( A ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . XopL or GFP constructs were co - infiltrated . RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities to the mean of control ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( P < 0 . 01 ) was shown by Student ' s t - test . The experiment was repeated more than 3 times by with similar results .

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(A) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. XopL or GFP constructs were co-infiltrated. RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities to the mean of control (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (P < 0.01) was shown by Student's t-test. The experiment was repeated more than 3 times by with similar results.


( B ) Immunoblot analysis of NBR1 and ATG8 protein levels in N . benthamiana plants transiently expressing GFP - XopL or GFP control at 2dpi verified with an anti - GFP antibody ( same blot was split for visualization purpose ) . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated at least three times with similar results .

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(B) Immunoblot analysis of NBR1 and ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL or GFP control at 2dpi verified with an anti-GFP antibody (same blot was split for visualization purpose). Ponceau Staining (PS) served as a loading control. The experiment was repeated at least three times with similar results.


( C ) Immunoblot analysis of ATG8 protein levels in N . benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment . Expression of GFP - XopL was verified with an anti - GFP antibody , while expression of GUS - HA was confirmed with an anti - HA antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

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(C) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment. Expression of GFP-XopL was verified with an anti-GFP antibody, while expression of GUS-HA was confirmed with an anti-HA antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( D ) GFP - ATG8g - labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA . Puncta were calculated from z - stacks ( 15 ) of n = 12 individuals using ImageJ . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * P < 0 . 01 , * * * P < 0 . 001 ) was determined by one way ANOVA . The experiment was repeated twice with similar results .

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(D) GFP-ATG8g-labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA. Puncta were calculated from z-stacks (15) of n=12 individuals using ImageJ. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (** P < 0.01, *** P < 0.001) was determined by one way ANOVA. The experiment was repeated twice with similar results.


( E ) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ , Xcv ΔxopQ ΔxopL or mock infected N . benthamiana plants at 2dpi . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

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(E) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ, Xcv ΔxopQ ΔxopL or mock infected N. benthamiana plants at 2dpi. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( F ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co - infiltrated with Agrobacteria containing the respective constructs . RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . The experiment was repeated 3 times with similar results .

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(F) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co-infiltrated with Agrobacteria containing the respective constructs. RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values (***P < 0.001) was revealed by Student's t-test. The experiment was repeated 3 times with similar results.


( A ) Interaction of XopL with SH3P2 in yeast two - hybrid assays . XopL fused to the GAL4 DNA - binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain ( AD ) in yeast strain Y190 . Cells were grown on selective media before a LacZ filter assay was performed . pSV40 / p53 served as positive control , while the empty AD or BD vector served as negative control . NtSH3P2 = Nicotiana tabacum SH3P2 . - LT = yeast growth on medium without Leu and Trp , - HLT = yeast growth on medium lacking His , Leu , and Trp , indicating expression of the HIS3 reporter gene . LacZ , activity of the lacZ reporter gene .

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(A) Interaction of XopL with SH3P2 in yeast two-hybrid assays. XopL fused to the GAL4 DNA-binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain (AD) in yeast strain Y190. Cells were grown on selective media before a LacZ filter assay was performed. pSV40/p53 served as positive control, while the empty AD or BD vector served as negative control. NtSH3P2 = Nicotiana tabacum SH3P2. -LT = yeast growth on medium without Leu and Trp, -HLT = yeast growth on medium lacking His, Leu, and Trp, indicating expression of the HIS3 reporter gene. LacZ, activity of the lacZ reporter gene.


( B ) Coimmunoprecipitation of GFP - XopL with AtSH3P2 - HA . GFP - XopL or GFP were transiently coexpressed with AtSH3P2 - HA in leaves of N . benthamiana . After 48 h , total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis using either anti - GFP or anti - HA antibodies . GFP blot was split for visualization purpose . AtSH3P2 = Arabidopsis thaliana SH3P2 . Two repetitions with similar results have been conducted .

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(B) Coimmunoprecipitation of GFP-XopL with AtSH3P2-HA. GFP-XopL or GFP were transiently coexpressed with AtSH3P2-HA in leaves of N. benthamiana. After 48 h, total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis using either anti-GFP or anti-HA antibodies. GFP blot was split for visualization purpose. AtSH3P2 = Arabidopsis thaliana SH3P2. Two repetitions with similar results have been conducted.


( C ) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay . Yellow fluorescent protein ( YFP ) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173 - XopL in combination with AtSH3P2 - VenusC155 . A positive control showing the dimerization of fructose - 1 , 6 - bisphosphatase ( FBPase ) within the cytosol . White arrows indicate reconstitution of YFP fluorescence . The red structures indicate autofluorescence of chloroplasts . The combination of VenusN173 - XopL with FBPase - VenusC155 or VenusN173 - FBPase with AtSH3P2 - VenusC155 do not induce YFP fluorescence and serve as negative controls . Bars = 20 μm .

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(C) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay. Yellow fluorescent protein (YFP) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173-XopL in combination with AtSH3P2-VenusC155. A positive control showing the dimerization of fructose-1,6-bisphosphatase (FBPase) within the cytosol. White arrows indicate reconstitution of YFP fluorescence. The red structures indicate autofluorescence of chloroplasts. The combination of VenusN173-XopL with FBPase-VenusC155 or VenusN173-FBPase with AtSH3P2-VenusC155 do not induce YFP fluorescence and serve as negative controls. Bars = 20 μm.


( D ) Colocalization analysis of GFP - XopL with SH3P2 - RFP , RFP - ATG8e and RFP - ATG8g in N . benthamiana leaves . Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells ( scale bars = 20 μm and 10 μm , lower panel ) . White arrows indicate colocalization of GFP and RFP signals . The experiment was repeated twice with similar results .

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(D) Colocalization analysis of GFP-XopL with SH3P2-RFP, RFP-ATG8e and RFP-ATG8g in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (scale bars = 20 μm and 10 μm, lower panel). White arrows indicate colocalization of GFP and RFP signals. The experiment was repeated twice with similar results.


( E ) Total proteins were extracted 48 hpi with A . tumefaciens harboring the respective GFP - XopL , HA - XopL and SH3P2 - GFP expression constructs . SH3P2 - GFP protein levels ( lower band ) were detected using an anti - GFP antibody . Expression of the XopL was verified using an anti - HA or anti - GFP antibody . Expression of GUS - HA served as a control . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results .

