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1.
A genomic library of Erwinia amylovora isolate T was constructed in the cosmid pLAFR3 and maintained in Escherichia coli. Clones were transferred individually by conjugation into the non-pathogenic isolate P66 of E. amylovora. Transconjugants were screened for restoration of pathogenicity to pear by stab inoculation into sections of immature pear fruits. Three clones complemented P66 restoring pathogenicity and ability to cause the hypersensitive reaction (HR) in Phaseolus vulgaris. Restriction mapping and hybridization experiments showed that the three clones had a common 3·7 kb fragment of E. amylovora DNA. Sub-cloning and insertion mutagenesis with Tn5-lac confirmed that a determinant of pathogenicity and ability to cause the HR (hrp gene) was located on a 2·1 kb HindIII/BamHI fragment within the common DNA. Hybridization experiments using the 2·1 kb HindIII/BamHI fragment as a probe demonstrated that the hrp gene was located in the chromosome of isolate T and that homologous sequences were present in the non-pathogenic isolates P66 and S. Clones which restored hrp function did not affect the growth of isolate P66 in minimal or nutrient-rich media. Transconjugants of Pseudomonas syringae pv. phaseolicola race 1 harbouring the hrp gene(s) cloned from E. amylovora did not cause the HR in susceptible cultivars of bean but symptoms developed more slowly than in the absence of the clones or with pLAFR3 alone.  相似文献   

2.
Extracellular endopolygalacturonase, purified from the pathogenic fungus Fusarium moniliforme, consists of four molecular forms (38, 41·5, 45, and 48·5 kDa, respectively. Three forms (38, 41·5, and 45 kDa) were purified to homogencity by FPLC on a Mono S column followed by electroelution after SDS-PAGE. The N-terminal amino acid sequences of each of the three forms, and of a mixture containing all four forms were shown to be identical to that predicted from the nucleotide sequence of the endopolygalacturonase gene previously cloned from F. moniliforme. Enzymatic deglycosylation experiments revealed the presence of N-linked, high mannose oligosaccharide side-chains on all four forms of endo polygalacturonase. Hydrogen fluoride catalysed chemical deglycosylation of the polygalacturonase mixture yielded a single polypeptide with an apparent molecular mass of 36·2 kDa. Southern blot analysis, carried out at high stringency with an endopolygalacturonase-specific probe on genomic DNA digested with three different restriction enzymes, showed a single hybridizing restriction fragment in all three digests. A single 2·0 Mb chromosome hybridized with the endo polygalacturonase-specific probe, as shown by Southern blot analysis of F. moniliforme chromosomes separated by CHEF electrophoresis. Northern blot analysis revealed only one mRNA species 1350 nt encoding endo polygalacturonase. These data indicate that a single gene encodes the endopolygalacturonases of F. moniliforme.  相似文献   

3.
Infection of groundnut leaves with the early leaf spot pathogen Cercospora arachidicola leads to a marked increase in extracellular 1,3-β-glucanase activity, limited to the infected tissue. Three isoforms of low molecular weight and extreme pI values, typical of pathogenesis-related proteins, were induced. These β-glucanases, when acting together, were capable of degrading the pathogen cell wall in vitro. Glucanases from homogenates of infected leaf tissue were partially purified by ion-exchange chromatography to give enzymes with molecular weights of 35, 32 and 20 kDa and pI values of 3·8, 3·6 and > 9, respectively. They were electrophoretically identical to the β-glucanases found in the intercellular washing fluid. Treatment of groundnut plants with 200 μM mercuric chloride induced the accumulation of identical extracellular β-glucanases. During the course of the infection an increase in peroxidase activity was also observed, but chitinase activity remained more or less constant.  相似文献   

