Bacterial alarmone (p)ppGpp mediates the pathogenicity of Clavibacter michiganensis via a dual mechanism that affects both enzyme production and the Tat secretion system

The gram-positive bacterium Clavibacter michiganensis (Cm) causes bacterial canker of tomato. The infection process of Cm is of considerable interest, and the role of the alarmone guanosine tetraphosphate or pentaphosphate [(p)ppGpp], a global regulator, has been strongly implicated in bacterial survival and pathogenicity. Transcriptome analysis comparing a (p)ppGpp-deficient strain (Δ rel ) to the wild-type strain demonstrated that (p)ppGpp down-regulates the expression of many genes encoding ribosomal components and ABC transporter proteins, while up-regulating genes associated with amino acid metabolism, biofilm synthesis, and the production and secretion of cell-wall degrading enzymes. Biochemical assays showed reduced biofilm synthesis and extracellular activity of cell-wall degrading enzymes such as amylase and xylanase. Moreover, the results of microscale thermophoresis and electrophoretic mobility shift assays indicated that (p)ppGpp interacts with the transcription factor Vatr1 to directly suppress expression of the xylanase gene xysB . Meanwhile, amidase reporter assays revealed that xylanase secretion depends on the Tat secretion system, which was significantly inhibited in Δ rel , leading to intracellular accumulation of the xylanase protein. Taken together, these results indicate that (p)ppGpp plays a complex role in the pathogenicity of C. michiganensis , mediating, among other things, not only the production of cell-wall degrading enzymes but also their transport and secretion. IMPORTANCE This study reveals the pivotal role of the bacterial alarmone guanosine tetraphosphate and guanosine pentaphosphate in the pathogenicity of Clavibacter michiganensis , a significant plant pathogen. Through the identification of its dual mechanisms in regulating enzyme production and the Tat secretion system, we uncover key insights into bacterial virulence strategies. Our findings not only advance the understanding of bacterial stress response systems but also offer new opportunities for developing targeted interventions to combat plant bacterial diseases, ultimately contributing to agricultural sustainability and food security.