The oomycete Phytophthora infestans is the most harmful pathogen of potato. It causes the disease late blight, which generates increased yearly costs of up to one billion euro in the EU alone and is difficult to control. We have performed a large-scale quantitative proteomics study of six P. infestans life stages with the aim to identify proteins that change in abundance during development, with a focus on preinfectious life stages. Over 10 000 peptides from 2061 proteins were analyzed. We identified several abundance profiles of proteins that were up- or downregulated in different combinations of life stages. One of these profiles contained 59 proteins that were more abundant in germinated cysts and appressoria. A large majority of these proteins were not previously recognized as being appressorial proteins or involved in the infection process. Among those are proteins with putative roles in transport, amino acid metabolism, pathogenicity (including one RXLR effector) and cell wall structure modification. We analyzed the expression of the genes encoding nine of these proteins using RT-qPCR and found an increase in transcript levels during disease progression, in agreement with the hypothesis that these proteins are important in early infection. Among the nine proteins was a group involved in cell wall structure modification and adhesion, including three closely related, uncharacterized proteins encoded by PITG_01131, PITG_01132, and PITG_16135, here denoted Piacwp1–3. Transient silencing of these genes resulted in reduced severity of infection, indicating that these proteins are important for pathogenicity. Our results contribute to further insight into P. infestans biology, and indicate processes that might be relevant for the pathogen while preparing for host cell penetration and during infection. The mass spectrometry data have been deposited to ProteomeXchange via the PRIDE partner repository with the data set identifier PXD002446.

Potato early blight is caused by the necrotrophic fungus Alternaria solani and can result in yield losses of up to 50% if left uncontrolled. At present, the disease is controlled by chemical fungicides, yet rapid development of fungicide resistance renders current control strategies unsustainable. On top of that, a lack of understanding of potato defences and the quantitative nature of resistance mechanisms against early blight hinders the development of more sustainable control methods. Necrotrophic pathogens, compared to biotrophs, pose an extra challenge to the plant, since common defence strategies to biotic stresses such as the hypersensitive response and programmed cell death are often beneficial for necrotrophs. With the aim of unravelling plant responses to both the early infection stages (i.e., before necrosis), such as appressorium formation and penetration, as well as to later responses to the onset of necrosis, we present here a transcriptome analysis of potato interactions with A. solani from 1 h after inoculation when the conidia have just commenced germination, to 48 h post inoculation when multiple cell necrosis has begun. Potato transcripts with putative functions related to biotic stress tolerance and defence against pathogens were upregulated, including a putative Nudix hydrolase that may play a role in defence against oxidative stress. A. solani transcripts encoding putative pathogenicity factors, such as cell wall degrading enzymes and metabolic processes that may be important for infection. We therefore identified the differential expression of several potato and A. solani transcripts that present a group of valuable candidates for further studies into their roles in immunity or disease development.

Summary: Transglutaminases (TGases) are enzymes highly conserved among prokaryotic and eukaryotic organisms, where their role is to catalyse protein cross-linking. One of the putative TGases of Phytophthora infestans has previously been shown to be localised to the cell wall. Based on sequence similarity we were able to identify six more genes annotated as putative TGases and show that these seven genes group together in phylogenetic analysis. All of the seven proteins are predicted to contain transmembrane helices and both a TGase domain and a MANSC domain, the latter of which was previously shown to play a role in protein stability. Chemical inhibition of transglutaminase activity and silencing of the entire family of the putative cell wall TGases are both lethal to P. infestans indicating the importance of these proteins in cell wall formation and stability. The intermediate phenotype obtained with lower drug concentrations and less efficient silencing displays a number of deformations to germ tubes and appressoria. Both chemically treated and silenced lines show lower pathogenicity than the wild type in leaf infection assays. Finally, we show that appressoria of P. infestans possess the ability to build up turgor pressure and that this ability is decreased by chemical inhibition of TGases.

Pathogenic isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot of tomato, secrete cell death in tomato 1 (CDiT1), a homodimeric protein of 35 kDa inducing cell death after infiltration into the leaf apoplast of tomato. CDiT1 was purified by fast protein liquid chromatography, characterized by mass spectrometry and cDNA cloning. Its activity was confirmed after infiltration of an affinity-purified recombinant fusion of the protein with a C-terminal polyhistidine tag. CDiT1 is highly expressed during tomato root infection compared with axenic culture, and has a putative ortholog in other pathogenic Pleosporales species producing proteinaceous toxins that contribute to virulence. Infiltration of CDiT1 into leaves of other plants susceptible to P. lycopersici revealed that the protein affects them differentially. All varieties of cultivated tomato (Solanum lycopersicum) tested were more sensitive to CDiT1 than those of currant tomato (S. pimpinellifolium). Root infection assays showed that varieties of currant tomato are also significantly less prone to intracellular colonization of their root cells by hyphae of P. lycopersici than varieties of cultivated tomato. Therefore, secretion of this novel type of inducer of cell death during penetration of the fungus inside root cells might favor infection of host species that are highly sensitive to this molecule.

Transglutaminases (TGases) are enzymes highly conserved among prokaryotic and eukaryotic organisms, where their role is to catalyze protein cross-linking. One of the putative TGases of Phytophthora infestans has previously been shown to be localized to the cell wall. Based on sequence similarity, we were able to identify six more genes annotated as putative TGases and show that these seven genes group together in phylogenetic analysis. These seven proteins are predicted to contain both a TGase domain and a MANSC domain, the latter of which was previously shown to play a role in protein stability. Chemical inhibition of TGase activity and silencing of the entire family of the putative cell wall TGases are both lethal to P. infestans, indicating the importance of these proteins in cell wall formation and stability. The intermediate phenotype obtained with lower drug concentrations and less efficient silencing displays a number of deformations to germ tubes and appressoria. Both chemically treated and silenced lines show lower pathogenicity than the wild type in leaf infection assays. Finally, we show that appressoria of P. infestans possess the ability to build up turgor pressure and that this ability is decreased by chemical inhibition of TGases. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .