DOI: 10.5061/dryad.jq2bvq8jp
title: Lemon zinc finger protein ClSUP induces the accumulation of reactive oxygen species and inhibits citrus yellow vein-clearing virus infection via interactions with ClDOF3.4

description: Citrus yellow vein-clearing virus (CYVCV) is an increasing threat to
 citrus cultivation. Notably, the role of zinc finger proteins (ZFPs) in
 mediating viral resistance in citrus plants is unclear. In this study, we
 demonstrate that ZFPs ClSUP and ClDOF3.4 enhance citrus
 defense responses against CYVCV in Eureka lemon. ClSUP
 interacted with the coat protein (CP) of CYVCV to reduce CP accumulation
 and inhibit its silencing suppressor function. Overexpression of
 CISUP triggered reactive oxygen species (ROS) and
 salicylic acid (SA) pathways, and enhanced resistance to CYVCV
 infection. In contrast, ClSUP-silencing resulted in
 increased CP accumulation and down-regulated ROS and SA-related genes.
 ClDOF3.4 interacts with ClSUP
 to facilitate its interactions
 with CP. Furthermore, ClDOF3.4 synergistically
 regulated the accumulation of ROS and SA with ClSUP and
 accelerated the down-regulation of CP accumulation. Transgenic
 plants co-expressing ClSUP and ClDOF3.4 remarkedly decrease the
 CYVCV. These findings provide a new reference for
 understanding the interaction mechanism between the host and CYVCV.
description: These data were obtained by RT-qPCR, ELISA assay, transcriptome
 sequencing analysis and other methods.
description: # Lemon zinc finger protein ClSUP induces the accumulation of reactive
 oxygen species and inhibits citrus yellow vein-clearing virus infection
 via interactions with ClDOF3.4
 [https://doi.org/10.5061/dryad.jq2bvq8jp](https://doi.org/10.5061/dryad.jq2bvq8jp) We have submitted our raw data of the figures (Data of figures.xls), raw data of the supporting figures (Data of supporting figures.xls), and raw data of the second reference genes (Data of the second reference genes.xls). #### **Description of data** **Data of figures.xls** ·Fig. 2D Relative expression level of *CP*: the relative expression level of *CP* at GFP+CP+GUS-His and GFP+CP+ClSUP-His infiltrated patches, GFP+CP+GUS-His was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 2G Relative expression level of *ClSUP*: the relative expression level of *ClSUP* in pGBi:GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 0, 4, 8, 12, and 16 dpi, the data of 0 dpi pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 2H and 2K Relative expression level of *CP*: the relative expression level of *CP* in pGBi:GFP, pGBi:ClSUP-GFP, pGBi:ClSUP-P1-GFP and pGBi:ClSUP-P5-GFP infiltrated Eureka lemon leaves at 0, 3, 6, 9, and 12 days after agro-infiltrated with pGBi:CP, the data of 0 dpi pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 3B and 3C Relative expression level of ROS and SA related genes: the relative expression level of ROS and SA related genes in pGBi:GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi, the date of pGBi:GFP were used as controls; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 3D H2O2 level (μmol/g): the H2O2 contents of pGBi:GFP, pGBi:ClSUP-GFP, pGBi:BAX, and Buffer infiltrated Eureka lemon leaves at 8 dpi. ·Fig. 3E MDA content (nmol/g FW): the MDA contents of pGBi:GFP, pGBi:ClSUP-GFP, pGBi:BAX, and Buffer infiltrated Eureka lemon leaves at 8 dpi. ·Fig. 3F SA content: the SA contents of pGBi:GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi. ·Fig.3G-I: the chlorophyll a, b, and total chlorophyll contents of pGBi:GFP, pGBi:ClSUP-GFP, pGBi:BAX, and Buffer infiltrated Eureka lemon leaves at 8 dpi. ·Fig. 3J Conductivity (μS/cm): the conductivity of pGBi:GFP, pGBi:ClSUP-GFP, pGBi:BAX, and Buffer infiltrated Eureka lemon leaves at 0, 2, 4, 6, 8, and 10 dpi. ·Fig. 4A Relative expression level of *ClSUP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 4E Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 4H and 4I Relative expression level of ROS and SA related genes: the relative expression level of ROS and SA related genes in *ClSUP *transgenic Eureka lemon, the data of