Isaac Barash

Differential Involvement of Indole-3-Acetic Acid Biosynthetic Pathways in Pathogenicity and Epiphytic Fitness of Erwinia herbicola pv. gypsophilae

Erwinia herbicola pv. gypsophilae (Ehg), which induces galls on Gypsophila paniculata, harbors two major pathways for indole-3-acetic acid (IAA) synthesis, the indole-3-acetamide (IAM) and indole-3-pyruvate (IPyA) routes, as well as cytokinin biosynthetic genes. Mutants were generated in which the various biosynthetic routes were disrupted separately or jointly in order to assess the contribution of IAA of various origins and cytokinins to pathogenicity and epiphytic fitness. Inactivation of the IAM pathway or cytokinin biosynthesis caused the largest reduction in gall size. Inactivation of the IPyA pathway caused a minor, nonsignificant decrease in pathogenicity. No further reduction in gall size was observed by the simultaneous inactivation of both IAA pathways only or in combination with that of cytokinin production. However, inactivation of the IPyA pathway caused a 14-fold reduction in the population of Ehg on bean plants. Inactivation of the IAM pathway or cytokinin production did not affect epiphytic fitness. While the apparent transcriptional activity of iaaM-inaZ fusion increased slightly in cells of Ehg on bean and gypsophila leaves, compared with that in culture, very high levels of induction were observed in cells injected into gypsophila stems. In contrast, moderate levels of induction of ipdC-inaZ in Ehg were observed on leaves of these plants and in gypsophila stems, when compared with that in culture. These results suggest that the IAM pathway is involved primarily in gall formation and support the main contribution of the IpyA pathway to the epiphytic fitness of this bacterial species.

Biosynthesis of indole-3-acetic acid via the indole-3-acetamide pathway in Streptomyces spp.

Various Streptomyces spp. including S. violaceus, S. scabies, S. griseus, S. exfoliatus, S. coelicolor and S. lividans secrete indole-3-acetic acid (IAA) when fed with L-tryptophan (Trp). Production of IAA was detected in Streptomyces strains causing potato scab as well as in non-pathogenic strains. The pathways for IAA synthesis from Trp were investigated in S. violaceus and S. exfoliatus. Indole-3-acetamide (IAM), indole-3-lactic acid (ILA), indole-3-ethanol (IEt) and IAA were identified by HPLC and GC-MS. Streptomyces cells were capable of catabolizing IAM, ILA, IEt and indole-3-acetaldehyde (IAAId) into IAA. Incorporation of radioactivity into IAM, IAA and ILA but not IEt was detected when cells were fed with L-[3-14C]tryptophan. Results indicate the presence of the IAM pathway (Trp-->IAM-->IAA) and the possible presence of additional pathways for IAA biosynthesis in Streptomyces.

Tomato Transcriptional Changes in Response to Clavibacter michiganensis subsp. michiganensis Reveal a Role for Ethylene in Disease Development

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive actinomycete, causing bacterial wilt and canker disease in tomato (Solanum lycopersicum). Host responses to gram-positive bacteria and molecular mechanisms associated with the development of disease symptoms caused by Cmm in tomato are largely unexplored. To investigate plant responses activated during this compatible interaction, we used microarray analysis to monitor changes in host gene expression during disease development. This analysis was performed at 4 d postinoculation, when bacteria were actively multiplying and no wilt symptoms were yet visible; and at 8 d postinoculation, when bacterial growth approached saturation and typical wilt symptoms were observed. Of the 9,254 tomato genes represented on the array, 122 were differentially expressed in Cmm-infected plants, compared with mock-inoculated plants. Functional classification of Cmm-responsive genes revealed that Cmm activated typical basal defense responses in the host, including induction of defense-related genes, production and scavenging of free oxygen radicals, enhanced protein turnover, and hormone synthesis. Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response. After inoculation with Cmm, Never ripe (Nr) mutant plants, impaired in ethylene perception, and transgenic plants with reduced ethylene synthesis showed significant delay in the appearance of wilt symptoms, compared with wild-type plants. The retarded wilting in Nr plants was a specific effect of ethylene insensitivity, and was not due to altered expression of defense-related genes, reduced bacterial populations, or decreased ethylene synthesis. Taken together, our results indicate that host-derived ethylene plays an important role in regulation of the tomato susceptible response to Cmm.

