ALD1 accumulation in Arabidopsis epidermal plastids confers local and non-autonomous disease resistance

Abstract

The Arabidopsis plastid-localized ALD1 protein acts in the lysine catabolic pathway that produces infection-induced pipecolic acid (Pip), Pip derivatives, and basal non-Pip metabolite(s). ALD1 is indispensable for disease resistance associated with Pseudomonas syringae infections of naïve plants as well as those previously immunized by a local infection, a phenomenon called systemic acquired resistance (SAR). Pseudomonas syringae is known to associate with mesophyll as well as epidermal cells. To probe the importance of epidermal cells in conferring bacterial disease resistance, we studied plants in which ALD1 was only detectable in the epidermal cells of specific leaves. Local disease resistance and many features of SAR were restored when ALD1 preferentially accumulated in the epidermal plastids at immunization sites. Interestingly, SAR restoration occurred without appreciable accumulation of Pip or known Pip derivatives in secondary distal leaves. Our findings establish that ALD1 has a non-autonomous effect on pathogen growth and defense activation. We propose that ALD1 is sufficient in the epidermis of the immunized leaves to activate SAR, but basal ALD1 and possibly a non-Pip metabolite(s) are also needed at all infection sites to fully suppress bacterial growth. Thus, epidermal plastids that contain ALD1 play a key role in local and whole-plant immune signaling.

Keywords: Pseudomonas syringae; ALD1; Arabidopsis; dexamethasone-inducible expression; epidermal plastid; plant immunity; systemic acquired resistance.

Fig. 1.

Leaf-specific expression of ALD1 transcripts after treatment with DEX painting. (A) Diagram of the molecular structure of native ALD1 transcripts. The binding sites of primers used for ALD1 expression analysis in (D) are indicated: primers 1F and 1R on the ALD1 CDS is for ‘Total ALD1’ revealing both native ALD1 and transgenic ALD1:GFP transcripts in (B); primers 2F and 2R are for ‘ALD1-3'UTR’, revealing native ALD1 transcripts. Gray boxes represent exons, and the black box represents the 3'-untranslated region (UTR). Primer 1R spans the intron. pALD1, native promoter of ALD1 in Arabidopsis. 5', 5'-UTR. (B) Diagram of the pBAV150 plant expression vector containing the DEX-inducible promoter (pDEX)-driven GFP-tag-fused ALD1 sequence in pDEX::ALD1 transgenic plants. Primer pair 2F and 3R is used for analysis of ALD1:GFP in (E), revealing ALD1:GFP transgene transcripts. (C) Cartoon showed that the third to fifth leaves (lower leaves) were used as local leaves which will be immunized during the primary infection, and the sixth to eighth leaves (upper leaves) were used as distal leaves which will be challenged in the secondary infection in SAR experiments. Local leaves were painted with 30 μM DEX for ~1 d, while distal leaves were untreated. (D) Relative normalized expression of total ALD1 and ALD1-3'UTR transcripts by qPCR in local leaves of the indicated genotypes: wild type (WT), ald1-T2 (ald1), and pDEX::ALD1 transgenic lines #6 and #10. The transgenic pDEX::ALD1 lines #6 and #10 are in the ald1 mutant background. (E) Relative normalized expression of DEX-inducible transgenic ALD1:GFP transcripts by qPCR in treated local leaves and in untreated distal leaves of the indicated genotypes. 1° DEX ‘+’ or ‘–’ in (D) and (E) indicates that local leaves were treated with DEX or mock, respectively. (F) Semi-quantitative RT-PCR of DEX-induced transgenic ALD1:GFP levels in DEX-treated local leaves (shown as ‘L’) and in untreated distal leaves (shown as ‘D’) of different genotypes shown in (E). Error bars indicate the SEM from three biological replicates and three technical replicates. Different letters indicate statistically significant differences (P<0.05, ANOVA, Fisher’s LSD test). In (D–F), ACTIN was used as the internal control.

Fig. 2.

