Protease Activities Triggered by Ralstonia solanacearum Infection in Susceptible and Tolerant Tomato Lines

Abstract

<jats:p>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 <jats:italic>R. solanacearum</jats:italic>, 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 <jats:italic>R. solanacearum</jats:italic>. Here, we explore the possibility that the limited <jats:italic>R. solanacearum</jats:italic> 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 <jats:italic>R. solanacearum</jats:italic> 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 <jats:italic>R. solanacearum</jats:italic> 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 <jats:italic>R. solanacearum</jats:italic>, 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 <jats:italic>R. solanacearum</jats:italic>.</jats:p>

Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation

“…Missing values were then imputed from the normal distribution using the standard values in Perseus (36) (downshift 1.8 and width 0.3). To screen for potential cathepsin targets, we analyzed the data set using the moderated t statistics as demonstrated in Planas-Marque's et al , (42). This method outperforms the standard t statistics (38) as it reduces the sample variance toward an estimated value, based on the average protein variance.…”

Interrogating Plant-Microbe Interactions with Chemical Tools: Click Chemistry Reagents for Metabolic Labeling and Activity-Based Probes

“…Vacuolar processing enzymes (VPEs) [ 43 ] and serine hydrolases were also studied in this pathogen-plant system using the aza-epoxide probe AMS101 and a fluorophosphonate with rhodamine reporter tag, respectively [ 49 ]. Similarly, DCG-04 and FP-biotin were used to examine protease activities in disease-resistant and -susceptible varieties of tomato infected with the soil-borne bacterial pathogen Ralstonia solanacearum [ 50 ]. ABPs have also been applied to profiling fungal enzymes [ 51 ] and enzyme activities in response to fungal species, including pathogens such as Cladosporium fulvum [ 52 , 53 ].…”

The Apoplast: A Key Player in Plant Survival

“…Apoplastic papain-like cysteine proteases (PLCP) play an important role in apoplastic immunity by releasing small peptides that serve as damage-associated molecular patterns (DAMPs) [183]. In addition, it is worth noting that PLCPs and serine hydrolases (SHs) protease families are up-regulated upon pathogen infection or targeted by pathogen-derived inhibitors [184]. For example, Ziemann et al [185] pointed out that Zip1 peptide, which is released in the presence of PLCP, leads to SA accumulation in maize leaves.…”

“…The recognition of microbe-associated molecular patterns (MAMPs) triggers several cellular response processes, such as alteration of ionic current between cell membranes which alkalizes intercellular space, increasing the cytoplasmic calcium ion concentration [184], and the biosynthesis of ethylene stress hormone which is activated by 1-aminocyclopropane-1-carboxylic acid-synthase (ACC-synthase). Other primary responses include activation of mitogen-activated protein kinases (MAPKs), which in turn activate transcription factors [205].…”