4.
Stemphylium botryosum pathogenic on alfalfa (Medicago sativa) produced a phytotoxin in a defined liquid medium which caused symptoms similar in sequence and appearance to stemphylium leafspot lesion development on attached alfalfa leaflets. The molecular weight of the phytotoxin was estimated to be 19500 by gel filtration chromatography. Treatment of partially purified phytotoxin with proteinase K or subtilisin resulted in loss of phytotoxic activity. Action of the proteases in inducing loss of phytotoxicity was inhibited by phenylmethylsulphonyl fluoride. Concentrated culture filtrates were fractionated by chromatofocusing, HPLC ion-exchange and gel filtration. Gel filtration also was followed by non-denaturing electrophoresis or isoelectric focusing (IEF). Analysis of sequential fractions from each procedure by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that phytotoxic activity was consistently coincident with the presence of two major polypeptides (Mr = 26900 ± 2%; 19500 ± 4%) and a triplet of polypeptides (Mr = 15500). The constant association between the three polypeptides suggested an intermolecular interaction during fractionation. The phytotoxic activity eluted from the chromatofocusing column between pH 5·79 and 5·43 whereas IEF estimated the isoelectric point to be between 5·40 and 5·02.Immobilized concanavalin A and wheat germ lectin failed to bind phytotoxicity. Proteolytic activity in some phytotoxic gel filtration fractions was reduced by addition of phenyl-methylsulphonyl fluoride and separated from phytotoxicity by non-denaturing electrophoresis, indicating that phytotoxicity was not due to serine protease activity.Pathogenicity tests of the cool-temperature biotype on 20 plant species representing eight plant families resulted in a compatible interaction only on M. sativa and M. polymorpha. In contrast, sensitivity to the phytotoxin was observed on both pathogen-resistant and susceptible alfalfa clones along with all other plants tested with the exception of Triticum aestivum, Zea mays, Apium graveolens and Melilotus officinalis, indicating non-host-specificity of the phytotoxic polypeptides produced by the cool-temperature biotype.  相似文献   

5.
Filtrates from shake-cultures of Fusarium oxysporum f. sp. lycopersici race 1, concentrated to 20% of the original volume, caused cell death in tomato leaf protoplasts from near-isogenic lines corresponding to the compatible cultivar/race reactions of whole plants. Maximum activity was found in late log phase cultures on Czapek-Dox supplemented with 2% casamino acids. Selective toxicity was associated only with the protein fraction of the culture filtrate. LD50 values for susceptible Ace and Moneycross to F. oxysporum f. sp. lycopersici race 1 culture filtrates were 1·92 and 0·36 μg protein ml−1. Corresponding values for cvs Royal Ace and MM161, each containing the I-gene conferring resistance to race 1, were >350. Culture filtrates from F. oxysporum f. sp. lycopersici race 2 gave LD50 values of 2·34 and 2·08 μg protein ml−1 on cvs Ace and Royal Ace, both susceptible to race 2. The LD50 of cv. Ace to a non-pathogenic isolate of F. xysporum f. sp. lycopersici was > 350. Culture filtrates from non-host formae of F. oxysporum were 9–149-fold less toxic on cv. Ace. Protoplasts from Pisum sativum, Lactuca sativa, Zea mays, Gossypium barbadense and Solanum melongena, all non-hosts of F. oxysporum f. sp. lycopersici, were 6–175 times less sensitive to F. oxysporum f. sp. lycopersici filtrates than susceptible tomato. The putative toxins lycomarasmin and fusaric acid showed no differential toxicity to I+ and I− tomato protoplasts. The results are discussed in the wider context of host-pathogen interaction in which specificity is considered as the recognition of susceptibility by a proteinaceous toxic metabolite of the pathogen. This hypothesis is further extended to include the specificity of F. oxysporum formae and races.  相似文献   

