Genome-Wide Identification and Characterization of the Sweet Orange (Citrus sinensis) GATA Family Reveals a Role for CsGATA12 as a Regulator of Citrus Bacterial Canker Resistance

Abstract

Citrus bacterial canker (CBC) is a severe bacterial infection caused by Xanthomonas citri subsp. citri (Xcc), which continues to adversely impact citrus production worldwide. Members of the GATA family are important regulators of plant development and regulate plant responses to particular stressors. This report aimed to systematically elucidate the Citrus sinensis genome to identify and annotate genes that encode GATAs and evaluate the functional importance of these CsGATAs as regulators of CBC resistance. In total, 24 CsGATAs were identified and classified into four subfamilies. Furthermore, the phylogenetic relationships, chromosomal locations, collinear relationships, gene structures, and conserved domains for each of these GATA family members were also evaluated. It was observed that Xcc infection induced some CsGATAs, among which CsGATA12 was chosen for further functional validation. CsGATA12 was found to be localized in the nucleus and was differentially upregulated in the CBC-resistant and CBC-sensitive Kumquat and Wanjincheng citrus varieties. When transiently overexpressed, CsGATA12 significantly reduced CBC resistance with a corresponding increase in abscisic acid, jasmonic acid, and antioxidant enzyme levels. These alterations were consistent with lower levels of salicylic acid, ethylene, and reactive oxygen species. Moreover, the bacteria-induced CsGATA12 gene silencing yielded the opposite phenotypic outcomes. This investigation highlights the important role of CsGATA12 in regulating CBC resistance, underscoring its potential utility as a target for breeding citrus varieties with superior phytopathogen resistance.

Keywords: GATA transcription factor; Xanthomonas citri subsp. citri (Xcc); abscisic acid (ABA); citrus bacterial canker (CBC); jasmonic acid (JA); reactive oxygen species (ROS); salicylic acid (SA).

Figure 1

CsGATA phylogenetic relationships and chromosomal locations. (A) MEGA XI was used to construct an ML phylogenetic tree comprising GATA family proteins from A. thaliana and C. sinensis (AtGATAs and CsGATAs), the latter of which are marked using black stars. The tree was constructed based on complete protein sequences using the Poisson model with 500 bootstrap replicates, and branches are color-coded according to the GATA subclasses. The number of substitutions per site is marked on the branches of the resultant tree. (B) The locations of CsGATA genes in the C. sinensis genome were visualized via MapChart v2.1. The scale indicates the sizes of chromosomes, and black lines denote CsGATA gene positions with corresponding numbers indicating their precise locations. Tandem duplication events are indicated using red rectangles, while red lines represent segmental and whole genome duplication.

Figure 2

Analyses of CsGATA collinearity, conserved motifs, and gene structures. (A) Collinearity among GATAs encoded by A. thaliana and C. sinensis was visualized. The red line represents the collinearity of GATA genes, while the gray lines represent collinearity of genomic segments. (B) GSDS v2.0 was used to analyze CsGATA-conserved motifs and gene structures. Introns, exons, and untranslated regions (UTRs) are, respectively, represented with yellow boxes, lines, and green boxes. Intron and exon lengths are indicated with the corresponding scale.

Figure 3

Analyses of the expression and localization of CsGATA12. (A) Kumquat and Wanjincheng leaves were infected with Xcc, and samples were collected at 0, 6, 12, and 24 hpi for qPCR analysis of CsGATA12 expression using CsGAPDH (CPDB ID: Cs_ont_5g044290) as a normalization control. Different letters indicate significant differences (p < 0.05, ANOVAs with Tukey’ s multiple range test). (B) The subcellular localization of CsGATA12 was evaluated. GFP, chloroplast luminescence (CH), brightfield (BF), and merged images are shown. Scale bar: 10 μm. 35S, cauliflower mosaic virus 35S promoter; NOS, NOS terminator; GFP, green fluorescent protein; LB: left border; RB: right border.

