Rice blast is caused by the fungus Magnaporthe grisea, which elaborates specialized infection cells called appressoria to penetrate the tough outer cuticle of the rice plant Oryza sativa. We found that the formation of an appressorium required, sequentially, the completion of mitosis, nuclear migration, and death of the conidium (fungal spore) from which the infection originated. Genetic intervention during mitosis prevented both appressorium development and conidium death. Impairment of autophagy, by the targeted mutation of the MgATG8 gene, arrested conidial cell death but rendered the fungus nonpathogenic. Thus, the initiation of rice blast requires autophagic cell death of the conidium.

One of the first responses of plants to microbial attack is the production of extracellular superoxide surrounding infection sites. Here, we report that Magnaporthe grisea, the causal agent of rice blast disease, undergoes an oxidative burst of its own during plant infection, which is associated with its development of specialized infection structures called appressoria. Scavenging of these oxygen radicals significantly delayed the development of appressoria and altered their morphology. We targeted two superoxide-generating NADPH oxidaseencoding genes, Nox1 and Nox2, and demonstrated genetically, that each is independently required for pathogenicity of M. grisea. ⌬nox1 and ⌬nox2 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. The initiation of rice blast disease therefore requires production of superoxide by the invading pathogen.O ne of the earliest manifestations of the plant defense response is the production of reactive oxygen species (ROS), including superoxide and its dismutation product, hydrogen peroxide (1) . These ROS can kill pathogens directly (2) but also strengthen plant cell walls through the oxidative cross-linking of cell wall structural proteins (3) and may function in the regulation of programmed cell death (4). Although numerous studies have documented the detection of plant-derived ROS (2, 3, 5-7), very little is known about the role of ROS generation in invading plant pathogenic microorganisms. However, the recent discovery of functional members of the superoxide-generating NADPH oxidase (Nox) family within filamentous fungi (8) has led to increased speculation regarding the possible role of ROS in pathogenic species.The most well characterized Nox remains that of the human phagocytic leukocyte, a multisubunit oxidase formed by the cytosolic regulatory components Rac, p67 phox , p47 phox , and p40 phox and the integral membrane protein flavocytochrome b 558 , composed of the catalytic subunit gp91 phox and p22 phox (9). In activated macrophages, Nox enzymes induce K ϩ influx, causing pH changes in the phagocytic vacuole, leading to the killing of pathogens through activation of neutral proteases (10). Mutations in the catalytic gp91 phox subunit result in chronic granulomatous disease, an immunological disorder in which macrophages are unable to prevent the spread of infection (11). Plants contain enzymes that are homologous to gp91 phox , designated respiratory burst oxidase homologues (Rboh) (12). Arabidopsis thaliana, for example, possesses 10 Rboh isoforms involved in a diverse range of plant processes. ROS generated by the A. thaliana RHD2/RBOHC regulate root hair growth through the activation of Ca 2ϩ channels (13), whereas RBOHD and RBOHF regulate stomatal closure, seed germination, and root elongation through abscisic acid signaling (14). Recent studies have shown that it is the activation of RBOHD and RBOHF that is responsible for ROS accumulation in several plant-microbe interactions (15). However, r...

Fungi are the principal degraders of biomass in terrestrial ecosystems and establish important interactions with plants and animals. However, our current understanding of fungal evolutionary diversity is incomplete and is based upon species amenable to growth in culture. These culturable fungi are typically yeast or filamentous forms, bound by a rigid cell wall rich in chitin. Evolution of this body plan was thought critical for the success of the Fungi, enabling them to adapt to heterogeneous habitats and live by osmotrophy: extracellular digestion followed by nutrient uptake. Here we investigate the ecology and cell biology of a previously undescribed and highly diverse form of eukaryotic life that branches with the Fungi, using environmental DNA analyses combined with fluorescent detection via DNA probes. This clade is present in numerous ecosystems including soil, freshwater and aquatic sediments. Phylogenetic analyses using multiple ribosomal RNA genes place this clade with Rozella, the putative primary branch of the fungal kingdom. Tyramide signal amplification coupled with group-specific fluorescence in situ hybridization reveals that the target cells are small eukaryotes of 3-5 μm in length, capable of forming a microtubule-based flagellum. Co-staining with cell wall markers demonstrates that representatives from the clade do not produce a chitin-rich cell wall during any of the life cycle stages observed and therefore do not conform to the standard fungal body plan. We name this highly diverse clade the cryptomycota in anticipation of formal classification.