pLGN were used as controls; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 5A Relative expression level of* ClDOF3.4*: the relative expression level of* ClDOF3.4* in *ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 5B Relative expression level of* ClSUP*: the relative expression level of* ClSUP* in *ClDOF3.4 *transgenic Eureka lemon, the data of pLGN was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 6F H2O2 level (μmol/g): the H2O2 contents of pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi. ·Fig. 6G MDA content (nmol/g FW): the MDA contents of pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi. ·Fig. 6H SA content: the SA contents of pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi. ·Fig. 6I-6K: the chlorophyll a, b, and total chlorophyll contents of pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi. ·Fig. 6L Relative expression level of genes: the relative expression level of genes in pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi, the data of pGBi:ClSUP-GFP were used as controls; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 6M Relative expression level of *CP*: the relative expression level of *CP* in pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 6 days after agro-infiltrated with pGBi:CP, the data of pGBi:ClSUP-GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 7A and 4B H2O2 level (μmol/g): the H2O2 contents of *ClSUP *transgenic Eureka lemon and *ClSUP*+*ClDOF3.4 *transgenic Eureka lemon. ·Fig. 7B and 4C MDA content (nmol/g FW): the MDA contents of *ClSUP *transgenic Eureka lemon and *ClSUP*+*ClDOF3.4 *transgenic Eureka lemon. ·Fig.7C and 4D SA content: the SA contents of *ClSUP *transgenic Eureka lemon and *ClSUP*+*ClDOF3.4 *transgenic Eureka lemon. ·Fig. 7D Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon and *ClSUP*+*ClDOF3.4 *transgenic Eureka lemon, the data of *ClSUP* was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 8D and 8E Relative expression level of genes: the relative expression level of ROS and SA related genes in *ClSUP *transgenic Eureka lemon and *ClSUP*+*ClDOF3.4 *transgenic Eureka lemon, the data of pLGN were used as controls; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method.  **Data of supporting figures.xls** ·Fig. S4A Relative expression level of *ClSUP*: the relative expression level of *ClSUP* in pGBi:GFP and pGBi:ClSUP-KO infiltrated Eureka lemon leaves at 0, 4, 8, 12, and 16 dpi, the data of 0 dpi pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S4B Relative expression level of *CP*: the relative expression level of *CP* in pGBi:GFP and pGBi:ClSUP-KO infiltrated Eureka lemon leaves at 0, 3, 6, 9, and 12 days after agro-infiltrated with pGBi:CP, the data of 0 dpi pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S5A and S5B Relative expression level of ROS and SA related genes: the relative expression level of ROS and SA related genes in pGBi:GFP and pGBi:ClSUP-KO infiltrated Eureka lemon leaves at 8 dpi, the date of pGBi:GFP were used as controls; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S5C H2O2 level (μmol/g): the H2O2 contents of pGBi:GFP, pGBi:ClSUP-KO, pGBi:BAX, and Buffer infiltrated Eureka lemon leaves at 8 dpi. ·Fig. S5D MDA content (nmol/g FW): the MDA contents of pGBi:GFP, pGBi:ClSUP-KO, pGBi:BAX, and Buffer infiltrated Eureka lemon leaves at 8 dpi. ·Fig. S5E SA content: the SA contents of pGBi:GFP and pGBi:ClSUP-KO infiltrated Eureka lemon leaves at 8 dpi. ·Fig. S7A Relative expression level of *CP*: the relative expression level of *CP* in *ClDOF3.4 *transgenic Eureka lemon leaves which infiltrated with pGBi:ClSUP-GFP, the date of pLGN:ClDOF3.4+pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S7B Relative expression level of *CP*: the relative expression level of *CP* in *ClDOF3.4 *transgenic Eureka lemon leaves which infiltrated with pGBi:ClSUP-KO, the date of pLGN:ClDOF3.4+pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S7C Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon leaves which infiltrated with pGBi:ClDOF3.4-GFP, the date of