Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease

Abstract

Citrus bacterial canker (CBC) disease occurs worldwide and incurs considerable costs both from control measures and yield losses. Bacteria that cause CBC require one of six known type III transcription activator-like (TAL) effector genes for the characteristic pustule formation at the site of infection. Here, we show that Xanthomonas citri subspecies citri strain Xcc306, with the type III TAL effector gene pthA4 or with the distinct yet biologically equivalent gene pthAw from strain XccA(w), induces two host genes, CsLOB1 and CsSWEET1, in a TAL effector-dependent manner. CsLOB1 is a member of the Lateral Organ Boundaries (LOB) gene family of transcription factors, and CsSWEET1 is a homolog of the SWEET sugar transporter and rice disease susceptibility gene. Both TAL effectors drive expression of CsLOB1 and CsSWEET1 promoter reporter gene fusions when coexpressed in citrus or Nicotiana benthamiana. Artificially designed TAL effectors directed to sequences in the CsLOB1 promoter region, but not the CsSWEET1 promoter, promoted pustule formation and higher bacterial leaf populations. Three additional distinct TAL effector genes, pthA*, pthB, and pthC, also direct pustule formation and expression of CsLOB1. Unlike pthA4 and pthAw, pthB and pthC do not promote the expression of CsSWEET1. CsLOB1 expression was associated with the expression of genes associated with cell expansion. The results indicate that CBC-inciting species of Xanthomonas exploit a single host disease susceptibility gene by altering the expression of an otherwise developmentally regulated gene using any one of a diverse set of TAL effector genes in the pathogen populations.

Fig. 1.

TAL effectors PthA4 and PthAw are required and sufficient for pustule formation in sweet orange and grapefruit. (A) Experimental design scheme for this study. (B) Loss of pthA4 eliminated pustule formation in sweet orange cultivar (cv.) Valencia (Left) and grapefruit cv. Duncan (Right), which was restored by PthA4 and PthAw. Panels: 1, inoculations with wild-type Xcc306; 2, pthA4 deletion mutant Xcc306∆pthA4; 3, water (mock) inoculation; 4, triple-deletion mutant Xcc306∆pthA1∆pthA2∆pthA3 (with intact pthA4); 5, Xcc306∆pthA4::pthA4; 6, Xcc306∆pthA4::pthAw. The leaves were photographed 5 d after infiltration.

Fig. 2.

CsSWEET1 and CsLOB1 are induced by PthA4. The expression level of CsSWEET1 and CsLOB1 reached peak levels at 24 h postinoculation of Xcc306 on sweet orange. Xcc306∆pthA4 did not induce either gene. Total RNA was isolated at 6, 12, 24, and 48 h after inoculation. The expression was normalized to housekeeping gene EF1α. Data represent the mean ± SD; different lowercase letters represent significant differences (P ≤ 0.01) using ANOVA analysis and Tukey test.

Fig. 3.

Identification of CsSWEET1 substrates through HEK293T cell–FRET sensor uptake assay. Sucrose/glucose transport activity for CsSWEET1 was measured by coexpression with cytosolic FRET sucrose sensor FLIPsuc90μΔ1V (A) and cytosolic glucose sensor FLIPglu600μD13V (B) in HEK293T cells. A drop in intensity ratios reflects uptake of the indicated sugar and loss of FRET fluorescence. Individual cells were analyzed by quantitative ratio imaging of CFP and Venus emission (acquisition interval 10 s). HEK293T cells transfected with sensor only (control, light blue) or with the sensor and the Arabidopsis SWEET12 (suc) or SWEET1 (glc; blue) as positive controls. CsSWEET1 shows sucrose and glucose influx (red). Bars are 1 SD unit.

Fig. 4.