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(E) Total proteins were extracted 48 hpi with A. tumefaciens harboring the respective GFP-XopL, HA-XopL and SH3P2-GFP expression constructs. SH3P2-GFP protein levels (lower band) were detected using an anti-GFP antibody. Expression of the XopL was verified using an anti-HA or anti-GFP antibody. Expression of GUS-HA served as a control. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( F ) Growth of Xcv and Xcv ΔxopL strains in roq1 N . benthamiana plants silenced for SH3P2 ( pTRV2 - SH3P2 ) compared to control plants ( pTRV2 ) . Leaves were dip - inoculated with a bacteria suspension at OD600 = 0 . 2 and bacteria were quantified at 3 and 6 dpi . Experimental repeats are indicated by data points in red ( n = 8 ) and yellow ( n = 6 ) data points . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Different letters indicate statistically significant differences ( P < 0 . 05 ) as determined by one - way ANOVA .

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(F) Growth of Xcv and Xcv ΔxopL strains in roq1 N. benthamiana plants silenced for SH3P2 (pTRV2-SH3P2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2 and bacteria were quantified at 3 and 6 dpi. Experimental repeats are indicated by data points in red (n=8) and yellow (n=6) data points. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically significant differences (P < 0.05) as determined by one-way ANOVA.


( A ) SH3P2 - GFP was transiently coexpressed together with GUS - HA and GFP - XopL in N . benthamiana using agroinfiltration . At 42 hpi , 200 μM MG132 was infiltrated into A . tumefaciens - inoculated leaves , and leaf material was collected 48 hpi . Expression of SH3P2 - GFP ( lower band ) and GFP - XopL ( upper band ) was detected using an anti - GFP antibody . GUS - HA expression was confirmed with an anti - HA antibody . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results .

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(A) SH3P2-GFP was transiently coexpressed together with GUS-HA and GFP-XopL in N. benthamiana using agroinfiltration. At 42 hpi, 200 μM MG132 was infiltrated into A. tumefaciens-inoculated leaves, and leaf material was collected 48 hpi. Expression of SH3P2-GFP (lower band) and GFP-XopL (upper band) was detected using an anti-GFP antibody. GUS-HA expression was confirmed with an anti-HA antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( B ) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL . GST - XopL , and MBP - SH3P2 were tested using the Arabidopsis His - AtUBA1 and His - AtUBC8 . Lanes 2 to 4 are negative controls . Proteins were separated by SDS - PAGE and detected by immunoblotting using the indicated antibodies . Asterisks indicate bands that are not ubiquitinated an present in controls . The experiment was repeated twice with similar results .

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(B) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL. GST-XopL, and MBP-SH3P2 were tested using the Arabidopsis His-AtUBA1 and His-AtUBC8. Lanes 2 to 4 are negative controls. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. Asterisks indicate bands that are not ubiquitinated an present in controls. The experiment was repeated twice with similar results.


( C ) SH3P2 - GFP was transiently coexpressed together with GFP , GFP - XopL and GFP - XopL ΔE3 in N . benthamiana using agroinfiltration . GFP protein levels were detected with an anti - GFP antibody . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results .

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(C) SH3P2-GFP was transiently coexpressed together with GFP, GFP-XopL and GFP-XopL ΔE3 in N. benthamiana using agroinfiltration. GFP protein levels were detected with an anti-GFP antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( D ) Immunoblot analysis of ATG8 protein levels in N . benthamiana plants transiently expressing GFP - XopL , GFP - XopL ΔE3 or GFP control at 2dpi . Expression of binary constructs was verified with an anti - GFP antibody . GFP blot was split for visualization purpose . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

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(D) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL, GFP-XopL ΔE3 or GFP control at 2dpi. Expression of binary constructs was verified with an anti-GFP antibody. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( E ) RLUC - ATG8a constructs were coexpressed with internal control FLUC in N . benthamiana . GFP - XopL , GFP - XopL ΔE3 or GFP control were co - infiltrated together with RLUC / FLUC mixture . Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * P < 0 . 5 , * * P < 0 . 01 ) was revealed by Student ' s t - test . The experiment was repeated 3 times .

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(E) RLUC-ATG8a constructs were coexpressed with internal control FLUC in N. benthamiana. GFP-XopL, GFP-XopL ΔE3 or GFP control were co-infiltrated together with RLUC/FLUC mixture. Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (* P < 0.5, ** P < 0.01) was revealed by Student's t-test. The experiment was repeated 3 times.


( A ) GFP - XopL was coexpressed with GFP or AIMp - RFP . Proteins were separated by SDS - PAGE and detected by immunoblotting using the indicated antibodies . GFP blot was split for visualization purpose . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results .

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(A) GFP-XopL was coexpressed with GFP or AIMp-RFP. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( B ) Colocalization of RFP - XopL with GFP - Joka2 in N . benthamiana leaves . Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells ( bars = 20 μm ) . The experiment was repeated twice with similar results .

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(B) Colocalization of RFP-XopL with GFP-Joka2 in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (bars = 20 μm). The experiment was repeated twice with similar results.


( C ) FRET FLIM measurements of GFP - Joka2 and RFP - XopL in N . benthamiana leaves . The freeRFP construct served as a negative control and RFP - ATG8E ( n = 9 ) as a positive control . Scattered points show individual data points , color indicates biological repeats . The lifetime ( in ns ) of GFP - Joka2 ( donor , n = 41 ) was significantly reduced in the presence of RFP - XopL ( n = 40 ) but not in the presence of freeRFP ( n = 35 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Significant differences were calculated using Wilcoxon rank sum test , with significantly different groups denoted by different letters . The experiment was repeated three times with similar results .

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(C) FRET FLIM measurements of GFP-Joka2 and RFP-XopL in N. benthamiana leaves. The freeRFP construct served as a negative control and RFP-ATG8E (n = 9) as a positive control. Scattered points show individual data points, color indicates biological repeats. The lifetime (in ns) of GFP-Joka2 (donor, n = 41) was significantly reduced in the presence of RFP-XopL (n = 40) but not in the presence of freeRFP (n = 35). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Wilcoxon rank sum test, with significantly different groups denoted by different letters. The experiment was repeated three times with similar results.


( D ) Immunoprecipitation ( IP ) of GFP - XopL reveals association with NBR1 . Immunoblots of input and IP samples from N . benthamiana plants transiently expressing GFP or GFP - XopL were probed with anti - GFP and anti - NBR1 antibodies . GFP blot was split for visualization purpose .

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(D) Immunoprecipitation (IP) of GFP-XopL reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP or GFP-XopL were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.


( E ) Immunoprecipitation ( IP ) of GFP - XopL and GFP - XopL ΔE3 reveals association with NBR1 . Immunoblots of input and IP samples from N . benthamiana plants transiently expressing GFP , GFP - XopL and GFP - XopL ΔE3 were probed with anti - GFP and anti - NBR1 antibodies . GFP blot was split for visualization purpose .