Epidermal cell-specific accumulation of ALD1:GFP fusion proteins after DEX treatments of leaves with or without infection. (A) Diagram of leaf structure and chloroplasts in different cell types. The size of chloroplasts in upper and lower epidermal cells is much smaller than that of the chloroplasts in mesophyll cells. (B–E) Laser scanning confocal micrographs of DEX-induced ALD1:GFP fusion protein in leaves of Arabidopsis transgenic lines pDEX::ALD1 #6 or #10. GFP fluorescence is shown in green and chlorophyll autofluorescence is shown in red. Scale bar=20 μm. (B) Expression and localization of DEX-induced ALD1:GFP fusion protein in the abaxial side of a half peeled leaf in pDEX::ALD1 #10. Above the dotted line are the mesophyll cells after removing the lower epidermis, while the part below is the original leaf with epidermis. Leaves were soaked in 30 μM DEX for 2 d before peeling. Four times (4×) enlarged images of selected insets are shown in the lower panels. Single layer scanning images were used. Similar results were observed in three independent experiments (n≥6 biological replicates for each experiment). (C) ALD1:GFP fusion protein in the epidermis or mesophyll of line pDEX::ALD1 #6 after pathogen Pma infection. Leaves were first sprayed with 30 μM DEX for 2 d, then inoculated by PmaDG6, PmaDG3 (OD₆₀₀=0.01), or 10 mM MgSO₄ for 18 h. Epidermal strips were peeled from the abaxial surface of the leaf, and the mesophyll layer was from the corresponding peeled region. Maximum intensity projections of Z-series images are used for epidermis data. Similar results were observed in two independent experiments (n≥6 biological replicates for each experiment). (D and E) Maximum intensity projections of the leaf with orthogonal projections to the XY, XZ, and YZ planes. Arrows indicate the same plastid. Leaves of line pDEX::ALD1 #6 were pre-treated with perfluorodecalin. Similar results were observed in two independent experiments (n≥6 biological replicates for each experiment). (D) Leaves of a 28-day-old plant were infiltrated with 30 μM DEX for 2 d. Images were taken from the adaxial surface of the leaf before infection. (E) Leaves of a 24-day-old plant were sprayed with 60 μM DEX for 1.5 d, and then infiltrated with PmaDG6 (OD₆₀₀=0.01) for 18 h. Images were taken from the abaxial surface of the leaf after infection.

Fig. 3.

Accumulation of ALD1:GFP 4 d post-treatment only in leaves directly painted with DEX. Confocal Z-series maximum intensity projection showing images of DEX-inducible ALD1:GFP fusion protein in transgenic pDEX::ALD1 lines #6 and #10. DEX-treated local leaves (Local-DEX) and no-treatment distal leaves (Distal-NT) were collected at 4 d after 30 μM DEX painting on local leaves. The ald1-T2 mutant was used as a negative control. Chlorophyll autofluorescence is shown in red, and GFP fluorescence is shown in green. Scale bar=20 μm. Biological replicates: local leaves, n=6; distal leaves, n=3. White arrowheads indicate the representative chloroplasts and ALD1:GFP signals showing co-localization in the merged images. Similar results were observed in other independent experiments after 2 d DEX painting as shown in Supplementary Fig. S3.

Fig. 4.

