Cauliflower mosaic virus, a compatible pathogen of Arabidopsis, engages three distinct defense-signaling pathways and activates rapid systemic generation of reactive oxygen species

Abstract

We analyzed expression of marker genes for three defense pathways during infection by Cauliflower mosaic virus (CaMV), a compatible pathogen of Arabidopsis (Arabidopsis thaliana), using luciferase reporter transgenes and directly by measuring transcript abundance. Expression of PR-1, a marker for salicylic acid signaling, was very low until 8 d postinoculation and then rose sharply, coinciding with the rise in virus levels. In contrast, as early as 2 h postinoculation, transcriptional up-regulation of GST1-a marker for reactive oxygen species-and PDF1.2-a marker for jasmonic acid/ethylene defense signaling-was detectable in the virus-inoculated leaf and systemically. In parallel with the activation of GST1, H(2)O(2) accumulated locally and systemically in virus- but not mock-inoculated plants. However, in plants inoculated with infectious CaMV DNA rather than virus particles, the onset of systemic luciferase activity was delayed by 24 to 48 h, suggesting that virion structural proteins act as the elicitor. This phenomenon, which we term the rapid systemic response, preceded virus movement from the inoculated leaf; therefore, the systemic signal is not viral. Systemic, but not local, H(2)O(2) accumulation was abolished in rbohDF double mutants and in etr1-1 and ein2-1 mutants, implicating NADPH oxidase and ethylene signaling in the generation and transduction of the response. Ethylene, but not rbohDF mutants, also showed reduced susceptibility to CaMV, whereas in NahG transgenics, virus levels were similar to wild type. These findings implicate reactive oxygen species and ethylene in signaling in response to CaMV infection, but suggest that salicylic acid does not play an effective role.

Figure 1.

A, Levels of CaMV DNA in plants at intervals after inoculation. Levels, measured by quantitative real-time PCR, are expressed in arbitrary units as a proportion of total DNA (see “Materials and Methods”). Error bars indicated sds of mean for triplicate samples each composed of DNA from three plants. B, Levels of CaMV 19S plus 35S RNA in plants at intervals after inoculation. Levels, measured by quantitative hybridization in slot blots and expressed in arbitrary units, are expressed as a proportion of total RNA (see “Materials and Methods”). Error bars indicated sds of mean for triplicate samples each composed of RNA from three plants. C, Percentage of plants that developed symptoms of infection following removal of the inoculated leaf at the time indicated. Non-cut indicates control in which the infected leaf was not removed. Infection was determined on the basis of symptom development by 16 dpi. Error bars indicate sd of mean based on three groups of 30 plants for each time point.

Figure 2.

Expression of defense-related genes PR-1 (A), PR-2 (B), PR-5 (C), and GST1 (D) at intervals after inoculation with CaMV. Levels of transcripts in noninoculated leaves were determined by quantitative hybridization in slot blots and are expressed in arbitrary units, which differ according to the probe used. Results were corrected for any differences in loading by quantitating the amount of RNA on each spot from the stained filter as described in “Materials and Methods.” ▪, CaMV inoculated; ♦, mock inoculated; PR-2 and PR-5 mRNAs were not detectable in mock-inoculated plants; zero values are not shown. Error bars indicate sds of mean for triplicate samples each composed of RNA from noninoculated leaves from three plants.

Figure 3.

LUC activity in virus- and mock-inoculated plants. Luminescence was determined at the time after inoculation indicated above each image, as described in “Materials and Methods.” Luminescence levels are shown in pseudocolor using the logarithmic scale shown in the legend. Where appropriate, the inoculated leaf is indicated by a red arrow. A, Luminescence in PR-1a::LUC transgenic plants. The image labeled partially symptomatic shows the pattern of luminescence activity at 19 dpi in a plant that failed to develop symptoms in the apical region. For comparison, the adjacent image marked 19 d shows a comparable fully symptomatic plant. The shoot apical region is indicated with a white arrow. B, Luminescence in GST1::LUC transgenic plants. An image of an uninoculated plant is shown for one time point (2 hpi) only. At later time points, patterns of LUC activity in uninoculated plants were similar to mock-inoculated plants and are not shown. C, Luminescence in GST1::LUC transgenic plants after inoculation with CaMV virus and CaMV DNA. Mock-inoculated controls are shown for comparison. D, Luminescence in PDF1.2::LUC transgenic plants. An image of an uninoculated plant is shown for one time point (2 hpi) only. At later time points, patterns of LUC activity were similar to mock-inoculated plants and are not shown.

Figure 4.

Accumulation of H₂O₂ in tissues as determined by DAB staining. The presence of H₂O₂ results in the deposition of a brown pigment. Where appropriate, the inoculated leaf is indicated by a red arrow and the cotyledons are indicated by blue arrows. A, DAB staining of virus- and mock-inoculated Col-0 plants and untreated (uninoculated) controls at the times after inoculation indicated above each image. B, DAB staining of virus- and mock-inoculated Col-0 and mutants at 1 dpi. The genotype of the mutant is shown above each image. Like uninoculated Col-0, none of the uninoculated mutants exhibited any obvious staining and images are not shown.

Figure 5.

Levels of GST1 mRNA at intervals after inoculation, assayed by quantitative real-time RT-PCR; ▪, CaMV-inoculated plants; ♦, mock-inoculated plants. From left to right, Col-0, rbohDF, and etr1-1. Each point represents the mean value (± sd) derived from duplicate assays of two batches of seven plants. Levels, expressed in arbitrary units, have been normalized using ACT2 as an internal reference.

Figure 6.

Levels of CaMV DNA accumulating in uninoculated leaves from infected wild type and mutants. Levels of CaMV DNA at 14 dpi (gray bars) and 24 dpi (white bars), expressed in arbitrary units, were measured by quantitative real-time PCR. Values were corrected by normalization to a standard (Arabidopsis 18S rDNA; see “Materials and Methods”). Mean levels of virus DNA were determined from three independent experiments. For each experiment, DNA was quantified (for each genotype) in two biological samples each comprising the pooled tissue from three infected plants. Error bars show sds of the mean.

Figure 7.

Bacterial titers (colony forming units/mL) in different leaves 64 h after vacuum infiltration with P. syringae pv maculicola strain ES4326. Col-0 or rbohDF seedlings were inoculated on a single true leaf with CaMV or mock inoculated with water and challenged with bacteria 6 hpi or 24 hpi later. VI, Virus-inoculated leaf; MI, mock-inoculated leaf; VO, virus-inoculated seedling—opposite leaf; MO mock-inoculated seedling—opposite leaf. Results show mean values from 10 plants. Error bars show sds of the mean.

Figure 8.

Effect of treatments designed to potentiate defense. Ler-0 plants were treated as described (“Materials and Methods”) and assessed visually for the presence of systemic symptoms of CaMV infection (vein clearing, stunting, mosaics) at 16 dpi. Shown: 0.1% DMSO (control); 1 mm salicylic acid (SA); 50 μm antimycin A in 0.1% DMSO (AA). Data shown are mean ± sd for duplicate groups of 40 plants.