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Novel Phakopsora pachyrhizi extracellular proteins are ideal targets for immunological diagnostic assays.

ABSTRACT: Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots.

SUBMITTER: Luster DG

PROVIDER: S-EPMC3346385 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Novel Phakopsora pachyrhizi extracellular proteins are ideal targets for immunological diagnostic assays.

Luster Douglas G DG McMahon Michael B MB Edwards H Herb HH Boerma Britney L BL Lewis Ivey Melanie L ML Miller Sally A SA Dorrance Anne E AE

Applied and environmental microbiology 20120323 11

Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (fo ...[more]

PMID: 22447596

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Project description: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.

Project description:The management of soybean rust (SBR) caused by the obligate fungus Phakopsora pachyrhizi mostly relies on the use of synthetic fungicides, especially in areas where the disease inflicts serious yield losses. The reliance on synthetic fungicides to manage this disease has resulted in resistance of P. pachyrhizi populations to most fungicides. In this study, bacteria isolated from diverse environments were evaluated for their biocontrol potential against P. pachyrhizi using soybean detached-leaf method and on-plant in the growth chamber, greenhouse, and field. Among 998 bacterial isolates evaluated using the detached-leaf method; 58% were isolated from plant-related materials, 27% from soil, 10% from insects, and 5% from other environments. Of the isolates screened, 73 were active (they had ⪖ 75% rust reduction) with an active rate of 7.3%. From the active isolates, 65 isolates were re-tested on-plant in the growth chamber for activity confirmation. In the confirmation test, 49 bacteria isolated from plant-related materials maintained their activity with a confirmation rate of 75%. The majority of bacteria with confirmed activity belonged to the taxonomic classes Bacilli and Gammaproteobacteria (70%). Active isolates were prioritized for greenhouse and field testing based on activity in the initial screen and confirmation test. Six bacterial isolates AFS000009 (Pseudomonas_E chlororaphis), AFS032321 (Bacillus subtilis), AFS042929 (Bacillus_C megaterium), AFS065981 (Bacillus_X simplex_A), AFS090698 (Bacillus_A thuringiensis_S), and AFS097295 (Bacillus_A toyonensis) were selected from those bacteria that maintained activity in the confirmation test and were evaluated in the greenhouse, and five among them were evaluated in the field. From the Alabama field evaluation, all bacterial isolates reduced rust infection as well as azoxystrobin (Quadris® at 0.3 L/ha) used as the fungicide control (P > 0.05). Moreover, the scanning electron micrographs demonstrated evidence of antagonistic activity of AFS000009 and AFS032321 against P. pachyrhizi urediniospores. Bacterial isolates that consistently showed activity comparable to that of azoxystrobin can be improved through fermentation and formulation optimization, developed, and deployed. These bacteria strains would provide a valuable alternative to the synthetic fungicides and could play a useful role in integrated disease management programs for this disease.

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Novel Phakopsora pachyrhizi extracellular proteins are ideal targets for immunological diagnostic assays.

Publications

Novel Phakopsora pachyrhizi extracellular proteins are ideal targets for immunological diagnostic assays.

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