The FUS3 MAPK signaling pathway of the citrus pathogen Alternaria alternata functions independently or cooperatively with the fungal redox-responsive AP1 regulator for diverse developmental, physiological and pathogenic processes

Abstract

Alternaria alternata, the fungus that causes citrus brown spot, invades its hosts primarily through the production and action of a host-selective ACT toxin that kills citrus cells prior to invasion. In this study, we show that, in the tangerine pathotype of A. alternata, a mitogen-activated protein kinase (MAPK)-mediated signaling pathway governs a number of biological functions, either separately or in a cooperative manner, with the AaAP1 gene encoding a transcription regulator. The reported MAPK is encoded by the AaFUS3 gene, which we show to be necessary for conidial development, resistance to copper fungicides, melanin biosynthesis, and particularly, for elaboration of the penetration process. In contrast, AaFUS3 negatively controls salt tolerance and production of several hydrolytic enzymes. AaFUS3 has no apparent role in the biosynthesis of host-selective toxin or in resistance to oxidative stress. Both AaAP1 and AaFUS3 are required for fungal resistance to 2,3,5-triiodobenzoic acid (TIBA), 2-chloro-5-hydroxypyridine (CHP), diethyl maleate (DEM), and many pyridine-containing compounds. A strain with mutations in both AaAP1 and AaFUS3 displayed an increased sensitivity to these compounds. Expression of the AaAP1 and AaFUS3 genes and phosphorylation of AaFUS3 were also induced by CHP, DEM, or TIBA. Expression of two genes coding for a putative MFS transporter was coordinately regulated by AaAP1 and AaFUS3. The AaAP1::sGFP (synthetic green fluorescent protein) fusion protein became localized in the nucleus in response to CHP or TIBA. Inactivation of the AaAP1 gene, however, promoted phosphorylation of AaFUS3. Taken together, our results indicate that A. alternata utilizes specialized or synergistic regulatory interactions between the AP1 and MAPK signaling pathways for diverse physiological functions.

Introduction

Alternaria alternata (Fr.) Keissler causes two important fungal diseases in citrus: Alternaria brown spot and Alternaria leaf spot. Alternaria brown spot is caused by the tangerine pathotype of A. alternata and affects tangerines (Citrus reticulata Blanco), grapefruit (Citrus paradisi Macfad.), their hybrids, and hybrids of tangerines and sweet oranges (Citrus sinensis (L.) Osbeck). Disease symptoms can be largely attributed to the production of host-selective ACT toxin (Akimitsu et al., 2003). In contrast, Alternaria leaf spot is caused by the rough lemon pathotype and affects only rough lemon (Citrus jambhiri Lush) and Rangpur lime (Citrus x limonia Osbeck). In this case, symptoms are due to a host-selective ACRL toxin.

Mitogen-activated protein kinase (MAPK) cascade pathways are composed of three serine/threonine protein kinases: MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK. These are important signal transduction pathways in all eukaryotes and function in the perception of environmental stimuli via phosphorylation and gene activation (Pelech and Sanghera, 1992, Robinson and Cobb, 1997). In response to various environmental and developmental stimuli, MAPKKK is first phosphorylated within the TXY (threonine/X/tyrosine) motif. The phosphorylated MAPKKK subsequently phosphorylates the downstream MAPKK, which in turn phosphorylates MAPK (Gustin et al., 1998, Kültz, 1998). The phosphorylated MAPK then activates a set of genes via regulation of appropriate transcription factors. MAPK pathways are well conserved among yeasts and fungi, but the biological functions of each component kinase may vary considerably in different species, showing high dependence on the species lifestyles and environment (Bardwell, 2006).

In the budding yeast Saccharomyces cerevisiae, phosphate transfer systems modulated by five MAPK-mediated signaling pathways with divergent functions are known to control mating, cell-wall reconstruction, formation of pseudo-hyphae, as well as response to osmotic stress (Schwartz and Madhani, 2004). Similarly, filamentous fungi often have three analogous MAPK protein kinases: the high-osmolarity glycerol (HOG1) homolog, the cell wall integrity (SLT2) homolog, and the FUS3/KSS1 homolog. These are necessary for a wide range of biological processes, including appressorium formation, conidial germination, stress response, osmotic regulation, and pathogenicity (Xu, 2000, Zhou et al., 2007).

In phytopathogenic fungi, the FUS3/KSS1 homologs, belonging to the yeast and fungal extracellular signal-regulated kinase (YERK1) subfamily (Kültz, 1998), have been intensively investigated. In the rice blast fungus Magnaporthe grisea, the FUS3/KSS1 homolog, designated PMK1, is able to complement an S. cerevisiae mutant defective in mating and is absolutely required for appressorium formation and pathogenicity (Xu and Hamer, 1996). The FUS3/KSS1 homolog also plays a central role in virulence of other phytopathogenic fungi, including Alternaria brassicicola, Botrytis cinerea, Claviceps purpurea, Cochliobolus heterostrophus, Colletotrichum lagenarium, Fusarium spp., Mycosphaerella graminicola, and Stagonospora nodorum (reviewed by Zhou et al., 2007).

Although MAPK kinases are central in the regulation of gene expression, cross talk often occurs between MAPK pathways and other signal transduction pathways through G protein-, cAMP-, calcium/calcineurin-, two-component histidine kinase-, and/or YAP1-mediated signaling cascades (Kronstad et al., 1998, Lengeler et al., 2000, Aguirre et al., 2005, Aguirre et al., 2006, Bahn et al., 2007). These types of interactions depend on which particular microorganism or which environmental stimulus encountered. Divergent specificity may also be involved in the membrane sensors that act upstream or the target transcriptional factors that function downstream of these signaling pathways. Furthermore, the specificity of each cascade may rely, in part, on the antagonistic inhibition of cross talk between the pathways, even though the MAPK signaling cascades often share common components. For example, HOG1 has been reported to inactivate the FUS3/KSS1 signaling cascade during hyperosmotic stress in S. cerevisiae (O’Rourke and Herskowitz, 1998, Shock et al., 2009).

Previous studies have demonstrated that AP1-mediated detoxification of reactive oxygen species (ROS) is absolutely required for pathogenesis of A. alternata in citrus (Lin et al., 2009, Yang et al., in press). In the present study, we have isolated and characterized the AaFUS3 gene that encodes an ortholog of the yeast FUS3/KSS1-like MAP kinase, in an effort to understand the mechanism underlying oxidative stress resistance, as well as the developmental and physiological functions of this gene in A. alternata. Collectively, the present study highlights the subtle regulatory actions of the AaFUS3 gene product during vegetative growth and conidial formation, and in the production of hydrolytic enzymes and melanin. A substantial contribution of AaFUS3 to fungal pathogenicity and to effective penetration and tissue colonization of citrus hosts is also established. A role for resistance to copper fungicides, osmotic, and a broad spectrum of structurally diverse compounds is also evident for AaFUS3. Unlike the AaAP1 gene, AaFUS3 is not required for resistance to H₂O₂. However, both of the genes are involved in resistance to 2-chloro-5-hydroxypyridine (CHP), 2,3,5-triiodobenzoic acid (TIBA), and many other compounds. FUS3 MAPK has been intensively studied in many microorganisms, but its interaction with the redox-responsive AP1 transcription regulator remains largely unknown. The present study implies a possible link or a synergistic interaction between the AaFUS3- and AaAP1-mediated signaling cascades.