We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.

To analyze the compartmentation of nucleolar protein complexes, the mechanisms controlling targeting of nucleolar processing proteins onto rRNA transcription sites has been investigated. We studied the reversible disconnection of transcripts and processing proteins using digitonin-permeabilized cells in assays capable of promoting nucleolar reorganization. The assays show that the dynamics of nucleolar reformation is ATP/GTP-dependent, sensitive to temperature, and CK2-driven. We further demonstrate the role of CK2 on the rRNA-processing protein B23. Mutation of the major CK2 site on B23 induces reorganization of nucleolar components that separate from each other. This was confirmed in assays using extracts containing B23 mutated in the CK2-binding sites. We propose that phosphorylation controls the compartmentation of the rRNA-processing proteins and that CK2 is involved in this process. INTRODUCTIONThe nucleolus is a model organelle to study nuclear compartmentation (Strouboulis and Wolffe, 1996). In the nucleolus ribosomal RNAs (rRNAs) are synthesized, processed, and assembled with ribosomal proteins to form the small 40S and large 60S preribosome subunits (for a review, see Shaw and Jordan, 1995). The dynamic integration of these different processes generates a typical nucleolar organization. Three main specific components are observed by electron microscopy (Scheer and Hock, 1999): the fibrillar centers (FCs) that are light areas surrounded by a highly contrasted region, the dense fibrillar component (DFC), and the granular component (GC) in which the FCs and DFC are embedded. In the active nucleolus, the early rRNA-processing proteins associated with transcripts during elongation are localized in the central part of the nucleolus, i.e., in the DFC. The processing proteins associated with late steps of rRNA processing are localized in the external part of the nucleolus, i.e., in the GC.The organization of active nucleoli illustrates the coordination that exists between transcription and processing mechanisms and the recruitment of the nucleolar protein complexes at specific steps of ribosome biogenesis. However the mechanisms that control the compartmentation of nucleolar protein complexes are poorly understood. With this in mind, we undertook to determine whether phosphorylation drives the connection of the processing proteins on rRNAs.It has been established that nucleolar disorganization can be induced by DRB (5,6 dichloro-1-␤-d-ribofuranosylbenzimidazole; Granick, 1975aGranick, , 1975bHaaf et al., 1991;Le Panse et al., 1999). Typically the ribosomal genes extend into the nucleoplasm forming the nucleolar necklace, each of the beads of the necklace corresponding to individual transcription sites in association with early rRNA-processing proteins (Granick, 1975a;Haaf and Ward, 1996). More recently it was demonstrated that DRB also induces the formation of masses containing late rRNA-processing proteins at a distance from the transcription sites (DavidPfeuty et al., 2001;Louvet et al., 2005). Thus, there r...

No abstract