Plastidial fatty acid levels regulate resistance gene-dependent defense signaling in Arabidopsis

Abstract

In Arabidopsis, resistance to Turnip Crinkle Virus (TCV) depends on the resistance (R) gene, HRT, and the recessive locus rrt. Resistance also depends on salicylic acid (SA), EDS1, and PAD4. Exogenous application of SA confers resistance in RRT-containing plants by increasing HRT transcript levels in a PAD4-dependent manner. Here we report that reduction of oleic acid (18:1) can also induce HRT gene expression and confer resistance to TCV. However, the 18:1-regulated pathway is independent of SA, rrt, EDS1, and PAD4. Reducing the levels of 18:1, via a mutation in the SSI2-encoded stearoyl-acyl carrier protein-desaturase, or by exogenous application of glycerol, increased transcript levels of HRT as well as several other R genes. Second-site mutations in the ACT1-encoded glycerol-3-phosphate acyltransferase or GLY1-encoded glycerol-3-phosphate dehydrogenase restored 18:1 levels in HRT ssi2 plants and reestablished a dependence on rrt. Resistance to TCV and HRT gene expression in HRT act1 plants was inducible by SA but not by glycerol, whereas that in HRT pad4 plants was inducible by glycerol but not by SA. The low 18:1-mediated induction of R gene expression was also dependent on ACT1 but independent of EDS1, PAD4, and RAR1. Intriguingly, TCV inoculation did not activate this 18:1-regulated pathway in HRT plants, but instead resulted in the induction of several genes that encode 18:1-synthesizing isozymes. These results suggest that the 18:1-regulated pathway may be specifically targeted during pathogen infection and that altering 18:1 levels may serve as a unique strategy for promoting disease resistance.

Fig. 1.

ssi2- and cpr5-mediated resistance signaling in various double mutant backgrounds. (A) Percentage of TCV-resistant plants in various genetic backgrounds. The number of plants tested is indicated above each bar. All plants were analyzed 3 weeks after inoculation. Asterisks indicate 100% susceptibility. (B) RT-PCR analyses showing HRT transcript levels in indicated genotypes. The level of β-tubulin was used as an internal control to normalize the amount of cDNA template. (C) Typical morphological phenotypes of TCV-inoculated plants; susceptible plants showed crinkling, stunted bolt development, and drooping of bolts. The resistant plants were morphologically similar to the mock-inoculated plants. The plants were photographed 2 weeks after inoculation. (D) Systemic spread of TCV to uninoculated tissue in TCV-inoculated plants. RNA was extracted from the uninoculated tissues at 18 days after inoculation and analyzed for the presence of the viral transcripts (TCV-U). Ethidium bromide staining of rRNA was used as a loading control.

Fig. 2.

HRT promoter assay and HRT transcript levels, 18:1 content, TCV resistance, and viral replication in leaves containing normal or higher oleate levels. (A) Transient GUS assay. Leaves were infiltrated with untransformed cells (-ve) or Agrobacterium transformed with HRT–GUS fusion construct. The sid2 plants were treated with water (−) or SA (+) for 2 days before infiltration. Leaf discs (sid2) or whole leaves (ssi2 sid2) were processed for GUS histochemical staining as described before (45). (B) RT-PCR analyses showing basal-level expression of HRT in HRT ssi2 act1 and HRT ssi2 gly1 plants. The level of β-tubulin was used as an internal control to normalize the amount of cDNA template. The 18:1 levels are a mean of six independent replicates. (C) Systemic spread of TCV to uninoculated tissue (TCV-U) in TCV-inoculated plants. RNA was extracted from the uninoculated tissues at 18 days after inoculation and analyzed for the presence of the viral transcripts. Ethidium bromide staining of rRNA was used as a loading control. (D) Typical morphological phenotypes of mock- and TCV-inoculated HRT ssi2 act1 and HRT ssi2 gly1 plants. The susceptible plants showed crinkling, stunted bolt development, and drooping of bolts. Plants were photographed at 8 days after inoculation. (E) Effect of 18:1 infiltrations on viral replication in the inoculated leaf. Oleic acid (O, 1 mM) or water (W) was injected 24 h before or after (last two lanes) TCV inoculation, and the samples were harvested 72 h after inoculation. Ethidium bromide staining of rRNA was used as a loading control..

