UC Berkeley

Humans rely on plants for food, fiber, fuel and medicines. Understanding plant-pathogen interactions is critical to maintaining and improving the health of crops. This is particularly true in light of future pressures on agricultural systems. Broadly, plant-pathogen interactions can be viewed through three lenses: microbial pathogenesis, plant immunity, and crosstalk between pathogenesis and resistance. This work consists of three studies, each focused through one of these three lenses. The first addresses a possible role for microbial production of the small molecule salicylic acid in crosstalk signaling. The second investigates the mechanism of microbial iron acquisition from host plant tissues. The third characterizes the effects of the immune regulatory phytohormone salicylic acid in root tissues, an area that was previously largely unexplored. Together, these three studies contribute new knowledge to our understanding of plant-pathogen interactions and new tools for future investigations.

Salicylic acid (SA, 2-hydroxybenzoic acid) is a small molecule with numerous known bioactivities in organisms ranging from bacteria to humans to plants. In plants the compound is considered a phytohormone because it acts at low concentrations to signal a major reprogramming of cellular activities and does so both locally and at sites distal from the site of synthesis. The best-characterized outcome of SA signaling is an activation of a plant innate immune response effective against numerous biotrophic pathogens. One such pathogen, Pseudomonas syringae pv tomato DC3000 infects Lycopersicon esculentum and Arabidopsis thaliana hosts and is predicted to synthesize SA as an intermediate for yersiniabactin (Ybt) biosynthesis. As a siderophore, Ybt is expected to function in high affinity iron acquisition, and several studies indicated that siderophore function is required for pathogenesis of microbial and fungal phytopathogens. Thus, if Ybt is synthesized for iron acquisition in planta, then active synthesis of SA by DC3000 may impact pathogenesis. Alternatively, plant SA could be sequestered by DC3000 through conversion to Ybt, thereby reducing host immunity. Indeed, SA is produced by DC3000 for Ybt synthesis under iron limited culture conditions (Chapter 2). However, results from a genetic approach described in Chapter 2 demonstrate that SA and Ybt from DC3000 are unlikely to play a significant role in pathogenesis.

Although iron acquisition through siderophores is well established as a key virulence determinant in many mammalian pathosystems, fewer examples exist for plant pathogens. As mentioned above, the Ybt siderophore is unlikely to play a role in DC3000 pathogenesis (Chapter 2). However, DC3000 has two predicted siderophore systems for high affinity iron uptake, Ybt and pyoverdin (Pvd), raising the possibility that Pvd production is sufficient for iron acquisition from plant hosts. Therefore, we continued with a genetic investigation into DC3000 siderophores in order to determine mechanism of pathogenic iron nutrition (described in Chapter 3). Results from this approach show that loss of both siderophores did not reduce pathogenesis, but also revealed the presence of a third DC3000 siderophore, citrate. However, despite the importance of iron-citrate uptake in iron limited culture, a triple DC3000 mutant lacking all three siderophores was no less pathogenic than wt DC3000. Further experiments combined with data from others lead to a questioning of the necessity for high affinity iron uptake in plant pathogenesis, and suggest that low affinity iron uptake is sufficient for growth in planta. The finding that DC3000 siderophores are not virulence factors and the view of the plant intercellular environment as iron replete, rather than iron-limited, changes our understanding of iron physiology for DC3000 and iron relations in plant-pathogen interactions in general.

Experiments described in Chapter 4 show that physiologically relevant concentrations of SA inhibit root growth in Arabidopsis primarily through a dramatic reduction in cell elongation. We could not demonstrate that this SA treatment alters auxin signaling in the root, but we do demonstrate that SA reduces root accumulation of the hydroxyl radical and other reactive oxygen species such as hydrogen peroxide (Chapter 4). Interestingly, the canonical NPR1 mediated SA immune response is not required for the inhibition of root growth. In fact, an npr1 mutant is more sensitive to SA (Chapter 4). A further screening of known defense mutants revealed a role for SA responsive transcription factor WRKY38 and paralog WRKY62 in modulating SA root inhibition. The wrky38wrky62 double mutant is more resistant to SA root inhibition and WRKY38 overexpressor mutants are more sensitive (Chapter 4). Intriguingly, WRKY38 is expressed in root tips without exogenous SA application, suggesting a possible role for endogenous SA in regulating root development. Together, the results from this investigation provide first insights into the little understood root inhibition activity of SA, and establish this tractable system for future investigations into cellular targets of SA action.