Molecular interplay among the redox-responsive regulator AaAP1, the two-component histidine kinase and the mitogen-activated protein (MAP) kinases in Alternaria alternata of citrus

Alternaria brown spot is caused by the tangerine pathotype of Alternaria alternata. The disease affects tangerine, grapefruit and their hybrids, resulting in severe agronomic and economic losses in Florida. This research determined the important roles of signaling pathways that are mediated by three MAP kinases (AaHOG1, AaSLT2 and AaFUS3), a redox-responsive transcription regulator (AaAP1), and a two-component histidine kinase (AaHSK1) in the life cycle of A. alternata.;The results revealed that AaAP1 is necessary for cellular response and adaption to oxidative stresses. Disruption of the AaAP1 gene in A. alternata abolished antioxidant activities and increased sensitivity to H2O2. The AaAP1 null mutant failed to induce any visible necrotic lesions on citrus leaves, primarily due to its inability to detoxify ROS produced by the host plant.;Molecular characterization of a conserved AaFUS3 gene, encoding a FUS3-type MAP kinase, from A. alternata revealed that AaFUS3 is required for vegetative growth, conidiation, pathogenicity, and production of several hydrolytic enzymes. Two genes encoding putative Major Facilitator Superfamily (MFS) transporters were identified from a suppression subtractive hybridization library. Expression of these two genes was coordinately regulated by AaAP1 and AaFUS3, suggesting a synergistic regulation between AaFUS3 MAP kinase and redox-responsive regulator AaAP1.;Furthermore, the AaHSK1 gene encoding a group III "two-component" histidine kinase, the AaHOG1 gene encoding a HOG1-type MAP kinase, and the AaSLT2 gene encoding a SLT2 MAP kinase, were also cloned and characterized in A. alternata. AaHSK1 is a primary regulator for cellular resistance to sugar osmotic stress and for sensitivity to dicaboximide or phenylpyrrole fungicides. AaHOG1, which conferred cellular resistance to salts and oxidative stress, bypasses AaHSK1 even though deletion of AaHSK1 affected AaHOG1 phosphorylation. These functions are likely modulated by unknown mechanisms rather than directly by the AaHOG1-mediated pathway.;AaSLT2 is necessary for conidiation, maintenance of cell-wall integrity, and fungal virulence but is dispensable for toxin production. As with AaAP1 and AaFUS3, AaHOG1 and AaSLT2 are necessary for fungal pathogenicity; yet AaHSK1 is completely dispensable for pathogenicity. Fungal mutants impaired in AaHSK1, AaHOG1, AaAP1, AaSLT2 or AaFUS3 were all hypersensitive to 2-chloro-5-hydroxypyridine (CHP) or 2,3,5-triiodobenzoic acid (TIBA). Overall, this study highlights the dramatic flexibility and uniqueness in the signaling pathways that are involved in pathogenicity and respose to diverse environmental stimuli in Alternaria alternata.