Fig. 2 | Nature Communications

Fig. 2

From: Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase

Fig. 2The alternative text for this image may have been generated using AI.

NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N. benthamiana. Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N. benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N. benthamiana leaves were co-infiltrated with A. tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody

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