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We developed PCR primers and assay methods to detect and differentiate three Phytophthora species which infect potatoes and cause late blight (Phytophthora infestans) and pink rot (P. erythroseptica and P. nicotianae) diseases. Primers based on sequence analysis of internal transcribed spacer region 2 of ribosomal DNA produced PCR products of 456 bp (P. infestans), 136 bp (P. erythroseptica), and 455 bp (P. nicotianae) and were used to detect the pathogens in potato leaf (P. infestans) and tuber (P. infestans, P. erythroseptica, and P. nicotianae) tissue with a sensitivity limit of 1 to 10 pg of DNA. Leaf and tuber tissue were processed for PCR by a rapid NaOH method as well as a method based on the use of commercially available ion-exchange columns. P. infestans primers and the rapid NaOH extraction method were used to detect late blight in artificially and naturally infected tubers of potato cultivar Red LaSoda. In sampling studies, P. infestans was detected by PCR from artificially infected tubers at 4 days postinoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect Phytophthora pathogens in potato seedlots and storages and thus limit the transmission and spread of new, aggressive strains of P. infestans in U.S. potato-growing regions.

Pseudomonas syringae pv. phaseolicola produces the tripeptide N8(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginine (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicoiana tabacum byAgwbacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.Bacterial pathogens of agronomic plants often produce low-molecular-weight toxins that function as virulence or pathogenicity determinants (15,53,61,62). Toxigenic strains (Tox+) of Pseudomonas syringae pv. phaseolicola growing at temperatures of 18 to 22°C produce phaseolotoxin [N6(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl-homoarginine (55)], which functions as a chlorosis-and growth retardationinducing toxin in the bean halo blight disease (53). This toxin and its nonpeptide derivative [N6(N'-sulfo-diaminophosphinyl)-ornithine, trivial name octicidine (55)] are potent inhibitors of ornithine carbamoyltransferases (OCTs) (15,53). Octicidine is a transition-state analog, causing irreversible inhibition of OCT, while phaseolotoxin inhibits the enzyme reversibly (53, 82). A total of 17 different OCTs that have been tested (7 bacterial, 9 from plants, and the rat liver enzyme) are all sensitive to octicidine and/or to phaseolotoxin in vitro and/or in vivo (18).Toxigenic strains ofP. syringae pv. phaseolicola possess a unique form of OCIT that is tolerant to phaseolotoxin and octicidine, in addition to an OCT that is sensitive to these inhibitors (15,19,79). The two enzymes (here designated ROCT and SOCT, respectively) are encoded by different genes, which were previously cloned in this laboratory (66).Several lines of evidence suggest that the chief function of ROCT is to confer phaseolotoxin tolerance in the producing strains. These strains grow normally in media lacking arginine, even when they produce phaseolotoxin at maximal rates (at 18°...

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CagA protein contributes to pro-inflammatory responses during H. pylori infection, following its intracellular delivery to gastric epithelial cells. Here, we report for the first time in an isogenic background, on the subtle role of CagA phosphorylation on terminal EPIYA-C motifs in the transcriptional activation and expression of IL-8. We utilized isogenic H. pylori mutants of P12 reference strain, expressing CagA with varying number of EPIYA-C motifs and the corresponding phosphorylation defective EPIFA-C motifs while preserving intact the CM multimerization motifs. These mutants had been previously closely scrutinized in terms of type IV secretion system functionality, CagA translocation and its subsequent phosphorylation. Following infection of gastric epithelial cell lines, transcriptional activation of IL-8 gene and secreted IL-8 levels were found to be strictly dependent upon the functionality of the EPIYA-C phosphorylation motifs, as EPIFA-C phosphorylation-deficient CagA expression failed to induce full IL-8 transcriptional activity. Interestingly, levels of IL-8 gene activation and of secreted IL-8 were the same, irrespective of the number of EPIYA-C terminal repeats. We monitored IkBα phosphorylation and confirmed CagA involvement in NF-kB activation. Furthermore, we observed that presence of EPIYA-C functional phosphorylation motifs contributed to NF-kB activation. NF-kB upstream signaling events, such as early ERK1/2 and AKT activation were confirmed to be independent of EPIYA-C phosphorylation. On the contrary, use of TAK1 specific inhibitor 5Z-7-Oxozeaenol resulted in complete arrest of IL-8 secretion, in a dose-dependent manner, irrespective of CagA status. H. pylori-infected TAK1-/- mouse embryonic fibroblasts (MEFs) failed to induce NF-kB activity, unlike the respective control MEFs. CagA and TAK1 were found to immunoprecipitate together, irrespective of CagA EPIYA-C status, thus confirming earlier reports of TAK1 and CagA protein interaction. Our data suggest that CagA may potentially interfere with TAK1 activity during NF-kB activation for IL-8 induction in early H. pylori infection.

Reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunocapture (IC)-RT-PCR protocols were developed and optimized for the sensitive detection of Onion yellow dwarf virus, Leek yellow stripe virus and allexiviruses infecting Allium species. Polyvalence of the designed primers was successfully demonstrated, using samples of dierent plant species and geographic origins. Dierent sample preparation procedures were evaluated for their suitability to provide appropriate PCR templates. IC-PCR, RT-PCR with plant tissue extracts, and RT-PCR with total RNA, proved to be 10 2 ±10 4 times more sensitive than double-antibody sandwich-enzymelinked immunosorbent assay (ELISA). Furthermore, à one step' IC-RT-PCR assay was developed using plant leaf extract as template source, which proved to be 102 times more sensitive than ELISA, and convenient for testing large numbers of leaf samples.