Cytoplasmic- and extracellular-proteome analysis of Diplodia seriata: a phytopathogenic fungus involved in grapevine decline

Abstract

BackgroundThe phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome.ResultsIntracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism.ConclusionsThis study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity process. In fact, several of the identified proteins have been reported as pathogenicity factors in other phytopathogenic fungi. Moreover, this proteomic analysis supposes a useful basis for deepening into D. seriata knowledge and will contribute to the development of the molecular biology of this fungal strain as it has been demonstrated by cloning the gene Prx1 encoding mitochondrial peroxiredoxin-1 of D. seriata (the first gene to be cloned in this microorganism; data not shown).

Toxicity of extracellular proteins from Diplodia seriata and Neofusicoccum parvum involved in grapevine Botryosphaeria dieback

“…Moreover, Luini et al (2010) hypothesised that the secreted proteins of Pch and Pal, associated with esca disease, modify plant cell (V. vinifera 41 B cells) metabolism and may act as virulence factors. A report on the extracellular proteome analysis of D. seriata has shown 75 secreted proteins between 12 and 84 kDa (Cobos et al 2010). Only 16 proteins have been identified, including 3 necrosis-and ethylene-inducing proteins (Nep) at 26.0, 33.8 and 35.7 kDa (Cobos et al 2010).…”

“…Some of these proteins have been identified as virulence factors in Nectria haematococca and Sclerotinia sclerotiorum but their contribution to pathogenicity was not precisely analysed (Cobos et al 2010). Ramirez-Suero et al (2014) demonstrated that the total extracellular compounds produced by N. parvum were more aggressive on calli than those of D. seriata and produced higher amounts of mellein.…”

Profiling the Secretome and Extracellular Proteome of the Potato Late Blight Pathogen Phytophthora infestans

“…This approach has frequently been applied in fungi to elucidate the proteome and secretome under various conditions including plant-pathogen interactions (27,28). Using this technology, however, only limited information has been gained in Phytophthora.…”

Proteomics Methodology Applied to the Analysis of Filamentous Fungi - New Trends for an Impressive Diverse Group of Organisms

“…In spite of this fact, several fungi which are producers of severe infections in fruit trees (fruit blight) and grapevines, like Diplodia seriata, keep fully unsequenced. Despite this limitation, 2D electrophoresis application combined with de novo sequencing and BLAST similarity search by a 4800 MALDI-TOF/TOF Analyzer (Applied Biosystems) led to the protein identification of virulence factors described in other fungal strains when they grew in medium supplemented with carboxymethyl cellulose (Cobos et al, 2010). This fact shows the wide range of Proteomics applications.…”

“…Thus, the use of liquid nitrogen to decrease warming of the disruption systems is a widely used method. The main cell-breaking system is the traditional pre-chilled mortar grinding due to its efficiency against the fungal cell wall (Cobos et al, 2010;Lu et al, 2010;Vödisch et al, 2011), although waring blender machines (Lim et al, 2001), or glass bead beating systems, either combined with a 10 mM Tris-HCl buffer (Oh et al, 2010) or with a phenol buffer (Coumans et al, 2010;Vodisch et al, 2011), have been successfully applied. After the breaking step, the protein solubilisation buffer always includes a protease inhibitor [e.g.…”