Label-free quantitative secretome analysis of Xanthomonas oryzae pv. oryzae highlights the involvement of a novel cysteine protease in its pathogenicity.

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases resulting in a huge loss of the total rice productivity. The initial interaction between rice and Xoo takes place in the host apoplast and is mediated primarily by secretion of various proteins from both partners. Yet, such secretory proteins remain to be largely identified and characterized. This study employed a label-free quantitative proteomics approach and identified 404 and 323 Xoo-secreted proteins from in vitro suspension-cultured cells and in planta systems, respectively. Gene Ontology analysis showed their involvement primarily in catalytic, transporter, and ATPase activities. Of a particular interest was a Xoo cysteine protease (XoCP), which showed dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence. Besides, a parallel analysis of in planta rice-secreted proteins resulted in identification of 186 secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. Identified secretory proteins were exploited to shed light on their possible role in the rice-Xoo interaction, and that further deepen our understanding of such interaction. BIOLOGICAL SIGNIFICANCE: Xanthomonas oryzae pv. oryzae (Xoo), causative agent of bacterial blight disease, results in a huge loss of the total rice productivity. Using a label-free quantitative proteomics approach, we identified 727 Xoo- and 186 rice-secreted proteins. Functional annotation showed Xoo secreted proteins were mainly associated with the catalytic, transporter, and ATPase activities while the rice secreted proteins were mainly associated with the catalytic, antioxidant, and electron carrier activities. A novel Xoo cysteine protease (XoCP) was identified, showing dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence.

Secretome analysis of the rice bacterium Xanthomonas oryzae (Xoo) using in vitro and in planta systems.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice, and that severely affects yield loss (upto 50%) of total rice production. Here, we report a proteomics investigation of Xoo (compatible race K3)-secreted proteins, isolated from its in vitro culture and in planta infected rice leaves. 2DE coupled with MALDI-TOF-MS and/or nLC-ESI-MS/MS approaches identified 139 protein spots (out of 153 differential spots), encoding 109 unique proteins. Identified proteins belonged to multiple biological and molecular functions. Metabolic and nutrient uptake proteins were common up to both in vitro and in planta secretomes. However, pathogenicity, protease/peptidase, and host defense-related proteins were highly or specifically expressed during in planta infection. A good correlation was observed between protein and transcript abundances for nine proteins secreted in planta as per semiquantitative RT-PCR analysis. Transgenic rice leaf sheath (carrying PBZ1 promoter::GFP cell death reporter), when used to express a few of the identified secretory proteins, showed a direct activation of cell death signaling, suggesting their involvement in pathogenicity related with secretion effectors. This work furthers our understanding of rice bacterial blight disease, and serves as a resource for possible translation in generating disease resistant rice plants for improved seed yield.