Pseudomonas syringae Type III Effector HopBB1 Promotes Host Transcriptional Repressor Degradation to Regulate Phytohormone Responses and Virulence

Abstract

Independently evolved pathogen effectors from three branches of life (ascomycete, eubacteria, and oomycete) converge onto the Arabidopsis TCP14 transcription factor to manipulate host defense. However, the mechanistic basis for defense control via TCP14 regulation is unknown. We demonstrate that TCP14 regulates the plant immune system by transcriptionally repressing a subset of the jasmonic acid (JA) hormone signaling outputs. A previously unstudied Pseudomonas syringae (Psy) type III effector, HopBB1, interacts with TCP14 and targets it to the SCF^COI1 degradation complex by connecting it to the JA signaling repressor JAZ3. Consequently, HopBB1 de-represses the TCP14-regulated subset of JA response genes and promotes pathogen virulence. Thus, HopBB1 fine-tunes host phytohormone crosstalk by precisely manipulating part of the JA regulon to avoid pleiotropic host responses while promoting pathogen proliferation.

Keywords: HopBB1; JAZ; Pseudomonas syringae; RNA-seq; TCP; hormone crosstalk; jasmonic acid; plant immunity; sub-nuclear foci; virulence effector.

Figure 1. TCP14 represses JA response and promotes disease resistance

(A) Three-week-old plants of Col-0, tcp14-6, UBQ::YFP-TCP14-3 and UBQ::YFP-TCP14-4. Bar=5mm (B) tcp14 mutants are more susceptible to Hpa Emwa1 than Col-0. Overexpressing TCP14 in Arabidopsis enhances disease resistance against Hpa Noco2. The means represent the numbers of sporangiophores (sp) on each cotyledon (n>50). (C) Mutation in TCP14 enhances the virulence of DC3000 cor-. Two-week-old plants were dip inoculated with indicated bacteria strains at OD₆₀₀=0.05. CFU: Bacterial colony formation units. Error bars represent ±SD. (D) JA-responsive genes are repressed in two-week-old TCP14-overexpressing plants. Hierarchical clustering of the 203 genes identified as differentially expressed in the pairwise comparison between Col-0 and UBQ::YFP-TCP14-3, UBQ::YFP-TCP14-4 and the coi1-16 mutant were also included. MeJA or BTH/SA-responsive genes (Table S1) are indicated on the right. Cluster 1: genes repressed in UBQ::YFP-TCP14 and coi1-16; Cluster 2: genes repressed in UBQ::YFP-TCP14 but not in coi1-16; Cluster 3: genes upregulated in UBQ::YFP-TCP14. (E) Genes down-regulated in UBQ::YFP-TCP14-3 are enriched for JA markers. Dashed lines represent the proportion of genes that belong to each group in the Arabidopsis genome. Geno. (Genome): Expected proportion of MeJA (or BTH/SA) markers in Arabidopsis genome. Up- /Down- : Proportion of MeJA (or BTH/SA) markers in genes up-regulated or down-regulated by UBQ::YFP-TCP14. (F) Elevated expression of JA-responsive genes in steady-state tcp14 mutants. *: FDR<0.0001 (G) Summary of transcriptional alterations in tcp14-6, UBQ::YFP-TCP14 overexpression line and in coi1-16 plants 24h after DC3000 cor- infection. The table depicts the number of differentially expressed genes in each line relative to infected wild-type plants. (H) Gene Ontology terms (biological processes) enriched in the set of 277 genes down-regulated in both UBQ::YFP-TCP14 and coi1-16 when compared to wild-type plants after infection. Note the prevalence of terms associated with the JA pathway. Gene names are labeled in red. (I) Gene Ontology terms (biological processes) enriched in the set of 183 genes up-regulated in both UBQ::YFP-TCP14 and coi1-16 when compared to wild-type plants after infection. Note the prevalence of terms associated with the SA pathway. Gene names are labeled in red. The complete GO enrichment results for (H) and (I) are shown in Table S2. See also Figure S1 and Tables S1 and S2.

