Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress

Zijing Pu,Asumi Tago,Satoshi Natsume,Hisashi Hirano,Motoki Shimizu,Yoko Ino,Keiichi Okazaki,Kentaro Kaneko,Yoshitaka Sano,Eigo Fukai,Daniel J Shea,Ryo Fujimoto,Yayoi Kimura,Shin-Ichi Fuji

doi:10.3389/fpls.2016.00031

Abstract

Fusarium oxysporum f.sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change > = 2-fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and 10 of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.

Highlights

The xylem vessel is formed via the programmed death of xylem tracheary elements, followed by connection of the elements to long tubes
Ligat et al (2011), in their study using liquid chromatographytandem mass spectrometry (LC-MS/MS) and Brassica EST and cDNA sequences, classified 189 B. oleracea xylem sap proteins into eight of the nine functional classes previously defined for Arabidopsis thaliana cell wall proteins; most of them belong to those acting on carbohydrates (e.g., β-1,3-glucanases and chitinase), oxido-reducatses and proteases (29.2, 23.8, and 17.1%, respectively), which are abundant/dominating protein classes in other plant xylem saps
In the susceptible cultivar (Delicious), subtle staining was observed at 1 dpi at the outer layers of the root apex and the lateral root primordial (LRP) (Figure 1A), supporting the previous study that the pathogen penetrates the surface of root apex and LRP, and traverses tissues outside the vascular cylinder in the primary phase of initial Foc penetration to A. thaliana (Diener, 2012)

Summary

Introduction

The xylem vessel is formed via the programmed death of xylem tracheary elements, followed by connection of the elements to long tubes. Earlier studies reported relatively abundant xylem sap proteins such as chitinase, peroxidase, and β-1,3-glucanases in cucumber (Masuda et al, 1999; Sakuta and Satoh, 2000) and tomato (Rep et al, 2002), using one-dimensional gel electrophoresis (1-DE). Ligat et al (2011), in their study using liquid chromatographytandem mass spectrometry (LC-MS/MS) and Brassica EST and cDNA sequences, classified 189 B. oleracea xylem sap proteins into eight of the nine functional classes previously defined for Arabidopsis thaliana cell wall proteins; most of them belong to those acting on carbohydrates (e.g., β-1,3-glucanases and chitinase), oxido-reducatses and proteases (29.2, 23.8, and 17.1%, respectively), which are abundant/dominating protein classes in other plant xylem saps. Other small protein classes reported in Ligat et al (2011), such as proteins related to lipid metabolism (4.9%), proteins with domains interacting with carbohydrates or proteins (e.g., lectin and protease inhibitor) (4.9%), miscellaneous proteins (8.5%) and proteins involved in signaling (5.5%), have been described in Glycine max, B. napus, and Oryza sativa (Kehr et al, 2005; Djordjevic et al, 2007; Aki et al, 2008)

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