PthA4 drives expression of CsSWEET1 and CsLOB1 promoter/uidA fusion genes. (A) Promoter constructs used in GUS transient expression assay. The predicted TAL EBEs are underlined. PD, deleted promoter; PM, mutated promoter; PT, truncated promoter; Pwt, wild-type promoter; and //, truncation. Base mutations are in lowercase letters, and red font represents putative TATAA box. Fragments including 5′ UTR and ∼100-bp coding sequences of the genes were fused to the ATG of the uidA coding sequence. (B) Transient GUS activity associated with CsSWEET1 and CsLOB1 promoters after inoculation with Xcc306 and derivative strains in sweet orange. Xanthomonas were inoculated 5 h after the inoculation with A. tumefaciens containing the GUS reporter constructs as indicated in A. N, empty vector without promoter fragment; + and −, with PthA4 and without PthA4, respectively; ^a, inoculation with Xcc306∆pthA4::pthA4. GUS activity was assayed 5 d after inoculation. SD values were calculated from three technical replicates of one experiment. The experiment was repeated twice with similar results.

Fig. 5.

dTALes-mediated induction of CsLOB1 or CsSWEET1. (A) RVDs of dTALes and the corresponding targeted EBE sequences in the host genome. dCsSWEET1.1 targets EBE_PthA4 in CsSWEET1 promoter but with a 3′ extension, whereas dCsSWEET1.2 targets a sequence 13 bp upstream of the predicted EBE_PthA4. dCsLOB1.1 targets a sequence 33 bp downstream of EBE_PthA4 in CsLOB1 promoter, whereas dCsLOB1.2 is the optimized dTALe for EBE_PthA4 in CsLOB1 promoter (exact consensus match). (B) Artificial dTALes induced expression of the corresponding targeted genes. The dTALes genes were introduced into Xcc306∆pthA4, and qRT-PCR analysis of host mRNA was conducted 48 h after the inoculation. Data represent the mean ± SD with three replications. (C) GUS activity assay using dCsSWEET1.1, dCsLOB1.1, and dCsLOB1.2 complementing Xcc306∆pthA4 strains. Agrobacterium and Xanthomonas were coinfiltrated into leaf tissue of sweet orange, and assays were conducted at 5 d after the infiltrations. Black columns indicate A. tumefaciens with CsLOBPwt::GUS constructs, gray columns indicate A. tumefaciens with CsSWPwt::GUS constructs. Inoculation with Xcc306∆pthA4::pthA4 was used as a positive control. (D) GUS staining assay in N. benthamiana leaves upon ectopic expression of either dCsSWEET1.2 or dCsLOB1.2, respectively, using the CaMV35S promoter to drive expression. Agrobacterium harboring 35S:dCsSWEET1.2 or 35S:dCsLOB1.2 was coinfiltrated with Agrobacterium containing CsSWPwt or CsLOBPwt promoter/uidA constructs as indicated in Fig. 4A.

Fig. 6.

dTALes targeting CsLOB1 promoter when expressed in Xcc306∆pthA4 restore pustule formation in citrus. (A) Lesion symptoms after inoculation with strains containing natural or artificial TAL effector genes. (Left) Sweet orange; (Right) grapefruit. Leaves were inoculated with a bacterial concentration of 5 × 10⁸ cfu/mL and photographed at 5 d after infiltration. Panels: 1, Xcc306; 2, Xcc306∆pthA4 (mutant); 3, Xcc306∆pthA4::dCsLOB1.1; 4, Xcc306∆pthA4::pthA4; 5, Xcc306∆pthA4::pthAw; 6, Xcc306∆pthA4::dCsSWEET1.1. The right table indicates presence or absence of the pustule symptoms with Xcc306∆pthA4 containing the gene for the indicated effector or Xcc306∆pthA4 alone. In pustule column, −, no pustule; +, pustule formation observed at 5 d. (B) Thin cross-section images of grapefruit leaves 5 d after inoculation with Xcc306∆pthA4 (Left), Xcc306∆pthA4::dCsLOB1.1 (Upper Right), and Xcc306∆pthA4:pthAw (Lower Right).