ALD1 accumulation at the site of infection fully restores defense responses in local leaves. (A) Treatment schemes in local leaves in (B–E). Local leaves (the third to fifth leaves) were painted with DEX (30 μM) or mock treated for 1 d, and then inoculated with PmaDG6. The primary (1°) local leaves were then collected at the indicated times for further analysis. (B) Titer of PmaDG6 in local leaves of the WT, ald1-T2 (ald1), and DEX- or mock-treated transgenic pDEX::ALD1 lines #6 and #10. Colony-forming unit (CFU) number was measured in local leaves on day 3 after infection with PmaDG6 (OD₆₀₀=0.0001). Error bars indicate the SEM of eight biological replicates. The experiment was repeated three times with similar results. Another experiment that employed DEX spraying also showed similar results. (C) PR1 gene expression level in DEX- (30 μM) painted local leaves at 0 h (no treatment, NT) and 9 h after PmaDG6 (DG6, OD₆₀₀=0.01) infection in the indicated genotypes: wild type (WT), ald1-T2 (ald1), and pDEX::ALD1#6 (#6). Error bars indicate the SEM from at least two biological replicates and three technical replicates. Each biological replicate consists of 6–9 leaves from at least three plants. The experiment was repeated twice with similar results. (D) Endogenous salicylic acid (SA) levels in local leaves were measured by HPLC in the indicated genotypes. DEX- (30 μM) or mock-painted local leaves were collected at 0 h (no treatment, NT) or 9 h after PmaDG6 (DG6, OD₆₀₀=0.01) infection. Free SA is shown in the left panel, and total SA is shown in the right panel. Error bars indicate the SEM of at least three biological replicates. Each biological replicate consists of 6–9 leaves from at least three plants. (E) Defense-related metabolite levels measured by GC-MS in local leaves of the indicated genotypes after 48 h infection. Error bars indicate the SEM from four biological replicates. (F) Pip and NHP levels in petiole exudates are not rescued in pDEX::ALD1 plants. Plants at ~4 weeks old of the WT, ald1, and pDEX::ALD1#6 (#6) were sprayed with 30 μM DEX for 1 d before infection. Petiole exudates were collected during 12–72 h post-local inoculation of the SAR-inducing PmaDG6 strain (OD₆₀₀=0.01). Metabolite levels measured by GC-MS. Results are the average with the SE from six biological replicates. Each biological replicate contains 12 leaves in 1.4 ml of 1 mM Na₂-EDTA (pH 8.0) solution. Different letters indicate statistically significant differences (P<0.05, ANOVA, Fisher’s LSD test). ND, not detected; hpi, hours post-infection.

Fig. 5.

Specific expression of ALD1 at the immunization site restores SAR in distal leaves. (A) Treatment schemes for specific expression of ALD1 at the immunization site during SAR establishment. Typically, local leaves (1°, the third to fifth leaves) were painted with 15–30 μM DEX or mock solution prior to SAR-triggering primary infection of an avirulent strain PmaDG6 (DG6, OD₆₀₀=0.01) or 10 mM MgSO₄. Then distal leaves (2°, the sixth to eighth leaves) without DEX treatment were inoculated with a virulent PmaDG3 (DG3, OD₆₀₀=0.0002) for the secondary infection. The quantification of DG3 growth in distal leaves was determined ~65–72 h later. (B) Titer of DG3 in distal leaves of the indicated genotypes. The number of colony-forming units (CFUs) of DG3 was measured in distal leaves. Error bars indicate the SEM of eight biological replicates (from eight plants). The result is representative of five independent experiments with similar results. Black triangles indicate SAR establishment under the corresponding treatment conditions. (C) Response gain of SAR associated with immunizing infection by 1° DG6 in local leaves with or without DEX treatment. Data for the line pDEX::ALD1 #6 (left panel) are the average of 2–3 experiments (DEX, three times; no DEX, twice), while data for the line pDEX::ALD1 line #10 (right panel) are the average of 1–2 experiments (DEX, twice; no DEX, once). Error bars indicate average uncertainties from the indicated experiments. Different letters indicate statistically significant differences (P<0.05, ANOVA, Fisher’s LSD test).

Fig. 6.

