charge coupled device ccd imaging apparatus  (Sangon Biotech)

Journal: bioRxiv

Article Title: Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding gene for canker formation

doi: 10.1101/2023.11.14.564028

Figure Lengend Snippet: Cs9g12620 and Cs9g12650 show different profiles of expression owing to the genetic variation in promoter. ( A ) The location of Cs9g12620 and Cs9g12650 in the Citrus sinensis chromosome. The location was revealed by a BLAST search of the Citrus Genome Database ( https://www.citrusgenomedb.org/ ). ( B ) Verification of the existence of Cs9g12620 and Cs9g12650 in the C. sinensis genome by PCR amplification. The specific primers for either gene were used for qRT-PCR analysis, which are shown in Supplementary file 1b. M, DL2000 DNA marker. ( C ) Semi-quantitative RT-PCR detection of the transcription of Cs9g12620 and Cs9g12650. CsActin was used as the internal control gene. A total of 2 μg of total RNA extracted from C. sinensis was used to synthesize the first single-stranded cDNA. The specific primers for each gene were the same as those of the qRT-PCR analysis and are shown in Supplementary file 1b. M, DL2000 DNA marker. ( D ) Luciferase assays of Cs9g12620 and Cs9g12650 promoter activity. The Cs9g12620 and Cs9g12650 promoter luciferase fusions were transiently expressed in the Nicotiana benthamiana leaves. Luciferase activity was measured with a CCD imaging system at 2 days post-agroinfiltration. The image on the right shows the quantification of luciferase signal using a microplate luminescence reader. Values are the means ± SD (n=3 biological replicates; Student’s t -test, **p□0.01). ( E ) Luciferase assays of Cs9g12620 and Cs9g12620- M with the deletion of 22 bp at the 3’-terminus. The Cs9g12620 and Cs9g12620- M promoter luciferase fusions were transiently expressed in the N. benthamiana leaves. Luciferase activity was measured with a CCD imaging system at 2 days post-agroinfiltration. The luciferase signal was quantified as described in D . CCD, charge-coupling device; qRT-PCR, real-time quantitative reverse transcription PCR; RT-PCR, reverse transcription PCR; SD, standard deviation. The online version of this article includes the following figure supplement(s) for : . Alignment of the promoter regions of Cs9g12620 and Cs9g12650 in Citrus sinensis . The promoter sequences were retrieved from the citrus genome database. The putative binding sites of CsLOB1 (LB1 and LB2) are shadowed with blue. The location of binding site of PthA4 (EBE) is indicated by an underline. A green shadow shows the location of predicted promoter core structure. The lack of 22 bp at the 3’ terminus is shown by a red box.

Article Snippet: For each assay at 2 dpi, the inoculated leaves were treated with 0.5 mM luciferin, kept in the dark for 1 min to quench the fluorescence, and then used to capture luciferase luminescence images by a cooled charge-coupled device (CCD) imaging apparatus (Roper Scientific, Trenton, NJ, USA).

Techniques: Expressing, Amplification, Quantitative RT-PCR, Marker, Luciferase, Activity Assay, Imaging, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Binding Assay

Journal: bioRxiv

Article Title: Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding gene for canker formation

