PthA4AT , a 7.5-repeats transcription activator-like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance

Abstract

Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant A^T (X. citri A^T ) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4^AT was identified within the pthA repertoire of X. citri A^T followed by assay of the effects on different hosts. The mode of action of PthA4^AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4^AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4^AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to 'classical' TALEs.

Keywords: Nicotiana benthamiana; Xanthomonas citri; citrus; hypersensitive response (HR); transcription activator-like (TAL) effectors.

Figure 1

Identification of variants of pthA genes in Xanthomonas citri A^T. (a) Southern blot analysis of X. citri A^T and X. citri 306 pthA genes. Plasmids were digested with EcoRI or BamHI and probed with a pthA PCR fragment. Fragments of 3.8, 3.2 and 2.4 kb correspond to pthA1, pthA2/3 and pthA4 from X. citri A^T, respectively. (b) Alignment of repeat variable di‐residue (RVD) sequences of PthA4 and PthA1. RVD sequences from X. citri isolated TALEs were compared with PthA4 and PthA1 from X. citri A^T. Dashed red lines show RVD deleted motif on PthA4^AT. The residues highlighted in red show the RVDs that differ in positions 13–15 on PthA1^AT. ^aAl‐Saadi et al. (2007), ^bGochez et al. (2018), ^cda Silva et al. (2002).

Figure 2

PthA4^AT triggers host defence response in Citrus limon leaves. (a) Symptoms induced by PthA1^AT and PthA4^AT on Xanthomonas citri 306. (b) and (c) Complementation of X. citri mutant strains (ΔpthA1, ΔpthA1,4 and ΔpthA4) with PthA1^AT or PthA4^AT. Leaves were infiltrated with the corresponding bacterial suspension and photographed at 15 days post‐inoculation (dpi). (d) Agrobacterium tumefaciens GV3101‐mediated transient expressions of PthA4^AT and PthA4 on C. limon leaves. Leaves were agroinfiltrated and symptoms were photographed at 10 dpi. HR, hypersensitive response; C, canker; NEC, non‐eruptive canker. Scale bar: 10 mm.

Figure 3

Nuclear localization of PthA4^AT is needed to trigger host defence response in Citrus limon. Phenotypic response of Xanthomonas citri 306 ∆pthA4 transformed with the different constructs was evaluated in C. limon leaves inoculated by pressure infiltration or cotton swab. Photographs were taken 15 days post‐inoculation and the insets show amplification of symptoms. NEC, non‐eruptive canker; HR, hypersensitive response. ∆NLS^AT:PthA4^AT derivative, with an 83‐amino acid deletion in the C‐terminal region, eliminating the three conserved nuclear localization signals (NLS); mutNLS^AT:PthA4^AT derivative mutated in the three NLS; ∆NLS^AT‐SV40:∆NLS^AT derivative containing the NLS of SV40. Scale bar: 10 mm.

Figure 4

DNA‐binding of PthA4^AT follows the TALE code. (a) Consensus predicted sequences for PthA4^AT obtained after protein binding microarray (PBM) assay (E‐score > 0.45) and TALgetter using Citrus sinensis promotorome for the analysis. (b) Relative binding of PthA4^AT to different DNA motifs. The box plot represents the distribution of intensities of the DNA probes containing the sequence element bound by PthA4^AT with the highest affinity (TATYRCCTT) and the intensities of probes covering the motif predicted by TALgetter (TAYTMCCTT), both in light blue. PthA4^AT recognizes both DNA elements, but binding to PBM‐derived motif is higher than to that from TALgetter. Red box corresponds to the distribution of intensities of all the probes in the PBM array. Statistical differences between the different distributions were calculated with the Wilcox exact test. Nucleotide codes are as follows: Y, C or T; M, A or C; R, A or G.

Figure 5

PthA4^AT activates transcription in planta. (a) Effector‐binding element (EBE) site of five Citrus sinensis (Cs) selected promoters. Underlined nucleotide indicates the 0 position of the EBE and the number in brackets indicates the position of last T nucleotide from the putative ATG. (b) GUS assay results after Agrobacterium co‐infiltration of Cs‐uidA promoters and 35S::PthA4^AT. (c) EBE site and base mutation (mEBE) on Cs4. GUS staining in Nicotiana benthamiana leaves was performed 48 h post‐inoculation. Cs1, Cs2, Cs3, Cs4 and Cs5 correspond to C. sinensis IDs orange1.1g019568, orange1.1g047725, orange1.1g038742, orange1.1g048430 and orange1.1g048684, respectively.

Figure 6

PthA4^AT‐mediated resistance depends on transcriptional activation. (a) PthA4^AT variant harbouring a deletion of 27 amino acids on the activation domain (∆AD^AT). Symptoms induced on Citrus limon leaves inoculated with X. citri 306 transformed with PthA4^AT or ∆AD^AT. HR: hypersensitive response: C, canker; NLS, nuclear localization signal; AD, activation domain. (b) Percentage of electrolytic leakage at 0, 48 and 72 h post‐inoculation of C. limon inoculated with ∆pthA4, transformed with PthA4^AT or ∆AD^AT. Values are expressed as means ±SD of three independent biological replicates. The dataset marked with an asterisk is significantly different as assessed by Tukey’s test (P < 0.05). (c) Symptoms induced on C. limon leaves inoculated with ∆pthA4 transformed with PthA4 or ∆AD from Xanthomonas citri 306 observed at different days post‐inoculation (dpi). ∆AD:PthA4 from X. citri 306 harbouring a deletion of 27 amino acids on the activation domain. NEC, non‐eruptive canker; C, canker. Scale bar: 10 mm.

Figure 7

Hypersensitive response (HR) induced by PthA4^AT in non‐host Nicotiana benthamiana model plant. (a) Agrobacterium tumefaciens GV3101‐mediated transient expressions of PthA4^AT on Arabidopsis thaliana, Solanum tuberosum and N. benthamiana leaves. Leaves were agroinfiltrated and symptoms were photographed 72 h post‐inoculation (hpi). (b) Phenotypic response of the transient expression of the different PthA4^AT constructs evaluated in N. benthamiana leaves. Photographs were taken at 24, 48 and 72 hpi. ∆NLS^AT:PthA4^AT derivative with an 83‐amino acid deletion in the C‐terminal region, eliminating the three conserved nuclear localization signals (NLS); mutNLS^AT:PthA4^AT derivative mutated in the three NLS; ∆NLS^AT‐SV40:∆NLS^AT derivative containing the NLS of SV40. ∆AD^AT:PthA4^AT derivative with a 27 amino acid deletion in the activation domain (AD) region.