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. 2017 Apr;18(3):405-419.
doi: 10.1111/mpp.12408. Epub 2016 Jun 10.

Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit

Alberto M Gochez  1   2 , Deepak Shantharaj  2 , Neha Potnis  2 , Xiaofeng Zhou  3 , Gerald V Minsavage  2 , Frank F White  2 , Nian Wang  3 , Jason C Hurlbert  4 , Jeffrey B Jones  2
Affiliations

Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit

Alberto M Gochez et al. Mol Plant Pathol. 2017 Apr.

Abstract

Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin-binding domain characteristic amino acid sequence motif (GPLL) and a C-terminal domain-containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two-hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2-SPLL and AvrGf2-AASL mutants showed weak and no interaction, respectively. Molecular modelling and in silico docking studies for the cyclophilin-AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin-binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C-terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site-directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.

Keywords: citrus canker; citrus cyclophilin; citrus resistance; hypersensitive response.

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Figures

Figure 1
Figure 1
(A) Three C‐terminal domains were predicted in AvrGf1 and AvrGf2 using MEME Suite. (B) Three C‐terminal domains were predicted in nine XopAG effectors (from five different bacterial genera, as mentioned in Table S1). (C) For all XopAG effectors included in this comparison, the ‘CLNAxYD’ site is highly conserved in Motif 1. (D) The following strains were infiltrated at 5 × 108 colony‐forming units (CFU)/mL into grapefruit leaves: Xc‐A:306, Xc‐A306:pLAFR3; Xc‐A:AvrGf2::CAvrGf1, Xc‐A306:NAvrGf2383::CAvrGf1; Xc‐A:AvrGf2, Xc‐A306:pLAFR3‐avrGf2; Xc‐A:CASA, Xc‐A306:avrGf2‐CASA. Note that the CLNA motif in avrGf2, when mutated to C448ASA and expressed in Xc‐306, did not result in the elicitation of a hypersensitive response.
Figure 2
Figure 2
Confocal laser scanning microscopy (CLSM) to visualize the localization of AvrGf2 (GPLL), AvrGf2 (SPLL) and AvrGf2 (AASL) mutants in Citrus cv. sweet orange. (A) Overexpression of AvrGf2(GPLLmotif)‐GFP using agro binary vector pGWB5 was assessed at 1 day post‐inoculation. Empty agro binary vector pGWB5 served as a negative control and 35S:sGFP expressed in agro binary vector pCambia2202 served as a positive control. Green fluorescent protein (GFP) fluorescence from AvrGf2(GPLL)‐GFP was observed in the cytoplasm and chloroplasts at 1 day after transient expression. Overexpression of AvrGf2(SPLL)‐GFP and AvrGf2(AASL)‐GFP using agro binary vector pGWB5 was assessed at 2 days post‐inoculation. GFP fluorescence from AvrGf2(SPLL)‐GFP was observed from cytoplasm and chloroplast. AvrGf2(AASL)‐GFP showed fluorescence expression in cytoplasm, but not in chloroplast. 35S:GFP, as control, was only found in the cytoplasm. Fluorescence detection was scanned at 488 nm for GFP and at 405 nm for chloroplast; the spectral detection window was selected as 505–550 nm (for GFP) and 600–747 nm (for chloroplast) for emission using a prism‐based spectrometer. White arrows indicate overlapping GFP fluorescence from AvrGf2(GPLL)‐GFP and chloroplast autofluorescence. Blue arrows indicate GFP fluorescence and green arrows indicate chloroplast autofluorescence. Bars correspond to image magnifications. (B) Co‐localization of AvrGf2 labelled with GFP in chloroplasts was analysed using Perkin‐Elmer volocity software version 6.3.0 (http://cellularimaging.com). The analysis of co‐localization was calculated using the automatic threshold setting, with a minimum object size of 10 μm3. Microscopy fluorescence settings: green channel for AvrGf2‐tagged GFP fluorescence and red channel for chloroplast autofluorescence. Co‐localization counts were measured per volume (µm3), where green and red fluorescence occupying the same voxel (three‐dimensional pixel) was calculated. The mean voxel counts were calculated from Z‐image series. The bars represent the mean with standard error of three different quantification experiments. *Significantly different voxel values (P < 0.0001) calculated by Tukey–Kramer honestly significant difference (HSD) test from the statistical package JMP® Pro 12.0.1.
Figure 3
Figure 3
The effect of site‐directed mutagenesis of the GPxL motif in avrGf2 on the elicitation of a hypersensitive response (HR) in Duncan grapefruit. (A) The following strains were infiltrated into grapefruit leaves at 5 × 108 colony‐forming units (CFU)/mL: Xc‐A, Xc‐A306:pLAFR3; avrGf2, Xc‐A306:avrGf2; AASL, Xc‐A306:avrGf2‐A356ASL; SPLL, Xc‐A306:avrGf2‐S356PLL. DAI, days after infiltration. (B) The following strains were infiltrated into grapefruit leaves at 5 × 105 CFU/mL: A306:pLAFR3 (Xc‐A306:pLAFR3, diamond); A306:avrGf2 (Xc‐A306:avrGf2, triangle); A306:AASL (Xc‐A306:avrGf2‐A 356 ASL, square); A306:SPLL (Xc‐A306:avrGf2‐S 356 PLL, circle). (C) Electrolyte leakage analysis in Duncan grapefruit leaves infiltrated with 5 × 108 CFU/mL using the same strains and including A306:avrGf1 (Xc‐A306:avrGf1, square). Each point represents the mean of one experiment with three replicates. Vertical lines represent the standard deviation of each mean.