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(E) Immunoprecipitation (IP) of GFP-XopL and GFP-XopL ΔE3 reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP, GFP-XopL and GFP-XopL ΔE3 were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.


( F ) GFP - XopL was transiently expressed in pTRV2 , pTRV2 - Joka2 and N . benthamiana WT plants . Expression of binary constructs was verified with an anti - GFP antibody . Joka2 silencing was verified using an anti - NBR1 antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(F) GFP-XopL was transiently expressed in pTRV2, pTRV2-Joka2 and N. benthamiana WT plants. Expression of binary constructs was verified with an anti-GFP antibody. Joka2 silencing was verified using an anti-NBR1 antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( G ) Growth of Xcv ΔxopQ in N . benthamiana plants silenced for Joka2 ( pTRV2 - Joka2 ) compared to control plants ( pTRV2 ) . Leaves were dip - inoculated with a bacteria suspension at OD600 = 0 . 2 . and bacteria were quantified at 3 and 6 dpi . Red , blue , and green data points represent repeats of the experiments . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Significant differences were calculated using Student ' s t - test and are indicated by : * * , P < 0 . 01 . The experiment was repeated three times with similar trends .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Growth of Xcv ΔxopQ in N. benthamiana plants silenced for Joka2 (pTRV2-Joka2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2. and bacteria were quantified at 3 and 6 dpi. Red, blue, and green data points represent repeats of the experiments. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Student's t-test and are indicated by: **, P < 0.01. The experiment was repeated three times with similar trends.


( H ) GFP - XopL , GFP - XopL ΔE3 were transiently expressed in N . benthamiana . RFP - AIMp was co - infiltrated to stabilize both XopL variants . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis of the precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a negative control . RFP - AIMp expression was verified by an anti - RFP antibody . GFP blot was split for visualization purpose . Asterisk indicates the GFP - XopL full - length protein . The experiment was repeated three times with similar results .

O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(H) GFP-XopL, GFP-XopL ΔE3 were transiently expressed in N. benthamiana. RFP-AIMp was co-infiltrated to stabilize both XopL variants. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a negative control. RFP-AIMp expression was verified by an anti-RFP antibody. GFP blot was split for visualization purpose. Asterisk indicates the GFP-XopL full-length protein. The experiment was repeated three times with similar results.


( I ) GFP - XopL was transiently expressed in N . benthamiana . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with the ubiquitin pan selector , followed by immunoblot analysis of the precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a control . Asterisk indicates the GFP - XopL full - length protein . GFP blot was split for visualization purpose . The experiment was repeated two times with similar results .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(I) GFP-XopL was transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with the ubiquitin pan selector, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a control. Asterisk indicates the GFP-XopL full-length protein. GFP blot was split for visualization purpose. The experiment was repeated two times with similar results.


( J ) Immunoblot analysis GFP - XopL and GFP - XopLK191A at 1 and 2 dpi using an anti - GFP antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results .

O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Immunoblot analysis GFP-XopL and GFP-XopLK191A at 1 and 2 dpi using an anti-GFP antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( K ) HA - XopL and GFP - Joka2 and its variants were transiently expressed in N . benthamiana . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis of the precipitates using either anti - GFP , anti - ubiquitin and anti - NBR1 antibodies . GFP served as a control .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O

(K) HA-XopL and GFP-Joka2 and its variants were transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP, anti-ubiquitin and anti-NBR1 antibodies. GFP served as a control.


C ) ELISA binding results of mAbs specific for hRSV F ( left panels ) or hMPV F ( right panels ) with the proteins indicated at left . mAbs specific of hRSV F bind epitopes of antigenic site II ( Mota and 47F ) or antigenic site IV ( 101F ) . mAs specific of hMPV F bind epitopes of antigenic site II ( MF9 , MF12 , MF14 and MF15 ) or antigenic site IV ( MF16 ) .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O

C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind epitopes of antigenic site II (Mota and 47F) or antigenic site IV (101F). mAs specific of hMPV F bind epitopes of antigenic site II (MF9, MF12, MF14 and MF15) or antigenic site IV (MF16).


Binding of Mz and 47F Fabs to the immobilized proteins indicated at left . Sensorgrams of eight different concentrations are shown in each panel . The calculated binding parameters are shown in the Table . RUs , response units .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Binding of Mz and 47F Fabs to the immobilized proteins indicated at left. Sensorgrams of eight different concentrations are shown in each panel. The calculated binding parameters are shown in the Table. RUs, response units.


Groups of BALB / c female mice ( n = 5 ) were inoculated twice ( four weeks apart ) i . m . with 10 µg / dose of the proteins indicated at the top of each panel , folded in their respective postfusion conformations . One week after the last dose , mice were sacrificed and their blood collected . A ) Each mouse serum ( identified by a number ) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom . The results represented in the y axis for each individual mouse serum correspond to the inverse dilution that gave 50 % of maximal binding , expressed in log10 units ( EC50 ( log10 ) ) . Each number represents an individual mouse . Short horizontal bars indicate mean values for each group . P values were calculated as indicated under Statistical Analysis . Only relevant P values are shown . * P = 0 . 00045 . Differences were considered significant when P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum (identified by a number) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom. The results represented in the y axis for each individual mouse serum correspond to the inverse dilution that gave 50% of maximal binding, expressed in log10 units (EC50 (log10)). Each number represents an individual mouse. Short horizontal bars indicate mean values for each group. P values were calculated as indicated under Statistical Analysis. Only relevant P values are shown. *P = 0.00045. Differences were considered significant when P < 0.05.


Groups of BALB / c female mice ( n = 5 ) were inoculated twice ( four weeks apart ) i . m . with 10 µg / dose of the proteins indicated at the top of each panel , folded in their respective postfusion conformations . One week after the last dose , mice were sacrificed and their blood collected . B ) Mouse sera were also tested in microneutralization assays with hRSV and hMPV , as indicated at the bottom . The results represented in the y axis correspond to the inverse dilution that inhibited 50 % of the viral infectivity ( IC50 ( log10 ) ) . P values : * P = 0 . 01001 and * * P = 0 . 00271 . Long horizontal lines in A and B indicate detection limits .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, as indicated at the bottom. The results represented in the y axis correspond to the inverse dilution that inhibited 50% of the viral infectivity (IC50 (log10)). P values: *P = 0.01001 and **P = 0.00271. Long horizontal lines in A and B indicate detection limits.


