|Keith, Ronald - OKLAHOMA STATE UNIV|
|Hernandez-Guzman, Gustavo - OKLAHOMA STATE UNIV|
|Uppalapati, Srinivasa - OKLAHOMA STATE UNIV|
|Bender, Carol - OKLAHOMA STATE UNIV|
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 13, 2003
Publication Date: April 20, 2003
Citation: Microbiology (2003) 149 pgs 1127 - 1138 Interpretive Summary: The plant pathogenic bacterium Pseudomonas syringae produces alginate, a capsular slime. The timing and signals to turn on alginate production in the plant are unknown. In this study, algD promoter fusions were constructed and used as a tool to determine when the alginate gene is turned on in host and nonhost plants infected with P. syringae pv. tomato DC3000. Collard greens spray-inoculated with DC3000 had water-soaked lesions and high levels of algD gene activity within 72 hours. Susceptible tomato plants had lower levels of gene activity and no water-soaked lesions. algD gene activity was also present on resistant tomatoes and could be seen microscopically within 12 hours after inoculation. The results indicate that alginate is produced on host and nonhost plants; however, the timing is different. Histochemical staining of the leaves indicated that the superoxide anion is a signal for alginate production in the plant. This is the first study showing that alginate is being produced within compatible and incompatible plants infected with bacteria.
Technical Abstract: Pseudomonas syringae produces the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. Although alginate has been isolated from plants infected by P. syringae, the signals and timing of alginate gene expression in planta have not been described. In this study, an algD::uidA transcriptional fusion designated pDCalgDP was constructed and used to monitor alginate gene expression in host and nonhost plants inoculated with P. syringae pv. tomato DC3000. When leaves of susceptible collard plants were spray-inoculated with DC3000(pDCalgDP), algD was activated within 72 hours post-inoculation (hpi) and was associated with the development of water-soaked lesions. In leaves of the susceptible tomato cv. Rio Grande-PtoS, algD activity was lower than in collard and was not associated with watersoaking. The expression of algD was also monitored in leaves of tomato cv. Rio Grande-PtoR, which is resistant to P. syringae pv. tomato DC3000. Within 12 hpi, a microscopic hypersensitive response (micro-HR) was observed in Rio Grande-PtoR leaves spray-inoculated with P. syringae pv. tomato DC3000(pDCalgDP). As the hypersensitive response progressed, histochemical staining indicated that individual bacterial cells on the surface of resistant tomato leaves were expressing algD. These results indicate that algD is expressed in both susceptible (e.g. collard, tomato) and resistant (Rio GrandePtoR) host plants. The expression of algD in an incompatible host-pathogen interaction was further explored by monitoring transcriptional activity in leaves of tobacco, which is not a host for P. syringae pv. tomato. In tobacco inoculated with DC3000(pDCalgDP), an HR was evident within 12 hpi, and algD expression was evident within 8-12 hpi. However, when tobacco was inoculated with a hrcC mutant of DC3000, the HR did not occur and algD expression was substantially lower. These results suggest that signals that precede the HR may stimulate alginate gene expression in P. syringae. Histochemical staining with nitroblue tetrazolium indicated that the superoxide anion (02-) is a signal for algD activation in planta. This is the first study to demonstrate alginate gene expression in planta, and the results indicate that alginate gene expression occurs in both compatible and incompatible plant host-pathogen interactions.