The N-glycoprotein quality control in filamentous fungal species
Eukaryotic cells produce a huge amount of proteins; the majority of them are glycoproteins, which are proteins contain a sugar chain (N-glycan). N-glycans play a significant role in protein function and stability, protecting them from proteolysis, facilitating the protein folding and secretion, affecting the protein solubility and the protein localization in the cells. It is also known that N-glycoproteins are structural componets of plasma membranes and fungal cell wall. N-glycoproteins are synthezised in the endoplasmic reticulum (ER) and Golgi apparatus and their glycan structures are processed as they proceed through the secretory pathway. Glycoprotein folding is one of the most important post-translational modifications in eukaryotic cells. When proteins consistently fail to be folded properly are trimmed by the ER-degradation enhancing α-mannosidase proteins (EDEMs) and are retrotranslocated to cytosol for further degradation by the ERAD pathway.
When misfolded glycoproteins are exported to cytosol the glycan chain must be cleaved from the protein in order to be degraded efficiently by the proteasome (10). This removal is conducted by the action of the ubiquitous, cytoplasmic peptide:N-glycanase (PNGase), releasing free N-glycans to the cytosol (Figure 1).
Figure 1. The deglycosylation activity of misfolded glycoproteins in eukaryotic cells.
This enzyme cleaves the amide bond in the side chain of glycosylated–asparagine residue generating free N-glycans with a chitobiose structure at the reducing terminus (GN2). Enzymatically active PNGases have been reported in a wide range of eukaryotic cells from mammals to yeasts. But the situation seems to be different in filamentous fungi, since data claim that the enzymatic function of the cytosolic neutral PNGases has been abolished. However, an acidic PNGase has been identified in many filamentous species but its function is completely unknown. When free N-glycans are created in cytosol they are further catabolized in order to be utilized possibly as a sugar source by cells. In cytosol two enzymes are responsible for free N-glycans degradation: the endo-β-N-acetyloglucosaminidases (ENGases) and the α-mannosidases. The role of ENGases in filamentous fungi seems to be crucial, since deletion of the cytosolic ones has a severe impact in fungal phenotype affecting among others the hyphal growth, conidiation, tolerance to abiotic stress, secretion etc. Despite the fact that ENGases have an important contribution in fungal biology many aspects remains unknown.
We study the mechanisms that filamentous fungi deploy in order to cope with ER stress caused by accumulation of misfolded glycoproteins. Our previous results showed that these organisms possibly utilize different mechanisms as compared to mammalian cells and to yeasts.
Effector proteins in soilborne pathogens
Plant pathogens are categorized in biotrophs, necrotrophs and hemibiotrophs, depending on their interaction established with their hosts. They deploy small-secreted proteins termed as effectors to manipulate plant defense responses. These proteins are usually host even lineage- specific.
Verticillium longisporum is a soil borne pathogen, infecting plants in Brassicaceae family, such as oilseed rape (Brassica napus), cauliflower (Brassica oleracea), turnip (Brassica rapa subsp rapa) etc (Figure 2). According to the data available by now, V. longisporum is assumed to be a hemibiotrophic pathogen.
Figure 2. Symptoms on Brassica napus plants infected by Verticillium longisporum (left) compared to healthy plants (right).
This pathogen is responsible for important yield losses worldwide. Any attempt to control this disease has a limited success since this pathogen forms special resting structures, able to survive in soil for a long time and under harsh environmental conditions. Furthermore, no resistant to V. longisporum cultivars have been developed by now.Our previous data show that V. longisporum contains a certain number of genes encoding putative effector proteins, but nothing is known about their contribution in virulence and pathogenicity. The main questions arisen in this project are:
- How the candidate effector proteins are regulated?
- What is their functional role in this pathosystem?
Rhizoctonia solani is also a soilborne Basidiomycete causing dumping-off disease in seedlings (Figure 3). It is assumed as a nectroph.
Figure 3. Infected sugar beet seedlings with Rhizoctonia solani (right) compared to healthy plants (left).
It forms microsclerotia, which are able to survive to the soil for long periods and under unfavourable environmental conditions. This pathogen is considered to have a necrotrophic lifestyle and only few effectors, promoting necrosis, have been identified. We focus on effectors that do not cause necrosis, and are induced on early-infection stages, studying their roles and their structure. A LysM effector recently has been characterized in this pathogen and it works as a chitin-binding protein, suppressing chitin-triggered immunity, similar to filamentous hemibiotrophic Ascomycetes
Different techniques are used, such as construction of deletion strains, heterologous expression of proteins, MS/MS spectrometry, confocal microscopy, genomic and transcription analyses etc.
