Marica Grujić

HFB7 – A novel orphan hydrophobin of the Harzianum and Virens clades of Trichoderma, is involved in response to biotic and abiotic stresses

Hydrophobins are small secreted cysteine-rich proteins exclusively found in fungi. They are able to self-assemble in single molecular layers at hydrophobic-hydrophilic interfaces and can therefore be directly involved in establishment of fungi in their habitat. The genomes of filamentous mycotrophic fungi Trichoderma encode a rich diversity of hydrophobins, which are divided in several groups based on their structure and evolution. Here we describe a new member of class II hydrophobins, HFB7, that has a taxonomically restricted occurrence in Harzianum and Virens clades of Trichoderma. Evolutionary analysis reveals that HFB7 proteins form a separate clade distinct from other Trichoderma class II hydrophobins and that genes encoding them evolve under positive selection pressure. Homology modelling of HFB7 structure in comparison to T. reesei HFB2 reveals that the two large hydrophobic patches on the surface of the protein are remarkably conserved between the two hydrophobins despite significant difference in their primary structures. Expression of hfb7 gene in T. virens increases at interactions with other fungi and a plant and in response to a diversity of abiotic stress conditions, and is also upregulated during formation of aerial mycelium in a standing liquid culture. This upregulation significantly exceeds that of expression of hfb7 under a strong constitutive promoter, and T. virens strains overexpressing hfb7 thus display only changes in traits characterized by low hfb7 expression, i.e. faster growth in submerged liquid culture. The hfb7 gene is not expressed in conidia. Our data allow to conclude that this protein is involved in defence of Trichoderma against a diversity of stress factors related to the oxidative stress. Moreover, HFB7 likely helps in the establishment of the fungus in wetlands or other conditions related to high humidity.

Esterase and peroxidase isoforms in different stages of morphogenesis in Fritillaria meleagris L. in bulb-scale culture

Morphogenesis in vitro is a complex and still poorly defined process. We investigated esterase and peroxidase isoforms detected in bulb scale, during Fritillaria meleagris morphogenesis. Bulbs were grown either at 4 °C or on a medium with an increased concentration of sucrose (4.5%) for 30 days. After these pre-treatments, the bulb scales were further grown on nutrient media that contained different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or thidiazuron (TDZ). Regeneration of somatic embryos and bulblets occurred at the same explant. The highest numbers of somatic embryos and bulblets were regenerated on the medium containing 2,4-D and KIN (1mg/L each), while morphogenesis was most successful at a TDZ concentration between 0.5 and 1mg/L. Monitoring of esterases and peroxidases was performed by growing bulb scales on a medium enriched with 2,4-D and KIN or TDZ (1mg/L), and the number and activity of isoforms were followed every 7 days for 4 weeks. In control explants, six isoforms of esterase were observed. Three isoforms of peroxidase were not detected in the control bulb scale, which has not begun its morphogenesis process.

Massive lateral transfer of genes encoding plant cell wall-degrading enzymes to the mycoparasitic fungus Trichoderma from its plant-associated hosts.

Unlike most other fungi, molds of the genus Trichoderma (Hypocreales, Ascomycota) are aggressive parasites of other fungi and efficient decomposers of plant biomass. Although nutritional shifts are common among hypocrealean fungi, there are no examples of such broad substrate versatility as that observed in Trichoderma. A phylogenomic analysis of 23 hypocrealean fungi (including nine Trichoderma spp. and the related Escovopsis weberi) revealed that the genus Trichoderma has evolved from an ancestor with limited cellulolytic capability that fed on either fungi or arthropods. The evolutionary analysis of Trichoderma genes encoding plant cell wall-degrading carbohydrate-active enzymes and auxiliary proteins (pcwdCAZome, 122 gene families) based on a gene tree / species tree reconciliation demonstrated that the formation of the genus was accompanied by an unprecedented extent of lateral gene transfer (LGT). Nearly one-half of the genes in Trichoderma pcwdCAZome (41%) were obtained via LGT from plant-associated filamentous fungi belonging to different classes of Ascomycota, while no LGT was observed from other potential donors. In addition to the ability to feed on unrelated fungi (such as Basidiomycota), we also showed that Trichoderma is capable of endoparasitism on a broad range of Ascomycota, including extant LGT donors. This phenomenon was not observed in E. weberi and rarely in other mycoparasitic hypocrealean fungi. Thus, our study suggests that LGT is linked to the ability of Trichoderma to parasitize taxonomically related fungi (up to adelphoparasitism in strict sense). This may have allowed primarily mycotrophic Trichoderma fungi to evolve into decomposers of plant biomass.

