Polyamine synthesis and biodisponibility has a mayor rol in the pathogenesis of Pseudomonas syringae DC3000

SOLMI L; ROMERO FM; CORIGLIANO MG; ROSSI FR; GÁRRIZ A

Chapadmalal

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiolgía General; 2022

Pathogenic bacteria use multiple molecular mechanisms to achieve tissue colonization and evade host defense effectively. Activation or repression of these mechanisms are tightly coordinated. Polyamines (PAs) such as putrescine (PUT) and spermidine (SPD) are cationic compounds that play a critical role in cell physiology and regulate virulenceassociated cellular processes in human pathogenic bacteria. However, their role in phytopathogenic bacteria remains unknown. In this study, we explore the function of PAs in Pseudomonas syringae pv. tomato DC3000 (Pto). We study virulence capability of wild-type and mutant strains unable to synthesized putrescine (∆speA∆speC) and spermidine (∆speE), the most abundant PAs found in this species. To entry into host tissue, Pto inhibits stomata closure through coronatine synthesis and secretion. ∆speE and ∆speA∆speC provoke minor effects on stomata closure compared to the WT strain. We then performed Arabidopsis thaliana seedling flood-inoculation to analyse the ability of bacteria to enter plant tissues. Measured cell growth in planta at different time revels that ∆speE only showed reduction in colony counts at 24 h pi, but these values recover at 48 h pi. In turn, population was significantly lower with the ∆speA∆speC mutant. We also conducted syringe-infiltration to bypass the entry process and evaluated bacteria proliferation inside the plant. ∆speA∆speC had significantly less cell number counts at all time points. In contrast, once ∆speE was inside the plant, it exhibited similar virulence as the WT strain. Activation of type 3 secretion system (T3SS) and effectors delivery are keys for successful pathogenesis. To test the activation of this system in the mutant strains, we inoculated the non-host Nicotiana benthamiana in order to visualize hypersensitive response (HR). Both mutants show a delay in HR-induction indicating less activation of T3SS or effector delivery. At last, apoplastic fluid extracted from healthy adult A. thaliana plants was used to supplement M9 minimal media and bacterial growth was measured by absorbance at 600nm. ∆speE responded to media supplementation, indicating that the SPD content in the apoplast is sufficient to promote cell growth. Our results show that putrescine and spermidine are involved in different mechanism associated with virulence in Pto DC3000. Synthesis of PUT is essential for Pto full-virulence. On the other hand, disruption of the SPD synthesis pathway affected some virulence mechanisms, but its bioavailability in plants enables bacterial to colonize host tissue correctly.