Functional Genomics of Early Infection by Phytophthora Sojae

NON-TECHNICAL SUMMARY: Phytophthora species and related oomycete pathogens cause tens of billions of dollars of damage each year to a huge range of agriculturally and ornamentally important plants. They also do severe damage to forests and threaten entire natural ecosystems. Because of their threat, several oomycetes are listed as bioterrorism agents. In order to develop improved methods for controlling Phytophthora infection, it is essential to understand the mechanisms by which these pathogens break down plants' defenses. The overall goal of this research is to identify the tool sets that the pathogen Phytophthora sojae uses to overcome the defenses of its host soybean. This project is focused on the identification and characterization of genes active at the earliest times during P. sojae infection, commencing with spore germination and penetration of the host, and proceeding through the first 16 hours of infection. These timepoints are the most crucial to a successful infection as the pathogen seeks to establish itself within the host tissue. The project will measure the activities of 15,800 pathogen genes and measure the abundance of 1000-2000 pathogen proteins immediately prior to penetration of the host. In addition, the activities of 100 master control genes will be measured following entry into the host tissue; technical issues currently prevent all 15,800 pathogen genes from being measured in the presence of plant tissue. Mutations will be obtained to disrupt the function of five to ten master control genes in or to determine in more detail how they control the infection process.

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APPROACH: Objective 1. Expression Profiling During Pre-penetration Events. We will prepare germinating cysts under two conditions, gentle shaking culture (100 rpm) or still culture in which the cysts have been allowed to settle on an impenetrable hydrophobic surface and germ tubes extending from the cysts form appressoria. We will use the recently released Affymetrix Soybean/P.sojae Genome Array, which contains probes corresponding to 15,800 P. sojae genes. We will use Linear Mixed Model statistical methodology to identify genes expressed at significantly higher levels during infection over time. The microarray (and proteomics) data will be housed and made publicly available in PhytophthoraDB, which is an implementation of the Genomics Unified Schema (GUS) developed at the University of Pennsylvania (www.gusdb.org/). Objective 2. Proteomic analysis of Pre-penetration Events. We will utilize Multidimensional Protein Identification Technology (MudPIT) to identify the 1000-2000 most abundant proteins expressed in cysts and appressoria. Cell lysates will be directly digested with trypsin to yield peptides that will be loaded onto a cation exchange/reverse phase multidimensional HPLC column in line with a tandem mass spectrometer. Spectra collected over an approximately twenty-four hour time period, encompassing 12 cycles of elution, will be queried against the P. sojae database using both commercial software packages and algorithms developed at ORNL and collated back into protein identifications. Objective 3. Real-time-PCR profiling of regulatory genes post-penetration. In order to identify potential master regulatory genes we will assay early expression of approximately 100 genes using RT-PCR analysis, chosen on the basis of their annotation as transcription factors, signal transduction components or receptors, in that order. Genes specifically expressed during pre-penetration or mid-to-late infection will be selected first. We will isolate mRNA from infection sites 0, 4, 10 and 16 hr after inoculation. We will replicate the time course of infection and RNA extraction three times. RNA will be extracted by a Trizole (Invitrogen) method and treated with DNase to remove residual genomic DNA. Objective 4. Isolation of P. sojae mutants with mutations in selected master regulatory genes. TILLing (Targeted Induced Local Lesions IN Genomes) uses high throughput PCR screening to identify mutations in genes of interest. We will screen for mutations in five to ten genes, as time permits. We will focus on genes encoding transcription factors or signal transduction pathway genes that are expressed during early in infection. P. sojae mutants will be assessed for growth rate and zoospore production, as well as for morphological differences during cyst germination and appressorium production. Then the mutants will be tested for virulence using a several standard infection assays. We will also use the Affymetrix GeneChips to assay global gene expression profiles in selected mutants, with a preference for transcription factor genes. We will focus on genes that are expressed prior to penetration.
