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Background: The phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation., Results: The mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production., Conclusions: The results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.

Background: The type III secretion systems (T3SSs) encoded by Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are important for invasion of epithelial cells during development of Salmonella-associated enterocolitis and for replication in macrophages during systemic infection, respectively. In vitro studies have previously revealed hierarchical transport of different SPI-1 factors and ordered synergistic/antagonistic relationships between these proteins during Salmonella entry. These results suggest that the level and timing of the expression of these proteins dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established., Results: In this study, we have constructed bacterial strains that contain a FLAG epitope inserted in frame to SPI-1 genes prgI, sipA, sipB, sopE2, spaO, and sptP, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. The tagged Salmonella strains were inoculated intraperitoneally or intragastrically into mice and recovered from various organs. Our results provide direct evidence that PrgI and SipB are expressed in Salmonella colonizing the spleen and cecum of the infected animals at early and late stages of infection. Furthermore, this study demonstrates that the SpaO protein is expressed preferably in Salmonella colonizing the cecum but not the spleen and that SptP is expressed preferably in Salmonella colonizing the spleen but not the cecum., Conclusion: These results suggest that Salmonella may express different SPI-1 proteins when they colonize specific tissues and that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the cecum and systemic infection in the spleen.