6.
In recent studies, we found that Apll (a PthA homologue) bound to three citrus proteins. Amino acid sequence analysis revealed that one of the target proteins was homologous to that of S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT), an enzyme which is specific for the substrate trans-caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA. From the consensus nucleotide sequences of CCoAMT genes, primers were chosen for PCR amplification of this gene from citrus total DNA. Two selected DNA fragments of 1.0 kb and 2.0 kb were obtained. These fragments were used as the probe to screen a citrus library. One clone, pCCl00, contained a 1.0-kb SalI fragment that hybridized to the probes. The nucleotide sequence of this fragment was determined in both directions. In this fragment, there was an open reading frame of 232 amino acids interrupted by an intron of 106 nt, and the deduced amino acid sequence had 95.9% homology to tobacco CCoAMT. Southern blot analysis of total citrus DNA showed that four EcoRI fragments hybridized to the probes, suggesting the presence of more than one copy of CCoAMT in the citrus DNA. Received 4 November 1999/ Accepted in revised form 26 November 1999  相似文献   

7.
为探讨昆虫生长调节剂氟铃脲和虱螨脲对黑腹果蝇Drosophila melanogaster和斑翅果蝇D.suzukii的毒性机制,采用室内生测法测定氟铃脲、虱螨脲对2种果蝇2龄幼虫的毒力,以及其在亚致死浓度LC_(10)、LC_(20)下对2种果蝇体内几丁质酶活性的影响。结果显示,经不同浓度的氟铃脲和虱螨脲处理后,随着时间的延长和浓度的增加,2种果蝇的死亡率均明显增加,氟铃脲处理可使2种果蝇的死亡率最高达到83.33%,明显高于虱螨脲处理后黑腹果蝇(65.00%)和斑翅果蝇(66.66%)的死亡率。亚致死浓度LC_(10)和LC_(20)的虱螨脲处理果蝇2龄幼虫24 h后,黑腹果蝇2龄幼虫体内的几丁质酶活性呈下降趋势,而斑翅果蝇2龄幼虫体内的几丁质酶活性则呈上升趋势。另外,亚致死浓度的氟铃脲可明显抑制黑腹果蝇体内几丁质酶的活性。表明昆虫生长调节剂氟铃脲和虱螨脲对果蝇有较强的毒力,氟铃脲的毒力高于虱螨脲,且果蝇体内几丁质酶的活性与氟铃脲和虱螨脲密切相关。  相似文献   

8.
We report the construction of a clone library of the Erwinia carotovora subsp. atroseptica genome and the isolation of a gene for endo-pectate lyase. The library, inserted into the PstI site of plasmid pBR322, contains approximately 1700 clones. Five of these produce pectolytic enzymes as detected by a plate screening assay. Using a cloned endo-pectate lyase gene from the related bacterium, E. carotovora subsp. carotovora, as a probe, we found that one pectolytic E. carotovora subsp. atroseptica clone had strong homology to the probe. We characterized that clone by restriction endonuclease mapping and studied its pectolytic protein product. The purified enzyme is an endo-pectate lyase with a cofactor preference for Co2+. The molecular weight of the protein is 31 000 and it has an isoelectric focusing point of 9·2, corresponding to an endo-pectate lyase produced by E. carotovora subsp. atroseptica, but not to the protein product of the E. carotovora subsp. carotovora probe DNA, which has a pI of 9·5. Restriction endonuclease site polymorphisms in the two cloned endo-pectate lyase genes suggest substantial sequence divergence between these two loci.  相似文献   

9.
The paper reported the synthesis and antifungal properties and mechanism of action of a series of 2-substituted methylthio-5-(2,4-dichlorophenyl)-1,3,4-oxadiazole/thiadiazole and their corresponding sulfones. The preliminary biological test showed these compounds exhibit moderate to good antifungal activity. Particularly, the compounds 7g and 7c inhibited mycelia growth by approximately 50% (EC50) at 2.6–59.2 μg/mL and 17.2–54.4 μg/mL respectively against nine kinds of fungi. The extent of inhibition induced by 7c on Rhizoctonia solani and underlying mechanism of action were studied in vitro. Docking simulation was performed to position selected compounds into the active site of family 18 chitinases. Variation in d-GlcNAc content and chitinase activity indicated that 7c can act as chitinase inhibitor for controlling fungal pathogens in plants.  相似文献   