Figure 4
Figure 4
Yeast two‐hybrid analyses of interactions of grapefruit cyclophilin (GF‐Cyp) with AvrGf1, AvrGf2 and AvrGf2 mutants with the GPLL motif mutated to SPLL and AASL. (A) Serial dilutions of yeast cells transferred with the bait (pDBLeu) and prey (pPC86) constructs indicated in the figure were assayed for growth on SD–Leu–Trp–His [synthetic medium lacking leucine (Leu), tryptophan (Trp) and histidine (His)] plates. The transformants bearing the pairs of plasmids pDBLeu + pPC86, pDBLeu + GF‐Cyp, pPC86:avrGf1 and pPC86:avrGf2 were used as negative controls. (B) Quantitative o‐nitrophenyl β‐d‐galactopyranoside (ONPG) assay of grapefruit Cyp interaction with AvrGf1 and AvrGf2. β‐Galactosidase units were calculated with three experimental replicates with standard error bars.
Figure 5
Figure 5
(A) Transient silencing experiment in young Duncan grapefruit leaves infiltrated separately with suspensions of Agrobacterium tumefaciens strains GV3101:pHellsGate (GV3101) and GV3101:pHellsGate‐grapefruit‐Cyp (sGF‐Cyp) adjusted to 5 × 106 colony‐forming units (CFU)/mL at day 0, and then infiltrated 0 and 6 days later with bacterial suspensions of Xc‐A306:pLAFR3 (top right part of the leaf) and Xc‐A306:avrGf2 (bottom left) adjusted to 5 × 108 CFU/mL. Leaves not infiltrated with Agrobacterium (NA) were used as a second control. DAI, days after infiltration. (B) Population dynamics in transiently silenced experiment in young grapefruit leaves infiltrated separately with suspensions of A. tumefaciens strain GV3101:pHellsGate empty (Agro) and GV3101:pHellsgate‐grapefruit‐Cyp (sCyp) at day 0 (DAI0) adjusted to 5 × 106 CFU/mL. Half of the leaves were then infiltrated at DAI0 (top) and the other half at DAI4 (bottom) with suspensions of 5 × 105 CFU/mL of Xc‐A306:pLAFR3 (A306) and Xc‐A306:avrGf2 (AGf2). Each point represents the mean of one experiment with three replicate measurements. (C) Electrolyte leakage in transient silenced young grapefruit leaves separately infiltrated with suspensions of 5 × 106 CFU/mL of A. tumefaciens strain GV3101:pHellsGate empty (Agro) or GV3101:pHellsGate‐grapefruit‐Cyp (sCyp) at day 0 (DAI0), and then infiltrated at DAI4 with suspensions of 5 × 108 CFU/mL of Xc‐A306:pLAFR3 (A306) or Xc‐A306:avrGf2 (AGf2). Each time point represents the mean from two experiments with three replicates. (D) Comparison of the areas under the curve (AUCs) of the electrolyte leakage analysis data performed in transient silenced young grapefruit leaves separately infiltrated with suspensions of 5 × 106 CFU/mL of A. tumefaciens strain GV3101:pHellsGate empty (Agro) or GV3101:pHellsGate‐grapefruit‐Cyp (sCyp) at day 0 (DAI0), and then infiltrated at DAI4 with suspensions of 5 × 108 CFU/mL of Xc‐A306:pLAFR3 (A306) or Xc‐A306:avrGf2 (AGf2). Values represent the mean and standard deviation (SD) from two experiments with three replicates. Vertical lines represent the standard error of the mean.
Figure 6
Figure 6
Structural superposition and structure‐based sequence alignment of three cyclophilins (Cyp) from Citrus sinensis. The crystal structure of CsCyp (PDB ID: 4JJM) is shown in mauve superposed with the homology models of Cyp19‐3 (XP_006487732.1) (tan) and peptidyl‐prolyl cis–trans isomerase‐like 1‐like (PPI‐1‐like) (XP_006487422.1) (light blue). Key residues delineating the active sites are identified with the amino acid numbers of CsCyp shown.
Figure 7
Figure 7
Predicted binding modes of the peptides tested to Citrus sinensis cyclophilin (CsCyp). The lowest energy conformation of the YSPSA peptide (tan), native AvrGf2 VAGPLL peptide (blue), AvrGf2 VASPLL peptide (green) and AvrGf2 VAAASL peptide (purple) are shown. In the lower left and lower right of the figure, the central image is rotated by 90º left and right, respectively. In the lower centre of the figure, Cyp is represented as a coulombic surface. Rosetta energy scores for each complex are shown in the top left corner.
Figure 8
Figure 8
Schematic representation of the interaction of AvrGf2 from Xanthomonas fuscans pv. aurantifolii type C in Citrus paradisi mesophyll cell. (1) Cis–trans activation of the AvrGf2 GPLL motif with the cytoplasmic prolyl isomerase, grapefruit‐cyclophilin (GQ853548.1). Interaction leads to cell death in Citrus paradisi. The importance of the GPLL motif of AvrGf2 was confirmed by amino acid substitution of the GPLL motif with SPLL and AASL, and transconjugation in X. fuscans pv. aurantifolii. The AvrGf2‐SPLL motif shows a reduced cyclophilin interaction with partial cell death (2), whereas AvrGf2‐AASL shows a complete loss of cyclophilin interaction with no cell death (3). ROS, reactive oxygen species.
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