Fig 1.
Atalantia buxifolia resistance to citrus canker.
The phylogenetic tree was constructed by using the maximum likelihood tree method and the substitution model GTRGAMMA in the RAxML software. Murraya koenigii was used as the outgroup. Fully expanded leaves were treated with Xcc (108 CFU/mL) to evaluate citrus canker resistance. The symptoms were observed at 7 and 12 d after inoculation. Red indicates canker susceptible citrus (Wild pummelo: purple pummelo; Cultivated pummelo: guanxi pummelo; Cultivated mandarin: Ponkan; Lemon: Eureka lemon). Purple indicates canker tolerant citrus (Wild mandarin: mangshan mandarin). Green indicates canker-resistant citrus. Bootstrap values greater than 60 are labelled at the node in the tree. Scale bars, 1 cm.
Fig 2.
Inability to grow of Xcc in Atalantia at low bacterial titer.
(A) Fully expanded mature leaves of Atalantia (ATL) and sweet orange (SWO) were treated with Xcc (106 CFU/mL). Photographs were taken at 7 and 12 d after inoculation. (B) Bacterial growth in ATL and SWO at 1, 7, and 12 d after Xcc inoculation. Error bars indicate standard deviation of three independent tests. Scale bars, 1 cm.
Fig 3.
Differentially expressed genes in Atalantia and sweet orange after Xcc (108 CFU/ml) inoculation.
(A) GO enrichment analysis of the up-regulated genes in Atalantia (ATL) and sweet orange (SWO) at 48 h after inoculation. (B) qRT-PCR validation of LOB1 expression in Atalantia and sweet orange at 48 h after inoculation with Xcc and sterile water. (C) Expression levels of four DEGs: pathogenesis-related gene 1 (PR1), Resistance to Pseudomonas syringae 5 (RPS5), HopW1-1-Interacting 2 (WIN2), and dirigent-like protein 22 genes, and their corresponding expression patterns in RNA-Seq data in Atalantia and sweet orange. The treatment group was inoculated with Xcc and the control group was inoculated with sterile water at 6 h, 24 h, and 48 h. All the gene expressions were normalized according to the gene expression of the sweet orange control group at 6 h post inoculation. The relative expression level was calculated by 2-△△Ct method with EF1a as the reference gene. Error bars indicate standard deviation of three independent repetitions.
Fig 4.
Interactions of AbTFIIAγ or CsTFIIAγ with TALE TFBs.
(A) Homology modeling of AbTFIIAγ protein. (B) Homology modeling of CsTFIIAγ protein. The structures of TFIIAγ proteins were homology modeled using SWISS-MODEL online service with default parameters (Fig 4A and 4B). (C) Interactions between AbTFIIAγ-nLuc or CsTFIIAγ-nLuc and TFBi-cLuc. (D) Interactions between AbTFIIAγ-nLuc or CsTFIIAγ-nLuc and TFBii-cLuc. Note: a, b, and d indicate the negative controls of tobacco leaves; c denotes the interactions between TFBi-cLuc or TFBii-cLuc and AbTFIIAγ-nLuc or CsTFIIAγ-nLuc in tobacco leaves in luciferase complementation assay (Fig 4C and 4D). (E) The interaction of AbTFIIAγ or CsTFIIAγ with TFBi in pull-down assays. (F) The interaction of AbTFIIAγ or CsTFIIAγ with TFBii in Pull-down assays. Note: GST-tagged AbTFIIAγ or GST-tagged CsTFIIAγ and His-tagged TFBi or His-tagged TFBii were incubated with immobilized glutathione S-transferase (GST) (Fig 4E and 4F). The experiments were repeated three times independently.
Fig 5.
Enhancement of sweet orange resistance to Xcc through CsTFIIAγ RNA interference.
(A) Relative expression levels of CsTFIIAγ in CsTFIIAγ-RNAi group and control group. (B) Symptoms of CsTFIIAγ-silenced lines (RNAi-1 and RNAi-4) and control leaves after inoculation with Xcc (108 CFU/mL). The photos were taken at 12 d after inoculation. (C) Disease lesion area on CsTFIIAγ-silenced lines and control leaves at 12 d after inoculation. The disease lesion area was calculated by ImageJ 2.0. (D) Bacterial growth in CsTFIIAγ-RNAi plants and control plants at 0, 7, and 12 d after inoculation. (E) Relative expression level of CsLOB1 at 48 h after inoculation. Data from three independent replicates were expressed as mean ± SD. Different letters above the bars represent significant differences (P < 0.05) in Duncan’s multiple range test.
Fig 6.
CsLOB1 gene expression in TFIIAγ-silenced sweet orange and tobacco hardly supported by AbTFIIAγ.
(A) Relative expression of TFIIAγ. (B) Relative expression of CsLOB1. Transient expression of AbTFIIAγ, CsTFIIAγ, and empty vector in CsTFIIAγ-RNAi sweet orange line (RNAi-1) at 4 days after Xcc (108 CFU/ml) inoculation (Fig 6A and 6B). (C) Albino phenotype exhibited by NbPDS-pTRV tobacco at 15 d after silencing NbPDS. (D) Relative expression level of NbTFIIAγ in NbTFIIAγ-pTRV and control (pTRV) tobacco. (E) Expression of vector pCsLOB1:GUS induced by 35S:pthA4 in NbTFIIAγ-pTRV plants after complementation of AbTFIIAγ, CsTFIIAγ, or empty vector. Data from three independent replicates were expressed as mean ± SD. Different letters above the bars represent significant differences (P < 0.05) in Duncan’s multiple range test.
Fig 7.
Significantly lower promoter activity of AbLOB1 than CsLOB1 induced by PthA4.
(A) Promoter activity of AbLOB1 and CsLOB1 induced by PthA4 in tobacco. (B) GUS staining assay in tobacco leaves. Agrobacteria containing pAbLOB1:GUS or pCsLOB1:GUS mixed with 35S: pthA4 or empty vector were infiltrated into tobacco leaves. Samples were collected at 2 days (Fig 7A and 7B). (C) Transient GUS activity related to pAbLOB1 and pCsLOB1 promoters after inoculation with Xcc or Xcc306ΔpthA4 in sweet orange. Samples were collected for further analysis at 4 days post inoculation. (D) GUS staining assay of sweet orange leaves upon ectopic expression of pAbLOB1:GUS or pCsLOB1:GUS post inoculation with Xcc or Xcc306ΔpthA4. Error bars indicate standard deviation of three independent replicates. Different letters above the bars represent significant differences (P < 0.05) in Duncan’s multiple range test.