SzMBL1, a metallo-β-lactamase gene in Sarocladium zeae, confers tolerance to the maize phytoanticipin 2-benzoxazolinone.

Abstract

Maize produces pre-formed secondary metabolites with antimicrobial and invertebrate anti-herbivory properties. Fusarium verticillioides, a mycotoxigenic maize pathogen, detoxifies the maize phytoanticipin 2-benzoxazolinone (BOA) through gene action of FvMBL1, which encodes a putative metallo-β-lactamase within the FDB1 gene cluster. This cluster is also present in other maize-colonizing fungi, including the pathogen Colletotrichum graminicola and, as reported here, the protective endophyte Sarocladium zeae, which exhibits antagonistic activity against mycotoxigenic fungi. The role of the FvMBL1 ortholog in S. zeae, designated SzMBL1, was assessed by gene deletion and add-back complementation. Deletion of SzMBL1 rendered S. zeae incapable of degrading BOA and therefore unable to grow on BOA-amended medium, whereas complemented add-back strains restored BOA tolerance and, in some cases, enhanced growth at higher concentrations of BOA. Quantitative PCR analyses revealed higher copy numbers and expression levels in all add-back transformants relative to the wild type, consistent with their enhanced growth. HPLC-MS/MS analysis further showed that SzMBL1 hydrolyzes BOA, ultimately resulting in biotransformation into the non-toxic metabolite N-(2-hydroxyphenyl)malonamic acid (HPMA). Together, this study confirms the conserved function of the SzMBL1 gene involved in BOA detoxification. This study suggests that the FDB1 gene cluster, harboring SzMBL1 in S. zeae, was likely transferred through horizontal gene transfer between maize-colonizing fungi and plays a role in the degradation of host-derived secondary metabolites that function as filters limiting which fungi are most frequently associated with maize.IMPORTANCEMaize produces secondary metabolites such as 2-benzoxazolinone (BOA) to help protect against microbes and insects. The pathogenic Fusarium verticillioides carries FvMBL1, a gene within the FDB1 gene cluster that helps break down BOA. This cluster is also present in other maize-associated fungi such as Colletotrichum graminicola and, as reported here, the protective fungus Sarocladium zeae. In our experiments, when a gene similar to FvMBL1, denoted SzMBL1, is deleted in S. zeae, the fungus could no longer break down BOA or grow on BOA-amended medium. Complementation restored BOA tolerance and growth phenotypes. Notably, some S. zeae add-back strains showed increased growth at higher BOA concentrations. Our analysis showed that these hypertolerant complemented strains had multiple, transcriptionally active copies of SzMBL1. The study highlights the role of SzMBL1 in BOA detoxification, thereby aiding fungal tolerance to antimicrobial compounds, and supports the hypothesis that the FDB1 gene cluster can be horizontally transferred among maize-colonizing fungi.