Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 03 Nov 2018 13:34:59 Calcineurin phosphatase and phospholipase C are required for developmental and pathological functions in the citrus fungal pathogen Alternaria alternata Hsieh-Chin Tsai 1 and Kuang-Ren Chung 1,2 Correspondence Kuang-Ren Chung krchung@ufl.edu Received 3 February 2014 Accepted 17 April 2014 1 Citrus Research and Education Center, Institute of Food and Agricultural Sciences (IFAS), University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USA 2 Department of Plant Pathology, IFAS, University of Florida, Gainesville, FL 32611, USA Excessive Ca 2+ or compounds interfering with phosphoinositide cycling have been found to inhibit the growth of the tangerine pathotype of Alternaria alternata, suggesting a crucial role of Ca 2+ homeostasis in this pathotype. The roles of PLC1, a phospholipase C-coding gene and CAL1, a calcineurin phosphatase-coding gene were investigated. Targeted gene disruption showed that both PLC1 and CAL1 were required for vegetative growth, conidial formation and pathogenesis in citrus. Fungal strains lacking PLC1 or CAL1 exhibited extremely slow growth and induced small lesions on calamondin leaves. Dplc1 mutants produced fewer conidia, which germinated at slower rates than wild-type. Dcal1 mutants produced abnormal hyphae and failed to produce any mature conidia, but instead produced highly melanized bulbous hyphae with distinct septae. Fluorescence microscopy using Fluo-3 dye as a Ca 2+ indicator revealed that the Dplc1 mutant hyphae emitted stronger cytosolic fluorescence, and the Dcal1 mutant hyphae emitted less cytosolic fluorescence, than those of wild-type. Infection assessed on detached calamondin leaves revealed that application of CaCl 2 or neomycin 24 h prior to inoculation provided protection against Alt. alternata. These data indicate that a dynamic equilibrium of cellular Ca 2+ is critical for developmental and pathological processes of Alt. alternata. INTRODUCTION The fungal pathogen Alternaria alternata causes brown spot disease in citrus. This pathogen produces a host-selective toxin that kills host cells before invasion, and acquires nutrients primarily from dead tissues (Akimitsu et al., 2003). Alt. alternata colonization in leaves of the citrus causes lipid peroxidation, increased accumulation of hydrogen peroxide (H 2 O 2 ) and cell death (Lin et al., 2011). Although toxin is absolutely required for fungal pathogenicity, Alt. alternata also relies on effective detoxification of H 2 O 2 and other reactive oxygen species (ROS) to ensure survival and colonization in the host plant (Chung, 2012). Alt. alternata lacks a sexual phase and propagates mainly through the production of conidia. Conidia are disseminated by rain splashes and are required for initiating Alternaria brown spot disease in citrus. Our previous studies have demonstrated that the cytoplasmic cAMP level, controlled by GTP-binding proteins (G-proteins), plays an important role in conidial formation by Alt. alternata (Wang et al., 2010). Conidial formation by the wild-type strain of Alt. alternata was suppressed by cAMP or its inhibitors (atropine, theophylline or 3-isobutyl-1-methylxanthine). An Alt. alternata strain impaired for the Ga subunit produced significantly fewer conidia than wild-type; however, applica- tion of cAMP or its inhibitors partially restored conidial formation by the Ga mutant. In contrast, the Alt. alternata strain impaired for the cAMP-dependent protein kinase A (PKA) catalytic subunit produced twofold more conidia than wild-type, and a strain lacking the PKA regulatory subunit failed to produce any conidia, suggesting a negative function of PKA for conidiation (Tsai et al., 2013). Studies with Alt. alternata revealed that conidial formation was also regulated by the FUS3 and SLT2 mitogen-activated protein kinase (MAPK)-mediated signalling pathways as well as NADPH oxidase (NOX), because fungal strains impaired for FUS3, SLT2 or NOX produced no conidia or significantly fewer than wild-type (Lin et al., 2010; Yago et al., 2011; Yang & Chung 2012, 2013). Abbreviations: CaM, calmodulin; G-protein, GTP-binding protein; IP 3 , inositol 1,4,5-triphosphate; MAPK, mitogen-activated protein kinase; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PKA, cAMP-dependent protein kinase A. The GenBank/EMBL/DDBJ accession numbers for the sequence data reported in this article are KF306218 (CAL1) and KF306219 (PLC1). One supplementary table and three supplementary figures are available with the online version of this paper. Microbiology (2014), 160, 1453–1465 DOI 10.1099/mic.0.077818-0 077818 G 2014 The Authors Printed in Great Britain 1453