Figure 2. HopBB1 interacts with TCP14 in planta

(A) Pto-delivered HopBB1, but not HopBB1_G126D associates with TCP14 in Arabidopsis. DC3000 cor- with empty vector (EV), HA-tagged HopBB1 or HopBB1_G126D were hand-infiltrated at OD₆₀₀=0.05 into leaves of four-week-old transgenic Arabidopsis expressing YFP-TCP14. Leaves were harvested 24 hrs after inoculation. (B) HopBB1_G126D loses interaction with TCP14 in yeast. (C) The C-terminus (111–283) of HopBB1 is sufficient to associate with TCP14 in N. benthamiana. (D) HopBB1 interacts with TCP14(aa180-216) in yeast. (E) HopBB1 associates with TCP14_180–216 in N. benthamiana. (F) TCP14_{RSAAST/NAAIRS} fails to interact with HopBB1 in yeast, but retains homodimerization. (G) TCP14_{RSAAST/NAAIRS} fails to associate with HopBB1 in N. benthamiana. Proteins were transiently expressed in N. benthamiana from a 35S promoter for (C), (E) and (G). See also Figure S2.

Figure 3. HopBB1 promotes bacteria growth and activates JA response

(A) Bacterial-delivered HopBB1 promotes the growth of DC3000 cor- in Col-0. Two-week-old plants were spray inoculated with a bacteria suspension at OD₆₀₀=0.2. CFU: Bacterial colony formation units. Error bars represent ±SD. (B) Summary of transcriptional changes in Col-0 plants 24h after the treatment with DC3000 (EV), MgCl₂ and the coronatine-deficient mutant strains carrying the empty vector (EV), HopBB1 or HopBB1_G126D. Numbers represent the differentially expressed genes relative to the DC3000 treatment. (C) Dendrogram constructed based on the entire transcriptome showing that the transcriptional signature of DC3000 cor- (EV), treated plants resembles that of the mock treatment; DC3000 cor- expressing either HopBB1 or HopBB1_G126D trigger similar transcriptional responses as DC3000 (EV). (D) A set of 253 JA marker genes are activated by DC3000 (EV) and/or DC3000 cor-(HopBB1); HopBB1_G126D has reduced ability to activate these genes. (E) Transgenic Arabidopsis plants expressing HopBB1 are morphologically indistinguishable from Col-0 wild-type. Bar=5mm (F) Plants expressing HopBB1 complement the growth defects of DC3000 cor-. (G) JA-responsive genes are activated in transgenic plants expressing HopBB1-myc. The z-score transformed expression of 672 JA responsive marker genes is shown for three biological replicates of Col-0 and transgenic plants expressing HopBB1-myc. (H) The distribution of HopBB1, HopX1, HopZ1a and coronatine biosynthesis pathway in 287 sequenced Pseudomonas syringae genomes. Arrowhead: genomes contain two JA-activating tools. N→Y: A polymorphism (N→Y) exists in the HopX1 allele. See also Figure S3 and Tables S3 and S4.

Figure 4. HopBB1-mediated degradation of TCP14 requires SCF^COI1 pathway

(A) Bacterial-delivered HopBB1, but not HopBB1_G126D, induced turnover of TCP14 during infection on Arabidopsis. Bacteria were hand-inoculated into leaves of four-week-old plants at an OD600=0.05. Samples were harvested 24 hrs after inoculation. (B) YFP-TCP14 is not subject to JA-mediated degradation in the absence of HopBB1 in Arabidopsis. Two-week old seedlings expressing UBQ::YFP-TCP14 were sprayed with mock or 50µM MeJA solution, and sampled at the indicated time. Each sample was pooled from eight seedlings. (C) Pto-delivered HopBB1 reduces TCP14 protein level in wild-type Col-0, but not in coi1-1 mutant. Experiments were performed as Figure 4A. YFP-TCP14 transcripts were quantified using real-time PCR. Error bars indicate ±SD

