Rapamycin is a lipophilic macrolide antibiotic which is famous for its immunosuppressive and anticancer activity. In recent years, rapamycin showed significant activity against various plant pathogenic fungi. However, the sensitivity of Colletotrichunm fungi to rapamycin is scarcely reported. In this study, we evaluated the sensitivity of 116 Colletotrichum isolates from tea-oil trees to rapamycin. Most isolates exhibited natural resistance with inhibition rates of 50 to 70% at 50 μg/mL. Three Colletotrichum camelliae isolates were found to be sensitive to rapamycin. No mutations were detected in the direct target FKBP12 and indirect target TOR-FRB domain of resistant and sensitive C. camelliae isolates. Notably, the expression of the TOR homolog (CcTOR) was higher in resistant C. camelliae isolates compared to the sensitive ones and overexpression of CcTOR in the sensitive isolate CcS1 resulted in decreased sensitivity to rapamycin. Moreover, ribosomal protein S6 phosphorylation was abolished in the sensitive isolate CcS1 but not in the resistant isolate CcR1 under rapamycin treatment. In addition, the expression levels of ribosome biogenesis genes and two other components of TORC1 were higher in CcR1 compared to CcS1 under the same treatment, which suggested that the abundance of TORC1 in CcR1 was greater than in CcS1, leading to more active TORC1 signaling in CcR1. These results provided a better understanding about natural resistance of C. camelliae isolates to rapamycin and could help for developing new TORC1 signaling-targeting fungicides.

Fusarium graminearum causes Fusarium head blight (FHB), a destructive disease of cereal crops worldwide. Carbendazim (methylbenzimidazol-2-ylcarbamate [MBC]) is widely used for controlling FHB. A previous study showed that the F240L mutation in the β2-tubulin of F. graminearum (Fgβ2-tubulin) confers hypersensitivity to MBC. Whether the substitution of phenylalanine by other amino acids in position 240 of the Fgβ2-tubulin gene also confers hypersensitivity to MBC is unknown. Moreover, the biological fitness of these mutants is poorly understood. In this study, we substituted position 240 of Fgβ2-tubulin with other amino acids. We found that the F240A, F240E, F240I, and F240Y mutations in Fgβ2-tubulin could also confer F. graminearum hypersensitivity to MBC, although the effective concentration resulting in 50% inhibition (EC50) differed among the mutations. The F240G mutation, in contrast, decreased the sensitivity to MBC. In addition, a molecular docking assay indicated that the binding affinity between Fgβ2-tubulin and MBC were increased by the F240A, F240E, F240I, and F240Y mutations but decreased by the F240G mutation. All mutants had normal conidial morphology, but the growth rates and pathogenicity of the F240A, F240E, F240G, F240I, and F240Y mutants were significantly decreased. Moreover, the F240A and F240G mutants produced twisted hyphae. In addition, microtubules were sparse and rarely observed in β2F240A-EGFP, β2F240E-EGFP, and β2F240G-EGFP. These results indicate that position 240 (phenylalanine) is not only vital to the function of Fgβ2-tubulin but also plays an important role in regulating the sensitivity of F. graminearum to MBC. Any mutation in this site would be detrimental to survival.

Fourteen quinones were isolated from endophytic fungus Fusarium sp. HJT-P-5 of Rhodiola angusta Nakai. The structures were determined by the chemical and spectroscopic methods. Compounds 1, 8, 9, and 14 are new compounds. The compounds 1, 6, 7, 8, 9, 10, 11, 12, 13 and 14 were evaluated for cytostatic activity against human liver cancer cells HepG2, human lung cancer cells A549, cervical cancer cells Hela, and human colorectal cancer cells HCT116.

