Proteome profile and genome refinement of the tomato-pathogenic bacterium Clavibacter michiganensis subsp. michiganensis

Authors: PERITORE-GALVE, F. CHRISTOPHER - Cornell University; SCHNEIDER, DAVID - Former ARS Employee; Yang, Yong; Thannhauser, Theodore - Ted; SMART, CHRISTINE - Cornell University; Stodghill, Paul

Submitted to: Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2019
Publication Date: 1/16/2019
Citation: Peritore-Galve, F., Schneider, D.J., Yang, Y., Thannhauser, T.W., Smart, C.D., Stodghill, P. 2019. Proteome profile and genome refinement of the tomato-pathogenic bacterium Clavibacter michiganensis subsp. michiganensis. Proteomics. https://doi.org/10.1002/pmic.201800224.
DOI: https://doi.org/10.1002/pmic.201800224

Interpretive Summary: The internationally quarantined bacterium, Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial canker of tomato, and can be economically devastating for growers in all tomato-growing regions of the world. The genome for Cmm has been sequenced and annotated using computational methods. Although some of its annotations have been validated experimentally, few attempts have been made to validate the entire annotation using high-throughput methods. This research generated proteomics datasets and used them to identify missing and misannotated genes in the Cmm genome. Thirty-four (34) new genes were identified, and 82 existing gene annotations were found to require changes. Several of these fall within a known pathogenicity island. Statistical analysis shows differences in digestion pattern of intra-cellular and extra-cellular proteins, which may be evidence of mechanisms that these bacteria use to evade plant defenses. The improved genome annotations and the evidence of extra-cellular protein degradation give us a better understanding of how Cmm might survive and cause disease in the plant. This knowledge will enable other scientists to develop better pest-resistant crop varieties and control strategies.

Technical Abstract: The internationally quarantined bacterium, Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial canker of tomato, and can be economically devastating for growers in all tomato-growing regions of the world. The genome for Cmm has been sequenced and annotated using computational methods. Although some of its annotations have been validated experimentally, few attempts have been made to validate the entire annotation using high-throughput methods. In this paper, we describe how we used a proteogenomic approach to validate the Cmm genome. We analyzed the pellet and supernatant from Cmm samples using nano-liquid chromatography and tandem mass spectrometry (nano-LC-MS/MS). Spectra were compared against a six-frame translation of the Cmm genome, and stringent FDR cutoffs were used to minimize the likelihood of incorrectly identified peptides. Multiple biological replicates were used to further enhance confidence in peptide predictions. Thirty-four (34) new genes were identified, 82 existing gene annotations were found to require changes, and one annotated psuedogene was found to produce a product. Several of these genes fall within a known pathogenicity island. The Kullback-Leibler (KL) divergence was applied to test the abundance of non-canonical residues at the N-terminus of non-tryptic peptides from the pellet and from the supernatant. In the pellet proteins, phenylalanine, histidine, tyrosine, and asparagine were over-represented, and in the supernatant, alanine, methionine, asparagine, and tyrosine were over-represented. The improved genome annotations and the evidence of extra-cellular protein degradation give us a better understanding of how Cmm might survive and cause disease in the plant.