Purification and characterization of pectolytic enzymes produced by virulent and hypovirulent isolates of Rhizoctonia solani Kuhn

Four pectolytic enzymes were purified from an isolate of Rhizoctonia solani (No. 82, AG-4) virulent on a wide range of hosts (Ichielvich-Auster et al. 1985, Phyloparasilica vol. 13, 103–112). The enzymes designated as endopolygalacturonase I and II (endoPG-I and endoPG-II), pectinesterase (PE) and endopectinlyase (endoPL) have been purified to homogeneity by a single chromatographic step on a cross linked polypectate column. These enzymes were identified also in two virulent isolates of R. zeae and two virulent binucleate Rhizoctonia spp. The endoPG-I, endoPG-II and PE but not endoPL were identified in three hypovirulent isolates of R. solani and two of R. zeae. These enzymes were purified to homogeneity from R. solani (No. 521, AG-4). The molecular weight (mol.wt), pH optimum, isoelectric point (pI) and optimal temperature (T) for each enzyme were endoPG-I, mol.wt 34 000, pH 4·8, pI 6·8, T 50°C); endoPG-II, mol.wt 37 000, pH 5·4, pI 7·4, T 42°C; PE, mol.wt 26 000, pH 7·7, pI 6·2, T 48°C; and endoPL (mol.wt. 45 500, pH 8·4, pI 8·1, T 53°C.

Recent Evolution of Bacterial Pathogens: The Gall-Forming Pantoea agglomerans Case

Pantoea agglomerans, a widespread epiphyte and commensal bacterium, has evolved into an Hrp-dependent and host-specific tumorigenic pathogen by acquiring a plasmid containing a pathogenicity island (PAI). The PAI was evolved on an iteron plasmid of the IncN family, which is distributed among genetically diverse populations of P. agglomerans. The structure of the PAI supports the premise of a recently evolved pathogen. This review offers insight into a unique model for emergence of new bacterial pathogens. It illustrates how horizontal gene transfer was the major driving force in the creation of the PAI, although a pathoadaptive mechanism might also be involved. It describes the crucial function of plant-produced indole-3-acetic acid (IAA) and cytokinines (CK) in gall initiation as opposed to the significant but secondary role of pathogen-secreted phytohormones. It also unveils the role of type III effectors in determination of host specificity and evolution of the pathogen into pathovars. Finally, it describes how interactions between the quorum sensing system, hrp regulatory genes, and bacterially secreted IAA or CKs affect gall formation and epiphytic fitness.

Characterization of a Clavibacter michiganensis subsp. michiganensis population in Israel

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected during the last decade from different locations in Israel, were analyzed by macrorestriction pulsed-field gel electrophoresis (PFGE). Fifty-eight strains from Israel and 18 from other sources were differentiated into 11 haplotypes with either VspI or DraI restriction enzymes. The strains from Israel formed four distinct groups among which groups A (16 strains) and B (32 strains) constituted the major clusters. These two groups originated from the Besor region, which is the main area for growing tomatoes under cover. Rep-PCR, with either ERIC or BOX primers, confirmed results obtained by PFGE. PCR with primers based on three genes – ppaA, chpC and tomA – that spanned the pathogenicity island of the reference strain NCPPB382, produced the expected products with the tested pathogenic strains. Plasmid analysis of representative strains revealed different profiles of one or two plasmids. However all the strains, including five non-pathogenic ones, reacted positively in PCR with primers based on celA gene, which resides on the plasmid pCM1 of NCPPB382. Southern hybridization of total DNA with a 3.2-kb BglII-fragment of pCM1 containing the celA gene was positive when carried out with 31 strains, but the size of the reacting band was not always the same as that of pCM1, suggesting that the plasmids carrying celA may differ in size. Comparison between the colonization rates of strain Cmm42 (group A) and of Cmm32 (group B) did not show any significant differences. The high diversity of the Cmm strains, on the one hand, and the presence of two persistent groups in the Besor region, on the other hand, suggests that the primary inoculum originated each year from residual plants in the soil rather than from infested seeds, in spite of extensive control measures taken by the growers in this area.