Groups of BALB / c mice ( n = 5 ) were inoculated three times , four weeks apart , with 20 µg of the postfusion F proteins indicated above each panel . One group ( n = 5 ) was inoculated only with the CpG adjuvant , as indicated . Two other groups of mice ( n = 8 ) were similarly inoculated with either F - 415 or F - 416 , as indicated at top of the panels . A ) Mouse sera , collected just before challenging with hRSV ( A2 strain ) , were used in microneutralization assays against the viruses indicated at bottom . Statistical results are shown as in Fig . 3B . * P = 0 . 00085 and * * P = 0 . 01986 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. A) Mouse sera, collected just before challenging with hRSV (A2 strain), were used in microneutralization assays against the viruses indicated at bottom. Statistical results are shown as in Fig. 3B. *P = 0.00085 and **P = 0.01986.


Groups of BALB / c mice ( n = 5 ) were inoculated three times , four weeks apart , with 20 µg of the postfusion F proteins indicated above each panel . One group ( n = 5 ) was inoculated only with the CpG adjuvant , as indicated . Two other groups of mice ( n = 8 ) were similarly inoculated with either F - 415 or F - 416 , as indicated at top of the panels . B ) Five days after virus inoculation , mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay . Mean virus titer for each group is indicated by short horizontal bar .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. B) Five days after virus inoculation, mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay. Mean virus titer for each group is indicated by short horizontal bar.


C ) ELISA binding results of mAbs specific for hRSV F ( left panels ) or hMPV F ( right panels ) with the proteins indicated at left . mAbs specific of hRSV F bind to epitopes of antigenic site Ø ( D25 ) , antigenic site II ( Mz and 47F ) or antigenic site IV ( 101F and 107F ) . mAs specific of hMPV F bind to epitopes of antigenic site II ( MF14 ) , antigenic site IV ( MF11 , MF16 and MF29 ) or an epitope shared by hRSV F and hMPV F ( MPE8 ) .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O

C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind to epitopes of antigenic site Ø (D25), antigenic site II (Mz and 47F) or antigenic site IV (101F and 107F). mAs specific of hMPV F bind to epitopes of antigenic site II (MF14), antigenic site IV (MF11, MF16 and MF29) or an epitope shared by hRSV F and hMPV F (MPE8).


F . Groups of BALB / c female mice ( n = 5 ) were inoculated twice with 10 µg of the proteins indicated at the top of each panel , folded in either the prefusion conformation for hRSV F , F - 410 and F - 411 or in the postfusion conformation for hMPV F . One week after the last dose , mice were sacrificed and their blood collected . A ) Each mouse serum was tested in ELISA for antibody binding to the F proteins indicated at the bottom of each panel . * P = 0 . 00046 and * * P = 0 . 02459 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum was tested in ELISA for antibody binding to the F proteins indicated at the bottom of each panel. *P = 0.00046 and **P = 0.02459.


Groups of BALB / c female mice ( n = 5 ) were inoculated twice with 10 µg of the proteins indicated at the top of each panel , folded in either the prefusion conformation for hRSV F , F - 410 and F - 411 or in the postfusion conformation for hMPV F . One week after the last dose , mice were sacrificed and their blood collected . B ) Mouse sera were also tested in microneutralization assays with hRSV and hMPV , indicated at the bottom . * P = 0 . 04138 , * * P = 0 . 04831 and * * * P = 0 . 00339 . The results in A and B are presented as in Fig . 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, indicated at the bottom. *P = 0.04138, **P = 0.04831 and ***P = 0.00339. The results in A and B are presented as in Fig. 3.


A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆ rapZ mutant Z28 under normal growth and GlcN6P starvation conditions . Both strains , which also carried a chromosomal glmS ' - lacZ fusion , were treated with 60 μg / ml Nva - FMDP or H2O ( " mock " ) . Samples were harvested hourly for Northern analysis and determination of β - galactosidase activity . Growth curves are shown in Appendix Fig S1 . Blots were re - probed using a 5S rRNA specific probe to provide loading controls .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆rapZ mutant Z28 under normal growth and GlcN6P starvation conditions. Both strains, which also carried a chromosomal glmS'-lacZ fusion, were treated with 60 μg/ml Nva-FMDP or H2O ("mock"). Samples were harvested hourly for Northern analysis and determination of β-galactosidase activity. Growth curves are shown in Appendix Fig S1. Blots were re-probed using a 5S rRNA specific probe to provide loading controls.


B The purification profile of Strep - RapZ from the ∆ glmS strain Z904 under GlcN6P replete and depletion conditions is shown ( top ) . The cleared lysate ( CL ) , flow through ( FT ) , washing steps ( W ) and the elution fractions ( E1 - 3 ) from StrepTactin affinity chromatography were separated on 12 . 5 % SDS - PAA gels and stained with Coomassie blue . Metabolites were extracted from E2 and analyzed by HILIC - MS / MS . The extracted ion chromatograms of the LC - MS analysis targeting GlcN6P ( retention time 16 . 6 minutes ) are shown below . The samples derived from purification of Strep - RapZ ( panels i and ii ) or Strep - KdpE ( panels iii and iv ) were analyzed with the SRM transition m / z 258 . 1 to m / z 97 in the negative ion mode . The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg / μl ( panel v ) .

O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

B The purification profile of Strep-RapZ from the ∆glmS strain Z904 under GlcN6P replete and depletion conditions is shown (top). The cleared lysate (CL), flow through (FT), washing steps (W) and the elution fractions (E1-3) from StrepTactin affinity chromatography were separated on 12.5 % SDS-PAA gels and stained with Coomassie blue. Metabolites were extracted from E2 and analyzed by HILIC-MS/MS. The extracted ion chromatograms of the LC-MS analysis targeting GlcN6P (retention time 16.6 minutes) are shown below. The samples derived from purification of Strep-RapZ (panels i and ii) or Strep-KdpE (panels iii and iv) were analyzed with the SRM transition m/z 258.1 to m/z 97 in the negative ion mode. The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg/μl (panel v).


C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites . The Strep - tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite ( i . e . 100 , 500 , 1000 , 2500 and 5000 nM ) were injected using a single cycle kinetics approach .

