Ralstonia solanacearum is a bacterial vascular pathogen causing devastating bacterial wilt. In the field, resistance against this pathogen is quantitative and is available for breeders only in tomato and eggplant. To understand the basis of resistance to R. solanacearum in tomato, we investigated the spatio-temporal dynamics of bacterial colonization using non-invasive live monitoring techniques coupled to grafting of susceptible and resistant varieties. We found four ‘bottlenecks’ that limit the bacterium in resistant tomato: root colonization, vertical movement from roots to shoots, circular vascular bundle invasion, and radial apoplastic spread in the cortex. Radial invasion of cortical extracellular spaces occurred mostly at late disease stages but was observed throughout plant infection. This study shows that resistance is expressed in both root and shoot tissues, and highlights the importance of structural constraints to bacterial spread as a resistance mechanism. It also shows that R. solanacearum is not only a vascular pathogen but spreads out of the xylem, occupying the plant apoplast niche. Our work will help elucidate the complex genetic determinants of resistance, setting the foundations to decipher the molecular mechanisms that limit pathogen colonization, which may provide new precision tools to fight bacterial wilt in the field.

Xylem vascular wilt pathogens cause some of the most devastating diseases in plants. Proliferation of these pathogens in the xylem tissue causes massive disruption of water and mineral transport, resulting in severe wilting and death of the infected plants. Upon reaching the xylem vascular tissue, these pathogens multiply profusely and later spread vertically within the xylem sap and horizontally between vessels and to the surrounding tissues. Plant resistance to these pathogens is very complex. One of the most effective defense responses in resistant plants is the formation of physico-chemical barriers in the xylem tissue upon pathogen perception. Vertical spread within the vessel lumen is restricted by structural barriers namely, tyloses and gels. Further, horizontal spread to the apoplast and surrounding healthy vessels and tissues is prevented by vascular coating of the colonized vessels mainly with lignin and suberin. Both vertical and horizontal barriers compartmentalize the pathogen at the site of infection and contribute to their elimination. Induction of these defenses must be tightly coordinated, both in time and space to avoid detrimental consequences for the plant such as cavitation and embolism. Here we discuss the current knowledge on mechanisms underlying plant inducible structural barriers against major xylem colonizing pathogens. This knowledge may be applied to engineering metabolic pathways of vascular coating compounds in specific cells, to produce resistant plants against xylem colonizers.

Phytaspases are Asp-specific subtilisin-like plant proteases that have been likened to animal caspases with respect to their regulatory function in programmed cell death (PCD). We identified twelve putative phytaspase genes in tomato that differed widely in expression level and tissue-specific expression patterns. Most phytaspase genes are tandemly arranged on tomato chromosomes one, four, and eight, and many belong to taxon-specific clades, e.g. the P69 clade in the nightshade family, suggesting that these genes evolved by gene duplication after speciation. Five tomato phytaspases (SlPhyts) were expressed in N. benthamiana and purified to homogeneity. Substrate specificity was analyzed in a proteomics assay and with a panel of fluorogenic peptide substrates. Similar to animal caspases, SlPhyts recognized an extended sequence motif including Asp at the cleavage site. Clear differences in cleavage site preference were observed implying different substrates in vivo and, consequently, different physiological functions. A caspase-like function in PCD was confirmed for five of the seven tested phytaspases. Cell death was triggered by ectopic expression of SlPhyts 2, 3, 4, 5, 6 in tomato leaves by agro-infiltration, as well as in stably transformed transgenic tomato plants. SlPhyts 3, 4, and 5 were found to contribute to cell death under oxidative stress conditions.

The causal agent of bacterial wilt, Ralstonia solanacearum, is a soilborne pathogen that invades plants through their roots, traversing many tissue layers until it reaches the xylem, where it multiplies and causes plant collapse. The effects of R. solanacearum infection are devastating, and no effective approach to fight the disease is so far available. The early steps of infection, essential for colonization, as well as the early plant defense responses remain mostly unknown. Here, we have set up a simple, in vitro Arabidopsis thaliana–R. solanacearum pathosystem that has allowed us to identify three clear root phenotypes specifically associated to the early stages of infection: root-growth inhibition, root-hair formation, and root-tip cell death. Using this method, we have been able to differentiate, on Arabidopsis plants, the phenotypes caused by mutants in the key bacterial virulence regulators hrpB and hrpG, which remained indistinguishable using the classical soil-drench inoculation pathogenicity assays. In addition, we have revealed the previously unknown involvement of auxins in the root rearrangements caused by R. solanacearum infection. Our system provides an easy-to-use, high-throughput tool to study R. solanacearum aggressiveness. Furthermore, the observed phenotypes may allow the identification of bacterial virulence determinants and could even be used to screen for novel forms of early plant resistance to bacterial wilt.

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.