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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOME SEQUENCE-BASED STRATEGIES FOR DETECTION & IDENTIFICATION OF PLANT PATHOGENIC PHYTOPLASMAS & SPIROPLASMAS, & VASCULAR WALLED BACTERIA

Location: Molecular Plant Pathology

Title: Functional analysis of a lipolytic protein, a potential phytoplasma pathogenicity factor

Authors
item Gedvilaite, Alma -
item Jomantiene, Rasa -
item Dabrisius, Jonas -
item Norkiene, Milda -
item Davis, Robert

Submitted to: Microbiological Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 7, 2013
Publication Date: N/A

Interpretive Summary: Very small bacteria known as phytoplasmas cause serious diseases in hundreds of plant species in agricultural and natural ecosystems around the world, but their mechanisms of pathogenicity are little understood. Phytoplasma genomes are some of the smallest known among pathogenic bacteria, a feature likely accounting for the continued inability to isolate phytoplasmas in laboratory culture. Yet, phytoplasma genomes are characterized by regions of repeated sequences that occur in genomic islands termed sequence-variable mosaics (SVMs). In this study, we hypothesized that an enzyme (lysophospholipase, LPL), that we predicted to be encoded by a gene (lpl) that is present in the genomic SVM region of Malaysian periwinkle yellows (MPY) phytoplasma, could behave as a phytoplasma virulence factor that acts upon phospholipid, a major constituent of cell membranes in the phytoplasma’s plant and insect hosts. For heterologous expression of the phytoplasmal lpl gene, we employed two expression/protein synthesis systems, eukaryotic (yeast) and prokaryotic (bacterial), and we assayed enzymatic activity of the LPL enzyme proteins synthesized in both systems. The results revealed that the MPY lpl gene encodes an enzymatically active protein that exhibits catalytic activities on phospholipids. This study provides the first direct, in vitro functional analysis of a putative virulence factor protein encoded by a phytoplasma genome, and the work reveals a newly recognized phytoplasmal factor potentially significant in pathogen-host interactions. This report will be of interest to research scientists nationally and internationally for pursuing the lpl gene and LPL protein as therapeutic targets, immediately impacting efforts to attain sustainable agricultural and forest ecosystems.

Technical Abstract: Wall-less bacteria known as phytoplasmas are obligate transkingdom parasites and pathogens of plants and insect vectors. These unusual bacteria possess some of the smallest genomes known among pathogenic bacteria, and have never been successfully isolated in artificial culture. Disease symptoms induced by phytoplasmas in infected plants include abnormal growth and often severe yellowing of leaves, but mechanisms involved in phytoplasma parasitism and pathogenicity are little understood. A phage based genomic island (sequence variable mosaic, SVM) in the genome of Malaysian periwinkle yellows (MPY) phytoplasma harbors a gene encoding membrane-targeted proteins, including a putative lysophospholipase (LPL), potentially important in pathogen-host interactions. Since some phytoplasmal disease symptoms could possibly be accounted for, at least in part, by damage and/or degradation of host cell membranes, we hypothesize that the MPY phytoplasma LPL is an active enzyme that acts as a phytoplasmal virulence factor. To test this hypothesis in part, functional analysis of the MPY lpl gene-encoded protein was carried out in vitro after its expression in bacterial and yeast hosts. The results demonstrated that the heterologously expressed phytoplasmal LPL is an active lipolytic enzyme and could possibly act as a pathogenicity factor in the plant, and/or insect, host.

Last Modified: 12/22/2014
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