The Arabidopsis gene SIGMA FACTOR-BINDING PROTEIN 1 plays a role in the salicylate- and jasmonate-mediated defence responses

Abstract

The chloroplast-localized SIB1 protein was previously identified by its interaction with SIGMA FACTOR 1 (SIG1), a component of the RNA polymerase machinery responsible for transcription of plastid genes. The physiological function of SIB1 is little known. We found that expression of SIB1 is induced by infection with Pseudomonas syringae, suggesting its possible involvement in the defence response. The sib1 loss-of-function mutation compromises induction of some defence-related genes triggered by pathogen infection and the treatments with salicylic acid (SA) and jasmonic acid (JA), two key signalling molecules in the defence response. Conversely, constitutive over-expression of SIB1 causes the plants to hyper-activate defence-related genes following pathogen infection or the SA and JA treatments, leading to enhanced resistance to infection by P. syringae. SIB1 is a member of the large plant-specific VQ motif-containing protein family, and might act as a link to connect defence signalling with chloroplast function.

Figure 1

Pathogen-triggered expression of the SIB1 gene. (a) An RNA blot probed with the SIB1 gene. RNA was isolated from the uninfected and Pst avrRpm1-infected plants. The pathogen-infected wild-type (wt) plants accumulated a 0.85 kb SIB1 transcript, whereas the sib1 mutant did not produce a detectable level of the normal SIB1 transcripts. Col: the Columbia ecotype used as wt plants. A portion of the ethidium bromide-stained agarose gel is shown as an RNA loading control. (b) An RNA blot revealed that SIB1 expression was induced by both Pst and Pst avrRpm1, and its expression was compromised in npr1, enhanced disease susceptibility 5 (eds5) and NahG plants. (c) The transcript levels of SIB1 in uninfected and Pst avrRpm1-infected wt, npr1, eds5 and NahG plants determined by qPCR analysis. (d) An RNA blot showing SIB1 transcript levels following infection with the Pst hrcC mutant strain and Pst avrRpm1. (e and f) Transcript levels of At2g41180 in wt, sib1 and SIB1 over-expression line (see below) determined by RNA blotting (e) and qPCR analysis (f). Leaves were inoculated with Pst avrRpm1. Mock (M) represents RNA samples from leaves after mock (H₂O) inoculation.

Figure 2

A sequence alignment of the VQ domain-containing regions from 25 plant proteins. Sequences were aligned using the ClustalW algorithm of the VectorNTI software. Identical residues are in black background, and similar/conserved residues are in grey background. Included in the alignment are 17 Arabidopsis proteins, two rice proteins, two grape proteins (CAO63044 and CAN64541), two moss proteins (XP_001760459 and XP_001771471) and two spike moss sequences (SmSeq1 and SmSeq2).

Figure 3

Phenotype analyses of loss- and gain-of-function mutations of SIB1. (a) The RNA blot shows the SIB1 transcript was undetectable from the sib1-2 mutant. The RNA samples were extracted from uninfected leaves (0 h) and leaves inoculated with Pst avrRpm1 at 6 hpi. (b) Morphological phenotypes of 4-week-old plants. (c) In planta growth of Pst in wild-type (wt), sib1 and 35S∷SIB1-1 leaves. Each data point represents the average of three replicates ± SD. (d) The RNA blot result showing the SIB1 transcript levels in uninfected leaves of the two SIB1 over-expression lines and wt. (e) The relative amount of chlorophyll in leaves of wt, sib1 and the SIB1 over-expression lines. Values are the average of eight replicates ± SD.

Figure 4

Loss- and gain-of-function mutations of SIB1 affect expression of defence-related genes triggered by pathogen infection and the SA treatment. (a) An RNA blot probed with PR1 and PR2 genes. Leaves were inoculated with Pst avrRpm1. SIB1 over-expression led to quicker and stronger induction of these genes. The sib1 mutant accumulated a slightly lower level of these transcripts at 24 hpi. (b, c) PR1 transcript levels in wild-type (wt), sib1 and the SIB1 over-expression lines following the SA treatment determined by RNA blot analysis (b) and qPCR analysis (c). (d) The qPCR result revealed that sib1-2 was also defective in SA-mediated induction of PR1.

Figure 5

Loss- and gain-of-function mutations of SIB1 alter expression of jasmonic acid (JA)-responsive genes following pathogen infection and the JA treatment. RNA was extracted from leaves taken at different time-points after inoculation with Pst avrRpm1 (a) and the JA treatment (b), and the transcript levels of pdf1.2a and pdf1.2b were determined by qPCR analysis.

Figure 6

Determination of transcript levels of SIG1, psaA and psaB. RNA samples were extracted from uninfected and Pst avrRpm1-infected leaves (1.5 and 6 hpi) of wild-type (wt), sib1 and the SIB1 over-expression line. The transcript levels of SIG1 (a), psaA (b) and psaB (c) were determined by qPCR.