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PeerJ
2016 Jan 01;4:e1734. doi: 10.7717/peerj.1734.
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Unravelling potential virulence factor candidates in Xanthomonas citri. subsp. citri by secretome analysis.
Ferreira RM
,
Moreira LM
,
Ferro JA
,
Soares MRR
,
Laia ML
,
Varani AM
,
de Oliveira JCF
,
Ferro MIT
.
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Citrus canker is a major disease affecting citrus production in Brazil. It''s mainly caused by Xanthomonas citri subsp. citri strain 306 pathotype A (Xac). We analysed the differential expression of proteins secreted by wild type Xac and an asymptomatic mutant for hrpB4 (ΔhrpB4) grown in Nutrient Broth (NB) and a medium mimicking growth conditions in the plant (XAM1). This allowed the identification of 55 secreted proteins, of which 37 were secreted by both strains when cultured in XAM1. In this secreted protein repertoire, the following stand out: Virk, Polyphosphate-selective porin, Cellulase, Endoglucanase, Histone-like protein, Ribosomal proteins, five hypothetical proteins expressed only in the wild type strain, Lytic murein transglycosylase, Lipoprotein, Leucyl-tRNA synthetase, Co-chaperonin, Toluene tolerance, C-type cytochrome biogenesis membrane protein, Aminopeptidase and two hypothetical proteins expressed only in the ΔhrpB4 mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isomerase and seven hypothetical proteins were detected in both strains, suggesting that there was no relationship with the secretion mediated by the type III secretory system, which is not functional in the mutant strain. Also worth mentioning is the Elongation factor Tu (EF-Tu), expressed only the wild type strain, and Type IV pilus assembly protein, Flagellin (FliC) and Flagellar hook-associated protein, identified in the wild-type strain secretome when grown only in NB. Noteworthy, that FliC, EF-Tu are classically characterized as PAMPs (Pathogen-associated molecular patterns), responsible for a PAMP-triggered immunity response. Therefore, our results highlight proteins potentially involved with the virulence. Overall, we conclude that the use of secretome data is a valuable approach that may bring more knowledge of the biology of this important plant pathogen, which ultimately can lead to the establishment of new strategies to combat citrus canker.
Figure 2. Comparative analysis of Xac secretome.(A) Comparison highlighting 55 total detected proteins, correlating culture mediums (XAM1 × NB—A1) and strains (Xac and Δ hrpB4—A2) analysed. The two circles represent the total proteins detected. (B) Categorization of annotated protein functions, adapted from Da Silva et al. (2002): I, Intermediary metabolism; II, Biosynthesis of small molecules; III, Metabolism of macromolecules; IV, Cell structure; V, Cellular processes; VI, Mobile genetic elements, VII, Pathogenicity; virulence, and adaptation, VIII, Hypothetical/Conserved hypothetical genes; XI, Without a defined function. (C) Venn Diagram highlighting each of the proteins detected under the four different conditions tested. Proteins annotated as hypothetical are highlighted in red. The same Venn diagram is shown on a smaller scale but with only the numbers of the proteins detected in each condition.
Figure 3. Model highlighting proteins related to virulence characterized in comparative proteomics.Three of these proteins (A×21, FliC, and Ef-Tu) are characterized as PAMPs capable of inducing a PTI response, which can consequently lead to the increased synthesis of chorismate. Chorismate is a precursor in the synthesis of IAA, Trp, and SA, which are involved in the ROS response and induction of plant defence. However, the secretion of PheA may reduce plant defence responses by shifting the metabolism of chorismate to prephenate synthesis. Also highlighted is the Peh-1 protein, which is an endopolygalacturonase regulated by HrpX and cellulase (Egl), and endo-1,4-beta-glucanase (EngXca), which is regulated by RpfF. Endopolygalacturonase and cellulase degrade pectin polymers and cellulose in the plant cell wall, respectively. Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan; IAA, indole-3-acetic acid; SA, salicylic acid; PTI, (PAMP) -triggered immunity; PAMPs, pathogen-associated molecular pattern; ROS, reactive oxygen species; EF-Tu, elongation factor Tu, EFR - EF-Tu receiver; A×21, Ax21-triggered immunity; FliC, flagellin; FLS2, flagellin sensitive receiver 2; PheA, chorismate mutase; Peh-1, endopolygalacturonase; Egl and EngXca, cellulase. HrpG/HrpX and RpfC/RpfF, two component systems (sensory and regulatory proteins, respectively); Black, detected in Xac and ΔhrpB4 grown in XAM1; Red, detected exclusively in Xac grown in XAM1; Orange, detected in Xac grown in BOTH media (NB and XAM1); Blue, detected exclusively in Xac grown in NB. Dotted arrows indicate regulation.
Figure 4. Model highlighting proteins related to adaptation in comparative proteomics.These proteins were grouped into five groups: lipoproteins, porins, outer membrane proteins, secreted proteins, and ribosomal proteins. HrpG/HrpX, PhoQ/PhoP and PhoR/PhoB—two component systems (sensory and regulatory proteins, respectively); Black, detected in Xac and ΔhrpB4 grown in XAM1; Red, detected exclusively in Xac grown in XAM1; Orange, detected in Xac grown in both media (NB and XAM1); Blue, detected exclusively in Xac grown in NB. Dotted arrows indicate regulation; Pi/PolyPi, inorganic phosphate and inorganic polyphosphate.
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