Fig. 5
Subcellular localization, transcriptional activities and function of CsGRAS9. A Phylogenetic tree of CsGRAS9, its closest homolog (CsGRAS3), homologous proteins in Citrus spp., and orthologous proteins in Arabidopsis, rice, and tomato, constructed using MEGA X. Branch lengths represent the number of substitutions per site. B Analysis of CsGRAS9 transcriptional activity in yeast. The CDS of CsLOB1 and CsGRAS9 were fused to pGBKT7 vector, respectively. The pGBKT7-CsGRAS9 construct was co-transformed with pGADT7 into yeast strain AH109. Yeast cells expressing BD-CsGRAS9 were spotted on SD/-Trp/-Leu, SD/-Trp/-Leu/-His media, and selection media containing X-α-gal. pGBKT7-CsLOB1 and pGADT7 served as positive and negative controls, respectively. C Subcellular localization of CsGRAS9 in N. benthamiana leaves. CsGRAS9 was fused with YFP in the pHB vector (35S::CsGRAS9-YFP). Fluorescence (yellow for YFP, blue for DAPI) was observed using a confocal microscope. Scale Bars: 50 and 75 μm. D CsGRAS9 specifically activated by dTALEs. The region targeted by dGRAS9 in the CsGRAS9 promoter is highlighted in yellow. Red nucleotides represent the translation start site. E Grapefruit leaves were infiltrated with Xcc suspensions (OD600 = 0.6) of Xcc049E/EV and Xcc049E/dGRAS9. Citrus canker symptoms and bacterial growth were evaluated at 16 days post-inoculation. Error bars represent the standard deviation of three biological replicates. Asterisks indicate statistically significant differences (****p < 0.0001) using Student’s t-test. F Relative expression of CsGRAS9 in grapefruit leaves was measured 48 h post-inoculation (OD600 = 1.0). CsEf1a was used as the internal control. Data from three biological replicates are presented as mean ± SEM values. Asterisks denote significant differences using Student’s t-test (*p < 0.05, **p < 0.01, ****p < 0.0001). G Grapefruit leaves were inoculated using a pinprick method with Xcc suspensions (OD600 = 1.0) of mixed suspensions Xcc003 + Xcc049E/EV, Xcc003 + Xcc049E/dGRAS9, Xcc086 + Xcc049E/EV, and Xcc086 + Xcc049E/dGRAS9 in a 1:1 ratio. Positive controls included inoculations with Xcc003 + Xcc049E/EV and Xcc086 + Xcc049E/EV suspensions. Citrus canker symptoms were assessed 8 days after inoculation. H-I Grapefruit leaves were infiltrated with Xcc suspensions (OD600 = 1.0). Xcc049E/EV and Xcc049E/dGRAS9 inoculations were performed, alongside Xcc003 + Xcc049E/EV (H) and Xcc086 + Xcc049E/EV (I) inoculations, to evaluate citrus canker severity. Symptoms were assessed 12 days post-inoculation. J Bacterial growth of Xcc strains in grapefruit leaves was monitored at 1, 4, 7, 10 days post-inoculation. Data are presented in mean colony-forming units (CFU)/cm2. Error bars represent standard deviations from three biological replicates. Different letters denote statistically significant differences using two-way ANOVA with Tukey's test (p < 0.05)