Hypoxia response in Arabidopsis roots infected by Plasmodiophora brassicae supports the development of clubroot

Abstract

Background: The induction of alcohol fermentation in roots is a plant adaptive response to flooding stress and oxygen deprivation. Available transcriptomic data suggest that fermentation-related genes are also frequently induced in roots infected with gall forming pathogens, but the biological significance of this induction is unclear. In this study, we addressed the role of hypoxia responses in Arabidopsis roots during infection by the clubroot agent Plasmodiophora brassicae.

Results: The hypoxia-related gene markers PYRUVATE DECARBOXYLASE 1 (PDC1), PYRUVATE DECARBOXYLASE 2 (PDC2) and ALCOHOL DEHYDROGENASE 1 (ADH1) were induced during secondary infection by two isolates of P. brassicae, eH and e2. PDC2 was highly induced as soon as 7 days post inoculation (dpi), i.e., before the development of gall symptoms, and GUS staining revealed that ADH1 induction was localised in infected cortical cells of root galls at 21 dpi. Clubroot symptoms were significantly milder in the pdc1 and pdc2 mutants compared with Col-0, but a null T-DNA insertional mutation of ADH1 did not affect clubroot susceptibility. The Arg/N-end rule pathway of ubiquitin-mediated proteolysis controls oxygen sensing in plants. Mutants of components of this pathway, ate1 ate2 and prt6, that both exhibit constitutive hypoxia responses, showed enhanced clubroot symptoms. In contrast, gall development was reduced in quintuple and sextuple mutants where the activity of all oxygen-sensing Group VII Ethylene Response Factor transcription factors (ERFVIIs) is absent (erfVII and prt6 erfVII).

Conclusions: Our data demonstrate that the induction of PDC1 and PDC2 during the secondary infection of roots by P. brassicae contributes positively to clubroot development, and that this is controlled by oxygen-sensing through ERFVIIs. The absence of any major role of ADH1 in symptom development may also suggest that PDC activity could contribute to the formation of galls through the activation of a PDH bypass.

Keywords: ADH1; Arabidopsis; Clubroot; ERFVII; Ethanol fermentation; Hypoxia; N-end rule pathway; PDC2; Plant gall disease; Plasmodiophora.

Fig. 1

qPCR analysis of the induction of fermentation-related genes during clubroot infection PDC1 and PDC2 = PYRUVATE DECARBOXYLASE 1 and 2. ADH1 = ALCOHOL DEHYDROGENASE1. NI = non-inoculated plants. eH & e2 are two isolates of P. brassicae. Data are means of 4 independent biological replicates. dpi = days post inoculation. Quantitative data were normalized with the expression of PP2A (At1G13320). Y axis is on log scale base 2. Bars represent standard errors. Stars indicate statistically significant differences following Student test (P < 0.05)

Fig. 2

Clubroot-induced regulation of the ADH1 promoter visualized through GUS staining. a-c GUS stained upper part of pivotal roots sampled at 21 dpi. a non-inoculated plant b eH isolate c e2 isolate. Rosette leaves were cut after staining for a better visualization of galls. Inserts show the GUS coloration in the lowest part of the root system only in non-inoculated plants. d roots infected with eH (21 pi) cut with a razor blade to show the GUS coloration in root cortical tissues e microscope observation of GUS coloration in a 3 μm slice of infected root (21 dpi). The arrow indicates the vascular structures. f-h GUS stained roots sampled at 7 dpi. f non-inoculated plants g eH isolate, and h GUS induction in non-inoculated plants subjected to a 24-h flooding treatment (positive control). Scale bars indicate 4 mm in a, b and c, 100 μm in e, and 0.5 mm in f-h

Fig. 3

Effect of clubroot infection on the respiration activity in root tissues at 7 dpi. Non-inoculated root samples (NI), and roots from plants inoculated with isolates eH or e2. Data are means of 4 biological repetitions. Bars represent standard errors. No statistical differences were detected between these three experimental conditions

Fig. 4

Clubroot susceptibility in lines defective for fermentation-related genes. a Impact of mutations adh1, pdc1 and pdc2 on the development of clubroot symptoms (eH isolate). b Impact of mutations on the number of spores of P. brassicae per plant at 21 dpi. Data are means of 4 biological repetitions (>12 plant per repetition). Ga/La clubroot disease index is an estimation of the ratio between gall and leaf rosette size from image analysis, as described in Materials and Methods. Bars represent standard errors. Stars represent statistically significant differences between conditions (student T-test, p < 0.05). The number of large spores (>3 μm) was determined using flow cytometry as described in the material & methods section

Fig. 5

Development of clubroot gall symptoms in Arabidopsis lines with constitutively induced or repressed hypoxia response. a Diagrammatical representation of the Arg/N-end rule pathway regulated stability of ERFVII’s and induction of hypoxia-related gene expression. Blue oval, ERFVII substrate proteins showing amino-terminal residues (single letter code); MAP, Met Aminopeptiase; PCO, Plant Cysteine Oxidase; NO, nitric oxide; ATE Arginyl transferase; PRT6, Proteolysis6; C*, oxidised Cysteine. b Clubroot symptom index (GA/LA) at 21 dpi. Hypoxia responses are constitutively induced in mutant lines ate1 ate2 and prt6-1, and constitutively repressed in erfVII and prt6-1 erfVII (erfVII = rap2.12 rap2.2 rap2.3 hre1 hre2). Data are means of 3 independent biological repetitions. For each repetition, clubroot symptom index was evaluated from >10 infected plants. Error bars represent SE. Stars indicate statistically significant difference with Col, from the Student test (P < 0.05) c Illustration of the impact of mutations on clubroot symptoms

Fig. 6

Transcriptional regulation of a set of 23 hypoxia and N-end rule-regulated genes, in Arabidopsis plants infected with gall-inducing pathogens. Data are from available transcriptome datasets from the literature [, , –22] and are expressed here as log2 ratios between inoculated vs non-inoculated conditions for disease responses [, –22], or as indicated for hypoxia and N-end rule driven responses [10]. Stars indicate datasets where only statistically significant regulations are given