Search

Heterologous expression of the putative 1,3,6,8-tetrahydroxynaphthalene synthase gene ChPKS from Colletotrichum higginsianum in Aspergillus nidulans led to the formation of at least eight new compounds. LC-MS analysis proved them as coupling products of 1,3,6,8-tetrahydroxynaphthalene with an intermediate of the cichorine biosynthetic pathway. Comprehensive NMR analysis confirmed the structures of the two predominant products higginidulans A and B. Deletion of the backbone gene of the cichorine pathway in host strain Aspergillus nidulans abolished the formation of higginidulans. Heterologous expression of ChPKS in the alternative Penicillium crustosum expression host resulted in the formation of the expected product 1,3,6,8-tetrahydroxynaphthalene, which was confirmed by acetylation and structural elucidation. This study provides an additional example of unexpected natural product formation by crosstalk of biosynthetic pathways derived from different species. Moreover, it highlights the importance of using alternative host systems for gene expression., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Small GTPase of the Rab family functions as molecular switch in vesicle trafficking, regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In our ongoing efforts to study the pathogenesis of Colletotrichum fructicola, the causal agent of anthracnose in edible-oil plant Camellia oleifera, we identified CfRab6 as the Rab GTPase and characterized its roles in C. fructicola. Consistent with our hypothesis, targeted gene deletion revealed that the ΔCfrab6 mutant displays defects in vesicle trafficking, including endocytosis and autophagy. These combined effects led to the impairments in growth, conidia, and pathogenicity. Moreover, we demonstrated the critical importance of the GDP/GTP motifs are crucial for the normal function of CfRab6. Additionally, our findings demonstrated that CfRic1 and CfRgp1 act as conserved GEFs for CfRab6, supported by their interactions with CfRab6 and the partial restoration of the active GTP-bound CfRab6, which alleviated phenotypic defects in the ΔCfric1 and ΔCfrgp1 mutants. In conclusion, our study sheds new light on the significance of CfRab6-mediated vesicle trafficking in the physiology and pathogenicity of C. fructicola, which might offer new potential targets for the management of anthracnose disease., Competing Interests: Declaration of competing interest The authors have declared that no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Efficient degradation of lignocellulosic biomass is key for the production of value-added products, contributing to sustainable and renewable solutions. This study employs a two-step approach to evaluate lignocellulolytic enzymes of Ceratocystis paradoxa, Colletotrichum falcatum, and Sporisorium scitamineum. First, an in silico genomic analysis was conducted to predict the potential enzyme groups produced by these fungi. Second, a 2³ full factorial design of solid-state cultivation was employed to investigate the cultivation conditions that optimize enzyme activity. In silico analysis of phytopathogen genomes identified proteins with the potential for biomass degradation. Cellulase and phenoloxidase activities were assessed in culture medium and solid-state cultivation. A 2³ full factorial design was employed for solid-state cultivation to evaluate the cellulose, endoglucanase, and laccase activities. In silico analysis shows that C. falcatum has the most diverse enzyme set for lignocellulosic biomass degradation. In vitro assays corroborate this, demonstrating that C. falcatum produces the highest enzyme quantities, except for cellulase, where C. paradoxa outperforms it. Both C. paradoxa and C. falcatum exhibit cellulase and phenoloxidase activities, but only C. falcatum shows laccase activity. Most favorable enzyme production in solid-state cultivation occurred with 85-95 g 100 g - 1 bagasse moisture and 5 g 100 g - 1 yeast extract, with four-day cultivation period needed for cellulase and endoglucanase in C. paradoxa and 12 days for endoglucanase and laccase in C. falcatum. The in silico and in vitro assays demonstrated that C. falcatum can produce a diverse enzyme set, including laccase, cellulase, and endoglucanase, making it a promising candidate for enzymatic industrial applications., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Conflict of interest: No potential conflict of interest was reported by the authors., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Phytopathogens secrete numerous molecules into the environment to establish a microbial niche and facilitate host infection. The phytopathogenic fungus Colletotrichum fructicola, which causes pear anthracnose, can colonize different plant tissues like leaves and fruits, which are occupied by a diversity of microbes. We speculate that this fungus produces antimicrobial effectors to outcompete host-associated competitive microorganisms. Herein, we identified two secreted ribonucleases, CfRibo1 and CfRibo2, from the C. fructicola secretome. The two ribonucleases both possess ribonuclease activity and showed cytotoxicity in Nicotianan benthamiana without triggering immunity in an enzymatic activity-dependent manner. CfRibo1 and CfRibo2 recombinant proteins exhibited toxicity against Escherichia coli , Saccharomyces cerevisiae , and, importantly, the phyllosphere microorganisms isolated from the pear host. Among these isolated microbial strains, Bacillus altitudinis is a pathogenic bacterium causing pear soft rot. Strikingly, CfRibo1 and CfRibo2 were found to directly antagonize B. altitudinis to facilitate C. fructicola infection. More importantly, CfRibo1 and CfRibo2 functioned as essential virulence factors of C. fructicola in the presence of host-associated microorganisms. Further analysis revealed these two ribonucleases are widely distributed in fungi and are undergoing purifying selection. Our results provide the first evidence of antimicrobial effectors in Colletotrichum fungi and extend the functional diversity of fungal ribonucleases in plant-pest-environment interactions., Importance: Colletotrichum fructicola is emerging as a devastating pathogenic fungus causing anthracnose in various crops in agriculture, and understanding how this fungus establishes successful infection is of great significance for anthracnose disease management. Fungi are known to produce secreted effectors as weapons to promote virulence. Considerable progress has been made in elucidating how effectors manipulate plant immunity; however, their importance in modulating environmental microbes is frequently neglected. The present study identified two secreted ribonucleases, CfRibo1 and CfRibo2, as antimicrobial effectors of C. fructicola . These two proteins both possess toxicity to pear phyllosphere microorganisms, and they efficiently antagonize competitive microbes to facilitate the infection of pear hosts. This study represents the first evidence of antimicrobial effectors in Colletotrichum fungi, and we consider that CfRibo1 and CfRibo2 could be targeted for anthracnose disease management in diverse crops in the future., Competing Interests: The authors declare no conflict of interest.

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides., Competing Interests: Declarations of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Multiunit-flavoenzyme NADPH oxidases (NOXs) play multiple roles in living cells via regulating signaling pathways. In several phytopathogenic fungi, NOXs are required for the polarized growth of hyphal tips and pathogenicity to host plants, but the possible mechanisms are still elusive. In our previous study, CgNOXA, CgNOXB, and CgNOXR were identified as components of the NOX complex in Colletotrichum gloeosporioides . The growth and the inoculation assays revealed that CgNOXA/B and CgNOXR regulate vegetative growth and are required for the full pathogenicity of C. gloeosporioides to Hevea leaves. We further demonstrated that the vital roles of CgNOXB and CgNOXR in appressorium formation and the development of invasion hyphae account for their functions in pathogenicity. Moreover, CgNOXB and CgNOXR regulate the production and distribution of ROS in hyphal tips and appressoria, control the specialized remodeling of F-actin in hyphal tips and appressoria, and are involved in fungal cell wall biosynthesis. Taken together, our findings highlight the role of NOXs in fungal pathogenicity through the organization of the actin cytoskeleton., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Wang, He, Luo, An and Wang.)

All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene 1 . This approach is fundamentally different from the biosynthesis of short-chain (C 10 -C 25 ) terpenes that are formed from polyisoprenyl diphosphates 2-4 . In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature., (© 2022. The Author(s).)