Discrimination of Arabidopsis PAD4 activities in defense against green peach aphid and pathogens

Abstract

The Arabidopsis (Arabidopsis thaliana) lipase-like protein PHYTOALEXIN DEFICIENT4 (PAD4) is essential for defense against green peach aphid (GPA; Myzus persicae) and the pathogens Pseudomonas syringae and Hyaloperonospora arabidopsidis. In basal resistance to virulent strains of P. syringae and H. arabidopsidis, PAD4 functions together with its interacting partner ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defenses. By contrast, dissociated forms of PAD4 and EDS1 signal effector-triggered immunity to avirulent strains of these pathogens. PAD4-controlled defense against GPA requires neither EDS1 nor SA. Here, we show that resistance to GPA is unaltered in an eds1 salicylic acid induction deficient2 (sid2) double mutant, indicating that redundancy between EDS1 and SID2-dependent SA, previously reported for effector-triggered immunity conditioned by certain nucleotide-binding-leucine-rich repeat receptors, does not explain the dispensability of EDS1 and SID2 in defense against GPA. Mutation of a conserved serine (S118) in the predicted lipase catalytic triad of PAD4 abolished PAD4-conditioned antibiosis and deterrence against GPA feeding, but S118 was dispensable for deterring GPA settling and promoting senescence in GPA-infested plants as well as for pathogen resistance. These results highlight distinct molecular activities of PAD4 determining particular aspects of defense against aphids and pathogens.

Figure 1.

Redundancy between EDS1 and SID2 is not important for controlling GPA infestation. A, Constitutive OE of EDS1 curtails GPA population. The no-choice assay shows GPA numbers on wild-type (WT) Ws, eds1-1, pad4-5, and eds1-1 pad4-5 mutants, and plants constitutively overexpressing EDS1 (EDS1-OE) or PAD4 (PAD4-OE) from the 35S promoter. This experiment was conducted three times with similar results. B, RT-PCR analysis of PAD4 and EDS1 expression in leaves of GPA-infested (+ GPA) plants of the indicated genotypes. Uninfested (− GPA) plants provided negative controls. ACT8 expression served as a control for RT-PCR. This experiment was conducted twice with similar results. C, Aphid population size is not impacted by simultaneous deficiency of EDS1 and SID2. The no-choice assay shows GPA numbers on wild-type accessions Ws and Col, pad4-5, eds1-1, sid2-1, and eds1-1 sid2-1 mutant plants (top panel), and wild-type Col, pad4-1, eds1-22, sid2-1, and eds1-22 sid2-1 mutant plants in accession Col (bottom panel). The pad4-5 and eds1-1 alleles and sid2-1 are in the Ws and Col backgrounds, respectively. These experiments were conducted twice with similar results. In A and C, GPA population size was determined 2 dpi (n = 10). Error bars represent se. ANOVA of GPA populations on different plant genotypes was conducted using PROC GLM (SAS Institute). Means were separated using the lsd procedure. Different letters above bars indicate values that are significantly different (P < 0.05) from each other.

Figure 2.

Amino acid sequence of PAD4 and homology to key regions of fungal lipases. A, Amino acid sequence of PAD4. Residues S118, D178, and H229 are in boldface. The underlined sequence corresponds to the GXSXG motif. B, Conservation of amino acid sequences around the S118, D178, and H229 residues between PAD4 and other putative fungal lipases. S118, D178, and H229 residues in PAD4 are underlined, invariant residues are in boldface, and asterisks identify conserved amino acids. RhTGL, Triacylglcyerol lipase precursor 1 from Rhizomucor miehei; FhTGL, triacylgylcerol lipase from Fusarium heterosporum; TlLIP, lipase from Thermomyces lanuginosus.

Figure 3.

PAD4 and SAG13 transcript and PAD4 protein accumulation in plants expressing pad4^S118A, pad4^D178A, and pad4^H229A variants. A, Time course of PAD4 and SAG13 transcript accumulation in uninfested (−GPA) and GPA-infested (+GPA) leaves of wild-type (WT) Ws, pad4-5, and pad4-5 mutant plants transformed with PAD4^WT (P4^WT) or the pad4^S118A (p4^S118A), pad4^D178A (p4^D178A), or pad4^H229A (p4^H229A) mutant constructs expressed from the PAD4 promoter. ACT8 expression served as a control for RT-PCR. B, Western-blot analysis of the PAD4 protein. Total protein extracted from leaves of uninfested and GPA-infested (24 hpi) wild-type Ws, pad4-5, PAD4^WT, pad4^S118A, pad4^D178A, and pad4^H229A plants was used for monitoring the accumulation of the transgene-encoded cMyc epitope-tagged PAD4 variants. An anti-cMyc antibody was used as the primary antibody. Coomassie blue-stained Rubisco large subunit (Rbc-L) is shown as a loading control. MW, Molecular mass markers in kD. These experiments were conducted twice with similar results.

Figure 4.

