Paranoid potato: phytophthora-resistant genotype shows constitutively activated defense

Abstract

Phytophthora is the most devastating pathogen of dicot plants. There is a need for resistance sources with different modes of action to counteract the fast evolution of this pathogen. In order to better understand mechanisms of defense against P. infestans, we analyzed several clones of potato. Two of the genotypes tested, Sarpo Mira and SW93-1015, exhibited strong resistance against P. infestans in field trials, whole plant assays and detached leaf assays. The resistant genotypes developed different sizes of hypersensitive response (HR)-related lesions. HR lesions in SW93-1015 were restricted to very small areas, whereas those in Sarpo Mira were similar to those in Solanum demissum, the main source of classical resistance genes. SW93-1015 can be characterized as a cpr (constitutive expressor of PR genes) genotype without spontaneous microscopic or macroscopic HR lesions. This is indicated by constitutive hydrogen peroxide (H₂O₂) production and PR1 (pathogenesis-related protein 1) secretion. SW93-1015 is one of the first plants identified as having classical protein-based induced defense expressed constitutively without any obvious metabolic costs or spontaneous cell death lesions.

Figure 1. Whole plant assay in the greenhouse performed on Desiree, SW93-1015 and Sarpo Mira after spraying with P. infestans sporangia (15,000/ml). (A) Graph showing scoring of P. infestans infection development, represented as percentage of plant area infected based on the Malcolmson 1–9 scale. Readings were taken after 1, 3, 5, 7 and 9 dpi in three independent experiments where the respective plant had the same score at a given time point in all three experiments. (B) Picture taken at 9 dpi after P. infestans treatment of Sarpo Mira, Desiree and SW93-1015 (from left to right). (C) Graph showing infection lesion diameter (mm) in SW93-1015, Sarpo Mira and S. demissum at 1, 3, 5, 7 and 9 dpi.

Figure 2. Detached leaf assay showing infection lesion or resistance reaction HR against P. infestans. (A) Desiree showing necrosis after infection, while S. demissum, Sarpo Mira and SW93-1015 displayed HR at 9 dpi. (B) Graph showing number of lesions formed per leaf on Sarpo Mira and SW93-1015 at 3 and 9 dpi. Error bars indicate standard deviation of the mean. (C) Graph showing mean size of lesions on Sarpo Mira and SW93-1015 at 3 and 9 dpi. Error bars indicate standard deviation of the mean.

Figure 3. Microscopic analysis of hypersensitive response (HR). Trypan blue staining performed on Desiree, SW93-1015 and Sarpo Mira. (A) Trypan blue staining did not show any HR in uninfected leaves of the three genotypes. (B) Phytophthora zoospores at 6 hpi in Desiree (left), SW93-1015 (middle) and Sarpo Mira (right). (C) Phytophthora zoospore germination in Desiree (left), scattered HR in SW93-1015 (middle) and Sarpo Mira (right) indicated by blue coloration at 24 hpi. (D) Intense hyphal growth in Desiree (left), scattered HR in SW93-1015 (middle) and increased cell death in Sarpo Mira (right) at 3 dpi. Size bars represent 100 µm.

Figure 4. 3,3-Diaminobenzidine (DAB) staining performed on Desiree, Sarpo Mira and SW93-1015 to detect H₂O₂ produced in detached leaves after P. infestans inoculation. H₂O₂ detected in (A) Desiree, (B) Sarpo Mira and (C) SW93-1015 on uninfected leaves at 6 hpi, 24 hpi and 72 hpi (from left to right). Size bar represents 15 mm.

Figure 5. (A) SDS-PAGE showing apoplast proteins from untreated Desiree, SW93-1015 and Sarpo Mira. (B) Venn diagram showing number of proteins identified by mass spectrometry in the three genotypes, also showing number of proteins overlapping and unique for the genotypes.