Functional characterization of the citrus canker susceptibility gene CsLOB1

Abstract

Xanthomonas citri ssp. citri (Xcc) is an important plant-pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator-like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1-GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX-treated wild-type plants and in non-treated CsLOB1-GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1-GR transgenic plants following DEX treatment. When CsLOB1-GR-transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX-treated transgenic plant leaves, but not on mock-treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX-induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4-type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.

Keywords: C3HC4-type RING finger protein; CsLOB1; Xanthomonas; citrus canker; susceptibility gene.

Figure 1

Tissue expression pattern and subcellular localization analyses of the CsLOB1 gene. (A) Expression pattern of the CsLOB1 gene in different tissues of Duncan grapefruit. The endogenous housekeeping gene used was GAPDH (Cs2g14940, glyceraldehyde 3‐phosphate dehydrogenase). Each value represents the mean ± standard deviation (SD) of three replicates. YL, young leaves, 15 days after flushing; ML, mature leaves, over 30 days after flushing; FL, folded flowers; ST, stems. Roots, fresh lateral root. All tissues in each replicate are from the same plant. (B) Subcellular localization of CsLOB1. 35S:EYFP‐CsLOB1 was transformed into citrus protoplast via polyethylene glycol (PEG)‐calcium‐mediated transfection. CsLOB1 localization to the nucleus was confirmed by co‐localization with Hoechst 33342. The fluorescence figures were taken 10 min after Hoechst dye addition to the protoplast at the rate of 1 : 600. The green colour indicates the fluorescence of enhanced yellow fluorescent protein (EYFP). The blue colour indicates the nuclear localization stained by Hoechst. Scale bar represents 10 μm.

Figure 2

Ectopic CsLOB1 expression in citrus induces pustule formation. (A) Diagram of the 35S:CsLOB1‐GR plasmid used to construct CsLOB1 transgenic plants. The 35S:CsLOB1‐GR construct contains green fluoresent protein (GFP) and neomycin phosphotransferase II (NptII) to facilitate the screening of transgenic plants. (B) Validation of CsLOB1 gene expression in different CsLOB1 transgenic lines (T1, T2 and T3) without dexamethasone (DEX) treatment by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Each value represents the mean ± standard deviation (SD) of three replicates. The housekeeping gene GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) was used as the endogenous control. Each value represents the fold change (transgenic plants vs. wild‐type plants) ± SD. T1, T2, T3, three different transgenic lines. (C) CsLOB1 transgenic plant leaves show canker‐like symptoms at about 1 month post‐treatment with DEX. The leaves were injected with DEX (100 μm) or mock solution (equivalent volume of solvent without DEX). The photograph was taken at 1 month post‐inoculation. T1, T2, T3, different transgenic plant lines; WT, wild‐type plant. (D) Pustules on CsLOB1 transgenic plant leaves at about 1 month post‐treatment with DEX. Left: pustules on CsLOB1 transgenic plant leaves show green fluorescence under a fluorescence microscope. Right: non‐transgenic plants observed under a fluorescence microscope; Scale bar represents 200 μm.

Figure 3

Ectopic CsLOB1 expression in citrus restores Xcc306ΔpthA4 canker symptoms. (A) Transgenic plant leaves were inoculated with Xcc306ΔpthA4 at 5 × 10⁸ colony‐forming units (cfu)/mL, and then with 20 μL of dexamethasone (DEX, 100 μm) or mock solution at 24 h after bacterial inoculation. The photograph was taken at 7 days post‐inoculation. (B) Overexpression of CsLOB1 promotes the growth of Xcc306ΔpthA4. Leaves were inoculated with Xcc306ΔpthA4 at a concentration of 5 × 10⁵ cfu/mL. The bacterial population was measured at the time points indicated. Error bar indicates the standard deviation for two replicates.

Figure 4

Citrus genes induced by CsLOB1. mRNA samples were extracted from CsLOB1 transgenic plant leaves treated with dexamethasone (DEX) solution and mock solution at 36 h post‐inoculation. The housekeeping gene GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) was used as an endogenous control. Each value represents the mean ± standard deviation (SD) of three replicates.

Figure 5

Electrophoretic mobility shift assay (EMSA) of CsLOB1 interaction with putative targets. (A) EMSA results revealed the interaction between biotin‐labelled LBD motif‐containing probes and CsLOB1 protein. (B) Competition for Cs2g20600 binding with the unlabelled probe was performed at 5× and 30× of the labelled probe. (C) Binding of the Cs2g20600 probe and mutant Cs2g20600 probe with CsLOB1. Unlabelled probe was added at 50× of the labelled probe. Cs2g20600‐m, mutant probe.