TRV) vector. At 15 dpi, the NbDDX3-silenced plants exhibited yellow colors on the leaves, similar to the silencing of phytoene desaturase gene (PDS) in positive control plants (Fig. 3a). The mottled phenotype of NbDDX3-silenced plants was observed on the first three leaves after inoculation with a TRV construct. The late budding leaves on plants were completely bleached when they became mature. The NbPSMD14-silenced plants exhibited a severe developmental abnormality. At 15 dpi, new budding leaves immediately died (Fig. 3a). In contrast, the silencing of Niben101Scf05290g02006.1 or Niben101Scf04231g02014.1 genes did not lead to distinctive growth phenotypes. The silencing efficiency of each gene was evaluated using qRT-PCR analysis (Fig. 3b).
Examination of the chlorosis induced by SDE1 in gene-silenced plants
To determine whether the screened genes were involved in SDE1-mediated chlorosis, SDE1 and SDE1mp were transiently expressed in gene-silenced plants. Owing to the leaf death in silencing of NbPSMD14, those plants were not analyzed in this trial. Similar to the phenotype in wild type, full length SDE1 did not induce chlorosis on all gene-silenced plants. The transient expression of SDE1mp induced chlorosis in both negative control and PDS-silenced plants (Fig. 4). The induction of chlorosis was difficult to discern on NbDDX3-silenced plants, since the leaves became mottled (Fig. 4). In the leaves from Niben101Scf05290g02006.1 or Niben101Scf04231g02014.1 gene-silenced plants, the transient expression of SDE1mp induced a chlorotic phenotype similar to that found in wild type (Fig. 4).
Transient expression of SDE1 suppresses the transcription of NbDDX3
The phenotypes on NbDDX3-silenced plants encouraged us to determine the expression pattern of NbDDX3 in response to SDE1. Therefore, the transcript levels of NbDDX3 were measured in N. benthamiana leaves that transiently expressed SDE1mp. The transcript level of NbDDX3 was reduced by 70% in plants that expressed SDE1mp in comparison with the uninoculated control at 2 dpi (Fig. 5). This demonstrated that the transcription of NbDDX3 gene was significantly suppressed when SDE1mp was transiently expressed in N. benthamiana. It appeared that the chlorosis induced by SDE1mp was caused by down-regulation of NbDDX3 transcription.
SDE1 Interacts With NbDDX3 At The Cell Membrane
The fused RFP-SDE1mp was found to be localized on cell membrane, nuclei, and cytoplasm (Fig. 1d). A GFP-NbDDX3 fusion was constructed to investigate its interaction with SDE1mp in vivo. Fluorescent signal detection revealed that GFP-NbDDX3 accumulated to high levels in cytoplasmic vesicles with a small amount localized to the cell membrane (Fig. 6a). To examine the spatial interaction, GFP-NbDDX3 and RFP-SDE1mp were transiently co-expressed in N. benthamiana cells. RFP-SDE1mp was observed in the cell membrane, nucleus, and cytoplasm Nevertheless, the subcellular location of GFP-NbDDX3 was altered. In comparison with expression alone, a portion of NbDDX3 was located in the nucleus, with the exception of the cell membrane and organelles. Furthermore, an additional number of NbDDX3 proteins were recruited to the cell membrane. GFP-NbDDX3 and RFP-SDE1mp were co-localized at the cell membrane and nucleus (Fig. 6b). A bimolecular fluorescence complementation assay was performed to further confirm the interaction between NbDDX3 and SDE1mp. This resulted in a clear yellow fluorescence that emitted from the cell membranes. In addition, the degrees of expression of SDE1mp-YC and NbDDX3-YN were analyzed using a western blot (Fig. 6c). RFP-SDE1mp was additionally co-expressed with GFP-XAC1347. GFP-XAC1347 is expressed in the membrane when transformed into N. benthamiana cells, implying that RFP-SDE1mp was truly localized to the cell membrane (Supplementary Table 1). In this case, yellow fluorescence was observed from the cell membrane (Supplementary Fig. 1). These results suggested that a fraction of NbDDX3 interacts with SDE1mp at the cell membranes.