Gene expression and proteomic analysis of the formation of Phakopsora pachyrhizi appressoria.
BACKGROUND
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 a host plant. 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 were used to generate a partial proteome of proteins present during appressoria formation.


RESULTS
Sequence analysis of 1133 expressed sequence tags (ESTs) revealed 238 non-redundant ESTs, of which 53% had putative identities assigned. Twenty-nine of the non-redundant ESTs were found to be specific to the appressoria-enriched cDNA library, and did not occur in a previously constructed germinated urediniospore cDNA library. Analysis of proteins against a custom database of the appressoria-enriched ESTs plus Basidiomycota EST sequences available from NCBI revealed 256 proteins. Fifty-nine of these proteins were not previously identified in a partial proteome of P. pachyrhizi germinated 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% of ESTs and 24% of proteins matched only to hypothetical proteins of unknown function, or showed no similarity to sequences in the current NCBI database. Three novel Phakopsora genes were identified from the cDNA 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.


CONCLUSIONS
Several genes and proteins were identified that are expressed in P. pachyrhizi during appressoria formation. Understanding the role that these genes and proteins play in the molecular and biochemical processes in the infection process may provide insight for developing targeted control measures and novel methods of disease management.
USDA-Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Fort Detrick, MD 21702, USA.
Christine L
CL
Stone
Michael B
MB
McMahon
Laurie L
LL
Fortis
Alberto
A
Nuñez
Gary W
GW
Smythers
Douglas G
DG
Luster
Reid D
RD
Frederick
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D030541:Databases, Genetic; D020224:Expressed Sequence Tags; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics; D025301:Hyphae / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013025:Glycine max / Q000382:microbiology
D005656:Fungal Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger