Moreira LM , Soares MR , Facincani AP , Ferreira CB , Ferreira RM , Ferro MIT , Gozzo FC , Felestrino ÉB , Assis RAB , Garcia CCM , Setubal JC , Ferro JA , de Oliveira JCF .

	Fig. 1. Representative 2D proteome images of the Xac proteins. a Xac grown in nutrient broth (NB) medium. b Xac grown in inducing virulence medium (XAM1) for 24 h, simulating 1DAI in plant. c Xac exudates from host citrus plants at 3DAI. d Xac exudates from host citrus plants at 5DAI. All samples were separated in 18 cm IPG strips across a linear pH range (4–7) using IEF in the first dimension and 12.5% SDS-PAGE in the second. Gels were stained with Coomassie blue. Numbers indicate the protein spots identified by mass spectrometry analysis (Tables 1). All experiments were done in triplicate
	Fig. 2. Comparative analysis of functional protein profile of Xac under different conditions. a Comparison of differentially abundant proteins (up-regulated is shown in green and down-regulated in red) according to the categories used in the genome annotation [131]: I - Intermediary metabolism, II - Biosynthesis of small molecules, III - Macromolecule metabolism, IV - Cell structure, V - Cellular processes, VI - Mobile genetic elements, VII - Pathogenicity, virulence, and adaptation, VIII - Hypothetical, IX - ORFs with undefined category. b Differentially abundant proteins according to status of expression in infectious conditions and the number of proteins in each category. Three of these categories (A/B/LPS, IRON and REDOX) are common in both analyses. For details, check Table 1. Categories: A/B/LPS - Adhesion, biofilm, and LPS modulation; ROSd/OSM - Reactive species depletion and osmotic control; IRON - iron acquisition and metabolism; HYP - Hypothetical proteins; REDOX - Reduction and oxidation related proteins; UC - Uncharacterized class; A/L/PURPYR - Amino acids, lipids, purines and pyrimidines metabolism; ENER-MET - Energy metabolism; DEG-ENZ - Degrading enzymes; CELL-P - Cellular process
	Fig. 3. Functional overview of differential expression proteins in infection condition. The order of circles and colors represent the day where they were detected and expression level, respectively (see legend)
	Fig. 4. Analysis of the phenotypic profile of virulence in the mutant XAC3981, located in a putative operon with XAC3982 and XAC3983 genes (gray arrows). The mutant XAC3981 led to a marked reduction in the virulence phenotype in both Citrus sinensis and Citrus limonia after 14DAI. The growth curve of the mutant in vivo showed less bacterial titration along the infectious process, which could explain the absence of observed phenotypes. This mutated gene is flanked by htrA (upstream), and by two hypothetical proteins (downstream) whose orthologous pairs were found only in bacteria of c (See Additional file 6: Figure S4)
	Fig. 5. Integrated outlook of proteins secreted between 1-5DAI. a During the first hours of infection, secretion system structural proteins, and proteins related with motility and quorum sensing are secreted. After recognition of PAMPs, a cascade of reactions culminates in the activation of PTI in order to avoid penetration of bacteria inside plant tissues increasing the ROS production during POB. Subsequently, Xac secretes effector proteins to overcome plant defenses and promote bacterial virulence. These processes may occur between 0 and 24 h after infection. b After 24 h of infection, Xac secretes higher amounts of proteins related with reactive species depletion and osmotic adaptation (ROSd/OSM), iron uptake and metabolism, biofilm formation, and LPS modulation in order to protect themselves against the stress caused by POB. c After activation of virulence related proteins during the first 24 h (pink), a protein repertoire involved in ROS depletion, EPS biosynthesis and LPS modulation, iron uptake and metabolism, and biofilm formation is active and working in an integrated manner between 1 and 5 DAI

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