1a.Objectives (from AD-416):
SASL is conducting research into the mechanisms by which plant-beneficial bacteria, such as Pseudomonas fluorescens PF5, colonize subterranean plant parts and suppress plant disease. Currently we are studying the role of the global regulatory molecule Vfr in these two processes. As this is a global regulatory molecule a system-wide or "omics" approach is required to determine all of the proteins in the Vfr regulatory cascade. One of the most cost-effective methods to do this is via proteomics, where the total protein profiles of the wild-type strain (PF5) and a strain with a mutation in vfr (and thus a non-producer of Vfr) are identified and compared under identical growth conditions. Proteins that are present in different concentrations in the two protein profiles are directly or indirectly regulated by Vfr. SASL does not have the infrastructure, equipment, or expertise to perform the protein separation and identification procedures on this scale. The cooperator has the technology, personnel, and infrastructure necessary to complete this analysis in a timely and cost-effective manner.
1b.Approach (from AD-416):
Grow the wildtype (PF5) and mutant, containing a mutation in the vfr gene, under identical conditions in a synthetic cucumber root exudate medium. Harvest cultures from both bacterial strains at three separate time points and freeze on dry ice. Total intracellular protein from bacterial biomass from these three time points is differentially stained by strain and then separated by two-dimensional gel electrophoresis. Individual proteins differentially expressed in the two strains are individually harvested and identified by mass spectroscopy (MALDI-TOF).
The first objective of the project was to use proteomic approaches to investigate proteins involved in the colonization of subterranean plant parts by plant-beneficial bacteria, such as Pseudomonas fluorescens Pf-5, to suppress plant disease. Specifically, this objective dealt with determining the role of the regulatory protein Vfr in these processes. Proteomic analysis revealed a number of proteins regulated by Vfr including protein transporters for uptake of polyamines and other compounds related to stress tolerance in bacteria. Polyamine uptake and stress tolerance have previously been identified as traits important to colonization of plant roots. This partially explains why a derivative strain, containing a mutation in vfr, was greatly impacted in root colonization. The second objective of the project was to identify proteins involved in hypovirulence of the important plant pathogen Rhizoctonia solani. Hypovirulent strains of R. solani are dramatically diminished in pathogenesis, and proteomic analysis of hypovirulent isolates may give us clues as to mechanisms important for pathogenesis by this fungus. Work over the past year focused on the second objective of this project. ARS scientists in Beltsville, MD provided freeze dried mycelia of select strains of R. solani that differed in growth and virulence to Applied Biomics. Applied Biomics performed 2-D DIGE to separate the proteomes of these strains and used MALDI-TOF to identify proteins differentially produced by virulent and hypovirulent strains. Approximately two hundred proteins have been isolated that differ in levels of production between virulent and hypovirulent strains. We are currently working towards identifying these proteins.