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. 2011 May 6;23(5):1795–1814. doi: 10.1105/tpc.111.083261

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© 2011 American Society of Plant Biologists

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Figure 6. — Arabidopsis JAZ1 Affects the Interaction of GL3 with GL1 and the Interaction of TT8 with MYB75.

(A) BiFC assays to detect the interaction of GL3 (fused with cYFP) with GL1 (fused with nYFP). cYFP-GL3 and GL1-nYFP were coexpressed in leaves of N. benthamiana. YFP fluorescence was detected 50 h after infiltration. DAPI staining indicates the nuclei.

(B) BiFC assays showing that JAZ1 attenuates the interaction of GL3 with GL1. YFP fluorescence was detected 50 h after coexpression of GL1-nYFP/cYFP-GL3 (CK), JAZ1/GL1-nYFP/cYFP-GL3 (+JAZ1), or GUS/GL1-nYFP/cYFP-GL3 (+GUS). The identical amounts of Agrobacterium tumefaciens strains containing GL1-nYFP and cYFP-GL3 were used in these three combinations (CK, +JAZ1, and +GUS). The three combinations were infiltrated into the same leaf of N. benthamiana and detected under the microscope with identical gain settings (lasers, 514 nm, 8.0%; pinhole, 34 μm; master gain, 732). The experiment was repeated in more than 10 independent biological replicates with similar results. Bar = 100 μm.

(C) Quantitative analysis of YFP fluorescence intensity in (B). Fifty independent fluorescent spots were assessed for fluorescence intensity. Error bars represent se.

(D) Real-time PCR analysis of JAZ1, GUS, cYFP-GL3 (GL3), and GL1-nYFP (GL1) expression in (B). Total RNAs were extracted from leaves of N. benthamiana coinfiltrated with the combinations of various constructs in (B). The primers specific for amplification of GUS, Arabidopsis JAZ1, cYFP-GL3 (GL3), and GL1-nYFP (GL1) are shown in Supplemental Table 1 online. Nicotiana Actin was used as the internal control. Expression levels of genes in the CK treatment were standardized as one unit in the real-time PCR system. Error bars represent se.

(E) JAZ1 attenuates the interaction of TT8 with MYB75. YFP fluorescence was detected 50 h after coexpression of MYB75-nYFP/cYFP-TT8 (CK), JAZ1/MYB75-nYFP/cYFP-TT8 (+JAZ1), or GUS/MYB75-nYFP/cYFP-TT8 (+GUS) in the same leaf of N. benthamiana.

(F) Real-time PCR analysis of JAZ1, GUS, cYFP-TT8 (TT8), and MYB75-nYFP (MYB75) expression in (E). Error bars represent se.

(G) COR attenuates JAZ1 inhibition of the interaction between GL3 and GL1. N. benthamiana leaves were coinfiltrated with Agrobacterium strains carrying the indicated constructs. CK, treatment with buffer on coexpression of GL1-nYFP/cYFP-GL3; +JAZ1, treatment with buffer on coexpression of JAZ1/GL1-nYFP/cYFP-GL3; +JAZ1+COR, treatment with 5 μM COR on coexpression of JAZ1/GL1-nYFP/cYFP-GL3. Fifty independent fluorescent spots were assessed for fluorescence intensity to generate the quantitative data. Error bars represent se.

(H) COR attenuates JAZ1 inhibition of the interaction between MYB75 and TT8. CK, treatment with buffer of coexpression of MYB75-nYFP/cYFP-TT8; +JAZ1, treatment with buffer of coexpression of JAZ1/MYB75-nYFP/cYFP-TT8; +JAZ1+COR, treatment with 5 μM COR of coexpression of JAZ1/MYB75-nYFP/cYFP-TT8.