Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis

Abstract

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.

Figure 1

Expression of different nitrilase genes during clubroot development in Arabidopsis. Control (⋄) and infected (♦) roots of transgenic Arabidopsis plants harboring the uidA gene under the control of NIT1-4 promoters were analyzed at different time points (14–35 dpi) for GUS activity. Labeling of the y-axes (GUS activity) is meant for all panels except RNA gel blot. Values are means ± se from different plants of the same experiment. Total GUS activity was calculated by addition of the activities obtained for NIT1-4::uidA plants. At 32 dpi a RNA gel-blot analysis was conducted with control roots (C), separately harvested root (R), and hypocotyl (H) clubs, as well as roots from infested soil showing no symptoms (NS). Nitrilase was detected with a NIT1-cDNA probe. Equal sample loading was confirmed by hybridizing the same blot with an 18S-rRNA probe. MU, Methylumbelliferone.

Figure 2

Immunolocalization of nitrilase in longitudinal sections of infected roots of Arabidopsis using a specific antibody against NIT1. Secondary plasmodia (sP), immature spores (imS), and mature (mS) resting spores of P. brassicae as well as nuclei (N) and starch granules (G) of the cortical host plant cells are labeled. Bars indicate 20 μm. A and B, Light microscopic pictures of large secondary plasmodia (23 dpi) stained with toluidine blue and basic fuchsin (A) and plasmodia and resting spores (29 dpi) stained with ethidium bromide and DAPI using epifluorescence optics specific for DAPI (B). Using different staining techniques, different parts of the host cell and the pathogen can be distinguished. In A the plasmodia and the enlarged host cells are clearly visible using toluidine blue and basic fuchsin staining, while in B the multinucleate plasmodia and the nuclei of the resting spores are stained with DAPI and ethidium bromide. Immunosignals of nitrilase in infected cells at 21 dpi (F) and 35 dpi (G) using FITC-specific epifluorescence optics, and the corresponding controls only using the secondary antibody for staining at 21 dpi (C) and 35 dpi (E). Immunolocalization of nitrilase in non-infected roots of a nitrilase-overproducing line (35SNIT2) 28 d after germination is also shown (D).

Figure 3

Immunolocalization of GUS in longitudinal sections of infected roots of Arabidopsis using a monospecific antibody against GUS. Secondary plasmodia (sP) and mature resting spores (mS) of P. brassicae as well as nuclei (N) of the cortical plant host cells are marked. GUS signals in NIT1::uidA lines 21 dpi (A) and 29 dpi (B) and in infected roots of a GUS-overproducing line (35S::uidA) 28 dpi (C). Bars indicate 10 μm.

Figure 4

Detection of nitrilase from genomic DNA of Arabidopsis and P. brassicae by DNA gel-blot analysis. Lanes A and B show the digested genomic DNA of Arabidopsis and P. brassicae, respectively, prior to blotting after separation on a 0.8% (w/v) agarose gel. From left to right: BamHI, EcoRI, and HindIII digest. Southern blot from genomic DNA of Arabidopsis (lane C) and P. brassicae (lane D) digested with EcoRI was performed with a NIT1-cDNA probe. No signals are visible in the lane with P. brassicae DNA.