Diversity and Function of Appressoria

Endophytic, saprobic, and pathogenic fungi have evolved elaborate strategies to obtain nutrients from plants. Among the diverse plant-fungi interactions, the most crucial event is the attachment and penetration of the plant surface. Appressoria, specialized infection structures, have been evolved to facilitate this purpose. In this review, we describe the diversity of these appressoria and classify them into two main groups: single-celled appressoria (proto-appressoria, hyaline appressoria, melanized (dark) appressoria) and compound appressoria. The ultrastructure of appressoria, their initiation, their formation, and their function in fungi are discussed. We reviewed the molecular mechanisms regulating the formation and function of appressoria, their strategies to evade host defenses, and the related genomics and transcriptomics. The current review provides a foundation for comprehensive studies regarding their evolution and diversity in different fungal groups.

AaPKAc Regulates Differentiation of Infection Structures Induced by Physicochemical Signals From Pear Fruit Cuticular Wax, Secondary Metabolism, and Pathogenicity of Alternaria alternata

Alternaria alternata, the casual agent of black rot of pear fruit, can sense and respond to the physicochemical cues from the host surface and form infection structures during infection. To evaluate the role of cyclic AMP-dependent protein kinase (cAMP-PKA) signaling in surface sensing of A. alternata, we isolated and functionally characterized the cyclic adenosine monophosphate-dependent protein kinase A catalytic subunit gene (AaPKAc). Gene expression results showed that AaPKAc was strongly expressed during the early stages of appressorium formation on hydrophobic surfaces. Knockout mutants ΔAaPKAc were generated by replacing the target genes via homologous recombination events. We found that intracellular cAMP content increased but PKA content decreased in ΔAaPKAc mutant strain. Appressorium formation and infection hyphae were reduced in the ΔAaPKAc mutant strain, and the ability of the ΔAaPKAc mutant strain to recognize and respond to high hydrophobicity surfaces and different surface waxes was lower than in the wild type (WT) strain. In comparison with the WT strain, the appressorium formation rate of the ΔAaPKAc mutant strain on high hydrophobicity and fruit wax extract surface was reduced by 31.6 and 49.3% 4 h after incubation, respectively. In addition, AaPKAc is required for the hypha growth, biomass, pathogenicity, and toxin production of A. alternata. However, AaPKAc negatively regulated conidia formation, melanin production, and osmotic stress resistance. Collectively, AaPKAc is required for pre-penetration, developmental, physiological, and pathological processes in A. alternata.

The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

The Ustilago hordei-barley interaction is a versatile system to characterize fungal effectors

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence/pathogenicity. In this study, we present the U. hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We have generated U. hordei solopathogenic strains, which form infectious filaments without presence of compatible mating partner. Solopathogenic strains are suitable as heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization were analyzed by transmission electron microscopy, which shows that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

Covering soybean leaves with cellulose nanofiber changes leaf surface hydrophobicity and confers resistance against Phakopsora pachyrhizi

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi, an obligate biotrophic fungal pathogen, is the most devastating soybean production disease worldwide. Currently, timely fungicide application is the only means to control ASR in the field. We investigated cellulose nanofiber (CNF) application on ASR disease management. CNF-treated leaves showed reduced lesion number after P. pachyrhizi inoculation compared to control leaves, indicating that covering soybean leaves with CNF confers P. pachyrhizi resistance. We also demonstrated that formation of P. pachyrhizi pre-infection structures including germ-tubes and appressoria, and also gene expression related to these formations, such as chitin synthases (CHSs), were significantly suppressed in CNF-treated soybean leaves compared to control leaves. Moreover, contact angle measurement revealed that CNF converts soybean leaf surface properties from hydrophobic to hydrophilic. These results suggest that CNF can change soybean leaf surface hydrophobicity, conferring resistance against P. pachyrhizi, based on the reduced expression of CHSs, as well as reduced formation of pre-infection structures. This is the first study to investigate CNF application to control field disease.

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of ChitosanIn VitroandIn Situ

ABSTRACTChitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed inEscherichia coliwas shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation.In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability forin situchitosan staining.