Figure 4

Analyses of the impact of CsGATA12 overexpression on CBC resistance, phytohormone levels, and ROS homeostasis. (A) CsGATA12 overexpression vector. 35S, cauliflower mosaic virus 35S promoter; NOS, NOS terminator; GUS: β-glucuronidase; NPTII: β-glucuronidase and NPT integrated coding genes. LB: left border; RB: right border. (B) The assessment of CsGATA12 expression following its transient overexpression, where CsGAPDH (CPDB ID: Cs_ont_5g044290) was set as a control for normalization. (C) Disease symptoms were evaluated at 10 dpi in Xcc-infected leaves overexpressing CsGATA12. Scale bar: 10 mm. (D,E) CBC lesion sizes (D) and disease index values (E) were assessed at 10 dpi in plants overexpressing CsGATA12. (F–K) Using CsGATA12-overexpressing plant samples, ABA (F), SA (G), JA (H), ET (I), ${\mathrm{O}}_2^{-}$ (J), and H₂O₂ levels (K). (L,M) The activity levels of CAT (L) and SOD (M) were assessed in the CsGATA12-overexpressing plant. Results were compared with two-tailed t-tests and presented as means ± SDs. pLGNe: Wanjincheng plants expressing the empty control pLGNe vector; pLGNe-CsGATA12: transgenic Wanjincheng plants overexpressing CsGATA12.

Figure 5

Evaluation of the effect of CsGATA12 silencing on CBC resistance. (A) A schematic overview of the VIGS plasmid used in this study. 35S, cauliflower mosaic virus 35S promoter; NOS, NOS terminator; GFP, green fluoresceny protein; CP: coat protein; LB: left border; RB: right border. (B) PCR-based confirmation of the successful transformation of VIGS plants. M: DNA ladder. −: negative control (ddH₂O); +: positive control (plasmid). (C) qPCR was used to assess relative CsGATA12 expression, using CsGAPDH (CPDB ID: Cs_ont_5g044290) as a normalization control. (D–F) VIGS plants were evaluated for Xcc infection and then at 10 dpi for disease symptoms (Scale bar: 10 mm) (D), lesion sizes (E), and disease index values (F). In (C,E–F), letters above bars represent the significance of the difference. TRV2-CsGATA12-1, -2, -3: CsGATA12 VIGS plants; TRV2: control plants transformed with the empty TRV2 vector (p < 0.05, ANOVAs with Tukey’ s multiple range test).

Figure 6

Biochemical analyses of the impact of CsGATA12 knockdown on sweet orange plant phenotypes. The levels of ABA (A), SA (B), JA (C), ET (D), ${\mathrm{O}}_2^{-}$ (E), and H₂O₂ (F) were analyzed after CsGATA12 knockdown via the VIGS approach. (G,H) The impact of CsGATA12 silencing on CAT (G) and SOD (H) activity levels were analyzed. TRV2-CsGATA12-1, -2, -3: CsGATA12 VIGS plants; TRV2: control plants transformed with the empty TRV2 vector (p < 0.05, ANOVAs with Tukey’ s multiple range test). Letters above bars represent the significance of the difference.

Figure 7

Evaluation of the impact of CsGATA12 silencing on phytohormone biosynthesis and signaling. (A–D) Relative ABA biosynthesis (A,B) and signaling (C,D) gene expression. (E) Relative ET biosynthesis-related gene expression. (F–J) Relative SA biosynthesis (F–H) and signaling (I,J) gene expression. (K–O) Relative JA biosynthesis (K–N) and signaling (O) gene expression. All qPCR analyses were performed using CsGAPDH (CPDB ID: Cs_ont_5g044290) as a reference control. TRV2-CsGATA12-1, -2, -3: CsGATA12 VIGS plants; TRV2: control plants transformed with the empty TRV2 vector (p < 0.05, ANOVAs with Tukey’ s multiple range test). Letters above bars represent the significance of the difference.