Regulation of catalase activity and gene expression during Phytophthora nicotianae development and infection of tobacco

Abstract

Plant defence against pathogen attack typically incorporates an oxidative burst involving elevated levels of reactive oxygen species such as hydrogen peroxide. In the present study, we have used an in-gel assay to monitor the activity of the hydrogen peroxide scavenging enzyme, catalase, during asexual development of Phytophthora nicotianae and during infection of host tobacco plants. In vitro, catalase activity is highest in sporulating hyphae; in planta, catalase activity increases dramatically about 8 h after host inoculation. We have cloned and characterized three catalase genes, designated PnCat1, PnCat2 and PnCat3, from P. nicotianae and identified their homologues in P. infestans, P. sojae and P. ramorum. In all three species, Cat2 is predicted to be targeted to the peroxisome and the other catalases are likely to be cytosolic. Quantitative real-time PCR assessment of catalase transcripts during development and infection indicates that peroxisomal PnCat2 is the gene predominantly expressed, with transcript levels peaking in vitro in sporulating hyphae and in planta increasing dramatically during the first 24 h after inoculation of susceptible tobacco seedlings. Levels of tobacco catalase gene expression are significantly down-regulated in susceptible tobacco 4, 8 and 24 h post-inoculation and in resistant plants at 24 h post-inoculation. Together, our results give evidence that during infection P. nicotianae increases its own peroxisomal catalase levels while concurrently down-regulating host catalase expression. This behaviour is consistent with a role of pathogen catalase in counterdefence and protection against oxidative stress and of pathogen-orchestrated enhanced plant cell death to support necrotrophic pathogen growth and plant colonization.

Figure 1

Catalase activity in P. nicotianae at different developmental stages. (a) A prominent catalase activity band occurs in extracts from hyphae sampled 8 h after induction of sporulation (8S). Activity bands of the same size but of lower intensity also occur in vegetative hyphae (V) and uninfected tobacco seedlings treated with H₂O₂ (T). A lower molecular weight activity band occurs in extracts from 2‐h germinated cysts (C). No catalase activity was detected in the zoospore protein extract (Z). (b) Catalase activity in germinated cysts, 4–24 h after germination. A band of catalase activity the same size as that from vegetative and sporulating hyphae extracts is present in cyst extracts 10–24 h after germination, with the highest level of activity occurring in the 20‐h sample. Two smaller bands of activity are visible in extracts from 16‐, 20‐ and 24‐h germinated cysts. The position of bovine catalase (232 kDa) is shown on the right (arrows).

Figure 2

Southern blot analysis of catalase genes in P. nicotianae gDNA cut with the restriction enzymes BamHI, EcoRI, HindIII, XhoI and PstI. The same blot was probed with PCR fragments from PnCat1, stripped and reprobed with PnCat2 and subsequently reprobed with PnCat3. Numbers on the left indicate the approximate size in kb.

Figure 3

Multiple sequence alignment of P. nicotianae predicted catalase proteins (GenBank accession numbers EU176856, EU176855 and EU258925) showing the degree of amino acid conservation. Amino acids in shaded regions are thought to be important for tetramer formation. The underlined region indicates the catalase proximal active site signature and the boxed region indicates the catalase proximal heme‐ligand signature motif. The PTS1 of the Pncat2 protein is shown in bold type.

Figure 4

Comparison of PnCat1 and PnCat2 expression in different developmental stages and in germinated cysts 2–24 h after encystment and germination. (A) PnCat1 is expressed at very low levels in all stages of development. PnCat2 expression is highest in sporulating hyphae and zoospores. Vegetative hyphae (V), hyphae sampled 8 h after induction of sporulation (8S), sporulating hyphae (S), zoospores (Z), 3‐h germinated cysts (C). Values show averages and standard deviations of transcript levels normalized against WS41 expression using comparative quantification from three biological replicates. (B) PnCat2 expression reaches a peak in cysts allowed to germinate for 4 h. The data are averages (and standard deviations) of values from two biological replicates. A third biological replicate had a similar expression profile but the peak occurred at 6 h.

Figure 5

Light microscopy images of tobacco cotyledons infected with P. nicotianae or mock inoculated with water after 48 h. N. tabacum cv Petit Gerard shows extensive colonization by P. nicotianae on the cotyledon surface (a) and throughout the tissue (c). N. tobacum cv NC2326 cotyledons show restricted hyphal growth on the surface of epidermal cells (b) and no penetration of the underlying mesophyll tissue (d). Hypersensitive cell death was only seen in the resistant tobacco, NC2326 (d). Mock inoculated seedlings of Petit Gerard (e) or NC2326 (f) show no signs of infection or cell death. Arrowheads indicate hyphae. Scale bars = 10 µm.

Figure 6

Catalase activity in susceptible (Petit Gerard) tobacco seedlings infected with P. nicotianae zoospores, 0–10 h after inoculation. Two bands of activity were seen in extracts of seedlings harvested at 8 and 10 h after inoculation. The larger of the activity bands was the same size as that seen for vegetative (V) and hyphae sampled 8 h after induction of sporulation (8S). No catalase activity could be seen in mock inoculated Petit Gerard seedlings over the same time period or in zoospore extracts (Z). The position of the 232 kDa bovine catalase is shown on the left (arrow).

Figure 7

PnCat2 catalase expression from 4‐, 8‐ and 24‐h germinated cysts (GC) grown in vitro and in infected susceptible and resistant tobacco, 4, 8 and 24 h after inoculation. PnCat2 expression increased substantially in the susceptible Petit Gerard (PG) and resistant NC2326 (NC) plants while expression in germinated cysts growing in vitro in nutrient medium declined over the same period. Values show averages of transcript levels normalized against WS41 expression using comparative quantification from three biological replicates. The increase in PnCat2 expression in planta was statistically significantly different from the decrease in expression in vitro at the 95% confidence level.

Figure 8

Comparison of tobacco catalase expression in infected and mock infected susceptible and resistant tobacco, 4, 8 and 24 h after inoculation. There was no consistent difference in levels of expression of tobacco catalase between susceptible Petit Gerard (PG mock) and resistant NC2326 (NC mock) mock‐infected tobacco or infected resistant tobacco (NC inf) at 4 and 8 h post‐inoculation. Tobacco catalase was down‐regulated in infected susceptible tobacco (PG inf) at all time points (statistically significant at the 95% confidence level). Tobacco catalase is also down‐regulated in inoculated resistant seedlings at 24 h (statistically significant at the 95% confidence level). Values show averages of transcript levels normalized against tobacco actin expression using comparative quantification from three biological replicates.