A large-scale genetic screen for mutants with altered salicylic acid accumulation in Arabidopsis

Abstract

Salicylic acid (SA) is a key defense signal molecule against biotrophic and hemibiotrophic pathogens in plants, but how SA is synthesized in plant cells still remains elusive. Identification of new components involved in pathogen-induced SA accumulation would help address this question. To this end, we performed a large-scale genetic screen for mutants with altered SA accumulation during pathogen infection in Arabidopsis using a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification method. A total of 35,000 M2 plants in the npr1-3 mutant background have been individually analyzed for the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326-induced SA accumulation. Among the mutants isolated, 19 had SA levels lower than npr1 (sln) and two exhibited increased SA accumulation in npr1 (isn). Complementation tests revealed that seven of the sln mutants are new alleles of eds5/sid1, two are sid2/eds16 alleles, one is allelic to pad4, and the remaining seven sln and two isn mutants are new non-allelic SA accumulation mutants. Interestingly, a large group of mutants (in the npr1-3 background), in which Psm ES4326-induced SA levels were similar to those in the wild-type Columbia plants, were identified, suggesting that the signaling network fine-tuning pathogen-induced SA accumulation is complex. We further characterized the sln1 single mutant and found that Psm ES4326-induced defense responses were compromised in this mutant. These defense response defects could be rescued by exogenous SA, suggesting that SLN1 functions upstream of SA. The sln1 mutation was mapped to a region on the north arm of chromosome I, which contains no known genes regulating pathogen-induced SA accumulation, indicating that SLN1 likely encodes a new regulator of SA biosynthesis. Thus, the new sln and isn mutants identified in this genetic screen are valuable for dissecting the molecular mechanisms underlying pathogen-induced SA accumulation in plants.

Keywords: Arabidopsis thaliana; NPR1; disease resistance; genetic screen; isn mutant; salicylic acid; sln mutant.

Figure 1

Pathogen-induced free SA levels in the SA accumulation mutants. (A) Luminescence from crude extracts of Psm ES4326-infected wild-type, npr1-3, pad4-1 npr1-3, eds5-1 npr1-3, sid2-1 npr1-3, and 19 putative mutant leaf tissues measured with the SA biosensor. (B) Free SA levels in Psm ES4326-infected wild-type, npr1-3, pad4-1 npr1-3, eds5-1 npr1-3, sid2-1 npr1-3, and 19 putative mutant plants detected by the HPLC-based method. Values are the mean of eight (A) or three (B) samples with standard deviation (SD). The experiments were repeated three times with similar results.

Figure 2

Pathogen growth in the SA accumulation mutants. Leaves of 4-week-old plants were inoculated with a Psm ES4326 suspension (OD₆₀₀ = 0.0001). The in planta bacterial titers were determined 3 days post-inoculation. Data represent the mean of eight independent samples with SD. cfu, colony-forming units. The experiment was repeated three times with similar results.

Figure 3

Further characterization of sln1 npr1-3. (A) Photos of 4-week-old soil-grown npr1-3 and sln1 npr1-3 plants. (B) Luminescence from crude extracts of Pst DC3000/avrRpt2-infected wild-type, npr1-3, pad4-1 npr1-3, sln1 npr1-3 leaf tissues measured with the SA biosensor. Values are the mean of eight independent samples with SD. Different letters above the bars indicate significant differences (P < 0.05, One-Way ANOVA). The experiment was repeated three times with similar results.

Figure 4

Pathogen-induced SA levels and ICS1 expression in the sln1 single mutant. Leaves of 4-week-old soil-grown wild-type and sln1 plants were infiltrated with a suspension of Psm ES4326 (OD₆₀₀ = 0. 001). The inoculated leaves were harvested 24 h post-inoculation (hpi) for SA measurement using HPLC or ICS1 expression analysis using qPCR. (A) Free SA levels in Psm ES4326-infected wild-type and sln1 plants. (B) Total SA levels in Psm ES4326-infected wild-type and sln1 plants. (C) ICS1 expression levels in Psm ES4326-infected wild-type and sln1 plants. Values are the mean of three independent samples with SD. Different letters above the bars indicate significant differences (P < 0.05, Student's t-test). The comparison was made separately for each time point. Expression of ICS1 in (C) was normalized against constitutively expressed UBQ5. The experiments were repeated three times with similar results.

Figure 5

Defense responses in the sln1 single mutant. (A) Disease symptoms of Psm ES4326-infected wild-type and sln1 leaves. Four-week-old soil-grown plants were inoculated with a suspension of Psm ES4326 (OD₆₀₀ = 0.0001). Photos were taken 3 days post-inoculation. (B) Growth of Psm ES4326 in wild-type and sln1 plants. Four-week-old soil-grown plants were inoculated with a suspension of Psm ES4326 (OD₆₀₀ = 0.0001). The in planta bacterial titers were determined immediately and 3 days post-inoculation. Data represent the mean of eight independent samples with SD. (C–E) Psm ES4326-induced PR1 (C), PR2 (D), and PR5 (E) gene expression in wild-type and sln1 plants. Four-week-old soil-grown plants were inoculated with a suspension of Psm ES4326 (OD₆₀₀ = 0.001). Total RNA was extracted from leaf tissues collected at 24 hpi and subjected to qPCR analysis. Data represent the mean of three independent samples with SD. An asterisk (^*) above the bars indicates significant differences (P < 0.05, Student's t-test). ns, not significant. All experiments were repeated three times with similar results.

Figure 6

Exogenous SA-induced PR gene expression and resistance in sln1. (A) Exogenous SA-induced PR1 expression in wild-type and sln1 plants. Four-week-old soil-grown wild-type and sln1 plants were soaked with an SA water solution (1 mM). Total RNA was extracted from leaf tissues collected at the indicated time points and analyzed for PR1 expression using qPCR. Values are the mean of three independent samples with SD. Different letters above the bars indicate significant differences (P < 0.05, One-Way ANOVA). The comparison was made separately for each genotype. (B) Exogenous SA-induced resistance to Psm ES4326 in wild-type and sln1 plants. Plants were treated as in (A). Twelve hours later, the plants were inoculated with a suspension of Psm ES4326 (OD₆₀₀ = 0.001). The in planta bacterial titers were determined 3 days post-inoculation. Values are the mean of eight independent samples with SD. An asterisk (^*) above the bars indicates significant differences (P < 0.05, Two-Way ANOVA). ns, not significant. These experiments were repeated three times with similar results.

Figure 7

Preliminary mapping of the sln1 mutation. A total of 74 F₂ progeny homozygous for sln1 were used to determine the approximate position of the sln1 mutation using bulked segregant analysis. The sln1 mutation was linked to the molecular marker PT1 on chromosome 1. Out of the 74 F₂ plants, six were heterozygous at gene At1g01448, and one was heterozygous at the molecular marker PAI1.2. The heterozygotes found by these two markers were mutually exclusive. No heterozygotes were found at PT1. The SLN1 gene is likely located in the vicinity of PT1, as indicated by the red bar. Rec., recombinant.