O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites. The Strep-tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite (i.e. 100, 500, 1000, 2500 and 5000 nM) were injected using a single cycle kinetics approach.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Strains Z197 and Z360 were used , which harbor glmY ' - lacZ and glmZ ' - lacZ fusions , respectively .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z197 and Z360 were used, which harbor glmY'-lacZ and glmZ'-lacZ fusions, respectively.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Expression of glmY ' - lacZ in strain Z197 and the ∆ rapZ mutant Z225 is compared .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Expression of glmY'-lacZ in strain Z197 and the ∆rapZ mutant Z225 is compared.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Strains Z190 and Z201 were addressed , which transcribe glmY ' - lacZ either from the σ54 - promoter or the σ70 promoter , respectively .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and Z201 were addressed, which transcribe glmY'-lacZ either from the σ54-promoter or the σ70 promoter, respectively.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Strains Z190 and the ∆ qseF mutant Z196 are compared , both of which transcribe the glmY ' - lacZ fusion solely from the σ54 promoter .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and the ∆qseF mutant Z196 are compared, both of which transcribe the glmY'-lacZ fusion solely from the σ54 promoter.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions . strains were grown in 96 - well plates and exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . Cells were harvested at indicated times and the β - galactosidase activities were determined . Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures . Strains Z197 and the ∆ rapZ mutant Z225 were used . Tested plasmids were pFDX4291 ( vector control for pFDX4324 and pYG82 = VC1 ) , pFDX4324 ( rapZ ) , pYG82 ( rapZquad ) , pBGG237 ( vector control for pBGG164 = VC2 ) and pBGG164 ( strep - rapZ ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures. Strains Z197 and the ∆rapZ mutant Z225 were used. Tested plasmids were pFDX4291 (vector control for pFDX4324 and pYG82 = VC1), pFDX4324 (rapZ), pYG82 (rapZquad), pBGG237 (vector control for pBGG164 = VC2) and pBGG164 (strep-rapZ).


A BACTH assay addressing interaction of RapZ with QseF and QseE . The following plasmid combinations were tested using reporter strain RH785 ( columns 1 - 6 ) : pBGG352 / pBGG349 , pBGG353 / pBGG348 , pYG199 / pBGG349 , pYG246 / pBGG348 , pKT25 / pUT18C ( negative control ) , pKT25 - zip / pUT18C - zip ( positive control ) . , β - galactosidase activities are presented as mean ± SD .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A BACTH assay addressing interaction of RapZ with QseF and QseE. The following plasmid combinations were tested using reporter strain RH785 (columns 1-6): pBGG352/pBGG349, pBGG353/pBGG348, pYG199/pBGG349, pYG246/pBGG348, pKT25/pUT18C (negative control), pKT25-zip/pUT18C-zip (positive control). , β-galactosidase activities are presented as mean ± SD.


B SPR spectroscopy experiments addressing interaction of RapZ with the cytoplasmic part ( aa 196 - 475 ) of kinase QseE and response regulator QseF . Strep - RapZ was captured onto a sensor chip and increasing concentrations of QseE ' - His10 or QseF - His10 were injected .

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B SPR spectroscopy experiments addressing interaction of RapZ with the cytoplasmic part (aa 196-475) of kinase QseE and response regulator QseF. Strep-RapZ was captured onto a sensor chip and increasing concentrations of QseE'-His10 or QseF-His10 were injected.


C To assess the role of RapZ for QseF activity , β - galactosidase activities were determined from strains Z196 ( ∆ qseF ) and Z1110 ( ∆ qseF ∆ rapZ ) at indicated times during growth . Strains harbored the glmY ' - lacZ fusion that is solely expressed from the σ54 promoter and the following plasmids : pKESK23 ( VC = vector control ; black and grey ; note that activities are too low for display ) , pYG89 ( qseF , green ) , pYG90 ( qseF - D56E , purple ) , pYG93 ( qseF - D56A , red ) . , β - galactosidase activities are presented as mean ± SD .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

C To assess the role of RapZ for QseF activity, β-galactosidase activities were determined from strains Z196 (∆qseF) and Z1110 (∆qseF ∆rapZ) at indicated times during growth. Strains harbored the glmY'-lacZ fusion that is solely expressed from the σ54 promoter and the following plasmids: pKESK23 (VC = vector control; black and grey; note that activities are too low for display), pYG89 (qseF, green), pYG90 (qseF-D56E, purple), pYG93 (qseF-D56A, red). , β-galactosidase activities are presented as mean ± SD.


D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10 - tagged QseE ' ( aa 196 - 475 ) in presence of various concentrations of RapZ . Samples were removed following [ γ - 32P ] - ATP addition at indicated times and separated on 12 . 5 % SDS - PAA gels , which were analyzed by phospho imaging .

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D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10-tagged QseE' (aa 196-475) in presence of various concentrations of RapZ. Samples were removed following [γ-32P]-ATP addition at indicated times and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.


E Analysis of QseE ' autophosphorylation ( lanes 1 - 6 ) and phosphoryl group transfer to QseF ( lanes 7 - 12 ) in absence or presence of 5 μM Strep - RapZ . To assess phosphoryl group transfer , 1 μM QseF - His10 was added to the assay .

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E Analysis of QseE' autophosphorylation (lanes 1-6) and phosphoryl group transfer to QseF (lanes 7-12) in absence or presence of 5 μM Strep-RapZ. To assess phosphoryl group transfer, 1 μM QseF-His10 was added to the assay.


F To analyse the role of GlcN6P , 5 μM RapZ or the equivalent volume of buffer was pre - incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE ' - His10 was added . Following an additional incubation for 5 min , [ γ - 32P ] - ATP was added and the reactions were stopped after 1 min .

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F To analyse the role of GlcN6P, 5 μM RapZ or the equivalent volume of buffer was pre-incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE'-His10 was added. Following an additional incubation for 5 min, [γ-32P]-ATP was added and the reactions were stopped after 1 min.


A β - Galactosidase activities of strains Z197 ( wild type ) and Z1118 ( ∆ glmY ∆ glmZ ) , which carry the chromosomal glmY ' - lacZ fusion , were determined during growth .

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A β-Galactosidase activities of strains Z197 (wild type) and Z1118 (∆glmY ∆glmZ), which carry the chromosomal glmY'-lacZ fusion, were determined during growth.


B Strains Z197 ( wild type ) and Z225 ( ∆ rapZ ) were transformed with the following plasmids expressing the mentioned sRNAs : pBR - plac ( vector control = VC ) , pYG83 ( glmY ) , pYG84 ( glmZ ) and pSD69 ( gcvB ) . sRNA expression was induced with 1 mM IPTG and β - galactosidase activities were determined in the exponential growth phase .

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B Strains Z197 (wild type) and Z225 (∆rapZ) were transformed with the following plasmids expressing the mentioned sRNAs: pBR-plac (vector control = VC), pYG83 (glmY), pYG84 (glmZ) and pSD69 (gcvB). sRNA expression was induced with 1 mM IPTG and β-galactosidase activities were determined in the exponential growth phase.


