Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization

Abstract

Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota.

Fig. 1.

Experimental setup and characteristics of SWATH assay libraries and SWATH measurements. Comprehensive SWATH assay libraries were generated for M. extorquens and S. melonis based on various sample types and proteomic changes during growth on the leaf surface was subsequently studied by SWATH MS (A). SWATH assay library coverage of M. extorquens PA1 and S. melonis Fr1 (B). Gray shadings indicate the number of unambiguous peptide identifications per protein. Percentage of quantified and differentially regulated target proteins per strain (C). For (B) and (C), 100% corresponds to all annotated proteins within the genome. Dynamic quantification range of the two sample types analyzed by SWATH MS measurements of both strains (D). The abundance of all identified proteins was estimated using the best flyer approach (see Methods). Overview of differentially regulated proteins of M. extorquens and S. melonis during colonization of leaves or minimal media plates (E). 100% corresponds to the total number of differentially regulated proteins per strain.

Fig. 2.

Metabolic responses of S. melonis and M. extorquens during growth on plants. Up-regulation of TCA cycle proteins during leaf colonization of S. melonis Fr1 (A). Several pathways feeding into this central metabolic cycle indicate which plant derived substrates might be utilized during phyllosphere growth. Pathway and involved proteins of oxalate metabolism by M. extorquens PA1 (B). Arrows are shown in blue (up-regulated) and yellow (down-regulated) if corresponding proteins are significantly regulated (fold change ≥ 2, p value ≤ 0.05). Fold changes indicated below the figures are logarithmized (log2); a star indicates a p value ≤ 0.05.

Fig. 3.

False color images of M. extorquens cells showing infrared autofluorescence indicative of aerobic anoxygenic photosynthesis during leaf colonization. Overlay (A) of pictures acquired from the same field of view in phase contrast (B) and infrared fluorescence channels (C). Scale bar corresponds to 5 μm. Methylobacterium cells grown on minimal medium agar plates in the dark do not show background infrared autofluorescence indicative of anoxygenic photosynthesis (supplemental Fig. S3).

Fig. 4.

Number and distribution of homologs of the identified target proteins in other leaf colonizing Methylobacteria and leaf microbiota members. We identified homologous proteins (Protein Blast; >70% query sequence coverage, >70% sequence identity) of the target proteins involved in phyllosphere colonization by M. extorquens PA1 in the genomes of 31 leaf colonizing Methylobacteria (Methylobacterium sp. Leaf85 to Methylobacterium sp. Leaf469) of a recently established bacterial phyllosphere strain collection (48) (A). Hidden Markov Models based on assigned KEGG Orthology (KO) terms were used to identify functionally similar proteins in more distantly related leaf microbiota members (B). The color scheme indicates the number of putative homologs identified within the queried genomes of the distinct target strains. Proteins are formyl-CoA transferase (KO term: K07749), oxalyl-CoA decarboxylase (K01577) and the major facilitator transporter (K08177) of the oxalate metabolism, subunits L, M and the cytochrome c subunit of the photosynthesis apparatus (K08928, K08929 and K13992, respectively) as well as the alkanesulfonate monooxygenase homologs SsuD (K04091) and SfnG (K17228).

Fig. 5.

Proteome comparison of M. extorquens PA1 and S. melonis Fr1. KO term assignments of all differentially regulated proteins of M. extorquens PA1 and S. melonis Fr1 and their functional categories based on the KEGG Brite database. Blue and yellow bars represent the percentage of up and down-regulated proteins, respectively for each functional category. Asterisks indicate significant enrichments (p ≤ 0.05; Hypergeometric test, Benjamini-Hochberg FDR correction) of up- or down-regulated proteins of each functional category.