The Xanthomonas type III effector XopD targets the Arabidopsis transcription factor MYB30 to suppress plant defense

Abstract

Plant and animal pathogens inject type III effectors (T3Es) into host cells to suppress host immunity and promote successful infection. XopD, a T3E from Xanthomonas campestris pv vesicatoria, has been proposed to promote bacterial growth by targeting plant transcription factors and/or regulators. Here, we show that XopD from the B100 strain of X. campestris pv campestris is able to target MYB30, a transcription factor that positively regulates Arabidopsis thaliana defense and associated cell death responses to bacteria through transcriptional activation of genes related to very-long-chain fatty acid (VLCFA) metabolism. XopD specifically interacts with MYB30, resulting in inhibition of the transcriptional activation of MYB30 VLCFA-related target genes and suppression of Arabidopsis defense. The helix-loop-helix domain of XopD is necessary and sufficient to mediate these effects. These results illustrate an original strategy developed by Xanthomonas to subvert plant defense and promote development of disease.

Figure 1.

XopD_Xcv Relocalizes MYB30 to Nuclear Foci and Represses MYB30 Transcriptional Activity. (A) and (B) Confocal images of epidermal cells of N. benthamiana leaves 36 h after agroinfiltration of the indicated constructs. Bars = 15 μm. (C) and (E) Transactivation of KCS1p by Arabidopsis MYB30 in N. benthamiana transient assays. Fluorimetric GUS assays in leaf discs 36 h after agroinfiltration of KCS1p:GUS (1), KCS1p:GUS + MYB30-TAP (2), KCS1p:GUS + MYB30-TAP + XopD_Xcv-YFPv (3), and KCS1p:GUS + MYB30-TAP + PopP2-YFPv (4) in (C) and of KCS1p:GUS (1), KCS1p:GUS + MYB96-TAP (2), and KCS1p:GUS + MYB96-TAP + XopD_Xcv-YFPv (3) in (E). Mean and se values were calculated from the results of eight independent experiments, with two to four replicates per experiment. Statistical significance according to a Student’s t test P value < 10⁻⁶ is indicated by letters. MU, methylumbelliferone; prot, protein. (D) and (F) Immunoblot analysis showing expression of TAP-tagged MYB30 or MYB96 and YFPv-tagged constructs. Ponceau S staining confirms equal loading. The interaction (+) or not (−) between CFP-tagged MYB proteins and YFPv-tagged constructs as determined by FRET-FLIM is indicated.

Figure 2.

Structure-Function Analysis of the Interaction between XopD_Xcv and MYB30. (A) Schematic representation of constructs. N-terminal extension, essential V and L residues in the HLH domain, tandemly repeated EAR motifs, conserved catalytic residues in the Cys protease (prot) domain, and NLS motif are shown. (B) Fluorimetric GUS assays in leaf discs 36 h after N. benthamiana agroinfiltration of KCS1p:GUS alone (white bar) or coexpressed with MYB30-TAP either alone (light gray bar) or together with the YFPv-tagged XopD constructs (dark gray bars) as indicated in (A). Mean and se values were calculated from the results of eight independent experiments, with two to four replicates per experiment. Statistical significance according to a Student’s t test P value < 10⁻⁷ is indicated by letters. MU, methylumbelliferone; prot, protein. (C) Immunoblot analysis showing expression of TAP-tagged MYB30 and YFPv-tagged constructs. Ponceau S staining confirms equal loading. (D) Confocal images of nuclei of epidermal cells of N. benthamiana leaves 36 h after agroinfiltration of MYB30-CFP expressed alone or together with the YFPv-tagged XopD constructs as indicated in (A). The interaction (+) between MYB30-CFP and YFPv-tagged XopD constructs as determined by FRET-FLIM is indicated. Bars = 15 μm.

Figure 3.