S118 in PAD4 is required for controlling GPA infestation. A, The no-choice assay shows GPA numbers on wild-type (WT) Ws, pad4-5, and two independently derived transgenic pad4-5 mutant lines expressing the PAD4^WT (P4^WT), pad4^S118A (p4^S118A), pad4^D178A (p4^D178A), and pad4^H229A (p4^H229A) constructs from the PAD4 promoter. GPA population size was determined 2 dpi (n = 12). This experiment was conducted three times with similar results. B, GPA numbers on a synthetic diet containing petiole exudate from PAD4^WT and pad4^S118A plants. Diet containing petiole exudate collected from the wild-type Ws and the pad4-5 mutant, and the buffer used to collect petiole exudates, provided controls for this experiment. Three adult aphids were introduced into each feeding chamber and allowed to feed on the diet, and the total numbers of aphids (nymphs plus adults) in each chamber were determined 4 d later (n = 3). This experiment was conducted three times with similar results. Error bars represent se. For details on statistical analysis, see legend to Figure 1. Different letters above the bars indicate values that are significantly different (P < 0.05) from each other.

Figure 5.

S118, D178, and H229 are not essential for the PAD4-determined deterrence of insect settling on Arabidopsis. In the choice tests, insects were given the choice of settling between plants of two genotypes by releasing 20 adult apterous GPA at the center of a pot containing one plant of each indicated genotype. The total numbers of adult GPA that had settled on eight plants of each genotype were determined 48 h later. Equal preference for each pair of genotypes was tested using the pooled χ² test. Asterisks indicate values that are significantly different (P < 0.05) from the other genotype. This experiment was conducted three times with similar results. For mean numbers of insects per plant with error bars for visual reference, see Supplemental Figure S2. WT, Wild type.

Figure 6.

S118, D178, and H229 are not essential for the PAD4-determined chlorosis in GPA-infested plants. A, Leaves of the wild-type (WT) Ws, pad4-5, and transgenic pad4-5 plants expressing the PAD4^WT (P4^WT) or pad4^S118A (p4^S118A), pad4^D178A (p4^S118A), and pad4^H229A (p4^H229A) transgenes 5 d after release of 20 GPA on each plant. Uninfested plants (−GPA) provided the negative controls. This experiment was conducted three times with similar results. B, Relative chlorophyll contents in GPA-infested leaves of plants of the indicated genotypes 5 d after release of 20 aphids on each plant. Values are relative to the chlorophyll contents in uninfested plants of the corresponding genotype (n = 5). Error bars represent se. Different letters above the bars indicate values that are significantly different (P < 0.05) from each other. This experiment was conducted twice with similar results.

Figure 7.

S118, D178, and H229 are not required for PAD4-mediated resistance to virulent or avirulent pathogens. A, Growth of virulent Pst DC3000 on wild-type (WT) Ws, eds1-1, pad4-5, eds1-1 pad4-5 double mutant, PAD4^WT (P4^WT), pad4^S118A (p4^S118A), pad4^D178A (p4^D178A), or pad4^H229A (p4^H229A) plants. Pathogen growth was monitored in two independently derived transgenic lines of each genotype. Pathogen-inoculated leaves were harvested at 0 and 3 dpi, and bacterial numbers were determined by plating dilutions of leaf extracts on selective medium. Bacterial numbers are represented as the log₁₀ of colony-forming units per unit area (cfu cm⁻²) of leaf (n = 3). Error bars represent se. Asterisks above the bars indicate values that are significantly different (P < 0.05; t test) from wild-type Ws at the equivalent time point. B, Representative Pst DC3000-inoculated leaves from plants of the indicated genotypes harvested 3 dpi. The extent of chlorosis is an indication of disease severity. C, Resistance to avirulent Hpa biotype Noco2 on wild-type Ws, pad4-5, and transgenic PAD4^WT, pad4^S118A, pad4^D178A, or pad4^H229A plants. Sixteen-day-old seedlings of the indicated genotypes were inoculated. At 6 dpi, 21 trypan blue-stained leaves per genotype were scored using the microscope for the presence of discrete hypersensitive response lesions (identified by red arrows) at infection sites or trailing necrosis (TN; identified by black arrows). Whereas extensive trailing necrosis was observed in approximately 50% of pad4-5 leaves (Table II), only the hypersensitive response was observed in the transgenic lines and Ws (representing more than 120 infection sites per line). Photographs of representative samples are shown. All infection assays were repeated at least twice with similar results.

Figure 8.

Model for different PAD4 molecular activities in Arabidopsis interaction with pathogens and GPA. At least two molecular activities of PAD4 are implicated in Arabidopsis interactions with biotrophic pathogens. PAD4, dissociated from EDS1, is required for ETI conditioned by TIR-NB-LRR-type receptors. Here, PAD4 and EDS1 activate a hypersensitive response involving localized host cell death and the restriction of pathogen growth. A different activity of PAD4 bound to EDS1 in a complex promotes the expression of SA biosynthetic and other genes (including PAD4 itself), leading to defense amplification (e.g. transcription of the PR1 gene) in basal resistance against virulent pathogens. In Arabidopsis interactions with GPA, PAD4 confers defenses without measurable EDS1 involvement. One PAD4 activity that does not require S118 deters insect settling and promotes leaf senescence, characterized by chlorophyll loss and increased SAG13 expression. This activity also promotes PAD4 expression in GPA-infested tissues. A different PAD4^S118-dependent activity deters insect feeding from the sieve elements and promotes the accumulation of an antibiosis factor in petiole exudates.