Fig. 3.

PR-1 and R gene expression levels, 18:1 levels, HR formation, and TCV resistance in plants treated with water (W), SA, or glycerol (G). (A) PR-1 gene expression and 18:1 content in treated plants. The glycerol-treated HRT ssi2 plants were harvested 24 h after treatment. Ethidium bromide staining of rRNA was used as a loading control. The 18:1 levels are a mean of six independent replicates. (B) RT-PCR analyses showing expression of HRT and SSI4 genes in treated plants. The level of β-tubulin was used as an internal control to normalize the amount of cDNA template. (C) Visible HR formation in treated plants at 3 days after inoculation. (D) Percentage of TCV-resistant plants obtained after exogenous application of water, SA, or glycerol. Resistance was analyzed 3 weeks after inoculation. The number of plants tested is indicated above each bar. Asterisks indicate 100% susceptibility. (E) Systemic spread of TCV to uninoculated tissue in TCV-inoculated plants. RNA was extracted from the uninoculated tissues at 18 days after inoculation and analyzed for the presence of the viral transcripts (TCV-U). Ethidium bromide staining of rRNA was used as a loading control. (F) Typical morphological phenotypes of TCV-inoculated plants. The susceptible plants showed crinkling, stunted bolt development, and drooping of bolts. Plants were photographed at 18 days after inoculation.

Fig. 4.

Oleic acid-modulated expression of R genes and resistance to bacterial pathogen. (A) RT-PCR analysis of various R genes in WT (SSI2), ssi2, and ssi2 sid2 backgrounds. (B) RT-PCR analysis of various R genes in WT (HRT), HRT ssi2, HRT ssi2 sid2, and HRT ssi2 act1 backgrounds. (C) RT-PCR analysis of various R genes in water (W)- or glycerol (G)-treated sid2 plants. (D) RT-PCR analysis of various R genes in WT (HRT), HRT cpr5, and HRT cpr5 sid2 backgrounds. (E) RT-PCR analysis of various R genes in WT (SSI2), ssi2, ssi2 eds1, and ssi2 pad4 backgrounds. (F) RT-PCR analysis of various R genes in water (W)- or glycerol (G)-treated WT (RAR1), eds1, or rar1 plants. The levels of β-tubulin were used as internal control to normalize the amount of cDNA template in experiments shown in A–F. (G) Growth of P. syringae on WT (HRT or hrt), HRT ssi2, HRT sid2, and HRT ssi2 sid2 leaves. The Nössen ecotype was also tested and showed resistance similar to that seen in Di-17 and Col-0 plants (data not shown). Four leaf discs were harvested from infected leaves 3 days after inoculation and ground in 10 mM MgCl₂, and the bacterial numbers were titered. The bacterial numbers ± SD (n = 4) are presented as log of cfu per cm². The experiment was independently performed twice with similar results.

Fig. 5.

Oleic acid and S-ACP-DES transcript levels after mock and TCV inoculation. (A) The inoculated leaves from Di-17 plants were sampled at 0–72 h after inoculation and processed for FA levels. The values are a mean of six independent replicates. The error bars represent SD. (B) S-ACP-DES transcript levels in mock- and TCV-inoculated plants 72 h after inoculation. Data from two independently extracted RNA samples are shown here. The level of β-tubulin was used as an internal control to normalize the amount of cDNA template. Transcript levels of S-ACP-DES1, S-ACP-DES2, S-ACP-DES4, and S-ACP-DES6 either were not detected or showed no difference between mock- and TCV-inoculated plants.