The plant pathogenic fungus, Fusarium graminearum, is known to have two β-tubulin genes (named Fg-β1tub and Fg-β2tub). Mutations in Fg-β2tub rather than in Fg-β1tub have been shown to confer resistance to carbendazim (MBC), even though Fg-β1tub has higher homology than Fg-β2tub to the β-tubulin isotypes related to benzimidazole resistance in other fungi. However, sequence alignment of β-tubulin isotypes related to benzimidazole resistance showed that the number and position of introns in Fg-β2tub are more consistent than Fg-β1tub to those in other β-tubulin genes. In detail, Fg-β1tub lacks three introns, i.e., intron i3, i4, and i6 corresponding to positions in Fg-β2tub of F. graminearum. To investigate the effects of the divergence introns on the function of β-tubulins in F. graminearum, a strategy of intron deletion and insertion was used. Our results showed that deletion of the second intron from Fg-β1tub gene increased Fg-β1tub expression levels leading to increased sensitivity to MBC. Besides, inserting the divergence introns into Fg-β1tub can increase Fg-β1tub expression leading to increased sensitivity to MBC. In addition, intron-mediated Fg-β1tub gene expression requires a splicing-competent intron within the body of the host gene. Furthermore, the insertion and deletion of introns in Fg-β1tub gene have no significant effect on hyphal growth, conidiation and virulence in F. graminearum. Thus, we proposed that introns may be among the factors contributing to the evolution and functional divergence of two β-tubulin genes and also significantly regulate the expression of β-tubulin genes, which, in turn, affects sensitivity to MBC fungicides in F. graminearum.

Resistance to benzimidazole fungicides in many phytopathogenic fungi is caused by specific point mutations in the β-tubulin gene (β-tubulin). However, the mutated locus and genotype of β-tubulin differ among phytopathogenic fungi. To validate the point mutation in Fusarium asiaticum β2-tubulin that confers resistance to carbendazim and to analyze the molecular interaction between carbendazim and F. asiaticum β2-tubulin. In this study, a new point mutation (GAG→GCG, E198A) at codon 198 of β2-tubulin in a wild-type F. asiaticum strain was constructed by site-directed mutagenesis followed by a split marker strategy. The site-directed mutants were verified and exhibited a high level of resistance to carbendazim. In the absence of fungicide treatment, the biological characteristics did not differ between the site-directed mutants and the wild-type strain. Molecular docking between carbendazim and β2-tubulin was carried out using the Surflex-Dock program in Sybyl X-2.0 version and the results indicated that the E198A mutation altered the configuration of β2-tubulin, resulting in the change of the bonding sites and docking scores. We concluded that the point mutation of F. asiaticum β2-tubulin conferring carbendazim resistance may not always be the bonding site for carbendazim.

Although the roles of introns have been much debated in eukaryotic organisms, none of them have been functionally characterized in Fusarium graminearum. In this study, we characterized the roles of introns in regulation of fungicide-sensitivity of F. graminearum. β2 tub, cyp51A and myosin-5 are important target genes of benzimidazoles, triazoles and cyanoacrylates respectively. To explore the sensitivity regulation functions of introns in target genes, several detailed deletion studies were completed on the intronic regions of β2 tub, cyp51A and myosin-5. Phenotypic analyses showed that deletion of the fourth intron from β2 tub gene (designated β2 Δi4), the sole intron from cyp51A gene (cyp51A-Δi) and the second intron from myosin-5 gene (myo5-Δi2) exhibited an increased sensitivity to corresponding fungicides. In contrast, deletion of the first or second intron from β2 tub gene exhibited a decreased sensitivity to carbendazim. qRT-PCR showed that the mRNA transcript levels of target genes were significantly downregulated in β2 Δi4, cyp51A-Δi and myo5-Δi2 respectively. Meanwhile, Western blot assays revealed that the protein expression levels of β2 tub was also dramatically reduced in β2 Δi4, but accumulated in β2 Δi1 and β2 Δi2. Overall, our results indicate that introns in target genes significantly regulate the fungicide-sensitivity by influencing expression of the corresponding resident genes in F. graminearum.

To determine the mechanism of resistance to the fungicide phenamacril (JS399-19) in Fusarium graminearum, the causal agent of Fusarium head blight, we sequenced and annotated the genome of the resistant strain YP-1 (generated by treating the F. graminearum reference strain PH-1 with phenamacril). Of 1.4 million total reads from an Illumina-based paired-end sequencing assay, 92.80% were aligned to the F. graminearum reference genome. Compared with strain PH-1, strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes. We sequenced 22 functional annotated genes of another F. graminearum sensitive strain (strain 2021) and corresponding resistant strains. The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5 (point mutations at codon 216, 217, 418, 420, or 786). To confirm whether the mutations in myosin-5 confer resistance to phenamacril, we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange. The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril. These results indicate that mutations in myosin-5 confers resistance to phenamacril in F. graminearum.