Analysis of XopD_Xcc8004 and XopD_XccB100 and Their Effects on MYB30. (A) Expression analysis of XopD_Xcc8004 (left) and XopD_XccB100 (right). Strains Xcc8004* or XccB100* (1), Xcc8004* (XopD-HA) or XccB100* (XopD-HA) (2), and Xcc8004* ΔhrcV (XopD-HA) or XccB100* ΔhrcV (XopD-HA) (3) were incubated in MOKA rich medium (total extract) or secretion medium (supernatant). Total protein extracts (10-fold concentrated) and trichloroacetic acid–precipitated filtered supernatants (200-fold concentrated) were analyzed by immunoblotting using anti-HA antibodies to detect XopD or anti-GroEL antibodies to show that bacterial lysis had not occurred. (B) Schematic representation of constructs. N-terminal extension, essential V and L residues in the HLH domain, tandemly repeated EAR motifs, conserved catalytic residues in the Cys protease (prot) domain, and NLS motif are shown. (C) Fluorimetric GUS assays in leaf discs 36 h after N. benthamiana agroinfiltration of KCS1p:GUS alone (white bar) or coexpressed with MYB30 either alone (light gray bar) or together with the YFPv-tagged XopD constructs (dark gray bars) as indicated in (A). Mean and se values were calculated from the results of eight independent experiments, with two to four replicates per experiment. Statistical significance according to a Student’s t test P value < 10⁻⁷ is indicated by letters. MU, methylumbelliferone; prot, protein. (D) Immunoblot analysis showing expression of TAP-tagged MYB30 and YFPv-tagged constructs. Ponceau S staining confirms equal loading. (E) Confocal images of nuclei from epidermal cells of N. benthamiana leaves 36 h after agroinfiltration of MYB30-CFP expressed alone or together with the YFPv-tagged XopD constructs as indicated in (B). The interaction (+) or not (−) between MYB30-CFP and YFPv-taggged XopD constructs as determined by FRET-FLIM is indicated. Bars = 15 μm.

Figure 4.

XopD_XccB100, but Not XopD_Xcc8004, Is Able to Suppress Arabidopsis Defense. (A) Confocal images of nuclei from Arabidopsis epidermal cells 54 h after agroinfiltration of the indicated constructs. The interaction (+) or not (−) between MYB30-CFP and YFPv-tagged XopD constructs as determined by FRET-FLIM analysis is indicated. Bars = 15 μm. (B) Expression analysis of the MYB30 target genes KCS1, FDH, and HCD1 in Col-0 Arabidopsis plants after inoculation with the indicated strains (10⁷ colony-forming units/mL). Expression values of the individual genes were normalized using SAND family and β-tubulin4 as internal standards. Mean and se values were calculated from the results of four independent experiments with three individual plants (four leaves/plant). Statistical differences according to a Student’s t test P value < 0.05 are indicated by letters. a.u., arbitrary units. (C) and (D) Col-0 and MYB30ko Arabidopsis plants were inoculated with the indicated strains (5 × 10⁵ [C] and 10⁷ colony-forming units [CFU]/mL [D]) and bacterial growth was measured 0 (white bars) and 3 d after inoculation (gray bars). Data were collected from four independent experiments with six individual plants (four leaves/plant). Statistical differences using multiple-factor analysis of variance (P value < 10⁻⁴) are indicated by letters.

Figure 5.

XopD_XccB100-Mediated Suppression of Arabidopsis Defense in MYB30_OE Plants. (A) Representative symptoms developed by MYB30_OE Arabidopsis plants 48 h after infection with the indicated strains (10⁷ colony-forming units/mL). Three independent experiments with five plants (four leaves/plant) were performed. (B) Quantification of cell death by measuring electrolyte leakage before (white bars) and 24 h after inoculation (gray bars) of MYB30_OE Arabidopsis plants with the indicated strains (10⁷ colony-forming units/mL). Statistical differences using multiple-factor analysis of variance (P value < 10⁻⁴) are indicated by letters. (C) MYB30_OE Arabidopsis plants were inoculated with the indicated strains (5 × 10⁵ colony-forming units [CFU]/mL) and bacterial growth measured 0 (white bars) and 3 d after inoculation (gray bars). Data were collected from four independent experiments with six individual plants (four leaves/plant). Statistical differences using multiple-factor analysis of variance (P value < 10⁻⁴) are indicated by letters. (D) Expression analysis of the MYB30 target genes KCS1, FDH, and HCD1 in MYB30_OE Arabidopsis plants after inoculation with the indicated strains (10⁷ cfu/mL). Expression values of the individual genes were normalized using SAND family and β-tubulin4 as internal standards. Mean and se values were calculated from the results of four independent experiments with three individual plants (four leaves/plant). Statistical differences according to a Student’s t test P value < 0.05 are indicated by letters. a.u., arbitrary units.