Figure 5. HopBB1 interacts with JAZ3 and disrupts its association with MYC2

(A) HopBB1 interacts with JAZ3 in Arabidopsis. HopBB1-myc conditionally expressed from an estradiol-inducible promoter was transformed into Arabidopsis constitutively expressing JAZ3-HA from a 35S promoter. Three-week-old seedlings were induced with 50µM estradiol and sampled 6 hrs after induction. (B) The C-terminus (111–283) of HopBB1 is sufficient to associate with JAZ3 in N. benthamiana. (C) HopBB1 interacts with an uncharacterized JAZ3 domain (206–302) in yeast. (D) HopBB1 associates with an uncharacterized JAZ3 domain (206–302) in N. benthamiana. (E) HopBB1 reduces the association between JAZ3 and MYC2 in planta. Proteins were transiently co-expressed in N. benthamiana. HopBB1 was induced using a gradient of estradiol (0.01µM, 1µM, 10µM) 6 hrs after Agrobacteria infiltration. Samples were harvested 18 hrs after induction. (F) HopBB1 disrupts the BiFC signal generated from the association between JAZ3 and MYC2. The co-expression of HopBB1 or HopBB1_G126D reduces the efficiency from 80% to 20%. RFP-positive cells were examined for the presence of CFP and YFP signal in nuclei. For each combination, the ratios were calculated by counting approximately 200 nuclei collected from four independent samples. Error bars indicate ±SD. See also Figure S4.

Figure 6. TCP14 is subject to JA-mediated degradation in the presence of HopBB1 and JAZ3

(A) HopBB1 alone does not trigger TCP14 degradation without MeJA. Leaves were co-infiltrated with Agrobacteria delivering 35S::TCP14-myc or EST::HopBB1-YFP-HA genes. (B) TCP14 is subject to JA-mediated degradation in the presence of HopBB1 and JAZ3. Agrobacteria carrying vectors expressing each protein under 35S constitutive promoter were co-infiltrated into N. benthamiana leaves. 50µM of MeJA was hand-infiltrated into leaves 24 hours post inoculation. Numbers below western signal indicate the relative signal intensity. The same method was applied to (B–H). (C) MYC2 is not co-degraded with TCP14. (D) HopBB1-mediated degradation of TCP14 is blocked by the 26S proteasome inhibitor, MG132. 50µM MeJA and 50µM MG132 were co-infiltrated 24 hours post inoculation. (E) The recruitment of JAZ3 to SCF^COI1 is required for HopBB1-mediated degradation of TCP14. (F) HaRxL45 cannot mediate TCP14 degradation with the presence of JAZ3 and MeJA. (G) HopBB1_G126D cannot mediate TCP14 degradation with the presence of JAZ3 and MeJA. (H) TCP14_{RSAAST/NAAIRS} is not subject to HopBB1-mediated degradation. See also Figure S5.

Figure 7. HopBB1 recruits TCP14 to a JAZ3-containing degradation site

(A) Localization of HopBB1, TCP14 and JAZ3 in nuclei. HopBB1 is evenly distributed in nuclei, while TCP14 and JAZ3 form subnuclear foci. Bar=5µM Proteins were transiently expressed in N. benthamiana for (A)-(D), (G)-(I). (B) HopBB1 was re-localized to subnuclear foci by TCP14 (top) and JAZ3 (middle). However, TCP14 and JAZ3 localize in distinct nuclear foci (bottom). Histograms represent the intensity of fluorescent signal on the pathway of the lines in the “Merged” panel. (C) HopBB1 (top), but not CFP (bottom), drives TCP14 and JAZ3 into the same sub-nuclear foci. (D) HopBB1 cannot co-localize TCP14_{RSAAST/NAAIRS} -YFP into the same foci as JAZ3-RFP. (E) The formation of TCP14 foci depends on its binding DNA ability. YFP-TCP14, but not TCP14_{H121Q R130K L161N}, expressed under UBQ promoter forms subnuclear foci in transgenic Arabidopsis. (F) The formation of JAZ3 foci depends on its ability to associate with COI1. JAZ3-RFP expressed from a constitutive 35S promoter forms subnuclear foci in transgenic Arabidopsis Col-0, but not coi1-1 mutant. Images in (E) and (F) were taken from cotyledon epidermal cells in transgenic Arabidopsis. (G) JAZ3_{P302A R305A} cannot form subnuclear foci in N. benthamiana. (H) TCP14 cannot re-localize JAZ3_{P302A R305A}. (I) TCP14 re-located JAZ3_P302AR305A to nuclear foci in the presence of HopBB1. See also Figure S6.