Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

Abstract

In the filamentous phytopathogen Botrytis cinerea, the Ca 2؉ /calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The cch1 and mid1 genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca 2؉ -chelating agents EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N=,N=-tetraacetic acid (BAPTA). The impact of external Ca 2؉ was investigated by supplementing with CaCl 2 and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1-green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in B. cinerea.

Components of the Calcium-Calcineurin Signaling Pathway in Fungal Cells and Their Potential as Antifungal Targets

“…Moreover, the ecm7⌬/⌬ mutants of Candida albicans were shown to be sensitive to oxidative stress, which resulted in a defect in hyphal development and attenuated the ability of yeast cells to invade and diffuse in mouse kidneys compared with the phenotype of the wild-type strain (20). LACS, whose main important component or regulator is Fig1p (22), displays a 16-foldlower affinity for Ca 2ϩ than HACS does (40). Many other factors related to polarized morphogenesis and cell fusion, such as Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p, were also found to be necessary for LACS activity (22).…”

“…Most fungal plasma membranes contain at least two different Ca 2ϩ influx systems, the high-affinity Ca 2ϩ influx system (HACS) and the low-affinity Ca 2ϩ influx system (LACS) (40). The HACS consists of two putative proteins, Cch1p and Mid1p, which are expressed and colocalize to the plasma membrane in a variety of fungi, such as the saprophytes Schizosaccharomyces pombe and Neurospora crassa (41,42), the animal-pathogenic fungi Candida albicans and Cryptococcus neoformans (43)(44)(45), and the plantpathogenic fungi Gibberella zeae, Claviceps purpurea, and Uromyces appendiculatus (46)(47)(48)(49).…”

Roles of three Fusarium oxysporum calcium ion (Ca2+) channels in generating Ca2+ signatures and controlling growth

“…Both CCH1 and MID1 have been shown to be required for the extracellular Ca 2+ uptake in response to mating pheromone and also are involved in iron and cold tolerance in yeast (Peiter et al, 2005). In several filamentous fungi, CCH1 and MID1 orthologs have been shown to be important role for vegetative growth and development (Cavinder et al, 2011;de Castro, 2014;Hallen and Trail, 2008;Harren and Tudzynski, 2013;Jiang et al, 2014;Lew et al, 2008). The YVC1 channel, a homolog of the animal transient receptor potential protein 2, resides in the vacuolar membrane and participates in controlling vacuolar pressure (Chang et al, 2010;Palmer et al, 2001).…”

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca 2+ Homeostasis in Aspergillus nidulans

“…Two different calcium carrier systems have been identified in most fungal species: the high-affinity Ca 2ϩ influx system (HACS) and the low-affinity calcium influx system (LACS) (10)(11)(12)(13). The main components of the HACS are an ␣ subunit of the mammalian voltage-gated Ca 2ϩ -channel homolog Cch1 and a stretch-activated ␤ subunit called Mid1 (14)(15)(16). Loss of the HACS results in an inability to grow under low-calcium conditions.…”