10.
Analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis of the soluble proteins extracted from muskmelon (Cucumis melo L.), inbred line PI 124111F (PI) with resistance to Pseudoperonospora cubensis (Berk, et Curt.) Rost, and two susceptible cvs, Hemed and AnanasYokneam revealed the presence of a unique 45 kDa protein (P45) in the resistant but not in the susceptible plants. Subcellular fractionation indicated that P45 is a cytoplasmic soluble protein. It was detected in leaves and cotyledons but not in stems or roots. F1 hybrid plants (Hemed × PI) that displayed only a partial resistance against P. cubensis expressed P45 at an intermediate level, whereas plants of the inbred generations F5, F7 and F10 that displayed a high degree of resistance, expressed P45 at a level similar to the parental PI. Back-cross progeny plants [(Hemed × PI) × Hemed] segregate 1:2:1 partially resistant: susceptible: highly susceptible to the disease. The partially resistant plants showed an intermediate level of P45 (similar to F1) whereas the highly susceptible plants had no P45, thus indicating cosegregation of P45 with resistance. The resistance of PI to P. cubensis was found to decrease at a colonization temperature of 12 °C. 35S-methionine in vivo protein labelling revealed a reduction in the intensity of the P45 band in plants incubated for 11 h at 12 °C. The application of P45 in breeding programs of muskmelon for downy mildew resistance and its possible involvement in resistance to P. cubensis are discussed.  相似文献   

11.
We investigated the in vitro and in vivo effects of some pesticides on rainbow trout erythrocyte glucose-6-phosphate dehydrogenase enzyme. The enzyme was purified 1691-fold with a specific activity of 16.235 U/mg protein and a yield of 63%. Cypermethrin, and propoxur inhibited glucose-6-phosphate dehydrogenase enzyme in vitro and deltamethrin inhibited both in vivo and in vitro. The obtained IC50 values for deltamethrin, cypermethrin, and propoxur were 0.63, 1.02, and 12 mM, respectively. The activity of the control was determined as 5.17 ± 0.62 U/g Hb in in vivo studies. The enzyme activities of the groups treated with 0.25 g/L deltamethrin were measured at 6, 12, 24, 48, and 72 h, and found to be 4.32 ± 0.47, 3.57 ± 0.39, 3.47 ± 0.45, 2.86 ± 0.37, and 2.31 ± 0.32 U/g Hb. In vivo experiments showed that deltamethrin significantly inhibited the G6PD enzyme activity after the 48th h (p < 0.05).  相似文献   

12.
The polygalacturonases (PG) and oxalic acid produced by Sclerotinia sclerotiorum in infected soybean hypocotyls were investigated as elicitors of the phytoalexin glyceollin I.Purification to homogeneity through isoelectrofocusing and ion-exchange fast protein liquid chromatography revealed three endo-PG isoenzymes (PG-I, PG-II and PG-IV) and one exo-PG (PG-III) in 6-day-old etiolated soybean hypocotyls infected with the B-24 isolate of S. sclerotiorum.PG-I and PG-III, in the range of concentrations tested (0·15–1·2 reducing units ml−1), did not act as elicitors of glyceollin I synthesis. Some elicitor activity was shown by PG-II at 0·6–1·2 reducing units ml−1. PG-IV, at lower doses (0·038–0·30 reducing units ml−1), was even more effective in inducing phytoalexin synthesis. However higher concentrations of PG-IV induced tissue softening and decreased phytoalexin accumulation.PG-II and PG-IV released heat-stable elicitors from purified soybean cell walls supporting the evidence that uronides are intermediate inducers in elicitation by endo-PGs. Oxalic acid was an active elicitor of glyceollin I over the range of concentrations tested (0·31–20 m ) with the maximum at a concentration of 5 m . The inability of oxalic acid to release uronides from purified cell walls makes it unlikely that uronide intermediate elicitors are involved in elicitation by oxalic acid.  相似文献   