pLGN:ClSUP+pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S7D Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon leaves which infiltrated with pGBi:ClDOF3.4-KO, the date of pLGN:ClSUP+pGBi:GFP was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S8D Relative expression level of *ClSUP*: the relative expression level of* ClSUP* in *ClDOF3.4+ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S8E Relative expression level of *ClDOF3.4*: the relative expression level of* ClDOF3.4* in *ClDOF3.4+ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10A-C Relative expression level of genes: The relative expression level of *ClSUP*, *ClDOF3.4*, reactive oxygen species (ROS)-related genes, and salicylic acid (SA)-related genes in Eureka lemon with exogenous SA and SA inhibitor 1-aminobenzotriazole (ABT) treatments, the data of CK were used as controls; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10D SA content: the SA contents of exogenous SA and SA inhibitor 1-aminobenzotriazole (ABT) treatments Eureka lemon leaves. ·Fig. S10E Relative expression level of *CP*: the relative expression level of *CP *in Eureka lemon with exogenous SA and SA inhibitor 1-aminobenzotriazole (ABT) treatments, the data of CK was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10G Relative expression level of *ClSUP*: the relative expression level of *ClSUP *in *ClSUP*-silenced Eureka lemon leaves treated with exogenous SA, the data of pGBi:ClSUP-KO was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10H Relative expression level of *CP*: the relative expression level of *CP *in *ClSUP*-silenced Eureka lemon leaves treated with exogenous SA, the data of pGBi:ClSUP-KO was used as control; *Actin* was utilized as an internal control, analyzed by the 2−ΔΔCt method.  **Data of the second reference genes.xls** ·Fig. 2D Relative expression level of *CP*: the relative expression level of *CP* at GFP+CP+GUS-His and GFP+CP+ClSUP-His infiltrated patches, GFP+CP+GUS-His was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 2G Relative expression level of *ClSUP*: the relative expression level of *ClSUP* in pGBi:GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 0, 4, 8, 12, and 16 dpi, the data of 0 dpi pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 2H and 2K Relative expression level of *CP*: the relative expression level of *CP* in pGBi:GFP, pGBi:ClSUP-GFP, pGBi:ClSUP-P1-GFP and pGBi:ClSUP-P5-GFP infiltrated Eureka lemon leaves at 0, 3, 6, 9, and 12 days after agro-infiltrated with pGBi:CP, the data of 0 dpi pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 3B and 3C Relative expression level of ROS and SA related genes: the relative expression level of ROS and SA related genes in pGBi:GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi, the date of pGBi:GFP were used as controls; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 4A Relative expression level of *ClSUP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 4E Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 4H and 4I Relative expression level of ROS and SA related genes: the relative expression level of ROS and SA related genes in *ClSUP *transgenic Eureka lemon, the data of pLGN were used as controls; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 5A Relative expression level of* ClDOF3.4*: the relative expression level of* ClDOF3.4* in *ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 5B Relative expression level of* ClSUP*: the relative expression level of* ClSUP* in *ClDOF3.4 *transgenic Eureka lemon, the data of pLGN was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 6L Relative expression level of genes: the relative expression level of genes in pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 8 dpi, the data of pGBi:ClSUP-GFP were used as controls; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 6M Relative expression level of *CP*: the relative expression level of *CP* in pGBi:ClSUP+ClDOF3.4-GFP and pGBi:ClSUP-GFP infiltrated Eureka lemon leaves at 6 days after agro-infiltrated with pGBi:CP, the data of pGBi:ClSUP-GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 7D Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon and *ClSUP*+*ClDOF3.4 *transgenic Eureka lemon, the data of *ClSUP* was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. 