Label-free quantitative secretome analysis of Xanthomonas oryzae pv. oryzae highlights the involvement of a novel cysteine protease in its pathogenicity
Introduction
Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice, causing up to 80% yield loss in the early stages of the rice growth. Based on the pathogenicity of Xoo, the interaction between bacteria and rice can be compatible or incompatible. During compatible interactions, bacteria successfully invade the plants either through the natural openings like hydathodes or through the wounds where it divides in the extracellular spaces of the host including apoplast and xylem. The Xoo-rice interaction is mediated largely by the secretory proteins from both the partners (plant and pathogen) in the host apoplast. In general, pathogen-secreted proteins are involved in degradation of plant cell walls, suppression of plant defense responses, and delivery of bacterial DNA and proteins into the host cytoplasm [1], while plant-secreted proteins are mainly involved in the identification of pathogen-derived elicitors and eliciting the defense response [2].
Given the fact that proteins play important roles in determining the fate of the rice-Xoo interaction, a few studies were conducted to identify those proteins [3], [4]. A study on plasma membrane proteins isolated from Xa21-transgenic rice suspension-cultured cells (SCCs) inoculated with incompatible (PXO99A) and compatible (DY89031) races of Xoo led to the identification of 20 differentially-modulated proteins, including H-ATPase, protein phosphatase, ascorbate peroxidase, and zinc-finger and Ca(2 +)-binding motif containing (C2 domain protein-like) proteins [3]. Their protein abundance correlated well with their transcript levels upon Xoo infection. Another report investigated the xylem sap proteins of an Italian rice cultivar Baldo, infected with Xoo and identified 324 Xoo proteins in that sap [4]. Furthermore, the knock-out mutant of PruProtein (Xoo1982) showed 34.2% reduction in the virulence in rice leaf [4]. These studies though increased our knowledge on the total and xylem sap proteomes, apoplastic secretory proteins remain yet to be largely investigated, in particular, the Xoo-secreted proteins.
Previously, we reported 139 Xoo-secreted proteins from in vitro SCCs and in planta systems using a gel-based proteomics approach [5]. In this extended study, we employed a label-free quantitative proteomics approach for in-depth apoplastic secretome analysis of the rice-Xoo interaction using both the in vitro SCCs and in planta systems.
Section snippets
Plant material preparation
Rice (Oryzae sativa) cultivar Dongjin was used as source material for preparation of apoplastic secretory proteins. Rice cultivars IR24 and IRBB1 were used for pathogenicity assay. Rice seeds were imbibed in sterilized water for two days at 4 °C, transferred to soil for germination for 7 days in growth chamber, followed by transfer of seedling pots to the greenhouse. Mature plants at flowering stage were used for Xoo infection.
Bacterial culture condition and rice leaf infection
Xanthomonas oryzae pv. oryzae strain K3 (virulence to rice cultivar
Identification of Xoo-secreted proteins
Xoo was either cultured on the liquid PSA (peptone, sucrose and agar) medium or used to inoculate leaves of a susceptible rice cultivar (in planta growth). During in planta growth, Xoo faces nutrient-deficient conditions. Therefore, in order to imitate the nutrient deficient condition, Xoo was first cultured on the N-starvation medium. However, the Xoo growth was very slow, and hence it was impossible to harvest sufficient bacteria for preparation of secreted proteins. Thus, we shifted to
Discussion
The apoplast plays an important role in the communication between the host(s) and plant pathogen(s) [22], [23]. Generally, pathogen-released signals, such as bacterial PGN, LPS, flagellin, or fungal chitin are recognized by the plasma membrane receptor complex in plant, like FLS2/BAK1, CERK1 [23], [24], [25], [26], [27], [28]. However, due to technical limitations, the apoplastic protein composition during host-pathogen interaction is still poorly understood. In general, in vitro secreted
Conclusion
In this study, we applied an integrated SDS-PAGE coupled with MudPIT proteomics approaches to study the secreted proteins from Xoo under in vitro and in planta conditions, resulting in identification of 404 and 323 secretory proteins, respectively. A large proportion of proteins related to catalytic, transporter, and ATPase activities were highly enriched. Several membrane bound proteins related with the nutrient uptake, protein secretion, and polysaccharides outputs were highly detected, which
Author contributions
Y.W., R.G. and W.S. performed the proteomics experiments and analyzed the data. H.H.H, S.E.L, J.W. and S.R.P. maintained the bacterial cultures and performed infection treatments, knock-out, complementation line generation and pathogenicity assays. Y.W., R.G. wrote the paper and K.Y.K., S.R.P, S.T.K. designed the experiments. R.R. and G.K.A. discussed the results, and critically reviewed and edited the paper. All authors discussed the results and commented on the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Conflict of interest
The authors have declared that no competing interests exist.
Acknowledgements
This study was financially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2016H1D3A1937706) provided to RG and STK and “Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center, PJ011038)” Rural Development Administration, Republic of Korea, given to STK. YW was supported by Max Planck Institute for Plant Breeding Research, and Alexander-Bayer Fellowship from
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These authors contribute equally to this work.