Ca2+ affects the hyphal differentiation to sclerotia formation of Athelia rolfsii

Abstract

ABSTRACT RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+–chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2–ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.

Keywords

• Athelia rolfsii
• calcium ion
• Macrophomina phaseolina
• microsclerotia
• reactive oxygen species (ROS)
• RNA-Seq