Fig. 4

Sequence divergence of CsGRAS9 in citrus cultivars. A Leaves of Hong Kong kumquat (F. hindsii, Shan Jin Gan) were inoculated with Xcc049E/pthA4 (control) and a mixed suspension (OD₆₀₀ = 1.0) of Xcc049E/pthA4 + pthA5 and Xcc049E/pthA4 + pthA6 at a 1:1 ratio. Canker symptoms were observed 16 days post-inoculation. B Illustration of the 740-bp promoter region of CsGRAS9 and the 182-bp promoter region of FhGRAS9-P2 used for Y1H, GUS, and LUC assays. Pink blocks represent core promoter TATA-box/AT-TATA-box motifs, blue blocks represent predicted EBE regions and green blocks represent as-1 motifs. C Schematic of plasmids used in Y1H assays. The CDS of pthA5 and pthA6 were cloned into pB42AD vector as the effector, respectively. Truncated promoter fragments of CsGRAS9 (CsGRAS9-P1/FhGRAS9-P1) and FhGRAS9 (FhGRAS9-P2) were fused with pLacZi reporter genes. Constructs were co-transformed into yeast EGY48 strains, grown on SD/-Leu/-Trp medium, and evaluated on SD/-Leu/-Trp/X-gal plates. Negative controls were co-transformants with empty vectors pB42AD or pLacZi. D-E GUS staining and expression assays in N. benthamiana leaves transformed with labeled effector and reporter constructs via transient expression assays. The CDS of pthA5 and pthA6 were cloned into the pHB vector. Truncated promoter fragments (740-bp CsGRAS9-P1 in D and 182-bp FhGRAS9-P2 in E) were fused with pCAMBIA1381::GUS. Negative controls included empty pHB co-transformed with vectors pCsGRAS9-P1::GUS or pFhGRAS9-P2::GUS. Three biological replicates were presented as mean ± SEM values. Asterisks indicate statistically significant differences using one-way ANOVA followed by Tukey's test (****p < 0.0001, ***p < 0.001, **p < 0.01). F Relative expression of FhGRAS9 in Hong Kong kumquat (Shan Jin Gan) leaves 48 h post-inoculation (OD₆₀₀ = 1.0). CsEf1a was used as a reference gene. Mean ± SEM values from three biological replicates are shown. Asterisks indicate significant differences between bacterial-inoculated leaves using one-way ANOVA with Tukey's test (*p < 0.05, **p < 0.01). G Alignment of the allelic variations of the EBE regions of GRAS9 promoters in citrus species. Sequences show the 26-bp and a 12-bp deletion in the CsGRAS9 homologs of M. paniculata, C. australasica and C. medica. Conserved nucleotides are in blue, while nucleotide variations are highlighted in black or represented by black dots for missing nucleotides. H-I Binding of purified His-tagged proteins PthA6-His, PthA5-His, and PthA4-His to EBE regions in the promoters of GRAS9 homologs in the M. paniculata, C. australasica and C. medica. EMSA assays were performed with Cy5-labeled probes for ProCmGRAS9 (H) and ProMpGRAS9 (I) and cold competitors. Cold probes were used as competitors with 1 × or 100 × excess over Cy5-labeled probes. Specific shifts and free probes are indicated by arrows. Symbol “ + ” or “–” indicates the presence or absence of protein and probes. Results are from three independent experiments