Analysis of Arabidopsis JAZ gene expression during Pseudomonas syringae pathogenesis

Abstract

The jasmonates (JAs) comprise a family of plant hormones that regulate several developmental processes and mediate responses to various abiotic and biotic stresses, including pathogens. JA signalling is manipulated by several strains of the bacterial pathogen Pseudomonas syringae, including P. syringae strain DC3000, using the virulence factor coronatine (COR) as a mimic of jasmonyl-L-isoleucine (JA-Ile). To better understand the JA-Ile-mediated processes contributing to P. syringae disease susceptibility, it is important to investigate the regulation of JA signalling during infection. In Arabidopsis thaliana, JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of JA signalling. The transcription factor JASMONATE INSENSITIVE1 (JIN1/ATMYC2) has been implicated in the regulation of JAZ gene expression. To investigate the regulation of JAZ genes during P. syringae pathogenesis, we examined JAZ gene expression during infection of Arabidopsis by DC3000. We found that eight of the 12 JAZ genes are induced during infection in a COR-dependent manner. Unexpectedly, the induction of the majority of JAZ genes during infection was not dependent on JIN1, indicating that JIN1 is not the only transcription factor regulating JAZ genes. A T-DNA insertion mutant and an RNA interference line disrupted for the expression of JAZ10, one of the few JAZ genes regulated by JIN1 during infection, exhibited enhanced JA sensitivity and increased susceptibility to DC3000, with the primary effect being increased disease symptom severity. Thus, JAZ10 is a negative regulator of both JA signalling and disease symptom development.

Figure 1

JASMONATE ZIM‐DOMAIN (JAZ) gene expression during DC3000 infection. Arabidopsis (Col‐0) plants were dip inoculated in DC3000 or MgCl₂ for mock inoculation. Untreated plants were used for the 0‐h time point. Transcript levels were monitored by reverse transcription‐polymerase chain reaction (RT‐PCR) in RNA samples isolated from tissue harvested at the indicated times (hours post‐infection) after inoculation. UBQ10 (At4g05320) is shown as an internal control.

Figure 2

Induction of JASMONATE ZIM‐DOMAIN (JAZ) genes during infection is dependent on coronatine (COR). Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of JAZ gene expression in wild‐type plants after dip infection with DC3000 (squares), the DC3000 COR^– mutant DB29 (circles) or MgCl₂ for mock infection (diamonds). Untreated plants were used for the 0‐h time point. Gene expression at each time‐point is represented relative to the internal control PP2AA3 (At1g13320; note different scales). Error bars represent ±SD of two biological replicates. Bacterial growth over the course of infection for this experiment is shown in Fig. S1a. Similar results were obtained in a second independent experiment.

Figure 3

JASMONATE ZIM‐DOMAIN (JAZ) gene expression in jin1 mutant plants during DC3000 infection. Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of JAZ gene expression during DC3000 infection in wild‐type (squares) and jin1‐1 (triangles) plants, and mock (MgCl₂) inoculation of wild‐type (diamonds) and jin1‐1 (×) plants. Experiments were performed and analysed as described for Fig. 2. Error bars represent ±SE of three biological replicates. Bacterial growth over the course of infection for this experiment is shown in Fig. S1b. Similar results were obtained in three additional independent experiments, one of which is shown in Fig. S2.

Figure 4

Characterization of jaz1, jaz5 and jaz10 mutants. (a) Schematic diagrams of the JAZ1 (At1g19180) and JAZ10 (At5g13220) loci. Bars represent exons with untranslated regions in grey. Lines represent introns, with double lines indicating introns that are retained in some splice variants. The T‐DNA insertion in jaz1‐1 is approximately 80 bp downstream of the end of the translated region of the gene. The T‐DNA insertion in jaz10‐1 is located 20 bp from the 3′ end of the second exon. (b) Reverse transcription‐polymerase chain reaction (RT‐PCR) of the indicated transcripts in wild‐type and jaz mutant plants. Transcript levels were monitored in seedlings after 10 days of growth on 10 µm methyl jasmonate (MeJA). UBQ10 (At4g05320) was used as an internal control. For JAZ5, a minus reverse transcriptase (–RT) control was included to demonstrate that the PCR product corresponds to JAZ5 transcript, not genomic DNA. (c) Jasmonate (JA) root growth inhibition assay of wild‐type (circles) and jaz1‐1 (diamonds) seedlings using 0, 5 and 10 µm MeJA. The normalized root length of at least 34 plants per treatment was calculated as a percentage of the average root length of plants grown without MeJA. Error bars represent ±SE. Experiments were performed three times with similar results. (d) JA root growth inhibition assay of wild‐type (circles), jaz10‐1 (diamonds) and JAZ10 RNAi (triangles) seedlings at increasing concentrations of MeJA. The normalized root length of at least 28 plants was calculated as described in (c).

Figure 5

jaz10‐1 is more susceptible to Pseudomonas syringae infection. (a) Disease symptoms of wild‐type (JAZ10), jaz10 lines and sid2 leaves, 4 days after hand infiltration. The right halves (to mid‐vein) of the leaves of each genotype were infiltrated with DC3000 at 5 × 10⁵ colony‐forming units (CFU)/mL. Leaves shown represent the range of symptoms exhibited by each genotype, from the mildest on the left to the most severe on the right. The black marks on the leaves are from a pen used to mark treated leaves at the time of infection. (b) Representative wild‐type and jaz10‐1 mutant plants, 4 days after dip inoculation with DC3000. (c) Growth of DC3000 in wild‐type (squares), jaz10‐1 (grey diamonds), JAZ10 RNAi‐9 (triangles) and sid2‐2 (circles) plants after hand infiltration. Bacteria were isolated from leaves at the indicated times after inoculation. Data points represent the average of four samples ± SE. Similar results were obtained in two additional independent experiments. (d) Growth of DC3000 in wild‐type (squares), jaz10‐1 (grey diamonds) and JAZ10 RNAi‐9 (triangles) after dip inoculation. Data were collected and analysed as in (c). Similar results were obtained in two additional independent experiments.