C To assess the impact of GlmY * and GlmZ on stimulation of QseE ' autophosphorylation by RapZ , 1 μM QseE ' - His10 was incubated with [ γ - 32P ] - ATP in absence or presence of 5 μM Strep - RapZ and / or the sRNAs GlmY * , GlmZ and GcvB . In lanes 3 - 11 QseE ' was co - incubated with RapZ as well as the indicated sRNAs provided at 0 . 5 μM , 1 . 25 μM and 2 . 5 μM . In lanes 14 - 16 , QseE ' was incubated with 2 . 5 μM of each sRNA without RapZ . Samples were removed 1 min after addition of [ γ - 32P ] - ATP and separated on 12 . 5 % SDS - PAA gels , which were analyzed by phospho imaging .

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C To assess the impact of GlmY* and GlmZ on stimulation of QseE' autophosphorylation by RapZ, 1 μM QseE'-His10 was incubated with [γ-32P]-ATP in absence or presence of 5 μM Strep-RapZ and/or the sRNAs GlmY*, GlmZ and GcvB. In lanes 3-11 QseE' was co-incubated with RapZ as well as the indicated sRNAs provided at 0.5 μM, 1.25 μM and 2.5 μM. In lanes 14-16, QseE' was incubated with 2.5 μM of each sRNA without RapZ. Samples were removed 1 min after addition of [γ-32P]-ATP and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.


Northern blot experiments assessing the half - lives of GlmY * and GlmZ under normal growth and GlcN6P starvation conditions . Bacterial cultures were either treated with 100 μg / ml Nva - FMDP or H2O ( mock ) . Transcription was stopped by rifampicin addition when cultures attained OD600 = 1 . 0 and samples were removed at indicated times for Northern analysis . A Analysis of GlmY * and GlmZ decay in the wild type strain Z8 . B Analysis of GlmY * and GlmZ decay in the ∆ rapZ mutant Z28 . C Semi - logarithmic plots of GlmY * and full - length GlmZ decay for half - life determination .

O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O

Northern blot experiments assessing the half-lives of GlmY* and GlmZ under normal growth and GlcN6P starvation conditions. Bacterial cultures were either treated with 100 μg/ml Nva-FMDP or H2O (mock). Transcription was stopped by rifampicin addition when cultures attained OD600 = 1.0 and samples were removed at indicated times for Northern analysis. A Analysis of GlmY* and GlmZ decay in the wild type strain Z8. B Analysis of GlmY* and GlmZ decay in the ∆rapZ mutant Z28. C Semi-logarithmic plots of GlmY* and full-length GlmZ decay for half-life determination.


Northern blot experiments ( A - C ) assessing GlmY * half - life under GlcN6P replete ( + GlcN ) and depletion conditions ( - GlcN ) . Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis . A Analysis of the ∆ glmS strain Z1126 . The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub - cultures was resupplied with GlcN ( indicated by arrow ) . B The ∆ glmS ∆ rapZ double mutant Z1127 was tested . C Semi - logarithmic plots of GlmY * decay for half - life determination .

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Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis. A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow). B The ∆glmS ∆rapZ double mutant Z1127 was tested. C Semi-logarithmic plots of GlmY* decay for half-life determination.


D EMSA experiments addressing the role of GlcN6P for GlmY * / RapZ interaction . Radiolabelled GlmY * was incubated with incremental concentrations of RapZ ( left panel ) or RapZ - CTD ( right panel ) in absence or presence of 7 . 5 mM GlcN6P . Binding reactions were separated on native PAA gels and analyzed by phospho - imaging . The RapZ / GlmY * complex is indicated by an arrow .

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D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow.


E EMSA following incubation of GlmY * with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 ( lane 2 ) to 8 mM ( lane 10 ) . The fraction of GlmY * remaining in the gel pocket is marked with an asterisk . The RapZ / GlmY * complex is indicated by an arrow .

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E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow.


F EMSA following incubation of GlmY * with 1200 nM RapZ in absence or presence of 7 . 5 mM of the indicated metabolite . The RapZ / GlmY * complex is indicated by an arrow .

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F EMSA following incubation of GlmY* with 1200 nM RapZ in absence or presence of 7.5 mM of the indicated metabolite. The RapZ/GlmY* complex is indicated by an arrow.


Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781 _ N783 ( left ) or P824R ( right ) CSF - 1R and treated with CSF - 1 ( D , E ) or IL - 34 ( F , G ) for 10 minutes . Horizontal line indicates untreated cells , with increasing concentrations 10 , 50 and 100 ng / ml . Corresponding densitometry displays the ratio of phospho - ERK to total ERK , normalised to the ratio of the untreated control for the native receptor ( * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 01 , * p < 0 . 05 , one - way ANOVA with Sidak ' s post - test for multiple comparison , n = 3 biological replicates , , error bars indicate SEM ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781_N783 (left) or P824R (right) CSF-1R and treated with CSF-1 (D, E) or IL-34 (F, G) for 10 minutes. Horizontal line indicates untreated cells, with increasing concentrations 10, 50 and 100 ng/ml. Corresponding densitometry displays the ratio of phospho-ERK to total ERK, normalised to the ratio of the untreated control for the native receptor (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, , error bars indicate SEM).


Western blot for CSF - 1R in HEK293 cells transfected with native and ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R and treated with rapamycin . Horizontal line indicates untreated cells , with concentrations 0 . 1 , 0 . 5 , 1 , 5 , 10μM . Corresponding densitometry ( below ) displays the fold change of CSF - 1R relative to the untreated control for each transfection ( * * * * p < 0 . 0001 , one - way ANOVA with Sidak ' s post - test for multiple comparison to untreated , n = 3 biological replicates , error bars indicate SEM ) . Western blot for CSF - 1R in HEK293 cells transfected with native and ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R and treated with 3 - methyladenosine . Horizontal line indicates untreated cells , with increasing concentrations 0 . 1 , 0 . 2 , 0 . 5 , 1mM . Corresponding densitometry ( below ) displays the fold change of CSF - 1R relative to the untreated control for each transfection ( * * * * p < 0 . 0001 , * p = 0 . 0114 , one - way ANOVA with Sidak ' s post - test for multiple comparison to untreated , n = 3 biological replicates , error bars indicate SEM ) . Western blot for CSF - 1R and Actin in HEK293 cells transfected with native and ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R and treated with MG132 or PLX3397 . Horizontal line indicates untreated cells , 20μM PLX3397 , with increasing concentrations 0 . 1 , 0 . 5 , 0 . 1 , 5μM . Corresponding densitometry ( below ) displays the fold change of CSF - 1R relative to the untreated control for each transfection ( * * * p = 0 . 0002 , * * p = 0 . 002 , one - way ANOVA with Sidak ' s post - test for multiple comparison to untreated , n = 3 biological replicates , error bars indicate SEM ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).


