Gene expression and proteomic analysis of the formation of P. pachyrhizi appressoria

Authors: Stone, Christine; McMahon, Michael - Mike; Fortis, Laurie; Nunez, Alberto; SMYTHERS, GARY - National Cancer Institute (NCI, NIH); Luster, Douglas - Doug; Frederick, Reid

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2012
Publication Date: 6/22/2012
Citation: Stone, C.L., Mcmahon, M.B., Fortis, L.L., Nunez, A., Smythers, G.W., Luster, D.G., Frederick, R.D. 2012. Gene expression and proteomic analysis of the formation of P. pachyrhizi appressoria. Biomed Central (BMC) Genomics. 13:269.

Interpretive Summary: Phakopsora pachyrhizi is an obligate fungal pathogen causing Asian soybean rust (ASR). A dual approach was taken to examine the molecular and biochemical processes occurring during the development of appressoria, specialized infection structures by which P. pachyrhizi invades host plant tissue. Our dual approach consisted of identifying both the RNA transcripts that code for proteins expressed during appressorial formation, as well as, the proteins themselves. Several genes and proteins were identified that are expressed in P. pachyrhizi during appressorium formation. We discovered many proteins not previously identified in P. pachyrhizi germinating spores, representing the developmental stage that precedes appressorial formation. Three novel Phakopsora genes were identified, along with six genes that have only been found in related rust fungi. Eight proteins of unknown function were found to possess amino acid sequences that indicate the proteins could be secreted into the infection court surrounding the area penetrated by the fungus during infection. Two of these secreted proteins have sequences suggesting that they may play a role in the infection process, and in the response of the host plant to infection. Understanding the molecular and biochemical processes that these proteins play in the infection process may provide insight for developing targeted control measures and novel methods of disease management.

Technical Abstract: Phakopsora pachyrhizi is an obligate fungal pathogen causing Asian soybean rust (ASR). A dual approach was taken to examine the molecular and biochemical processes occurring during the development of appressoria, specialized infection structures by which P. pachyrhizi invades host plant tissue. Suppression subtractive hybridization (SSH) was utilized to generate a cDNA library enriched for transcripts expressed during appressoria formation. Two-dimensional gel electrophoresis and mass spectroscopy analysis was used to generate a partial proteome of proteins present during appressoria formation. Sequence analysis of 1133 expressed sequence tags (ESTs) revealed 238 non-redundant ESTs, of which, 53 percent had putative identities assigned. Twenty-nine of the non-redundant ESTs were found to be specific to this appressoria library, and did not occur in a previously constructed germinated urediniospore EST library. Analysis of proteins against a custom database of the appressoria-enriched ESTs plus Basidiomycota EST sequences available from NCBI revealed 253 proteins. Fifty-nine of these proteins were not previously identified in a partial proteome of P. pachyrhizi germinating urediniospores. Genes and proteins identified fell into functional categories of metabolism, cell cycle and DNA processing, protein fate, cellular transport, cellular communication and signal transduction, and cell rescue. However, 38 percent of ESTs and 24 percent of proteins matched only to hypothetical proteins of unknown function, or showed no similarity the current NCBI database. Three novel Phakopsora genes were identified from the EST library along with six potentially rust-specific genes. Protein analysis revealed eight proteins of unknown function which possessed classic secretion signals. Two of the extracellular proteins are reported as potential effector proteins. Several genes and proteins were identified that are expressed in P. pachyrhizi during appressorium formation. Understanding the molecular and biochemical processes that these proteins play in the infection process may provide insight for developing targeted control measures and novel methods of disease management.