doi: 10.1101/2023.11.14.564028

Figure Lengend Snippet: PthA4 binds to and suppresses the Cs9g12620 promoter. ( A ) A yeast one-hybrid assay shows that PthA4 interacts with the Cs9g12620 promoter. pGBKT7-pthA4 and pG221-P Cs9g12620 were co-transformed into the yeast strain EGY48 and screened on synthetic dropout SD/-Ura and SD/-Ura/-Trp media. The transformed cells were dissociated by repeated freeze-thaw treatment and then used for β-galactosidase assays on filters soaked with Z buffer that contained 20 μg/mL X-gal. The pGBKT7 and pG221 vectors were used as negative controls by co-transformation with the corresponding constructs. The columns below show the quantitative assay of β-galactosidase by the Miller method. Values are the means ± SD (n=3 biological replicates). (ANOVA, P <0.01). ( B ) An electrophoretic mobility shift assay (EMSA) shows that PthA4 binds to the Cs9g12620 promoter. Purified GST-PthA4 was incubated with 25 ng of promoter DNA in gel shift binding buffer at 28°C for 30 min and then analyzed in 6% non-denaturing PAGE. A GST tag was used as a negative control. ( C ) The overexpression of PthA4 suppressed the Cs9g12620 promoter. PthA4 was transiently co-expressed with a Cs9g12620 promoter GUS fusion in Nicotiana benthamiana. The promoter-driven GUS activity was assayed at 2 days post-agroinfiltration. The upper images show histochemical staining of the Cs9g12620 promoter-driven GUS activity. The bar chart below shows the qRT-PCR analysis of the transcript levels of gusA and pthA4 . The level of expression of each gene in the samples that expressed the promoter alone was set as 1, and the levels of the other samples were calculated relative to that. Values are the mean results from three biological replicates and are the means ± SD (ANOVA, P <0.01). ( D ) EMSA shows that PthA4 binds to the 19 bp predicted binding site (EBE) in the Cs9g12620 promoter. The pur fied GST-PthA4 was incubated with 25 ng of synthetic 19 bp EBE DNA fragment in gel shift binding buffer at 28°C for 30 min and then analyzed in 6% non-denaturing PAGE. ( E ) Luciferase assays of the activity of Cs9g12620 promoter with site-directed mutation in PthA4 EBE. The first nucleotide acid “T” in EBE was mutated into A, C, or G, which generated P Cs9g12620- M A , P Cs9g12620- M C, and P Cs9g12620- M G , respectively. The mutants were fused with luciferase and transiently overexpressed in N. benthamiana leaves. The luciferase activity was measured with a CCD imaging system at 2 days post-agroinfiltration. ( F ) PthA4 did not bind to P Cs9g12620- M A , P Cs9g12620- M C and P Cs9g12620- M G . The left image represents a yeast one-hybrid assay that did not show an interaction of PthA4 between P Cs9g12620- M A , P Cs9g12620- M C , and P Cs9g12620- M G . The experiments were conducted in the same manner as those in A . The right image represents an EMSA that shows that PthA4 does not bind to the promoter mutants. The analysis was the same as that described in B . Luciferase assays showing the suppression of PthA4 on P Cs9g12620- M A , P Cs9g12620- M C , and P Cs9g12620- M G . The promoter luciferase fusions were co-expressed with PthA4 in N. benthamiana . Co-expression with the empty binary vector pHB was used as the control. The analysis was the same as that described in E . The image on the right shows the quantification of luciferase signal using a microplate luminescence reader. Values are the means ± SD (n=3 biological replicates). ANOVA, analysis of variance; CCD, charge-coupling device; GST, glutathione-S transferase; PAGE, polyacrylamide gel electrophoresis; qRT-PCR, real-time quantitative reverse transcription PCR; SD, standard deviation. *p□0.05. **p□0.01 (Student’s t -test).

Article Snippet: For each assay at 2 dpi, the inoculated leaves were treated with 0.5 mM luciferin, kept in the dark for 1 min to quench the fluorescence, and then used to capture luciferase luminescence images by a cooled charge-coupled device (CCD) imaging apparatus (Roper Scientific, Trenton, NJ, USA).

Techniques: Y1H Assay, Transformation Assay, Construct, Electrophoretic Mobility Shift Assay, Purification, Incubation, Binding Assay, Negative Control, Over Expression, Activity Assay, Staining, Quantitative RT-PCR, Expressing, Luciferase, Mutagenesis, Generated, Imaging, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Standard Deviation

Journal: bioRxiv

Article Title: Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding gene for canker formation