Immunocytochemistry of microglia isolated from Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + P0 mouse pup brains . Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead + cells via microscopy . Cells were stained using MitoTracker ( red ) . Arrowheads ( white ) indicate bead + cells . Quantification of mouse microglial phagocytic activity expressed as percentage bead + cells ( n = 4 assays , error bars indicate SEM ) .

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Immunocytochemistry of microglia isolated from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ P0 mouse pup brains. Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker(red). Arrowheads (white) indicate bead+ cells. Quantification of mouse microglial phagocytic activity expressed as percentage bead+ cells (n = 4 assays, error bars indicate SEM).


L ) Immunocytochemistry of macrophages differentiated in vitro from Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mouse bone marrow . Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead + cells via microscopy . Cells were stained using MitoTracker ( red ) and DAPI ( blue ) . Arrowheads ( white ) indicate bead + cells . M ) Quantification of mouse macrophage phagocytic activity expressed as percentage bead + cells . ( * p = 0 . 024 , Student ' s t - test with Welch ' s correction , n = 4 assays , error bars indicate SEM ) .

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L) Immunocytochemistry of macrophages differentiated in vitro from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mouse bone marrow. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells. M) Quantification of mouse macrophage phagocytic activity expressed as percentage bead+ cells. (* p = 0.024, Student's t-test with Welch's correction, n = 4 assays, error bars indicate SEM).


A ) Immunohistochemistry of Csf - 1rflx / wt ; Cx3cr1 - Cre - ( top ) and Csf - 1rflx / wt ; Cx3cr1 - Cre + ( bottom ) mice unilaterally injected with Aβ1 - 42 in the hippocampus and stained for IB4 ( green ) , F4 / 80 ( white ) and Aβ ( red ) . Scale bars indicate 50 μm . B ) Quantification of F4 / 80 and IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . Quantification of immunopositivity in both injected and uninjected hippocampi of Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mice . ( Two - way ANOVA with Sidak ' s test for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of the uninjected hippocampus , obliques ( # ) indicate comparison between Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mice , # # or * * p < 0 . 003 , # # # # or * * * * p < 0 . 0001 , n = 5 mice per group , error bars indicate SEM ) .

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A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).


C ) Immunohistochemistry of Csf - 1rflx / wt ; Tie2 - Cre - ( top ) and Csf - 1rflx / wt ; Tie2 - Cre + mice ( ottom ) mice unilaterally injected with Aβ1 - 42 in the hippocampus and stained for IB4 ( green ) , F4 / 80 ( white ) and Aβ ( red ) . D ) Quantification of F4 / 80 and IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . Quantification of immunopositivity in both injected and uninjected hippocampi of Csf - 1rflx / wt ; Tie2 - Cre - and Csf - 1rflx / wt ; Tie2 - Cre + mice . ( n = 5 mice per group . Two - way ANOVA with Sidak ' s test for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of the uninjected hippocampus , * p < 0 . 01 , * * * p < 0 . 0005 , error bars indicate SEM ) ) .

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C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).


qPCR of wild type ( blue ) or Csf - 1r + / - ( red ) endothelial cells treated with control , Csf - 1r + / + MCM or Csf - 1r + / - MCM . Statistical analyses of inter - genotype changes . ( * * * * p < 0 . 0001 , * * p < 0 . 005 , Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings , Two - way ANOVA with multiple comparisons and Sidak post - test , error bars indicate SEM ) ) .

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qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of inter-genotype changes. (**** p < 0.0001, ** p < 0.005, Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM)).


IHC of ALSP patient post mortem tissue for amyloid - β ( green ) and CLD5 ( red ) , ( B ) human IgG ( green ) and PDGFRβ ( red ) , ( C ) GFAP ( green ) and CLD5 ( red ) . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present .

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IHC of ALSP patient post mortem tissue for amyloid-β (green) and CLD5 (red), (B) human IgG (green) and PDGFRβ (red), (C) GFAP (green) and CLD5 (red). ΔA781_N783 and P824R indicate the CSF-1R variant present.


( D ) Iba1 ( green ) and GFAP ( red ) , ( E ) amyloid - β ( green ) and CD68 ( red ) , ( f ) CD163 ( green ) and CLD5 ( red ) , imaging of an ALSP patient with the ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present .

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(D) Iba1 (green) and GFAP (red), (E) amyloid-β (green) and CD68 (red), (f) CD163 (green) and CLD5 (red), imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


( G ) Fibrinogen ( green ) and CLD5 ( red ) and ( H ) FLAIR imaging of an ALSP patient with the ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present .

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(G) Fibrinogen (green) and CLD5 (red) and (H) FLAIR imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


B : PK vs . PIPLC treatment to determine prion loads in infected cells in 384 - well plates . Both treatments discriminate between RML prion infected CAD5 and non - infectious brain homogenate ( NBH ) treated CAD5 cells . * * * * = p - value < 0 . 0001 ( Student ' s t - test ) . Shown are mean ± SD , n ≤ 30 individually cultured wells .

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B: PK vs. PIPLC treatment to determine prion loads in infected cells in 384-well plates. Both treatments discriminate between RML prion infected CAD5 and non-infectious brain homogenate (NBH) treated CAD5 cells. **** = p-value < 0.0001 (Student's t-test). Shown are mean ± SD, n ≤ 30 individually cultured wells.


C : The infectivity of PIPLC and PK - treated cell lysates was determined by infecting CAD5 cells with lysates as indicated . The signal intensity of the highest dilution was measured and compared to untreated RML CAD5 cell lysate . PIPLC - treated cells and PK - treated lysates showed similar infectivity titers . Prion - infected and NBH , as well as RML on CAD5ΔPrnp were used for control .

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C: The infectivity of PIPLC and PK-treated cell lysates was determined by infecting CAD5 cells with lysates as indicated. The signal intensity of the highest dilution was measured and compared to untreated RML CAD5 cell lysate. PIPLC-treated cells and PK-treated lysates showed similar infectivity titers. Prion-infected and NBH, as well as RML on CAD5ΔPrnp were used for control.


D : RML - infected CAD5 and GT - 1 / 7 cells were transfected with non - targeting ( NT ) or Prnp targeting siRNAs in a 384 - well plate and subjected to PK or PIPLC treatment . Z ' - factors were calculated for each condition . Shown are mean ± SD , n = 22 individually cultured wells .

