In Silico Evaluation, Phylogenetic Analysis, and Structural Modeling of the Class II Hydrophobin Family from Different Fungal Phytopathogens.

The class II hydrophobin group (HFBII) is an extracellular group of proteins that contain the HFBII domain and eight conserved cysteine residues. These proteins are exclusively secreted by fungi and have multiple functions with a probable role as effectors. In the present study, a total of 45 amino acid sequences of hydrophobin class II proteins from different phytopathogenic fungi were retrieved from the NCBI database. We used the integration of well-designed bioinformatic tools to characterize and predict their physicochemical parameters, novel motifs, 3D structures, multiple sequence alignment (MSA), evolution, and functions as effector proteins through molecular docking. The results revealed new features for these protein members. The ProtParam tool detected the hydrophobicity properties of all proteins except for one hydrophilic protein (KAI3335996.1). Out of 45 proteins, six of them were detected as GPI-anchored proteins by the PredGPI server. Different 3D structure templates with high pTM scores were designed by Multifold v1, AlphaFold2, and trRosetta. Most of the studied proteins were anticipated as apoplastic effectors and matched with the ghyd5 gene of Fusarium graminearum as virulence factors. A protein-protein interaction (PPI) analysis unraveled the molecular function of this group as GTP-binding proteins, while a molecular docking analysis detected a chitin-binding effector role. From the MSA analysis, it was observed that the HFBII sequences shared conserved 2 Pro (P) and 2 Gly (G) amino acids besides the known eight conserved cysteine residues. The evolutionary analysis and phylogenetic tree provided evidence of episodic diversifying selection at the branch level using the aBSREL tool. A detailed in silico analysis of this family and the present findings will provide a better understanding of the HFBII characters and evolutionary relationships, which could be very useful in future studies.

Secretome Analysis for a New Strain of the Blackleg Fungus Plenodomus lingam Reveals Candidate Proteins for Effectors and Virulence Factors.

The fungal secretome is the main interface for interactions between the pathogen and its host. It includes the most important virulence factors and effector proteins. We integrated different bioinformatic approaches and used the newly drafted genome data of P. lingam isolate CAN1 (blackleg of rapeseed fungus) to predict the secretion of 217 proteins, including many cell-wall-degrading enzymes. All secretory proteins were identified; 85 were classified as CAZyme families and 25 were classified as protease families. Moreover, 49 putative effectors were predicted and identified, where 39 of them possessed at least one conserved domain. Some pectin-degrading enzymes were noticeable as a clustering group according to STRING web analysis. The secretome of P. lingam CAN1 was compared to the other two blackleg fungal species (P. lingam JN3 and P. biglobosus CA1) secretomes and their CAZymes and effectors were identified. Orthologue analysis found that P. lingam CAN1 shared 14 CAZy effectors with other related species. The Pathogen-Host Interaction database (PHI base) classified the effector proteins in several categories where most proteins were assigned as reduced virulence and two of them termed as hypervirulence. Nowadays, in silico approaches can solve many ambiguous issues about the mechanism of pathogenicity between fungi and plant host with well-designed bioinformatics tools.

Native Rhizospheric and Endophytic Fungi as Sustainable Sources of Plant Growth Promoting Traits to Improve Wheat Growth under Low Nitrogen Input.

Wheat crops require effective nitrogen fertilization to produce high yields. Only half of chemical N2 fertilizers are absorbed into plants while the rest remains in the soil, causing environmental problems. Fungi could maximize nitrogen absorption, and from an environmental and biodiversity point of view, there is an urgent necessity for bioprospecting native fungi associated with wild plants growing in harsh environments, e.g., St. Katherine Protectorate (SKP) in the arid Sinai. Recovered taxa, either endophytic and/or rhizospheric, were screened for their plant growth-promoting (PGP) traits. Eighteen fungal isolates (15 rhizospheric and 3 endophytic) belonging to anamorphic ascomycetes were recovered from 9 different wild plants, and their PGP traits (indole-3-acetic acid [IAA] production, phosphate solubilization, siderophore production, and hydrolytic enzyme production) were measured. Rhizospheric isolate NGB-WS14 (Chaetosphaeronema achilleae) produced high levels of IAA (119.1 µg mL-1) in the presence of tryptophan, while NGB-WS 8 (Acrophialophora levis) produced high IAA levels (42.4 µg mL-1) in the absence of tryptophan. The highest phosphate-solubilizing activity (181.9 µg mL-1) was recorded by NGB-WFS2 (Penicillium chrysogenum). Endophytic isolate NGB-WFE16 (Fusarium petersiae) exhibited a high percentage level of Siderophore Unit (96.5% SU). All isolates showed variability in the secretion of extracellular hydrolytic enzymes. Remarkably, all isolates had antagonistic activity (55.6% to 87.3% suppression of pathogen growth) against the pathogenic taxon Alternaria alternata (SCUF00001378) in the dual-assay results. Out of the 18 isolates, 4 rhizospheric and 1 endophytic isolate showed significant increases in shoot dry weight and shoot nitrogen and chlorophyll content of wheat plants subjected to low inputs of chemical nitrogen (N) fertilizers (50% reduction) compared with the non-inoculated control in a pot experiment. Potent taxa were subjected to sequencing for molecular confirmation of phenotypic identification. The retrieved ITS sequences in this study have been deposited in GenBank under accession numbers from LC642736 to LC642740. This study considered the first report of endophytic fungi of Cheilanthes vellea, a wild plant with PGPF which improves wheat growth. These results recommend using PGPF as inoculants to alleviate low nitrogen fertilization.