Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin.

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.

Baseline Sensitivity and Potential Resistance Mechanisms for Fusarium pseudograminearum to Fludioxonil.

Fusarium crown rot (FCR), which is caused by Fusarium pseudograminearum, is one of the most important diseases affecting wheat production in the Huanghuai wheat-growing region of China. Although the phenylpyrrole fungicide fludioxonil is known to have a broad-spectrum activity against a wide range of plant pathogens, including F. pseudograminearum, it has not yet been registered for the control of FCR in China, and further research is needed to assess the biological characteristics and molecular mechanisms associated with fludioxonil resistance, and especially the potential for highly resistant isolates to emerge. The current study demonstrated that the baseline fludioxonil sensitivity of 61 F. pseudograminearum isolates collected from the Henan province of China during the summers of 2019 to 2021 conformed to a unimodal distribution with a mean effective concentration for 50% inhibition (EC50) value of 0.021 ± 0.003 µg/ml, which indicated that none of the isolates exhibited natural resistance to fludioxonil. Nevertheless, four fludioxonil-resistant mutants were attained after repeated exposure to fludioxonil under laboratory conditions. All resistant mutants exhibited significantly lower growth rates on potato dextrose agar (PDA) and lower levels of sporulation and pathogenicity in wheat seedlings. In addition, the resistant mutants also exhibited less growth on PDA amended with either 0.5 M mannitol, 0.5 M glucose, 0.5 M MgCl2, or 0.5 M NaCl, which indicated that they had greater sensitivity to osmotic stress. Molecular analysis of the proposed fludioxonil target protein FpOs1 indicated that the predicted sequences of the resistant mutants contained none of the characteristic amino acid changes previously associated with fludioxonil resistance in other species. Further investigation via quantitative real-time PCR analysis revealed that expression of the FpOs1 gene was significantly altered in the resistant mutants in both the absence and presence of fludioxonil. Meanwhile, plate assays found evidence of cross-resistance between fludioxonil and cyprodinil, as well as with the triazole fungicides tebuconazole and difenoconazole, but not with other commonly used fungicides including prochloraz, fluazinam, and carbendazim. Taken together, these results provide new insights into the mechanism and biological characteristics associated with fludioxonil resistance in F. pseudograminearum and indicate that fludioxonil could provide effective and sustained control of FCR during wheat production.