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D: RML-infected CAD5 and GT-1/7 cells were transfected with non-targeting (NT) or Prnp targeting siRNAs in a 384-well plate and subjected to PK or PIPLC treatment. Z'-factors were calculated for each condition. Shown are mean ± SD, n = 22 individually cultured wells.


A : Western blot showing Hnrnpk siRNA transfection ( 96 hrs . ) decreases Hnrnpk protein levels while increases PrPSc in RML prion infected GT - 1 / 7 . Prnp siRNAs suppressed both PrPC and PrPSc as expected . ⍺ : anti Quantifications are reported as normalized to Actin and in comparison , to NT . PK - western blot is quantified relative to NT .

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A: Western blot showing Hnrnpk siRNA transfection (96 hrs.) decreases Hnrnpk protein levels while increases PrPSc in RML prion infected GT-1/7. Prnp siRNAs suppressed both PrPC and PrPSc as expected. ⍺: anti Quantifications are reported as normalized to Actin and in comparison, to NT. PK-western blot is quantified relative to NT.


B : Western Blot of PSA - treated uninfected and infected GT - 1 / 7 cells . Increasing concentration of PSA leads to a more prominent reduction of PrPSc in mouse cells . PK - western blot is quantified relative to DMSO .

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B: Western Blot of PSA-treated uninfected and infected GT-1/7 cells. Increasing concentration of PSA leads to a more prominent reduction of PrPSc in mouse cells. PK-western blot is quantified relative to DMSO.


C : mRNA levels of Hnrnpk and Prnp following siRNA and PSA treatment . FPKM : Fragments per kilobase of transcript per million mapped reads . Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA levels as well as an increase in Prnp mRNA levels . No difference is seen between DMSO treated and PSA treated cells for either Hnrnpk levels of Prnp levels . n = 2 per treatment group .

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C: mRNA levels of Hnrnpk and Prnp following siRNA and PSA treatment. FPKM: Fragments per kilobase of transcript per million mapped reads. Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA levels as well as an increase in Prnp mRNA levels. No difference is seen between DMSO treated and PSA treated cells for either Hnrnpk levels of Prnp levels. n = 2 per treatment group.


D : Quantification of PrPSc levels in RML GT - 1 / 7 cells following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

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D: Quantification of PrPSc levels in RML GT-1/7 cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


E : Brightfield microscopy images of the effect of siRNA mediated HNRNPK downregulation on prion - induced vacuolation in PG127 hovS cells . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA . ovPRNP siRNA transfected , as well as uninfected cells were used as controls Downregulation of ovPRNP in the infected hovS eliminates the vacuoles ; Right panel : Quantification of vacuoles of NT , HNRNPK and ovPRNP siRNA treated PG127 hovS . Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells . Values represent mean ± SD . * p = 0 . 0113 , * * * * p = < 0 . 0001 ( Dunnett ' s multiple comparisons test ) . Scale bar = 100 µm . n = 3 technical replicates .

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E: Brightfield microscopy images of the effect of siRNA mediated HNRNPK downregulation on prion-induced vacuolation in PG127 hovS cells. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA. ovPRNP siRNA transfected, as well as uninfected cells were used as controls Downregulation of ovPRNP in the infected hovS eliminates the vacuoles; Right panel: Quantification of vacuoles of NT, HNRNPK and ovPRNP siRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. * p = 0.0113, **** p = <0.0001 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


F : Quantification of PrPSc levels in PG127 hovS cells following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

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F: Quantification of PrPSc levels in PG127 hovS cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


G : Same as in E , using shRNAs instead of siRNA . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA . Uninfected cells were used as controls Right panel : Quantification of vacuoles of NT and HNRNPK shRNA treated PG127 hovS . Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells . Values represent mean ± SD . * * * p = < 0 . 0009 ( Dunnett ' s multiple comparisons test ) . Scale bar = 100 µm . n = 3 technical replicates .

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G: Same as in E, using shRNAs instead of siRNA. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA. Uninfected cells were used as controls Right panel: Quantification of vacuoles of NT and HNRNPK shRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. *** p = <0.0009 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


H : Quantification of PrPSc levels in PG127 hovS cells following treatment with shRNA against HNRNPK and 1 µM PSA in comparison to DMSO and NT . Each dot represents an experiment . * = p - value < 0 . 027 ( Student ' s t - test ) . Shown are mean ± SD .

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H: Quantification of PrPSc levels in PG127 hovS cells following treatment with shRNA against HNRNPK and 1 µM PSA in comparison to DMSO and NT. Each dot represents an experiment. *= p-value < 0.027 (Student's t-test). Shown are mean ± SD.


I : Gene set overrepresentation analysis of differentially expressed genes ( log2FC - 0 . 25 ≥ or 0 . 25 ≤ and FDR ≤ 0 . 05 ) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT - 1 / 7 cells analyzed by RNAseq . Differentially regulated genes ( up in siRNA treatment and down in PSA or vice versa ) were overlapped and used for pathway analysis . No significantly enriched pathways are detected for upregulated genes in Hnrnpk and downregulated in PSA treatment . For the opposing direction , an enrichment of genes involved in glucose metabolism was detected .

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I: Gene set overrepresentation analysis of differentially expressed genes (log2FC -0.25 ≥ or 0.25 ≤ and FDR ≤ 0.05) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT-1/7 cells analyzed by RNAseq. Differentially regulated genes (up in siRNA treatment and down in PSA or vice versa) were overlapped and used for pathway analysis. No significantly enriched pathways are detected for upregulated genes in Hnrnpk and downregulated in PSA treatment. For the opposing direction, an enrichment of genes involved in glucose metabolism was detected.


A : Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO . PSA treatment was started two weeks after infection and continued until lysis . PSA reduced the amount of PrPSc . Right panel : Quantification of the Western Blot ; n = 5 biological replicates . Values : mean ± SD . * p = 0 . 0211 ( Student ' s t - test ) .

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A: Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO. PSA treatment was started two weeks after infection and continued until lysis. PSA reduced the amount of PrPSc. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values: mean ± SD. * p = 0.0211 (Student's t-test).


B : Western Blot analysis of NBH - treated COCS . PSA treatment was identical to the samples of A . PSA did not affect PrPC expression . Right panel : Quantification of the Western Blot ; n = 5 biological replicates . Values represent mean ± SD . n . s . = not significant ( Student ' s t - test ) . ⍺ :

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B: Western Blot analysis of NBH-treated COCS. PSA treatment was identical to the samples of A. PSA did not affect PrPC expression. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values represent mean ± SD. n.s. = not significant (Student's t-test). ⍺: anti