ALD1 accumulation at the site of an immunizing infection restores many distal leaf defenses but not Pip accumulation. (A) Treatment schemes for SA measurement in distal leaves in (B–D). After 1° DEX (30 μM) painting for 1 d, local leaves were infection by PmaDG6 (OD₆₀₀=0.01) or 10 mM MgSO₄. Then after 1° immunization infection for 2 d, distal leaves (without DEX treatment) were collected at 0 h (NT) or the indicated times after 2° challenge infection with PmaDG3 (OD₆₀₀=0.01). (B) SA levels in distal leaves of the indicated genotypes induced by immunizing infection before 2° infection (2° NT) and after 2° infection (2° DG3). SA levels were measured by HPLC in different genotypes after treatments. After 1° immunization infection for 2 d, distal leaves (without DEX treatment) were collected at 0 h (NT), or 9 h after 2° challenge infection with PmaDG3. Error bars indicate the SEM from four biological replicates. Each biological replicate consists of 6–9 leaves from three plants. (C) Response gain of SA in distal leaves of the indicated genotypes due to 1° PmaDG6 and 2° NT (1° DG6/2° NT, PN/MN), or due to 1° PmaDG6 and 2° PmaDG3 (1° DG6/ 2° DG3, PP/MP), corresponding to the SA data in (B). Meaning of symbols: PN, 1° PmaDG6 and 2° no treatment; MN, 1° MgSO₄ and 2° no treatment; PP, 1° PmaDG6 and 2° PmaDG3; MP, 1° MgSO₄ and 2° PmaDG3. (D) Expression levels of defense-related genes PR1 and FMO1 in distal leaves of the indicated genotypes induced by 1° immunizing infection and 2° challenge infection. The distal leaves were collected with no treatment (2° NT) or 2° PmaDG3 for 24 h. ACTIN was used as an internal reference. Error bars indicate the SEM from three biological replicates and two to three technical replicates. (E) Pip levels in distal leaves of the indicated genotypes measured by GC-MS. After 2 d immunization infection by PmaDG6, distal leaves (without DEX treatment) were collected at 0 h (NT), or 24 h after 2° infection with PmaDG3 (DG3). Error bars indicate the SEM from three biological replicates. Each biological replicate consists of 6–9 leaves from three plants. ND, not detected. Different letters indicate statistically significant differences (P<0.05, ANOVA, Fisher’s LSD test). For (C), the comparisons are within the DEX group or no DEX group, respectively.

Fig. 7.

ALD1 accumulation at the 2° distal challenge site does not restore SAR. (A) Treatment scheme for specific expression of ALD1 at the 2° challenge infection site in SAR. Local leaves (the third to fifth leaves) were infiltrated with DG6 (OD₆₀₀=0.01) or 10 mM MgSO₄ during immunizing infection. Then distal leaves (the sixth to eighth leaves) were painted with DEX at 1 d prior to 2° challenge infection by DG3 (OD₆₀₀=0.0001). The quantification of DG3 growth in distal leaves was measured 72 h later. (B) SAR response in distal leaves of the WT, ald1-T2, and pDEX::ALD1 #6 painted with 3 μM DEX. Growth of DG3 was measured in distal leaves. Error bars indicate the SEM from eight biological replicates. Another independent experiment with 15 μM DEX painting on distal leaves showed similar results. (C) Response gain of SAR in DEX-painted distal leaves due to 1° immunizing infection by DG6 (1° DG6/2° DG3). Results for pDEX::ALD1 #6 (left panel) and #10 (right panel) are each the average of two independent experiments. Different letters indicate statistically significant differences (P<0.05, ANOVA, Fisher’s LSD test).

Fig. 8.

Proposed ALD1 site of action model during local defenses and different stages of SAR. Step 1: basal defense status before 1° infection. ALD1 mainly regulates the basal level of pattern recognition receptor complex FLS2/BAK1 possibly mediated by non-Pip basal metabolites (Cecchini et al., 2015a). This study also shows that ALD1 predominantly in epidermal cells is sufficient to control infections with virulent and avirulent bacteria. Step 2: ALD1 at the 1° immunization site in epidermal cells is sufficient for the local defense responses (pathogen suppression, SA and ROS accumulation, callose deposition, defense gene expression, Pip biosynthesis, and other mobile immune signals for SAR establishment). Step 3: primed state at the distal leaf before 2° infection. ALD1 at the distal leaf is indispensable for most of the biosynthesis of Pip. 1° ALD1 in the epidermal cells at the immunization site contributes to controlling the majority of the accumulation of SA and defense genes (such as PR1) at the distal leaf. Step 4: 1° and 2° ALD1 expression regulate SAR output after 2° infection at the distal leaf. The local ALD1 predominantly in the chloroplasts of epidermal cells is sufficient to restore the systemic immunity. Blue, SAR-triggering bacteria at the 1° immunization site; red, virulent pathogenic bacteria infection at the 2° infection site. The ALD1 protein structure model (Sobolev et al., 2013) was downloaded from https://www.rcsb.org/structure/4FL0.