A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid.
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Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroidae, replicates and accumulates in the chloroplast. Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) with a multisubunit structure similar to the Escherichia coli enzyme and a single-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerases. On a different basis, sensitivity to tagetitoxin, two major RNA polymerase activities, tagetitoxin sensitive (TS) and resistant (TR), have been found in plastids. The most plausible candidates for the TS and TR RNA polymerases are PEP and NEP, respectively. To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations from ASBVd-infected leaves were assayed for their in vitro ability to transcribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, accD, and rpoB) of the three classes of chloroplastic genes according to their promoter structure. High concentrations of alpha-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5-10 microM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50-100 microM) was a 25% inhibition observed. These results suggest that NEP is the RNA polymerase required in ASBVd replication, although the participation of another TR RNA polymerase from the chloroplast cannot be excluded. (+info)
pH regulation of pectate lyase secretion modulates the attack of Colletotrichum gloeosporioides on avocado fruits.
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Growth of Colletotrichum gloeosporioides in pectolytic enzyme-inducing medium (PEIM) increased the pH of the medium from 3. 8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached 5.8, and the level of secretion increased up to pH 6.5. PL gene (pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or when C. gloeosporioides hyphae were transferred from PL-secreting conditions at pH 6.5 to pH 3.8. This behavior differed from that of polygalacturonase (PG), where pg transcripts and protein secretion were detected at pH 5.0 and continued up to 5.7. Under in vivo conditions, the pH of unripe pericarp of freshly harvested avocado (Persea americana cv. Fuerte) fruits, resistant to C. gloeosporioides attack, was 5.2, whereas in ripe fruits, when decay symptoms were expressed, the pericarp pH had increased to 6.3. Two avocado cultivars, Ardit and Ettinger, which are resistant to C. gloeosporioides attack, had pericarp pHs of less than 5.5, which did not increase during ripening. The present results suggest that host pH regulates the secretion of PL and may affect C. gloeosporioides pathogenicity. The mechanism found in avocado may have equivalents in other post-harvest pathosystems and suggests new approaches for breeding against and controlling post-harvest diseases. (+info)
Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure.
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Viroids replicate through a rolling-circle mechanism in which the infecting circular RNA and its complementary (-) strand are transcribed. The precise site at which transcription starts was investigated for the avocado sunblotch viroid (ASBVd), the type species of the family of viroids with hammerhead ribozymes. Linear ASBVd (+) and (-) RNAs begin with a UAAAA sequence that maps to similar A+U-rich terminal loops in their predicted quasi-rod-like secondary structures. The sequences around the initiation sites of ASBVd, which replicates and accumulates in the chloroplast, are similar to the promoters of a nuclear-encoded chloroplastic RNA polymerase (NEP), supporting the involvement of an NEP-like activity in ASBVd replication. Since RNA folding appears to be kinetically determined, the specific location of both ASBVd initiation sites provides a mechanistic insight into how the nascent ASBVd strands may fold in vivo. The approach used here, in vitro capping and RNase protection assays, may be useful for investigating the initiation sites of other small circular RNA replicons. (+info)
A mitogen-activated protein kinase kinase required for induction of cytokinesis and appressorium formation by host signals in the conidia of Colletotrichum gloeosporioides.
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Differentiation of fungal conidia of phytopathogens into the infection structure, appressorium, requires contact with a hard surface and host signals. The molecular signaling involved in the induction of this differentiation is poorly understood. We report the cloning of a mitogen-activated protein kinase kinase (MEK), CgMEK, from Colletotrichum gloeosporioides and its role in the induction of these developmental processes involved in pathogenesis. Disruption of CgMEK1 resulted in the loss of its ability to form appressoria in response to the host's signals and a loss of virulence. Results of confocal microscopic examination of germinating conidia of the gene-disrupted mutants were similar to those for wild-type conidia treated with an MEK inhibitor, suggesting that CgMEK1 is involved in two developmental processes in the differentiation into appressorium: (1) polarized cell division, with the preferential increase in F-actin in one of the daughter nuclei after nuclear division and the formation of septum; and (2) differentiation of the germ tube into an appressorium. CgMEK1 is required for the differentiation. (+info)
Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado.
