Functional genomics of extracellular proteins of Phytophthora infestans.

摘要

Genomics offers new perspectives for the discovery of novel genes in the oomycetes, a unique group of eukaryotic microbes comprising saprophytes as well as pathogens of plants and animals. Among the oomycetes, the most devastating plant pathogen is the potato and tomato late blight pathogen Phytophthora infestans. Currently tens of thousands of expressed sequence tags (ESTs) are available for P. infestans. The challenge of the post-genomic era is to link a sequence to a phenotype (functional genomics) with as little experimental effort as possible. In the first study, we combined EST data mining with functional assays to identify extracellular effector proteins from P. infestans, which can manipulate plant molecular and cellular processes to induce defense or disease like responses. We developed the PexFinder algorithm for automated identification of P&barbelow;hytophthora e&barbelow;x&barbelow;tracellular proteins (Pex). Using a virus based high throughput functional assay to express Pex cDNAs in Nicotiana benthamiana and tomato, we identified two novel necrosis inducing effectors crn1 and crn2, which are members of large gene family. crn1 and crn2 were demonstrated to be expressed during P. infestans infection on tomato. In addition, crn2 induced defense related genes in tomato.; In a second study, we characterized a Pex cDNA, pipg1 which represents the first endopolygalacturonase (endoPG) described from P. infestans. Generally, endopolygalacturonases are plant cell wall degrading enzymes primarily found and characterized extensively in plants and fungi. We showed that pipg1 is a member of a gene family and is expressed in both preinfection and infection stages. Phylogenetic analyses showed an affinity of pipg1 to fungal endoPGs, a feature that contrasts with phylogenies obtained with ribosomal sequences or compiled mitochondrial and chromosomal genes.; In a third study, We generated ESTs from a cDNA library constructed from mRNA of Saprolegnia parasitica, an oomycete pathogen that causes saprolegniasis in fish. We performed preliminary comparisons of abundance of selected ESTs between S. parasitica and P. infestans and assessed the implications for S. parasitica. The long-term objective of this project is to develop genomic resources from diverse oomycete pathogens for comparative genomics.