Downy mildew genomics
The downy mildews are a group of oomycete plant pathogens that cause foliage blights and belong to the family Peronosporaceae. They have an obligate biotrophic lifestyle and interact closely with living host cells through haustoria. These feeding structures do not only serve to obtain nutrients, they are also thought to allow the pathogen to modulate plant cell processes. The most striking example of the intimate interaction between the plant and the pathogen is the generation of the plant-derived membrane around the haustoria. In this research we are studying secreted proteins of the downy mildews Hyaloperonospora parasitica and Bremia lactucae. H. parasitica infects Arabidopsis thaliana, the model plant for which the complete genome sequence and many molecular tools are available, making it ideally suited to serve as a model system in oomycete - plant interactions (Figures 1 and 2).
Figure
1: [left] Arabidopsis thaliana infected with Hyaloperonospora parasitica
[right] Micrograph of P. parasitica hyphae and conidiophores, stained
with trypan blue, in an A. thaliana leaf.
Bremia lactucae, the downy mildew of lettuce (Lactuca sativa L.), forms a threat to lettuce production in temperate climates. A large number of resistance genes (Dm genes) have been used to protect lettuce cultivars from downy mildew disease, but Dm gene-dependent resistance is usually overcome by rapid adaptation of the pathogen. To find new sources of resistance to lettuce downy mildew, B. lactucae secreted proteins will be characterised. These proteins potentially interact with lettuce proteins and may induce a hypersensitive response in certain lettuce genotypes. To identify the required proteins we will collect downy mildew ESTs.
Figure 2: Schematic drawing of the early stages downy mildew infection. The spore germinates and produces a germ tube and appressorium. The hyphae grow in the intercellular space and haustoria branch off into adjacent mesophyll cells. Haustoria are separated from the cell cytoplasm by the plant-derived extrahaustorial membrane.
Downy mildew Expressed Sequences Tags
A large number of 5’ ESTs (expressed sequence tags) will be generated from directionally cloned cDNA libraries from downy mildew spores. In addition, ESTs from downy mildew-infected plants will be obtained to identify genes that are specifically expressed during the plant-pathogen interaction. As the relative abundance of H. parasitica mRNA in infected A. thaliana appears to be rather low, we will explore methods to select for downy mildew clones, such as enrichment and differential screening. Similarity to known sequences will be determined by blasting all ESTs against the public databases. The presence of signal peptides will be predicted by SignalP, using artificial neural network and hidden Markov models (Figure 3).
Figure 3: EST analysis pipeline. Blastn = nucleotide similarity search, Blastx = translated similarity search, nr = non-redundant database, est = EST database, pdb = protein database, NN = neural networks, HMM = hidden Markov model.
P. parasitica cDNAs will be subjected to further study using microarray technology: the cDNAs of interest will be spotted onto microarray slides and hybridised with mRNA isolated at different stages of the pathogen’s lifecycle. Furthermore, silencing and over-expression studies may be employed to elucidate the function of some promising cDNAs. These studies will be carried out in P. parasitica, if a transformation system is established, or in the A. thaliana - infecting oomycete Phytophthora brassicae. So far, 195 clones from the downy mildew spores cDNA libraries have been sequenced and results are promising: about 5% of the ESTs are predicted to encode putative secreted proteins and 10 to 20% show significant similarity to other oomycete sequences in the database. We have already found several ESTs that resemble proteins from plant pathogenic fungi and oomycetes that interact with the host plant.
Downy mildew genomics for new sources of plant resistance
The hypersensitive response (HR) is a localised induced cell death at the site of pathogen infection and is associated with the arrest of pathogen growth. This form of plant defence is triggered by the recognition of a pathogen-derived protein by resistant host plants. In order to find new lettuce genotypes that are resistant to B. lactucae, we will test a large panel of lettuce breeding lines for the ability to induce HR in response to a selection of secreted downy mildew proteins. For this, we will use Agrobacterium-mediated transient expression: A. tumefaciens is transformed with the cDNA of interest, cloned into a binary vector. Leaves are infiltrated with transgenic A. tumefaciens and then screened for localised necrosis (Figure 4). We have successfully established Agrobacterium mediated transient expression in lettuce using the GUS intron reporter gene.
Figure
4: A. tumefaciens-mediated transient expression of B. lactucae genes
in lettuce. Schematic overview of the experimental set-up for
Agrobacterium-mediated transient expression of B. lactucae cDNAs.
Through our downy mildew genomics research, we intend to identify new sources of disease resistance and provide insight into the role of downy mildew proteins in the infection of susceptible plants.
Acknowledgements
This research is funded by the Dutch Technology Foundation STW, and the
lettuce breeding companies Enza Zaden and RIJK ZWAAN.
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