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Avocado (Persea americana Mill. cv Lula) is a climacteric fruit that exhibits a rise in ethylene as the fruit ripens. This rise in ethylene is followed by an increase in abscisic acid (ABA), with the highest level occurring just after the peak in ethylene production. ABA is synthesized from the cleavage of carotenoid precursors. The cleavage of carotenoid precursors produces xanthoxin, which can subsequently be converted into ABA via ABA-aldehyde. Indirect evidence indicates that the cleavage reaction, catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED), is the regulatory step in ABA synthesis. Three genes encoding NCED cleavage-like enzymes were cloned from avocado fruit. Two genes, PaNCED1 and PaNCED3, were strongly induced as the fruit ripened. The other gene, PaNCED2, was constitutively expressed during fruit ripening, as well as in leaves. This gene lacks a predicted chloroplast transit peptide. It is therefore unlikely to be involved in ABA biosynthesis. PaNCED1 was induced by water stress, but expression of PaNCED3 was not detectable in dehydrated leaves. Recombinant PaNCED1 and PaNCED3 were capable of in vitro cleavage of 9-cis-xanthophylls into xanthoxin and C(25)-apocarotenoids, but PaNCED2 was not. Taken together, the results indicate that ABA biosynthesis in avocado is regulated at the level of carotenoid cleavage. (+info)
Modification of articular cartilage and subchondral bone pathology in an ovine meniscectomy model of osteoarthritis by avocado and soya unsaponifiables (ASU).
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OBJECTIVE: To examine the effect of an oral preparation of avocado and soya unsaponifiables (ASU) on the development of joint pathology in an ovine model of osteoarthritis (OA), using computer-assisted histomorphometric methods. DESIGN: OA was induced in ovine knee joints by bilateral lateral meniscectomy (N=32). ASU (900 mg/weekday) was given orally to half the group (MenX+ASU), the remainder receiving placebo (MenX). Sixteen animals were used as non-operated controls (NOC). At 3 and 6 months post-meniscectomy, histological sections from the medial and lateral femoral condyles (MFC, LFC), tibial plateaux (MTP, LTP) and trochlear groove (TG) were prepared from all joints. Sections were scored using traditional histopathological scales, and computerized image analysis, measuring total cartilage area, uncalcified cartilage (UCC) and subchondral bone plate (SCP) thickness, and intensity of articular cartilage toluidine blue staining (mean greyscale intensity, black=255) as an index of proteoglycan (PG) content. RESULTS: Computerized image analysis showed significant histological differences not detectable by traditional scoring methods. ASU-treated animals at 6 months showed reduced loss of toluidine blue stain in the MTP (P=0.015) and LTP (P=0.001), and significantly greater staining in the TG than either placebo or NOC groups (P=0.011). UCC thickness increased after meniscectomy, but tended to be highest in ASU-treated animals, significantly so in the middle zone of the LFC (MenX+ASU: 1.03+/-0.21mm vs MenX: 0.79+/-0.14 mm, P=0.018; NOC: 0.77+/-0.17 mm). Lateral compartment SCP thickness increased post-meniscectomy but was increased significantly less in the inner zone of the LTP in ASU-treated sheep (MenX+ASU: 1.37+/-0. 23 mm vs MenX: 1.68+/-0.28 mm, P=0.033; NOC=1.22+/-0.33 mm). CONCLUSIONS: In this model ASU treatment following meniscectomy appeared to confer a subtle but statistically significant protective effect on articular cartilage. Although the drug failed to prevent focal cartilage lesions, characteristic of this model, histomorphometric analysis demonstrated greater PG content and UCC thickness in adjacent joint regions of ASU-treated animals. In addition, a statistically significant reduction of subchondral bone sclerosis was noted in the LTP region of the drug-treated group. An anabolic effect on chondrocytes, resulting in the stimulation of matrix production in regions distant to the insult, was also suggested by the data. These findings support other studies which have proposed that ASU may exhibit disease-modifying anti-OA activity. (+info)
Hydrogel control of xylem hydraulic resistance in plants.
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Increasing concentrations of ions flowing through the xylem of plants produce rapid, substantial, and reversible decreases in hydraulic resistance. Changes in hydraulic resistance in response to solution ion concentration, pH, and nonpolar solvents are consistent with this process being mediated by hydrogels. The effect is localized to intervessel bordered pits, suggesting that microchannels in the pit membranes are altered by the swelling and deswelling of pectins, which are known hydrogels. The existence of an ion-mediated response breaks the long-held paradigm of the xylem as a system of inert pipes and suggests a mechanism by which plants may regulate their internal flow regime. (+info)
Inhibition by (+)-persenone [corrected] A-related compounds of nitric oxide and superoxide generation from inflammatory leukocytes.
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We have previously reported that persenone A, isolated from avocado fruit, is an effective inhibitor of both nitric oxide (NO) and superoxide (O2-) generation in cell culture systems. In this study, we have prepared four persenone A-related compounds and examined their inhibition of NO and O2- generation from inflammatory leukocytes. Some structural importance in persenone A to attenuate free radical generation is discussed. (+info)