Guanosine tetraphosphate modulates salicylic acid signalling and the resistance of Arabidopsis thaliana to Turnip mosaic virus

Abstract

Chloroplasts can act as key players in the perception and acclimatization of plants to incoming environmental signals. A growing body of evidence indicates that chloroplasts play a critical role in plant immunity. Chloroplast function can be regulated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In plants, (p)ppGpp levels increase in response to abiotic stress and to plant hormones which are involved in abiotic and biotic stress signalling. In this study, we analysed the transcriptome of Arabidopsis plants that over-accumulate (p)ppGpp, and unexpectedly found a decrease in the levels of a broad range of transcripts for plant defence and immunity. To determine whether (p)ppGpp is involved in the modulation of plant immunity, we analysed the susceptibility of plants with different levels of (p)ppGpp to Turnip mosaic virus (TuMV) carrying a green fluorescent protein (GFP) reporter. We found that (p)ppGpp accumulation was associated with increased susceptibility to TuMV and reduced levels of the defence hormone salicylic acid (SA). In contrast, plants with lower (p)ppGpp levels showed reduced susceptibility to TuMV, and this was associated with the precocious up-regulation of defence-related genes and increased SA content. We have therefore demonstrated a new link between (p)ppGpp metabolism and plant immunity in Arabidopsis.

Keywords: (p)ppGpp; TuMV; Turnip mosaic virus; chloroplast; guanosine tetraphosphate; pathogen; salicylic acid.

Figure 1

OX:RSH3.1 plants are more susceptible to infection with Turnip mosaic virus carrying a green fluorescent protein reporter (TuMV‐GFP). Forty‐two‐day‐old wild‐type (WT) and OX:RSH3.1 plants were agroinoculated on the first and second leaves with TuMV‐GFP. (A) Systemic TuMV‐GFP multiplication was visualized using fluorescence imaging at 12, 14 and 16 days post‐inoculation (dpi). Representative images are shown from one experiment. Fluorescence intensity is shown in false colour. (B) TuMV‐GFP multiplication was quantified in 20 inoculated plants of each genotype by calculating the area infected for each plant (area of GFP signal/area of plant), and the data are presented as a box plot. The boxes show the interquartile range and median, the mean is indicated by a cross and the whiskers delimit the 10th and 90th percentiles. *P < 0.05, **P < 0.01 for WT versus OX:RSH3.1, Kruskal–Wallis test, n = 20. Similar results were obtained in three independent repeats of the experiment.

Figure 2

Pathogen responses in wild‐type (WT) and OX:RSH3.1 plants infected with Turnip mosaic virus carrying a green fluorescent protein reporter (TuMV‐GFP). (A) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) of the indicated transcripts was performed on cDNA derived from the systemic leaves of mock‐inoculated (MOCK) or TuMV‐GFP‐inoculated (TuMV) plants 16 days after inoculation. Four biological replicates were used for MOCK and six for TuMV. Transcript abundance was normalized to APT1 and PP2A reference transcripts. (B) Immunoblots on equal quantities of total protein from plants 2 weeks after mock or TuMV inoculation using the indicated antibodies. The loading control is the RuBISCO large subunit stained with Coomassie brilliant blue (CBB). Free salicylic acid (SA) levels were determined in the aerial parts of healthy non‐inoculated plants (NI) grown under the same conditions as for TuMV‐GFP inoculation (n = 3 independent plants) (C) or TuMV‐GFP‐inoculated plants 2 weeks after inoculation (n = 6 independent plants (D). *P < 0.05, **P < 0.01 for WT versus OX:RSH3.1, analysis of variance (ANOVA). dwt, dry weight; error bars, standard error of the mean (SEM).

Figure 3

RSH mutants are more resistant to infection with Turnip mosaic virus carrying a green fluorescent protein reporter (TuMV‐GFP). Forty‐two‐day‐old wild‐type (WT), QM1 and QM2 plants were agroinoculated on the first and second leaves with TuMV‐GFP. (A) Systemic TuMV‐GFP multiplication was visualized using fluorescence imaging at 12, 14 and 16 days post‐inoculation (dpi). Representative images are shown from one experiment. Fluorescence intensity is shown in false colour. (B) TuMV‐GFP multiplication was quantified in 14 inoculated plants of each genotype by calculating the area infected for each plant (area of GFP signal/area of plant), and the data are presented as a box plot. The boxes show the interquartile range and median, the mean is indicated by a cross and the whiskers delimit the 10th and 90th percentiles. Statistical significance was tested by the Kruskal–Wallis test and significantly different groups are indicated by different letters for each time point (P < 0.05, n = 14). Similar results were obtained in three independent repeats of the experiment.

Figure 4

Pathogen responses in wild‐type (WT) and QM2 plants infected with Turnip mosaic virus carrying a green fluorescent protein reporter (TuMV‐GFP). (A) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) of the indicated transcripts was performed on cDNA derived from the systemic leaves of mock‐inoculated (MOCK) or TuMV‐GFP‐inoculated (TuMV) plants 16 days after inoculation. Four biological replicates were used for MOCK and six for TuMV. Note that no RSH1, RSH2 or RSH3 amplicons accumulated in QM2 because of the presence of T‐DNA insertions in these genes. Transcript abundance was normalized to APT1 and PP2A reference transcripts. Immunoblots on equal quantities of total protein from plants 2 weeks after mock or TuMV inoculation using the indicated antibodies (B) or from non‐inoculated plants of the same age (C). The loading control is the RuBISCO large subunit stained with Coomassie brilliant blue (CBB). Free salicylic acid (SA) levels were determined in the aerial parts of healthy non‐inoculated plants (NI) grown under the same conditions as for TuMV‐GFP inoculation (n = 3 independent plants) (D) or TuMV‐GFP‐inoculated plants 2 weeks after inoculation (n = 6 independent plants) (E). *P < 0.05, **P < 0.01 versus WT, analysis of variance (ANOVA). dwt, dry weight; error bars, standard error of the mean (SEM).

Figure 5

OX:RSH1 plants are more resistant to infection with Turnip mosaic virus carrying a green fluorescent protein reporter (TuMV‐GFP). Forty‐two‐day‐old wild‐type (WT) and OX:RSH1 plants were agroinoculated on the first and second leaves with TuMV‐GFP. (A) Systemic TuMV‐GFP multiplication was visualized using fluorescence imaging at 12, 14, 16 and 19 days post‐inoculation (dpi). Representative images are shown from one experiment. Fluorescence intensity is shown in false colour. (B) TuMV‐GFP multiplication was quantified in 24 inoculated plants of each genotype by calculating the area infected for each plant (area of GFP signal/area of plant), and the data are presented as a box plot. The boxes show the interquartile range and median, the mean is indicated by a cross and the whiskers delimit the 10th and 90th percentiles. Statistical significance was tested by the Kruskal–Wallis test (*P < 0.05, **P < 0.01, n = 24 plants). Similar results were obtained in three independent repeats of the experiment.