13.
The response of epidermal cells of cucumber leaf tissue infected by Sphaerotheca fuliginea was examined by light microscopy to understand how silicon in infected host cells affects host defence mechanisms. Leaf pieces from plants treated with nutrient solutions containing 0·05, 0·50 or 2·3 mm of silicon (Si) were harvested at intervals of 24, 48, 72, 96 and 120 h after inoculation with the pathogen and examined after staining with toluidine blue or aniline blue. Si treatments significantly reduced the time to initiation of production and/or accumulation of phenolic materials in infected host epidermal cells, and increased the number of infected cells that produced and/or accumulated phenolics. The number of haustoria produced per colony of S. fuliginea was significantly reduced over time, and conidiophore development was delayed on the leaves of cucumber treated with 2·3 mm Si nutrient solution.  相似文献   

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17.
Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat β 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into ‘Nantes Coreless’ carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing β-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20–50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10–20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants.  相似文献   

18.
Accumulation of pathogenesis-related proteins is thought to play a role in pathogen-induced plant defense responses. Although early accumulation of hydrolytic enzymes such as chitinase and β-1,3-glucanase has been associated previously with genetically-inherited and induced systemic resistance, their role in resistance in tomato(Lycopersicon esculentum)to the phytopathogenic fungusAlternaria solaniis not yet understood. Here we describe the accumulation patterns of specific isozymes of pathogenesis-related proteins in the resistant tomato genotypes 71B2, NC EBR-1, NC EBR-2 and the susceptible cultivar Piedmont. Western blot analysis demonstrated that four isozymes of chitinase (26, 27, 30, and 32kDa) were induced in all genotypes upon challenge withA. solani,but only resistant lines had significantly higher constitutive levels of the 30kDa isozyme as well as total chitinase activity. In addition, the 30kDa chitinase isozyme was found to accumulate to significantly higher levels in resistant lines during pathogenesis than the susceptible genotype. Two isozymes of β-1,3-glucanase (33 and 35kDa) were detected in all genotypes, but a slightly higher constitutive level was detectable in all resistant lines when compared to the susceptible. Similar accumulation patterns of these isozymes were observed in all genotypes during the course of pathogenesis. Purified preparations of acidic and basic tomato chitinase and β-1,3-glucanase isozymes were tested for their antifungal activity againstA. solani in vitro.Results presented in this study indicate that only basic isozymes of chitinase and β-1,3-glucanase were inhibitory toA. solaniwhereas, no inhibitory activity was observed with the acidic isozymes. The results of this study suggest that a higher constitutive level of chitinase and β-1,3-glucanase and the induction pattern of a 30kDa chitinase isozyme in early blight resistant breeding lines is related to genetically-inherited resistance of tomato toA. solani.  相似文献   

19.
It is our working hypothesis that suppression of the activity of glycoprotein non-specific elicitors (NSE) from fungal cell walls is required in establishment of basic compatibility in the Cladosporium fulvum-tomato system. A suppressor of NSE-induced necrosis on tomato leaves was partially purified from intercellular fluid (IF) obtained from C. fulvum infected, or uninoculated, leaves. The suppressor was stable to treatment with heat, protease, glucosidase, galactosidase, laminarinase, periodate, and mild acid (0·1 N HCl) and base (0·01 N NaOH). Addition of the chelators, EDTA (15 mM) or EGTA (2 mM), to IF resulted in a marked reduction in suppressor activity. Suppressor activity was partially reduced by dialysis of IF, but activity was lost upon dialysis by prior treatment of IF with urea, or with protease which was then inactivated by heating. Activity of a low molecular weight suppressor, partially purified by dialysis and Sephadex G-25 column chromatography, corresponded with a carbohydrate peak. Pectinase or pectinase-generated oligogalacturonides from polypectate suppressed activity of NSE. However, incubation of NSE with pectinase, followed by heat treatment before assay, did not affect NSE activity. It is suggested that low molecular weight suppressor molecules may originate from action of pectolytic enzymes on host cell walls. In addition to these low molecular weight suppressor, native IF, but not heat treated IF, contained proteins that on in vitro incubation with NSE slowly reduced its ability to induce necrosis.  相似文献   

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