Resumen de Quantitative proteomic approaches for the characterization of bacterial wilt resistance mechanisms in tomato. Lnsights on the role of p69 subtilisin-like proteases in defense
The plant pathogen Ralstonia solanacearum is the causal agent of the devastating disease known as bacterial wilt. This disease affects more than 200 plant species in over 50 families, including economically important crops such as tomato, potato, banana or pepper. R. solanacearum is a soil-borne and vascular pathogen. It enters its hosts through wounds in the roots and lateral root emerging sites. Then, the pathogen traverses the cortical apoplast until it reaches the vascular cylinder and colonizes to very high numbers the xylem vessels. The combination of bacterial growth and the secretion of a mucus-like exopolysaccharide clogs the infected vessels, blocking its water flow and causing the wilt disease symptoms that lead to the dead of the plant. To date, the most reliable management strategy to control R. solanacearum has been the use of genetic resistance. In this thesis we attempted to shed light on the old question of bacterial wilt resistance, which has haunted plant epidemiologists for more than one hundred and twenty years, using novel proteomic techniques. We thought that the best way to address this challenge was to follow a “divide and conquer” approach: by identifying the key bottlenecks of resistance (Chapter 1), we would be able to precisely dissect which proteins are involved in different tissues and stages of the infection (Chapters 2 and 3). Finally, in Chapter 4 we delved into the tomato P69 family of subtilisin-like proteases, as many of its members appeared upregulated during infection. In Chapter 1 we identified the plant tissues and organs that represented major constraints during R. solanacearum colonization. Using simple and very visual tools –bacterial reporter strains and plant grafting–, we highlighted the root system as the first and most conspicuous barrier. The roots consist of an epidermis and a cortical parenchyma that R. solanacearum must traverse before reaching the vasculature, a movement that occurs through the intercellular space or apoplast. In this chapter, we showed how the roots of resistant plants (H7996) effectively prevented R. solanacearum entry and invasion, single-handedly reducing the colonization and consequent development of wilt disease symptoms in grafted plants. A reduction in colonization was also evident within the xylem vessels of H7996. Grafting susceptible roots onto resistant hypocotyls allowed us to confront the resistant stems with high bacterial inoculum using the conventional –and more natural– soil-drench inoculation method. Once the bacteria faced the resistant tissue, a decrease in colonization was observed, indicating that H7996 can also restrict R. solanacearum movement/growth along the xylem in a root-independent manner. In addition, we observed how this resistant tomato variety impeded the escape of R. solanacearum from colonized xylem vessels towards the apoplastic space of the neighboring parenchymatic cells, a situation clearly promoted in susceptible plants (Marmande) and that facilitated tissue decay in the latest stages of infection. Altogether, the data presented in this chapter highlighted these two plant environments, the apoplast and the xylem, as the two main battlefields of infection. In Chapters 2 and 3 we used quantitative mass spectrometry approaches to investigate the apoplastic and xylem sap proteomes in response to R. solanacearum. In Chapter 2 we focused on the active proteome of the apoplast using activity-based molecular probes that covalently bound to the active site of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs). Upon infection, the activity of PLCPs and SHs was higher in the apoplast of H7996 than in Marmande. We found that two well-known PLCPs, Rcr3 and Pip1, and many SHs from distinct protease families experienced significant changes after R. solanacearum inoculation and in the comparison of the two tomato varieties. Additionally, a protein network analysis suggested that the apoplastic proteome of H7996 might be more naturally prepared to face intruders, as many protein interactions did not substantially change upon infection. In Chapter 3 we focused on the xylem sap proteome and, similar to the apoplast, we observed many differentially accumulated proteins (DAPs) in response to infection. A clear “downregulation” was detected in Marmande, which could either be due to a deliberated inhibition caused by R. solanacearum or to tissue damage resulting from the infection process. On the other hand, cell wall modifying enzymes, including peroxidases and glycoside hydrolases (GHs), were overaccumulated in H7996. Moreover, the overaccumulation of many tomato and R. solanacearum GHs both in the apoplast and the xylem highlighted the importance of the cell wall as an effective barrier in plant-pathogen interactions, particularly against R. solanacearum. Finally, in the survey of both proteomes –apoplast and xylem–, we identified members of the P69 family of subtilases to overaccumulate upon R. solanacearum infection. This 10 gene family of proteases was comprehensibly addressed in Chapter 4. We analyzed the conservation of this family and found that they are only present in Solanaceae, being P69A and P69D the two most conserved P69 genes. P69s were not only induced by R. solanacearum but responded to unrelated plant pathogens, as illustrated by several transcriptomic and proteomic studies. Additionally, their overall high percentage of identity (>71%) appeared to partially translate in their cleavage specificity, although some particularities could be drawn for P69A, P69B and P69D. Conversely, substrate specificity might also be determined by their differential post-translational N-glycosylation status, and/or by the higher sequence divergence within their protease- associated domain, which could influence protein-protein interactions. Finally, a CRISPR/Cas9 mutagenic approach was conducted to obtain a P69D single mutant and two separate P69-cluster deletion mutants. We assayed the susceptibility of the P69D single mutant and found a slight yet significant increase in susceptibility, probably caused by an increased growth of the pathogen in its root system. The potential role of P69D in defense and other aspects regarding P69 subtilases are addressed.