Fig. 7.

dCsLOB1.1 enhances growth of Xcc306∆pthA4 in sweet orange. Xcc306∆pthA4 and Xcc306∆pthA4::dCsSWEET1.1 have reduced bacterial leaf population compared with Xcc306∆pthA4::pthA4 and Xcc306∆pthA4::dCsLOB1.1. Leaves were inoculated at the concentration of 5 × 10⁵ cfu /mL, and the population was measured at the time points indicated. Error bars represent 1 SD. Significance between strains was assessed at final time point at P < 0.01 by using Tukey–Kramer HSD test for post-ANOVA analysis. Values at 15 dpi with the same letter do not differ at the significance level of P < 0.01. The experiment was repeated twice with similar results.

Fig. 8.

Multiple TAL effectors associated with pustule formation in CBC induce CsLOB1 and/or CsSWEET1 in sweet orange and grapefruit. Black columns represent the expression values of CsSWEET1, and white columns indicate expression values of CsLOB1. RNA was prepared 48 h after inoculation. Strains with genes for PthB and PthC did not induce CsSWEET1 in either species. Data represent the mean ± SD of three replications.

Fig. 9.

PthB and PthC drive CsLOB1 promoter, but not CsSWEET1 promoter, expression. (A) Consensus EBEs of PthA4, PthAw, PthA*, PthB, and PthC (in gray) and the corresponding nucleotide sequences are depicted in the CsLOB1 and CsSWEET1 promoters from sweet orange. Mismatches between predicted EBE and CsLOB1 promoter are indicated in bold red font and underlined; different bases in CsSWEET1 promoter compared with that of CsLOB1 are in green font. (B) GUS transient assays in sweet orange with the coinoculation of Xcc306 or derivative strains and A. tumefaciens harboring promoter/uidA fusion genes listed in Fig. 4A. Each set of columns is labeled with the specific TAL effector produced by the corresponding gene in strain Xcc306∆pthA4. Data bars represent the mean ± SD with three technical replicates of one experiment. The experiment was repeated twice with similar results. Columns for CsSWPwt with the same lowercase letters do not differ from each other at the significance level of P < 0.05 using the Tukey test.

Fig. 10.

Cell wall-related genes are coinduced by synthetic TAL effectors that target CsLOB1. Representative cell expansion or wall-related metabolism genes in sweet orange were activated by Xcc306∆pthA4::dCsLOB1.1 in comparison with Xcc306∆pthA4. Quantitative RT-PCR was conducted on host mRNA using gene-specific primers at 48 h after the infiltration. Expression values were normalized to housekeeping gene EF1α. The probe sets are labeled and annotated as follows: Cit.8700, Cit.8700.1.S1_at, extension; Cit.30858, Cit.30858.1.S1_at, expansin; Cit. 20509, Cit.20509.1.S1_at, pectate lyase; Cit. 2392, Cit.2392.1.S1_at, acidic cellulose; Cit. 3027, Cit.3027.1.S1_s_at, CsSWEET1; Cit. 37210, Cit.37210.1.S1_at, CsLOB1; Cit. 7877, Cit.7877.1.S1_at, expansin; Cit. 39387, Cit.39387.1.S1_at, pectate lyase.

Fig. 11.

Cell wall-related gene expression is sensitive to cycloheximide (CHX) and associated with transient CsLOB1 expression. (A) Semiquantitative RT-PCR at 36 h after the infiltration of mutant Xcc306∆pthA4 and wild-type Xcc306 in the presence (+) or absence (−) of CHX. (B) Quantitative RT-PCR assays were conducted 6 d after infiltration of Agrobacterium with 35S::CsLOB1 or empty vector. Expression values were calculated in relation to water infiltration. The representative cell wall-related genes were a subset of genes used in Fig. 10. Error bars represent 1 SD. Values between treatments were normalized to the housekeeping gene EF1α.