| Dickeya dadantii 3937 is a Gram negative phytopathogenic bacterium which causes soft-rot, wilt, and blight diseases on a wide range of plant species. During invasion, it secretes a set of effector proteins into plant cells through a type III secretion system (T3SS) which plays important roles in the early stage of the bacterial colonization in plant. To successfully suppress plant immune systems, avoid elicitation of the plant defense response and establish infection, a fine tune regulatory mechanism of T3SS is needed. In this work, we identified new regulators in D. dadantii that are linked to the T3SS regulatory network, and studied their modes of action. In addition, to give a comprehensive understanding of how T3SS genes are regulated in a D. dadantii population, we also studied the expression and regulation of T3SS genes at single cell level.;First, a green fluorescent protein (GFP) reporter screening system was established to screen for novel T3SS regulators. 19 genes encoding various functions were identified as putative T3SS regulatory genes in D. dadantii 3937.;Second, the regulatory mechanisms of some T3SS regulators identified in the screening, such as OpgH and polynucleotide phosphorylase (PNPase), were further characterized. We showed that PNPase down-regulates the transcription of T3SS structural and effector genes in D. dadantii by affecting the mRNA stability of hrpL (encoding a T3SS master regulator) and rpoN (encoding a sigma 54 sigma factor). In addition, by characterizing the relation between the integrity and biological activity of a small RNA RsmB, we showed that the regulation of PNPase on hrpL mRNA stability is probably through RsmB.;In the study of the T3SS gene expression in a D. dadantii population, we discovered that the expressions of T3SS genes in the HrpL regulon are bistable in a homogeneous D. dadantii population. The bistabilities of these T3SS genes are generated by a unique mechanism. Although the expression of the T3SS master regulatory gene hrpL is not bistable, it still plays important roles in the development of the bistable expressions of the HrpL-regulon genes. In addition, we also demonstrated that the T3SS bistability is a conserved population behavior in many Gram negative bacteria pathogens. |
