Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea

Abstract

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant. IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

Iron homeostasis in the absence of ferricrocin and its consequences in fungal development and insect virulence in Beauveria bassiana

“…Moreover, as reported above Δ ferS exhibited the increased virulence against the insect host. This is strikingly similar to the hypervirulence phenotype found in the mutant Δ fet1 knocked-out in the ferroxidase gene, a core component of the reductive iron assimilation system in the phytopathogen Botrytis cinera 20 .…”

The Botrytis cinerea Gene Expression Browser

“…Among all genes encoding NRPS (non-ribosomal peptide synthetases) and PKS–NRPS (polyketide synthetases–NRPS hybrids) in B. cinerea , we discovered that two genes ( bcnrps7 and bcpks5 ) display remarkably similar expression patterns across all experimental conditions. Highlighting the utility of BEB, the observed expression pattern supports the role of the uncharacterized bcpks5 in siderophore biosynthesis, a relevant iron acquisition mechanism in fungi [ 40 , 41 ]. Finally, taking advantage of BEB as a curated gene expression database, we propose a set of new reference genes that can be used in RT-qPCR studies.…”

“…Little is known about iron acquisition pathways and transcriptional regulatory mechanisms in B. cinerea [ 67 ]. However, this fungus is expected to synthesize at least nine siderophores to support metal acquisition [ 40 , 68 ] in addition to its membrane-bound reductive iron assimilation mechanism [ 41 ].…”

The Botrytis cinerea Gene Expression Browser

“…Little is known about B. cinerea ’s iron acquisition pathways and transcriptional regulatory mechanisms. However, this fungus is expected to synthesize at least nine siderophores to support this metal acquisition (Konetschny-Rapp et al, 1998; Bushley and Turgeon, 2010) in addition to the membrane-bound reductive iron assimilation mechanism that also participates in iron incorporation (Vasquez-Montaño et al, 2020).…”