Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX families of transcription factors

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast (Saccharomyces cerevisiae) one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10. Specific binding to a region in the untranslated leader sequence of Oskn2 was confirmed by yeast and in vitro binding assays. ProOskn2:β-glucuronidase reporter expression was down-regulated by OsGRF3 and OsGRF10 in vivo, suggesting that these proteins function as transcriptional repressors. Likewise, we found that the GRF protein BGRF1 from barley (Hordeum vulgare) could act as a repressor on an intron sequence in the KNOX gene Hooded/Barley Knotted3 (Bkn3) and that AtGRF4, AtGRF5, and AtGRF6 from Arabidopsis (Arabidopsis thaliana) could repress KNOTTED-LIKE FROM ARABIDOPSIS THALIANA2 (KNAT2) promoter activity. OsGRF overexpression phenotypes in rice were consistent with aberrant meristematic activity, showing reduced formation of tillers and internodes and extensive adventitious root/shoot formation on nodes. These effects were associated with down-regulation of endogenous Oskn2 expression by OsGRF3. Conversely, RNA interference silencing of OsGRF3, OsGRF4, and OsGRF5 resulted in dwarfism, delayed growth and inflorescence formation, and up-regulation of Oskn2. These data demonstrate conserved interactions between the GRF and KNOX families of transcription factors in both monocot and dicot plants

Video 6. Macrophage burst at the exponential phase of Mm infection.

Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm were imaged alive every ~63 sec from 1 dpi to 2 dpi by CLSM (Nikon A1). Macrophage burst and spreading of Mm (red) are shown, followed by the recruitment of GFP-positive (green) macrophages and DsRed-positive (blue) neutrophils. In the left bottom the macrophages and Mm are visualized separately. In the right bottom only the Mm are visualized, and the burst event at t = 5h is indicated by arrowheads. A neutrophil that phagocytized Mm after the burst event, indicated by an arrow in the upper panel at t= 8, is moving away from the infection site. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 50 µm

Video 9. Three-dimensional block-face SEM images of Mm infected tail fin.

A double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larva infected with M. marinum was fixed and block face SEM imaging was performed on an 80 by 80 µm region of interest using a Quanta FEG 250 with a Gatan 3View Ultramicrotome. The video shows 154 images acquired every 150 nm step size in z-direction (103 nm pixel size) at 10 frames per second. Scale Bar: 10 µm

Video 3. Cell death of Mm infected macrophages showing fragmentation morphology.

Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec using CLSM (Nikon A1). The GFP-positive (green) macrophage infected with Mm (red) indicated by white arrow undergoes cell death showing clear fragmentation at 57 minutes. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm

Video 10. Three-dimensional block-face SEM images of necrotic neutrophil undergoing netosis.

A double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larva infected with M. marinum was fixed and block face SEM imaging was performed on an 80 by 80 µm region of interest using a Quanta FEG 250 with a Gatan 3View Ultramicrotome. The video shows 70 images in z-direction (23 nm pixel size) of the neutrophil (NF2, Fig. 4). Note the partial intact nuclear envelope (arrow) and the vesicles (arrowheads) fusing with bacterial aggregate. The images were acquired with every 150 nm step size in z-direction and are in this video visualized at 10 frames per second. Scale Bar: 2 µm

Video 7. Extrusion of a Mm aggregate out of the tail fin.

Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm were imaged alive every ~60 sec from 1 hpi to 17 hpi by CLSM (Nikon A1). The GFP-positive (green) macrophages containing a Mm (red) aggregate undergoes cell death showing fragmentation. The bacterial content is clearly extruded from the tail fin 3 hours after the macrophage showing fragmentation (Fig. 3D). The corresponding transmission channel is shown below, showing a protuberance on the outer epithelial layer, which is eventually shed off. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm

Video 4. Cell death of Mm infected macrophages showing roundup morphology.

Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec using CLSM (Nikon A1). The GFP-positive (green) macrophage infected with Mm (red) undergoes cell death showing a roundup morphology at 50 minutes, and the GFP signal associated with Mm decreases gradually in 2 hours. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm

Video 1. Macrophage and neutrophil behavior at the lag phase of Mm infection.

Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec from 1 hpi to 12 hpi by CLSM (Nikon A1). In the top panel the dynamics of GFP-positive (green) macrophages and DsRed-positive (blue) neutrophils are shown, whereas in the bottom panels the trajectories for macrophages (left) and neutrophils (right) are indicated. The magenta and red lines represent infected macrophages and neutrophils, whereas the yellow and blue lines represent uninfected leukocytes. The infected cells remain in the tail fin, except for one of the neutrophils (red line), which undergoes reverse migration along the caudal vein. The uninfected macrophages show short trajectories in the tail fin and the neutrophils show longer tracks, of which one neutrophil (blue line) seems to be scanning a large area of the tail fin. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 100 µm

Video 5. Cell death of Mm infected macrophages showing rapid signal disappearance morphologies.

Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm (red) were imaged alive every ~60 sec using CLSM (Nikon A1). The GFP-positive (green) macrophage infected with Mm (red) indicated by white arrow undergoes cell death showing rapid signal disappearance at 54 minutes and immediate recruitment of another macrophage. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: 20 µm

Video 8. CLSM on macrophage and neutrophil behavior at the exponential phase of Mm infection.

Double transgenic Tg(mpeg1:EGFP) and Tg(lyz:DsRed2) larvae infected with Mm were imaged alive every ~60 sec at 3 dpi for 2.5 hours by CLSM (Nikon A1) before fixation. The two infected GFP-positive (green) macrophages and two infected DsRed-positive (blue) neutrophils that were correlated with 3D block-face SEM images are indicated by arrows. The indicated macrophages (MP1 and MP2) phagocytized Mm approximately 2 and 1.5 hours before fixation respectively and these cells remained GFP-positive after fixation. The neutrophil (NP1) containing Mm was observed 2.5 hours before fixation and undergoes cell death, observed as rapid (within ~1 min) disappearance of the fluorescent signal, ~30 min before fixation. The second neutrophil (NP2) was recruited to another infected neutrophil ~30 min before fixation. The maximum intensity projection from 11 CLSM images (step size in z-direction, 5.56 µm) is shown at 10 frames per second. Scale bar: right panel 100 µm and for the left panel 50 µm