The presence of hrp genes on the pathogenicity-associated plasmid of the tumorigenic bacterium Erwinia herbicola pv. gypsophilae.

The pathogenicity-associated plasmid (pPATH) of Erwinia herbicola pv. gypsophilae (Ehg), which is present only in pathogenic strains, contains a gene cluster encoding indole-3-acetic acid and cytokinin biosynthesis. The transposon-reporter Tn3-Spice was used to generate nonpathogenic mutants on two overlapping cosmids, pLA150 and pLA352, of the pPATH. A cluster of such mutations, which spanned 16 kb, mapped approximately 15 kb from the gene cluster involved in phytohormone biosynthesis. Non-pathogenic mutants also failed to elicit the hypersensitive reaction (HR) on tobacco. Pathogenicity and HR were restored concomitantly to these mutants by in trans complementation with wild-type Ehg DNA. A 3.8-kb HindIII DNA fragment that complemented the hrp mutants was sequenced and six complete and two partial open reading frames (ORFs) were identified. Comparison of the deduced amino acid sequences of the eight ORFs showed striking homology and co-linearity with hrp genes of E. amylovora as well as with other plant and mammalian pathogenic bacterial genes encoding proteins of the type III secretion system. Limited DNA sequencing at various sites on the remaining 11-kb region of pLA352 also showed high identity to Hrp proteins of E. amylovora, E. stewartii, and Pseudomonas syringae. These results suggest that hrp genes are mandatory for gall formation by E. herbicola pv. gypsophilae.

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.

Pathogenicity ofVerticillium lecanii to different developmental stages of the silverleaf whitefly,Bemisia argentifolii

Thirty-five strains ofVerticillium lecanii which originated from different hosts and geographical locations were tested as potential biocontrol agents against silverleaf whitefly,Bemisia argentifolii Bellows & Perring. All strains were tested for their pathogenicity to third-instar nymphs. Several isolates which exhibited high pathogenicity to nymphs were also tested against eggs, pupae and adults ofB. argentifolii. Eggs were found to be immune to infection, but mortality of hatching nymphs reached 95–98%. The rate of hatching nymphs’ infection depended on the age at which the eggs were inoculated and the strain’s virulence. Mortality of nymphs recorded on day 4 after inoculation varied from 0.5±0.3% to 83±2.4%; that of the control ranged from 2.5% to 10.2%. The most virulent strains, with LT50 ranging between 3.2 and 3.8 days, were isolated from aphids in Israel and probably have a similar origin. The pathogenicity ofV. lecanii strains to pupae 6 days after inoculation varied between 59± 12.1 % and 72.5± 13.1 %, as compared with natural mortality of 13.5±4%. The maximum adult mortality caused byV. lecanii strains was between 34.1±5.1% and 52.6±3.8%.

The Dual Function in Virulence and Host Range Restriction of a Gene Isolated from the pPATHEhg Plasmid of Erwinia herbicola pv. gypsophilae

The host range of the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) is restricted to gypsophila whereas Erwinia herbicola pv. betae (Ehb) attacks beet as well as gypsophila. Both pathovars contain an indigenous plasmid (pPATH(Ehg or pPATH(Ehb)) that harbors pathogenicity genes, including the hrp gene cluster. A cosmid library of Ehg824-1 plasmid DNA was mobilized into Ehb4188 and the transconjugants were screened for pathogenicity on beet. One Ehb transconjugant harboring the cosmid pLA173 of pPATHEb induced a hypersensitive-like response and abolished pathogenicity on beet. Transposon mutagenesis of an open reading frame (ORF) located on this cosmid eliminated its affect on pathogenicity. Marker exchange of this mutation into Ehg824-1 caused a substantial reduction in gall size on gypsophila and caused Ehg824-1 to extend its host range and incite galls on beet. The ORF (1.5 kb) was designated as pthG (pathogenicity gene on gypsophila). DNA sequence analysis of pthG revealed no significant homology to known genes in the data bank. Only remnants of the pthG sequences were identified on the pPATH of Ehb4188. The deduced protein lacked an N-terminal signal peptide but contained a short trans-membrane helix in its C terminus. The gene product, as determined by expression in Escherichia coli and Western blots (immunoblots), was a 56-kDa protein.