8D and 8E Relative expression level of genes: the relative expression level of ROS and SA related genes in *ClSUP *transgenic Eureka lemon and *ClSUP*+*ClDOF3.4 *transgenic Eureka lemon, the data of pLGN were used as controls; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S4A Relative expression level of *ClSUP*: the relative expression level of *ClSUP* in pGBi:GFP and pGBi:ClSUP-KO infiltrated Eureka lemon leaves at 0, 4, 8, 12, and 16 dpi, the data of 0 dpi pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S4B Relative expression level of *CP*: the relative expression level of *CP* in pGBi:GFP and pGBi:ClSUP-KO infiltrated Eureka lemon leaves at 0, 3, 6, 9, and 12 days after agro-infiltrated with pGBi:CP, the data of 0 dpi pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S5A and S5B Relative expression level of ROS and SA related genes: the relative expression level of ROS and SA related genes in pGBi:GFP and pGBi:ClSUP-KO infiltrated Eureka lemon leaves at 8 dpi, the date of pGBi:GFP were used as controls; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S7A Relative expression level of *CP*: the relative expression level of *CP* in *ClDOF3.4 *transgenic Eureka lemon leaves which infiltrated with pGBi:ClSUP-GFP, the date of pLGN:ClDOF3.4+pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S7B Relative expression level of *CP*: the relative expression level of *CP* in *ClDOF3.4 *transgenic Eureka lemon leaves which infiltrated with pGBi:ClSUP-KO, the date of pLGN:ClDOF3.4+pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S7C Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon leaves which infiltrated with pGBi:ClDOF3.4-GFP, the date of pLGN:ClSUP+pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S7D Relative expression level of *CP*: the relative expression level of *CP* in *ClSUP *transgenic Eureka lemon leaves which infiltrated with pGBi:ClDOF3.4-KO, the date of pLGN:ClSUP+pGBi:GFP was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S8D Relative expression level of *ClSUP*: the relative expression level of* ClSUP* in *ClDOF3.4+ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S8E Relative expression level of *ClDOF3.4*: the relative expression level of* ClDOF3.4* in *ClDOF3.4+ClSUP *transgenic Eureka lemon, the data of pLGN was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10A-C Relative expression level of genes: The relative expression level of *ClSUP*, *ClDOF3.4*, reactive oxygen species (ROS)-related genes, and salicylic acid (SA)-related genes in Eureka lemon with exogenous SA and SA inhibitor 1-aminobenzotriazole (ABT) treatments, the data of CK were used as controls; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10E Relative expression level of *CP*: the relative expression level of *CP *in Eureka lemon with exogenous SA and SA inhibitor 1-aminobenzotriazole (ABT) treatments, the data of CK was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10G Relative expression level of *ClSUP*: the relative expression level of *ClSUP *in *ClSUP*-silenced Eureka lemon leaves treated with exogenous SA, the data of pGBi:ClSUP-KO was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method. ·Fig. S10H Relative expression level of *CP*: the relative expression level of *CP *in *ClSUP*-silenced Eureka lemon leaves treated with exogenous SA, the data of pGBi:ClSUP-KO was used as control; *GAPDH* was utilized as an internal control, analyzed by the 2−ΔΔCt method.  **Table S5.xls** ·The DEGs enriched in the plant hormone signal transduction pathway. CK represented pLGN control plants, A represented *ClSUP* transgenic plants, AB represented *ClSUP* and *ClDOF3.4* co-expression transgenic plants. Analyzed under DESeq2_EBSeq, FDR = 0.01, FC = 2. Code/Software The aforementioned files are compatible with Excel and can be accessed using the program.