Abstract
Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum.
Highlights
Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum is one of the most destructive and economically damaging bacterial diseases, affecting over 200 plant species, including important crops such as tomato, potato and peanut [1, 2]
Protocols for the collection of large amounts of apoplastic fluid from the roots are yet to be optimized. To overcome these two limitations and considering that R. solanacearum moves through an apoplastic environment during the first stage of the infection, we used leaf apoplast to study the intercellular plant proteome triggered in response to the pathogen
The bacterial multiplication did not correlate with the decreased leaf necrosis observed in Hawaii 7996, the protein content in the infected apoplast of this variety was higher than that of Marmande, and some proteins were induced in the apoplast of the tolerant cultivar on infection
Summary
Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum is one of the most destructive and economically damaging bacterial diseases, affecting over 200 plant species, including important crops such as tomato, potato and peanut [1, 2]. No major R-genes have been identified in tomato [15], where resistance has been reported to be mainly quantitative, involving two major quantitative trait loci (QTLs) (Bwr-12 and Bwr-6), and three minor loci (Bwr-3, Bwr-4 and 8 Bwr-8) (16 –21). These QTLs, defined in the cultivar Hawaii 7996, were found to be both strain- and environment-specific [16, 21]. The apoplast is the first battlefield where the plant has to face the pathogen before it reaches the xylem. Recent research provides evidence of plant apoplastic proteases playing an important role in immunity, with their activity often targeted by pathogen-derived effectors [28, 29]
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