Forward and reverse genetics to identify genes involved in the age-related resistance response in Arabidopsis thaliana

Abstract

SUMMARY Age-related resistance (ARR) occurs in numerous plant species, often resulting in increased disease resistance as plants mature. ARR in Arabidopsis to Pseudomonas syringae pv. tomato is associated with intercellular salicylic acid (SA) accumulation and the transition to flowering. Forward and reverse genetic screens were performed to identify genes required for ARR and to investigate the mechanism of the ARR response. Infiltration of SA into the intercellular space of the ARR-defective mutant iap1-1 (important for the ARR pathway) partially restored ARR function. Inter- and intracellular SA accumulation was reduced in the mutant iap1-1 compared with the wild-type, and the SA regulatory gene EDS1 was also required for ARR. Combining microarray analysis with reverse genetics using T-DNA insertion lines, four additional ARR genes were identified as contributing to ARR: two plant-specific transcription factors of the NAC family [ANAC055 (At3g15500) and ANAC092 (At5g39610)], a UDP-glucose glucosyltransferase [UGT85A1 (At1g22400)] and a cytidine deaminase [CDA1 (At2g19570)]. These four genes and IAP1 are also required for ARR to Hyaloperonospora parasitica. IAP1 encodes a key component of ARR that acts upstream of SA accumulation and possibly downstream of UGT85A1, CDA1 and the two NAC transcription factors (ANAC055, ANAC092).

Figure 1

Disease symptoms in Col‐0 and iap1‐1 (homozygous and heterozygous). Leaves were inoculated with 10⁶ colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst) and photographed at 3 days post‐inoculation (dpi).

Figure 2

In planta bacterial levels in iap1‐1, Col‐0 and NahG. (a) Col‐0, iap1‐1 and NahG were inoculated with 10⁶ colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst) at 3 and 6 weeks post‐germination (wpg). In planta bacterial levels [cfu/leaf disc (ld)] were monitored at 3 days post‐inoculation (dpi) and are presented as the mean ± standard deviation (SD) of three samples. This experiment was repeated four times with similar results. (b) In planta bacterial growth in mature iap1‐1 and Col‐0 plants (6 wpg) was measured over 3 dpi (10⁶ cfu/mL Pst) and is presented as the mean of three samples ± SD. The bacterial density was significantly lower (Student's t‐test) in Col‐0 than iap1‐1 by day 3. This experiment was repeated three times with similar results.

Figure 3

Hyaloperonospora parasitica infection of iap1‐1. (a) Six‐week‐old Col‐0 and iap1‐1 plants were sprayed with the virulent H. parasitica isolate Noco 2. Photographs were taken 7 days after infection. (b) The number of H. parasitica spores per infected leaf was quantified using a haemocytometer. This experiment was repeated with similar results.

Figure 4

Salicylic acid (SA) application and SA levels in iap1‐1 and Col‐0. (a) Leaves of 5‐week‐old plants were inoculated with Pseudomonas syringe pv. tomato (Pst) at 5 or 24 h post‐SA infiltration (hpi; with 0.1 mM). In planta bacterial levels [colony‐forming units/leaf disc (cfu/ld)] were monitored 3 days after Pst inoculation (SA, Pst) and compared with control treatments (H, Pst), where water infiltration was followed by inoculation with Pst 24 h later. Asterisks indicate a significant decrease in bacterial density in SA‐treated plants vs. the corresponding water‐treated plants (Student's t‐test). The mean ± standard deviation (SD) of three replicate samples is shown. Experiments in which Pst was inoculated at 5 hpSAi were repeated twice with similar results. (b) Intercellular washing fluids (IWFs) were collected from mature leaves that were either mock‐inoculated or inoculated with Pst (10⁶ cfu/mL). SA levels were determined and are presented as ng/mL of IWF collected. Asterisks indicate a significant difference in SA levels in mock‐inoculated leaves compared with leaves inoculated with Pst (Student's t‐test). The mean ± SD of three replicate samples is shown. This experiment was repeated twice with similar results. (c) Mature leaves were mock‐inoculated or inoculated with Pst (10⁶ cfu/mL). IWFs were removed and total SA levels (SA plus SA glucosides) were determined in leaves and presented as ng/g fresh weight. Asterisks indicate a significant difference in SA levels in mock‐inoculated leaves compared with leaves inoculated with Pst (Student's t‐test). The mean ± SD of three replicate samples is shown. This experiment was repeated with similar results.

Figure 5

(a) In planta bacterial levels in eds1‐1, jar1‐1, jin1‐1, Col‐0 and Ws. Plants were inoculated with 10⁶ colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst) at 3 and 6 weeks post‐germination (wpg). In planta bacterial levels [cfu/leaf disc (cfu/ld)] were monitored at 3 days post‐inoculation (dpi) and are presented as the mean ± standard deviation (SD) of three samples. This experiment was repeated once for jin1 and twice for eds1‐1 and jar1‐1 with similar results. (b) eds1‐1 and Col‐0 plants (5‐week‐old) were infiltrated with water (H, Pst‐5) or salicylic acid (SA) (0.1 mM) (SA, Pst‐5) , followed by inoculation with Pst 5 h later, as in Fig. 4. This experiment was repeated twice with similar results.

Figure 6

(a) In planta Pseudomonas syringe pv. tomato (Pst) levels in young and mature Col‐0, anac092, anac055, cda1, ugt85A1 and sid2. Plants were inoculated with 10⁶ colony‐forming units (cfu)/mL Pst at 3, 5 or 6 weeks post‐germination (wpg). In planta bacterial levels [cfu/leaf disc (cfu/ld)] were monitored at 3 days post‐inoculation (dpi) and are presented as the mean ± standard deviation (SD) of three samples. Col‐0 controls are presented for each independent experiment. Significant differences between mature Col‐0 and mutants are indicated by an asterisk (P < 0.05 Student's t‐test). Different letters represent significant differences between mature genotypes in Expt. 3 (Tukey's honest significant difference, P < 0.05). These experiments were repeated at least twice with similar results. (b) Mature (6 wpg) Col‐0, anac092, anac055, cda1 and ugt85A1 were spray inoculated with Hyaloperonospora parasitica isolate Noco 2. The number of H. parasitica spores per infected leaf was quantified using a haemocytometer at 7 dpi. This experiment was repeated twice with similar results.

Figure 7

Age‐related resistance (ARR) gene expression analysis. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of leaves collected from 3 and 6 weeks post‐germination (wpg) Col‐0 at <5 min post‐inoculation (mpi) or 12, 24 and 48 h post‐inoculation (hpi) with 10⁶ colony‐forming units (cfu)/mL Pseudomonas syringe pv. tomato (Pst); 28 PCR cycles were used for ANAC055, ANAC092, CDA1, UGT85A1 and ACTIN primers. This experiment was replicated once with similar results.