Asexual spores (conidiospores) are dispersed by wind or water to leaves, where they attach via formation of an infection structure, the appresorium. An infection peg penetrates the epidermis, and gives rise to branching, filamentous hyphae (thread-like structures) that grow between cells in the mesophyll (interior leaf cells) to form a vegetative mycelium inside the leaf. Associations are formed between hyphae and host mesophyll cells through structures called haustoria that penetrate the host cell wall and invaginate (but do not penetrate) the host cell membrane. Uptake of nutrients through the haustorial and hyphal membranes is thought to provide the necessary components for parasite growth and reproduction. Hyphal tips emerge through open stomata and differentiate into tree-like sporangiophores, which collectively produce thousands of spores (protected reproductive cells) on a single leaf. These spores can be collected in sufficient quantities to isolate DNA and RNA for genomic and cDNA libraries. Hyaloperonospora arabidopsidis isolates do not have defined mating types (homothallic), but are capable of outcrossing. Oöspores (sexual spores) form in the mesophyll, following connection of hyphal tips. Oöspores are capable of persisting in the soil after the plant decays, and can infect the shoots or roots of newly germinating plants.
Hyaloperonospora arabidopsidis is a pathogen of more than 140 species in the family Cruciferae, including crops such as cabbage, oilseed rape, broccoli and cauliflower, as well as the plant model organism, Arabidopsis thaliana. H. arabidopsidis is representative of a large group of related microbes that cause downy mildew disease, which is typified by dense patches of aerial fruiting bodies that lend a “downy” appearance to the surface of infected leaves. Downy mildews are distributed worldwide and are estimated to parasitize ~15% of flowering plant families, including a wide range of monocot and dicot crops. Downy mildew diseases account for ~20% of the $5 billion global fungicide market, and two downy mildew pathogens of maize are listed among seven plant pathogens considered to be major US bioterror threats (Agricultural Bioterrorism Protection Act of 2002). H. arabidopsidis and other downy mildews are classified as oömycetes. Although oömycetes share superficial morphological resemblances with fungi (e.g. a filamentous growth habit and production of aerial spore-bearing structures), numerous lines of evidence place oömycetes within the kingdom Stramenopila, which includes golden- brown algae, diatoms, and brown algae such as kelp. Downy mildews are biotrophic, meaning that they extract nutrients only from living host tissue. Downy mildews cannot be cultured on synthetic media and are incapable of surviving apart from their hosts. Although many important plant pathogens employ biotrophic life styles, little is currently known about the molecular mechanisms that support biotrophy. Because H. arabidopsidis is the most frequently occurring eukaryotic pathogen of Arabidopsis, it has become one of the two most widely used model pathogens (along with the bacterium Pseudomonas syringae) for studies of Arabidopsis defense networks. The H. arabidopsidis-Arabidopsis system is also being developed as a model to explore the mechanisms by which biotrophic pathogens manipulate their hosts. Experimental tools include an H. arabidopsidis linkage map, ESTs, and a BAC library that facilitated molecular cloning of two avirulence loci. A genome sequence will be an important asset for understanding many aspects of oömycete biology.
The Hyaloperonospora arabidopsidis isolate selected for sequencing (Emoy2) is available from Dr. John McDowell or Dr. Jim Beynon. This isolate originally was identified by Dr. Eric Holub (Horticulture Research International, UK) from a naturally occurring population of H. arabidopsidis growing on Arabidopsis in East Malling, UK. The genome will be sequenced to a total of 8.5x whole genome coverage. A combination of whole genome shotgun plasmids, fosmids, and BAC end reads will be placed in the assembly. One round of automated sequence improvement (pre-finishing) will be done. A BAC fingerprint map is under construction and will be compared to the final assembly for further refinement of both the sequencing assembly and the fingerprint map. The NSF-USDA Interagency Microbial Genome Sequencing Program is funding the whole genome shotgun portion of this project (Dr. Brett Tyler, Virginia Bioinformatics Institute; Dr. John McDowell, Virginia Tech). BAC end-sequencing, physical map construction, and EST sequencing is being carried out in the United Kingdom, funded by the Biotechnology and Biological Sciences Research Council (Dr. Jim Beynon, Horticulture Research International; Dr. Jane Rogers, Sanger Centre). The data from the Sanger Centre will be incorporated into the final assembly, which then will be automatically annotated and imported into the oömycete community annotation database at the Virginia Bioinformatics Institute for further refinement.