FUNCTIONAL DIVERSITY IN THE PHYLLOSPHERE: A METAPROTEOMIC APPROACH

Eder da Costa dos Santos (ESALQ/USP); David Crowley (UCR); Marcio Rodrigues Lambais (ESALQ/USP)

The leaf surface of the trees (phyllosphere) contains dense, complex and diverse microbial communities, which are highly associated with the plant species. Genomic approaches have recently revealed the microbial composition in these habitats. However, the knowledge of microbial community structure in a given environment does not necessarily lead to useful information on the metabolism of the microbial populations.  Therefore, our goal was to develop a novel approach to directly identify microbial proteins in the phyllosphere of tree species from Atlantic Forest, in order to better understand the functional roles of the microorganisms in these environments. For this study, the total proteins in the microbial biofilm on the leaves were extracted using sonication for 2-DE analysis. Due to the presence of compounds interfering with the IEF separation of proteins, horizontal and vertical streaking was observed on the gels. Different cell recovery and purification methods were tested for protein extraction and purification prior to 2-DE analysis. A protocol has been optimized, allowing high resolution separation of more than 500 discrete proteins. The metaproteomes of four tree species from the Atlantic forest are being compared. Our results showed significant differences in protein profiles in the phyllosphere of the tree species evaluated. The spots with differential accumulation in the phyllospheres of different tree species will be excised from the gels, treated with trypsin and identified by MALDI-TOF/TOF peptide-mass fingerprint analysis, followed by de novo sequencing by tandem mass spectrometry and database comparison. The metaproteomic analyses carried out will allow us to tracking new functional genes and metabolic pathways and identifying proteins preferentially associated with specific stresses. This is the first report of a large-scale identification of proteins of the phyllosphere using a metaproteomic approach.