Vol. 7, No. 4
Anastomosis Is Required for Virulence of the Fungal Necrotroph Alternaria brassicicola
Kelly D. Craven,1† Heriberto Velez,1 Yangrae Cho,2 ´¨ Christopher B. Lawrence,2 and Thomas K. Mitchell1*
Center for IntegratedFungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina,1 and Virginia Bioinformatics Institute, Blacksburg, Virginia2
Received 19 November 2007/Accepted 18 February 2008
A fungal mycelium is typically composed of radially extending hyphal ﬁlaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination ofnutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula andA. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This ﬁnding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activatedprotein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola. Fungal colonial growth is often described as long branching ﬁlaments; however, in most cases it is better depicted as netlike or an interconnected web. A fungal colony (mycelium) is regarded as an individual and typically establishes itselfand grows by hyphal extension and branching as the organism explores the surrounding substrate. The process of vegetative hyphal fusion, or anastomosis, is a fundamental activity for the vast majority of ﬁlamentous fungi (9, 10, 15, 22, 26). The capacity of individual hypha within a colony to recognize, grow toward, and anastomose with each other (self-fusion or selfanastomosis) facilitates theinterconnectedness between sectors of the organism that enables nutrient distribution (13) and the transduction of chemical signals in response to the environment (21). However, the role of self-anastomosis in phytopathogenic fungi is largely unknown. Recent studies of Neurospora crassa suggest that prior to self-anastomosis, a complex interplay of extracellular signaling occurs among hyphae,resulting in directed growth (homing) and subsequent fusion (12, 23). Targeted mutagenesis of an N. crassa gene termed so (“soft”) resulted in a loss of preanastomosis homing and, consequently, a failure of adjacent hyphae to fuse (7). In addition to this cellular fusion defect, the so mutant exhibited shortened aerial hyphae, an altered conidiation pattern, and female sterility. Further characterizationhas revealed that the SO protein localizes to septal plugs within mycelia that are undergoing developmental changes associated with sexual structures or a programmed cell death response to non-self-recognition (8). The precise function of the SO protein is thus far unknown, but it contains a double-tryptophan (WW) domain that is commonly involved in mediating proteinprotein interactions byrecognizing proline-rich ligands (17). It has been proposed that so encodes part of the biochemical machinery involved in the synthesis and/or perception of an extracellular chemoelicitor leading to hyphal homing (7). The SO protein hypothetically functions via a phosphorylation cascade resulting in the transcription of genes required for anastomosis. Several mitogen-activated protein kinase (MAPK)...