Ca 2+ 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), H 2 O 2 , or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN 3 , and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca 2+ influx, therefore, glutathione culture assays with the combination of CaCl 2 , Ca 2+ –chelator egtazic acid, DETC, and H 2 O 2 were tested on A. rolfsii , as well as two other fungi ( Sclerotinia sclerotiorum and Macrophomina phaseolina ) for comparison. Although the addition of CaCl 2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl 2 –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 Ca 2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca 2+ and independent of ROS stimuli. IMPORTANCE Management 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 Ca 2+ 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 Ca 2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca 2+ , and the applicability of Ca 2+ in reducing sclerotia could be further assessed for disease management.