The protein 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).
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. The model fungal species Aspergillus nidulans is used in this project
Effector proteins in soilborne pathogens
Plant pathogens are categorized in biotrophs, necrotrophs and hemibiotrophs, depending on their interaction established with their hosts. Biotrophy defined the growth on living plant cells; hemibiotrophic pathogens establish first a biotrophic stage and then switch to necrotrophic, while necrotrophic pathogens grow on dead plant material. However, many necrotrophs rely on an initial short biotrophic phase in order to establish a successful infection. It is known that pathogens deploy small-secreted proteins termed as effectors to manipulate plant defense responses. These proteins are usually host even lineage- specific. There are also some effectors, which are more conserved, such as the necrosis and ethylene–inducing-like proteins (NLPs) and the LySM effectors, which are present in a broad range of organisms.
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 severe 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 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, X-ray crystallography, MS/MS spectrometry, electron and confocal microscopy, RNA-seq etc.
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2. Dolfors F., Holmquist L., Dixelius C., Tzelepis G. 2019. A LysM effector protein from the basidiomycete Rhizoctonia solani contributes to virulence through suppresion of chitin-triggered immunity. Molecular Genetics and Genomics DOI: 10.1007/s00438-019-01573-9.
3. Tzelepis G. and Karlsson M. 2019. Killer toxin-like chitinases in filamentous fungi: Structure, regulation and potential roles in fungal biology. Fungal Biology Reviews. 33: 123-132.
4. Dölfors F., Moschou N.P, Andersson L., Dixelius C and Tzelepis G. 2018. The Rhizoctonia solani LysM and RlpA-like effector proteins contribute to virulence suppressing chitin-triggered immunity and hypersensitive response. BioRxiv, https://doi.org/10.1101/395582
5. Nygren K., Dubey M., Zapparata, A., Iqbal M., Tzelepis G., Brandström Durling M., Funck Jensen D. and Karlsson M. 2018 The mycoparasitic fungus Clonostachys rosea responds with both common and specific gene expression during interspecific interactions with fungal prey. Evolutionary Applications. 1-19.
6. Fogelqvist J., Tzelepis G., Bejai., Ilbäck J., Schwelm A. and Dixelius C. 2018. Analysis of the hybrid genomes of two field isolates of the soil- borne fungal species Verticillium longisporum. BMC Genomics. 19:14
7. Singh K*., Tzelepis G*., Zouhar M., Ryšánek P. and Dixelius C. 2018. The immunophilin repertoire of Plasmodiophora brassicae and functional analysis of PbCYP3 cyclophilin. Molecular Genetics and Genomics. 293(2):381-390 (Corresponded author)* equal contribution.
8. Tzelepis G., Karlsson M. and Suzuki T. 2017 Deglycosylating enzymes acting on N-glycans in fungi: Insights from genome survey. BBA General Subjects, 1861:2551–2558
9. Tzelepis G., Bejai S., Sattar N., Schwelm A., Ilbäck J., Fogelqvist J. and Dixelius C. 2017. Detection of Verticillium species in Swedish soils by using real-time PCR. Archives of Microbiology. 199(10): 1383-1389.
10. Wibberg D., Andersson L., Tzelepis G., Rupp O. et al. 2016. Genome analysis of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB revealed high numbers in secreted proteins and cell wall degrading enzymes. BMC Genomics, 17:245.
11. Kamou N.N., Dubey M., Tzelepis G., Karlsson M., Lagopodi A. and Jensen D. F. 2016. Investigating the compatibility of the biocontrol agent Clonostachys rosea IK726 with Serratia rubidaea S55 and phenazine-producing Pseudomonas chlororaphis ToZa7. Archives of Microbiology, 198: 369-377.
12. Tzelepis G., Dubey M., Jensen D. F and Karlsson M. 2015. Identifying glycoside hydrolase family 18 genes in the mycoparasitic fungal species Clonostachys rosea. Microbiology-Sgm, 161: 1407-1419.
13. Kamou N.N., Karasali H., Menexes G., Kasiotis K.M., Bon M.C., Papadakis E.N., Tzelepis G., Lotos L., Lagopodi A. 2015. Isolation screening and characterisation of local beneficial rhizobacteria based upon their ability to suppress the growth of Fusarium oxysporum f. sp. radicis-lycopersici and tomato foot and root rot. Biocontrol Science and Technology, 25 (8): 928-949.
14. Karlsson M., Brandström-Durling M., Choi J., Lackner G, Tzelepis G., et al 2015. Insights on the evolution of mycoparasitism from the genome of Clonostachys rosea. Genome Biology and Evolution. 7 (2): 465-480.
15. Tzelepis G., Hosomi A., Hossain J.T., Hirayama H., Dubey M., Jensen D. F., Suzuki T. and Karlsson, M. 2014. Endo-β-N-acetylglucosamidases (ENGases) in the fungus Trichoderma atroviride: Possible involvement of the filamentous fungi-specific cytosolic ENGase in the ERAD process. Biochemical and Biophysical Research Communications 449: 256-261. (Corresponded author).
16. Tzelepis G., Melin P., Jensen D. F, Stenlid J. and Karlsson M. 2014. Functional analysis of the C-II subgroup killer toxin-like chitinases in the filamentous ascomycete Aspergillus nidulans. Fungal Genetics and Biology 64:58-66. (Corresponded author).
17. Strandberg R. Tzelepis G., Johannesson H. and Karlsson M. 2013. Co-existence and expression profiles of two alternative splice variants of the pheromone receptor gene pre-1 in Neurospora crassa. Archives of Microbiology 195:773-780.
18. Tzelepis G., Melin P, Jensen D. F, Stenlid J. and Karlsson M. 2012. Functional analysis of glycoside hydrolase family 18 and 20 genes in Neurospora crassa. Fungal Genetics and Biology. 49: 717-730. (Corresponded author).
19. Tzelepis G, Lagopodi AL. 2011. Interaction between Clonostachys rosea IK726 and Pseudomonas chlororaphis PCL 1391 against tomato foot and root rot caused by Fusarium oxysporium f. sp. radicis lycopersici. IOBC/wprs Bull 63, 75-79
20. Bardas G.A., Tzelepis G., Lotos L., Karaoglanidis G.S (2009). First report of Botrytis cinerea causing gray mold of pomegranate (Punica granatum) in Greece. Plant Disease 93:1346.
21. Bardas G.A., Tzelepis G., Lotos L., Karaoglanidis G.S (2009). First report of Penicillium glabrum causing fruit rot of pomegranate (Punica granatum) in Greece. Plant Disease 93:1347.