Henia Mor

The type III effectors HsvG and HsvB of gall-forming Pantoea agglomerans determine host specificity and function as transcriptional activators

Pantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) induces galls on both beet and gypsophila. The pathogenicity of both pathovars is dependent on the presence of a plasmid harbouring type III secretion system (TTSS) components and effectors. The HsvG TTSS effectors of Pag (HsvG‐Pag) and Pab (HsvG‐Pab) determine the host specificity of both pathovars on gypsophila. Here we describe a novel HsvG homologue, HsvB, which determines the host specificity of Pag and Pab on beet. HsvG requires two direct amino acid repeats for pathogenicity on gypsophila, whereas one repeat in HsvB is sufficient for pathogenicity on beet. Exchanging repeats between HsvG‐Pag and HsvB‐Pab resulted in a switch of host specificities. Transient expression of GFP–HsvG or GFP–HsvB fusions in gypsophila, beet or melon leaves showed that HsvG and HsvB were localized to the nuclei of host and non‐host plants. A yeast one‐hybrid assay revealed that a single repeat of HsvG or HsvB was sufficient to activate transcription. By employing random binding‐site selection and gel‐shift assay HsvG was demonstrated to be a double‐stranded DNA‐binding protein with an ACACC/aAA consensus binding site. These results suggest that HsvG and HsvB are host‐specificity determinants and bear the potential to affect the host transcriptional machinery.

The presence of diverse IS elements and an avrPphD homologue that acts as a virulence factor on the pathogenicity plasmid of Erwinia herbicola pv. gypsophilae.

The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) and Erwinia herbicola pv. betae (Ehb) is dependent on a native plasmid (pPATH(Ehg) or pPATH(Ehb)) that harbors the hrp gene cluster, genes encoding type III effectors, phytohormones, biosynthetic genes, and several copies of IS1327. Sequence analysis of the hrp-flanking region in pPATH(Ehg) (cosmid pLA150) revealed a cluster of four additional IS elements designated as ISEhel, ISEhe2, ISEhe3, and ISEhe4. Two copies of another IS element (ISEhe5) were identified on the upstream region of the indole-3-acetic acid operon located on the same cosmid. Based on homology of amino acids and genetic organization, ISEhe1 belongs to the IS630 family, ISEhe2 to the IS5 family, ISEhe3 and ISEhe4 to different groups of the IS3 family, and ISEhe5 to the IS1 family. With the exception of ISEhe4, one to three copies of all the other IS elements were identified only in pathogenic strains of Erwinia herbicola pv. gypsophilae and Erwinia herbicola pv. betae whereas ISEhe4 was present in both pathogenic and nonpathogenic strains. An open reading frame that exhibited high identity (89% in amino acids) to AvrPphD of Pseudomonas syringae pv. phaseolicola was present within the cluster of IS elements. An insertional mutation in the AvrPphDEh, reduced gall size in gypsophila by approximately 85%. In addition, remnants of known genes from four different bacteria were detected on the same cosmid.

Production of zinniol by Alternaria dauci and its phytotoxic effect on carrot

Zinniol was isolated from culture filtrates, mycelium and cell walls of Alternaria dauci. Its production was increased by a high carbon—nitrogen ratio and in the presence of L -asparagine or carrot leaves, as compared to nitrate. Zinniol could be detected during spore germination and early growth phases. Application of zinniol to carrot leaves was followed by the development of dark brown necrotic spots within 1 h. The symptoms caused by zinniol were identical with those produced by the fungus. The lowest phytotoxic dose was 1 mM. Both the hydroxy-methyl groups and the isoprenoid chain were essential for the phytotoxic activity of the zinniol molecule. The position of the 3-methyl-2-butenyl-oxy in the benzene ring was not significant for activity. [14C]zinniol was readily metabolized by carrot leaves and could not be detected in artificially infected leaves.

Characterization of siderophores produced by different species of the dermatophytic fungiMicrosporum andTrichophyton

The dermatophytic fungiTrichophyton spp andMicrosporum spp secrete ferrichrome type siderophores under low iron conditions. Three different species ofMicrosporum, i.e.M. qypseum, M. canis andM. audouinii, as well asT. rubrum produce ferrichrome C and ferricrocin, whereasT. mentagrophytes andT. tonsurans produce only ferrichrome. The identification of the siderophores was established by means of thin layer chromatography, high performance liquid chromatography and mass spectroscopy.

Uptake of iron byGeotrichum candidum, a non-siderophore producer

SummaryGeotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from59FeCl3 showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentKm = 3 μm,Vmax = 0.054 nmol · mg−1 · min−1). The maximal rate of Fe2+ uptake was higher than Fe3+ (Vmax = 0.25 nmol · mg−1 · min−1) but theKm was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (Km = 2 μM,Vmax = 0.22 nmol · mg−1 · min−1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either57FeCl3 or57FeCl2 accumulated unknown ferric and ferrous binding metabolites.

Characterization of siderophore-mediated iron transport in Geotrichum candidum, a non-siderophore producer.

SummaryGeotrichum candidum is capable of utilizing iron from hydroxamate siderophores of different structural classes. The relative rates of iron transport for ferrichrome, ferrichrysin, ferrioxamine B, fusigen, ferrichrome A, rhodotorulic acid, coprogen B, dimerium acid and ferrirhodin were 100%, 98%, 74%, 59%, 49%, 35%, 24%, 12% and 11% respectively. Ferrichrome, ferrichrysine and ferrichrome A inhibited [59Fe]ferrioxamine-B-mediated iron transport by 71%, 68% and 28% respectively when added at equimolar concentrations to the radioactive complex. The inhibitory mechanism of [59Fe]ferrioxamine B uptake by ferrichrome was non-competitive (Ki 2.4 μM), suggesting that the two siderophores do not share a common transport system. Uptake of [59Fe]ferrichrome, [59Fe]rhodotorulic acid and [59Fe]fusigen was unaffected by competition with the other two siderophores or with ferrioxamine B. Thus,G. candidum may possess independent transport systems for siderophores of different structural classes. The uptake rates of [14C]ferrioxamine B and67Ga-desferrioxamine B were 30% and 60% respectively, as compared to [59Fe]ferrioxamine B. The specific ferrous chelates, dipyridyl and ferrozine at 6 mM, caused 65% and 35% inhibition of [59Fe]ferrioxamine uptake. From these results we conclude that, although about 70% of the iron is apparently removed from the complex by reduction prior to being transported across the cellular membrane, a significant portion of the chelated ligand may enter the cell intact. The former and latter mechanisms seem not to be mutually exclusive.