Figure 1.
Subcellular distribution of the identified DBP1 putative targets.
Full-length coding sequences were fused to the N-terminus of green fluorescent protein (GFP) or to the C-terminus of yellow fluorescent protein (YFP), and transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Leaf sections were inspected by confocal microscopy 3 d after infiltration. Fusion proteins were expressed in epidermal cells. A large central vacuole occupies most of the cellular space, constraining cytosol to a narrow peripheral area. Co-expression with mRFP-tagged centromeric histone H3 reveals nuclei localization.
Figure 2.
Identified proteins interact in vivo with DBP1.
A, BiFC assay with YFP fusions transiently expressed in N. benthamiana leaves by agroinfiltration. Both reciprocal combinations are shown for each interaction. As a negative control, DBP1 and the unrelated transcription factor BREVIS RADIX (BRX) YFP fusions were used. Centromeric histone H3 fused to mRFP was co-expressed as a marker for nuclear localization. B, Coimmunoprecipitation validated the reported interactions. Relevant fusion proteins were transiently expressed in N. benthamiana leaves by agroinfiltration as indicated, and immunoprecipitated with anti-MaBP antibodies. Western blots of the immunoprecipitated fractions decorated with the indicated antibodies are shown. Anti-GFP antibodies allow for detection of YFP.
Figure 3.
MPK11 interacts with GRF6 and is negatively modulated by DBP1.
A, DBP1 inhibited MPK11 kinase activity. MPK11 and DBP1 were transiently expressed as translational fusions to YFP and MaBP, respectively, in N. benthamiana leaves by agroinfiltration. YFP-MPK11 was immunoprecipitated 3 d after infiltration with anti-GFP antibodies and MAPK activity was measured in the immunoprecipitate by Western blot with antibodies specifically recognizing phosphorylated MyBP (lower panel). A fraction of the immunoprecipitated protein was analyzed using anti-GFP (upper panel) and anti-MaBP (middle panel) antibodies. B, MPK11 interacted with GRF6 in vivo. BiFC analysis in N. benthamiana leaves showing specific MPK11-GRF6 interaction. The MPK11-GRF6 complex was detected predominantly in the nucleus. C, GRF6 and MPK11 co-immunoprecipitation. GRF6-HA was expressed in N. benthamiana leaves and immunoprecipitated with anti-HA antibodies. YFP-MPK11 was detected by Western blot with anti-GFP antibodies in the anti-HA immunoprecipitate when coexpressed with GRF6 (upper panel), confirming interaction between the two proteins. Lower panel shows GRF6-HA immunodetection in the immunoprecipitate by Western blot.
Figure 4.
MPK11 phosphorylated GRF6 promoting ubiquitination and increased turnover.
A, Detection of GRF6 phosphorylation. GRF6-HA was transiently expressed, alone or together with YFP-MPK11 and MaBP-DBP1 as indicated in N. benthamiana leaves by agroinfiltration, and immunoprecipitated using anti-HA antibodies. The three upper panels show detection by Western blot of the different expressed proteins with the indicated antibodies. The two lower panels show immunoprecipitation of GRF6-HA using anti-HA antibodies: an aliquot of the immunoprecipitated fraction was loaded above as a loading control, whereas in the bottom panel serine phosphorylation detected using anti-phosphoserine monoclonal antibody is shown. B, MPK11 mediated increased GRF6 turnover. GRF6-HA and YFP-MPK11 were expressed in N. benthamiana leaves by agroinfiltration. Western blot of protein extracts from infiltrated leaves 3 d post-infiltration immunodecorated with the indicated antibodies for specific expressed protein detection. Ponceau-S staining of the nitrocellulose membrane is shown at the bottom. C, GRF6 was ubiquitinated in vivo and MPK11 increased ubiquitination rate. The indicated proteins were transiently expressed in N. benthamiana leaves by agroinfiltration, and GRF6-HA was immunoprecipitated with anti-HA antibodies 3 d post-infiltration. Left panel, Western blot of the anti-HA immunoprecipitate immunodecorated with anti-ubiquitin antibodies. Right panel, the same blot shown in the left panel was stripped, and immunodecorated with anti-HA antibodies, indicating that the detected ubiquitinated protein corresponds to GRF6-HA and revealing unmodified GRF6.
Figure 5.
Putative DBP1 targets played a role in PPV infection, positively or negatively affecting PPV multiplication.
A, Progression of PPV infection in loss-of-function mutants. Plants of the indicated genotypes were inoculated with PPV-GFP and infection extent was analyzed by fluorescence microscopy to monitor GFP distribution. B, PPV accumulation in loss-of-function mutants. Viral accumulation was measured by RT-qPCR using primers specifically amplifying the viral coat protein gene. Data were normalized using ACT2/8 as a reference gene. Relative accumulation with respect to Col-0 is shown as the mean of three independent experiments ± SD. C, Gene expression analysis during the course of PPV-GFP infection in Col-0 plants. Inoculated leaf tissue showing productive infection was pooled into four different categories according to the degree of viral colonization determined under the fluorescence microscope. Leaf material with no visible (1), low (2), medium (3), and high (4) viral accumulation was harvested, and gene expression was analyzed by RT-qPCR using ACT2/8 as a reference gene. Expression data were normalized to those obtained for similar samples collected from uninfected plants. The mean of three independent experiments ± SD is shown. D, PPV accumulation in the selected infection categories. Viral accumulation was quantified by RT-qPCR as above, and referred to the value in (1). Although no visible signal was observed in these leaves when assessed microscopically, there was an incipient infection detectable by RT-qPCR.
Figure 6.
MPK11 promotes increased GRF6 protein turn-over during PPV infection.
A, Analysis of GRF6 accumulation in infection by PPV. GRF6-HA was transiently expressed in N. benthamiana leaves alone or together with MPK11 as indicated. Protein accumulation was analyzed 3 days post-infiltration in leaves that were not inoculated with PPV (0 dpi), leaves that were inoculated with PPV one day after GRF6-HA agroinfiltration (2 dpi; in these leaves, PPV infection is at day 2 when GRF6 protein accumulation was analyzed), and leaves that were inoculated with PPV one day before GRF6-HA agroinfiltration (4 dpi; in these leaves, PPV infection is at day 4 when GRF6-HA accumulation was recorded). As an internal control, accumulation of ascorbate peroxidase (APX) is shown. Ponceau-S staining of membrane indicates equal protein loading. PPV and ACTIN2/8 RNA accumulation was determined by RT-PCR. B, Quantification of GRF6 and APX bands in Western blot using ImageJ software. Values were referred to those in the absence of PPV and MPK11. The experiment was repeated three times with similar results. C, Proteasome mediates GRF6 degradation promoted by MPK11. GRF6-HA and YFP-MPK were transiently expressed in N. benthamiana leaves 4 days after inoculation of the same leaves with PPV. Two days later, leaves were infiltrated either with the proteasome inhibitor MG-132 or with the same concentration of the solvent DMSO, and after 24 h, leaf material was collected. Protein accumulation was analyzed by Western blot using specific antibodies as indicated.