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PROGRESS: 2005/04 TO 2009/04<BR>
OUTPUTS: The overall goal of our research is to identify the tool sets that the pathogen Phytophthora sojae uses to overcome the defenses of its host soybean, using both transcriptomics and proteomics. This proposal is focused on the earliest steps of infection prior to penetration of the host tissue, and during the first 24 hours of infection when the success of the infection is primarily determined. We determined global gene expression profiles using Affymetrix GeneChips, to identify sets of genes specifically expressed prior to and during infection. This work included improving the annotation of the Affymetrix soybean gene chip that contains P. sojae probes and developing new bioinformatic methods to normalize pathogen gene expression data obtained from RNA from infected tissue. We determined P. sojae gene expression profiles at 0, 3, 6 and 12 hr post-infection; the zero time point consisted of the mycelium used for inoculum. Recently we used ABI Solid technology to generate 50 million sequence tags from the 0 time point inoculum and 200 million tags from infected tissue 12 hr post-infection; these data are still being analyzed. We also used Affymetrix chips to determine P. sojae gene expression profiles of zoospore cysts following 0, 0.5, 1, 2, 4 and 8 hours of germination under conditions in which appressoria are formed (still culture) or not (gently shaking). Towards our goal of conducting proteomics analysis early infection structures, we carried out an analysis of the proteome of mycelia and germinating cysts using two-dimensional HPLC in conjunction with tandem mass spectrometry (MUDPit; Multidimensional Protein Identification Technology), in collaboration with Hayes McDonald at the Oak Ridge National Laboratory. <BR> PARTICIPANTS: Postdoctoral fellows Trudy Torto-Alalibo, Sucheta Tripathy, Dianjing Guo and Adriana Ferreira worked on this project. Graduate student Brian Smith and research associate Felipe Arredondo also worked on the project. Our partners in the project were Kurt Lamour (University of Tennessee, Knoxville) and Hayes McDonald (Oak Ridge national laboratory). Graduate student Alon Savidor (University of Tennessee, Knoxville), worked on this project, jointly supervised by these two partners. Collaborators were Yuanchao Wang (Nanjing Agricultural University). <P>
IMPACT: 2005/04 TO 2009/04<BR>
We found that some effector genes are strongly expressed at the outset of infection, and in pre-infection stages, while others are weakly expressed at the outset, but strongly induced. Some effectors that are expressed late trigger strong defense responses that can be suppressed by the effectors that are expressed earlier, suggesting coordinated programming of effector expression. Functional assays of the relevant effectors, carried out by the team of collaborator Yuanchao Wang, supported this hypothesis. Many stress-related responses were induced early in infection suggesting that stress might be a stimulus for some infection-induced genes. A bZIP transcription factor was identified that was induced early in infection, and a common sequence motif characteristic of bZIP transcription factor binding sites was found upstream of many effector genes. This transcription factor is being further analyzed. In the proteomics study, proteins produced in the vegetative (mycelium) and infective (germinating cyst) life stages of P. sojae were compared to similar proteins produced in the broad host range pathogen Phytophthora ramorum to identify candidate proteins involved in host range, early infection, and vegetative growth. Sixty-two candidates for early infection, 26 candidates for vegetative growth, and numerous proteins that may be involved in defining host specificity were identified. In addition, common life stage proteomic trends between the organisms were observed. In mycelia, proteins involved in transport and metabolism of amino acids, carbohydrates, and other small molecules were up-regulated. In the germinating cysts, up-regulated proteins associated with lipid transport and metabolism, cytoskeleton, and protein synthesis were observed. It appears that the germinating cyst catabolizes lipid reserves through the beta-oxidation pathway to drive the extensive protein synthesis necessary to produce the germ tube and initiate infection. Once inside the host, the pathogen switches to vegetative growth in which energy is derived from glycolysis and utilized for synthesis of amino acids and other molecules that assist survival in the plant tissue. These proteomic expression patterns were consistent with transcriptome patterns observed in the micorarray analysis and in earlier EST studies.