Enhancement of amylase production by Aspergillus sp. using carbohydrates mixtures from triticale

With the purpose of finding a suitable available inducer in com- bination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced the production of an amylase cocktail (α-amylase and glucoamylase) in Aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and TLC analysis of the carbohydrate mixtures before and after fermentation. Glucoamylase production by A. niger was the highest in the presence of the extract obtained after auto- hydrolysis of starch from triticale (95.88 U mL -1 ). Carbohydrate mixtures from triticale induced the production of α-amylase in A. oryzae. More α-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. A 48-h induction was the most efficient using a triticale extract (101.35 U mL -1 ). Carbohydrates from triticale extracts could be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by Aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation.

Enhancement of amylase production using carbohydrates mixtures from triticale in Aspergillus sp

For the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale was used as inducers and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced production of amylase cocktail (α-amylase and glucoamylase) in Aspergillus niger , unlike induction with well-known inducers which induce only glucoamylase, showed by zymogram and TLC analysis of carbohydrates mixtures before and after fermentations. Glucoamylase production by A. niger was highest in the presence of extract obtained after autohydrolysis of starch from triticale (95.88 U/mL). Carbohydrate mixtures from triticale induced production of α-amylase in A. oryzae. More α-amylase isoforms were detected upon using complex carbohydrate mixture, compared to induction with maltose or starch. The 48 h induction was the most efficient by using triticale extract (101.35 U/mL). Carbohydrates from triticale extracts can be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as the inducer in amylase production by Aspergillus sp, such a way it could be used as sole substrate in fermentation.

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4‐methylumbelliferyl‐β‐d‐cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates.

Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base

The aim of this study was to determine the enzymatic profile of esterases and peroxidases during early stages of somatic embryogenesis of Fritillaria meleagris L. Somatic embryogenesis was induced using the leaf base as explant on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Zymography showed the presence of different moieties, six isoforms of esterases and peroxidases, during morphogenesis as compared to control explants. One isoform of esterases was detected only during the process of somatic embryogenesis, and one isoform was detected in control explants. Analysis of esterases with 1-naphthyl butyrate proved that esterases, which participate in somatic embryogenesis of F. meleagris , belong to the family of aryl esterases. For the first time it was proved that five isoforms of esterases, which are involved in morphogenesis of F. meleagris , belong to the family of aryl esterases, while two isoforms are carboxyl esterases. One isoform of carboxyl esterases was visible in control explants. This is also the first description of peroxidases during the morphogenetic process, and of the difference between aryl and carboxyl esterases. More isoforms of esterases during morphogenesis as compared to control explants are probably responsible for some early physiological process during somatic embryogenesis of F. meleagris . https://doi.org/10.2298/ABS161130007P Received: November 30, 2016; Revised: January 10, 2017; Accepted: March 3, 2017; Published online: March 10, 2017 How to cite this article: Petric M, Subotic A, Jevremovic S, Trifunovic-Momcilov M, Tadic V, Grujic M, Vujcic Z. Esterase and peroxidase isoforms during initial stages of somatic embryogenesis in Fritillaria meleagris L. leaf base. Arch Biol Sci. 2017;69(4):619-25.