doi: 10.1101/2023.11.14.564028

Figure Lengend Snippet: The Cs9g12620 promoter is directly activated by CsLOB1. ( A ) A yeast one-hybrid (Y1H) assay shows that CsLOB1 interacts with the Cs9g12620 promoter. pGBKT7-CsLOB1 and pG221-P Cs9g12620 were co-transformed into yeast strain EGY48 and screened on synthetic dropout SD/-Ura and SD/-Ura/-Trp media. The transformed cells were dissociated by repeated freeze-thaw treatment and then used for β-galactosidase assays on filters soaked with Z buffer that contained 20 μg/mL of X-gal. pGBKT7 and pG221 vectors were used as the negative control by co-transformation with corresponding constructs. The columns below show the quantitative assay of β-galactosidase by the Miller method. Values are the means ± SD (n=3 biological replicates) (ANOVA, P <0.01). ( B ) An electrophoretic mobility shift assay (EMSA) shows that CsLOB1 binds to the Cs9g12620 promoter. Purified GST-CsLOB1 was incubated with 25 ng of promoter DNA in gel shift binding buffer at 28°C for 30 min and was then analyzed by 6% non-denaturing PAGE. ( C ) Cs9g12620 promoter activity is induced by CsLOB1. CsLOB1 was transiently co-expressed with the Cs9g12620 promoter GUS fusion in Nicotiana benthamiana. Promoter-driven GUS activity was assayed at 2 days post-agroinfiltration. The upper images show the histochemical staining of Cs9g12620 promoter-driven GUS activity. The bar chart below shows the qRT-PCR analysis of transcript levels of gusA and pthA4 . The level of expression of each gene in the samples that expressed the promoter alone was set as 1, and the levels of the other samples were calculated relative to that. Values are the mean results from three biological replicates and are the means ± SD (ANOVA, P <0.01). ( D ) A Y1H assay shows that CsLOB1 interacts with the Cs9g12620 promoter P Cs9g12620 and CsLOB1-binding site deletion mutants P Cs9g12620- M LB1 , P Cs9g12620- M LB2 , andP Cs9g12620- M LB1/2 . The upper diagram shows the putative CsLOB1 binding sites and the corresponding deletion mutants. The bar chart below represents a Y1H assay that shows the interaction of CsLOB1 with the promoter mutants. The analyses were same as those conducted in A . ( E ) An EMSA showed that CsLOB1 binds to Cs9g12620- M LB1 , P Cs9g12620- M LB2 , and P Cs9g12620- M LB1/2 . Purified GST-CsLOB1 was incubated with 25 ng of promoter DNA in gel shift binding buffer at 28°C for 30 min. The analyses were same as those in B . ( F ) Luciferase assays show the role of CsLOB1 on P Cs9g12620- M LB1 , P Cs9g12620- M LB2 , and P Cs9g12620- M LB1/2 . Promoter luciferase fusions were co-expressed with CsLOB1 in N. benthamiana . Co-expression with the empty binary vector pHB was used as the control. Luciferase activity was measured with a CCD imaging system at 2 days post-agroinfiltration. The image on the right shows the quantification of luciferase signal using a microplate luminescence reader. Values are the means ± SD (n=3 biological replicates, **p□0.01 [Student’s t -test]). ( G ) qRT-PCR analysis of the transcript level of Cs9g12620 in CsLOB1- silenced plants. CsLOB1 was silenced in Citrus sinensis using the citrus tristeza virus (CTV)-based gene silencing vector CTV33. The effective infection by CTV was evaluated by the expression of P23 gene harbored in CTV33. The silencing efficiency of CsLOB1 was verified by comparison with the levels of expression in the WT and empty CTV33-infected control plants. The reduced expression of Cs9g12620 is shown by comparison with the level of expression in the empty CTV33-infected plants. The qRT-PCR was performed as described in C . ANOVA, analysis of variance; CCD, charge-coupled device; GUS, β-glucuronidase; PAGE, polyacrylamide gel electrophoresis; qRT-PCR, real-time quantitative reverse transcription PCR; SD, standard deviation; WT, wild type.

Article Snippet: For each assay at 2 dpi, the inoculated leaves were treated with 0.5 mM luciferin, kept in the dark for 1 min to quench the fluorescence, and then used to capture luciferase luminescence images by a cooled charge-coupled device (CCD) imaging apparatus (Roper Scientific, Trenton, NJ, USA).

Techniques: Y1H Assay, Transformation Assay, Negative Control, Construct, Electrophoretic Mobility Shift Assay, Purification, Incubation, Binding Assay, Activity Assay, Staining, Quantitative RT-PCR, Expressing, Luciferase, Plasmid Preparation, Imaging, Virus, Infection, Comparison, Polyacrylamide Gel Electrophoresis, Standard Deviation