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Analysis of Promoters Recognized by HrpL, an Alternative σ-Factor Protein from Pantoea agglomerans pv. gypsophilae

July 2005 , Volume 18 , Number  7
Pages  634 - 643

Gal Nissan , 1 Shulamit Manulis , 2 Dan M. Weinthal , 1 , 2 Guido Sessa , 1 and Isaac Barash 1

1Department of Plant Sciences, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel; 2Department of Plant Pathology and Weed Research, ARO, Volcani Center, Bet Dagan 50250, Israel

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Accepted 4 February 2005.

HrpL, an alternative σ factor, activates the transcription of the Hrp regulon by its binding to a common “hrp box” promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5′-GGAACC-N15--16-CCACNNA-3′. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the -35 consensus motif (GGAAC) and the 3 nt of the -10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the -35 consensus motif eliminated HrpL binding, whereas mutations in the -10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P. agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.

Additional keywords: Hrp gene cluster , type III effectors .

© 2005 The American Phytopathological Society