Translational and post-translational protein modifications play a key role in the response of plants to pathogen
infection. Among the latter, phosphorylation is critical in modulating
protein structure, localization and interaction with other partners. In this work, we used a multiplex staining approach with 2D
gels to study quantitative changes in the
proteome and phosphoproteome of Flavescence dorée-affected and recovered 'Barbera' grapevines, compared to healthy plants.
RESULTS: We identified 48
proteins that differentially changed in abundance, phosphorylation, or both in response to Flavescence dorée phytoplasma infection. Most of them did not show any significant difference in recovered plants, which, by contrast, were characterized by changes in abundance, phosphorylation, or both for 17
proteins not detected in infected plants. Some
enzymes involved in the
antioxidant response that were up-regulated in infected plants, such as
isocitrate dehydrogenase and
glutathione S-transferase, returned to healthy-state levels in recovered plants. Others belonging to the same functional category were even down-regulated in recovered plants (
oxidoreductase GLYR1 and
ascorbate peroxidase). Our proteomic approach thus agreed with previously published biochemical and RT-qPCR data which reported down-regulation of scavenging
enzymes and accumulation of H2O2 in recovered plants, possibly suggesting a role for this molecule in remission from
infection. Fifteen differentially phosphorylated
proteins (| ratio | > 2, p < 0.05) were identified in infected compared to healthy plants, including
proteins involved in photosynthesis, response to stress and the
antioxidant system. Many were not differentially phosphorylated in recovered compared to healthy plants, pointing to their specific role in responding to
infection, followed by a return to a steady-state phosphorylation level after remission of symptoms. Gene ontology (GO) enrichment and statistical analysis showed that the general main category "response to stimulus" was over-represented in both infected and recovered plants but, in the latter, the specific child category "response to biotic stimulus" was no longer found, suggesting a return to steady-state levels for those
proteins specifically required for defence against pathogens.
CONCLUSIONS: Proteomic data were integrated into
biological networks and their interactions were represented through a hypothetical model, showing the effects of
protein modulation on primary metabolic ways and related secondary pathways. By following a multiplex-staining approach, we obtained new data on grapevine
proteome pathways that specifically change at the phosphorylation level during phytoplasma infection and following recovery, focusing for the first time on phosphoproteome changes during pathogen
infection in this host.