Isolation and Characterization of the Single beta-tubulin Gene in Phytophthora cinnamomi

Weerakoon, N. D.; Roberts, J. K.; Lehnen, Larry P.; Wilkinson, Jack; Marshall, Jerry S.

doi:10.2307/3761016

Cited by 17 publications

(12 citation statements)

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“…b-Tubulin of P. ultimum shares highest identity (96±97%) with homologs from Oomycetes, and is also highly related (90±93% identity) to b-tubulins from a diverse group of aquatic lower eucaryotic organisms including the green alga C. reinhardtii, the protozoan T. thermophila and the slime mold P. polycephalum. This observation supports the view that Oomycetes and the organisms listed above belong to the Kingdom Protoctista (Cameron et al 1990;Margulis et al 1990;Weerakoon et al 1998). …”

Section: Discussionsupporting

confidence: 80%

“…Homology among b-tubulins has been used as a measure of phylogenetic distances (Weerakoon et al 1998). The fact that b-tubulin from P. microspora shares highest amino acid identity (93±97%) with its counterparts in N. crassa, A.¯avus and A. nidulans (benA) supports its classi®cation in the fungal group Ascomycota (Table 1).…”

Section: Discussionmentioning

confidence: 93%

“…In A and B, experiments were conducted in duplicate, and data presented are representative of several experiments reports that fungi generally have one, or at most a few, b-tubulin genes. For instance, S. cerevisiae, Schizosaccharomyces pombe, N. crassa, A. klebsiana and P. cinnamomi each contain a single b-tubulin gene (Ne et al 1983;Hiraoka et al 1984;Orbach et al 1986;Cameron et al 1990;Weerakoon et al 1998), while A. nidulans, Colletotrichum graminicola and Trichoderma viride each contain two b-tubulin genes (May et al 1987;Panaccione et al 1988;Goldman et al 1993). In contrast, vertebrates have 6±7 a or b-tubulins that exhibit complex patterns of tissue-speci®c or developmental expression (Sullivan 1988).…”

Section: Discussionmentioning

confidence: 97%

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Analysis of β-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

Bollon

Sidhu

1999

Mol Gen Genet

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The anti-cancer drug taxol binds to beta-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC50 greater than 11.7 microM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC50 0.1 microM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding beta-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of beta-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of beta-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [3H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [3H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various beta-tubulin sequences showed that beta-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but beta-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and beta-tubulin.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 97%

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Analysis of β-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

Bollon

Sidhu

1999

Mol Gen Genet

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“…Elicitin is also known to have multiples copies (49). By contrast, β-tubulin is usually found in fewer copies (3,57). Using genes in multiple copies allows detection of smaller amounts of target DNA compared with assays based on single copy genes.…”

Section: Discussionmentioning

confidence: 99%

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

Bilodeau¹,

Lévesque²,

Cock³

et al. 2007

Phytopathology®

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Bilodeau, G. J., Lévesque, C. A., de Cock, A. W. A. M., Duchaine, C., Brière, S., Uribe, P., Martin, F. N., and Hamelin, R. C. 2007. Molecular detection of Phytophthora ramorum by real-time polymerase chain reaction using TaqMan, SYBR Green, and molecular beacons. Phytopathology 97:632-642.Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), β-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using β-tubulin and seemed to be more sensitive.

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“…Localisation of the VATPase to the spongiome but not to the bladder membrane gives strong support to one of the models of water expulsion vacuole function which envisages that the spongiome and bladder are discrete systems which undergo repeated connection and disconnection during each pulsation cycle (Linder and Staehelin 1979). Protists that possess water expulsion vacuoles include species of Oomycetes, a distinct class of organisms traditionally classified with the fungi but which has close affinities with the algae and a variety of other protists (Gunderson et al 1987, Gaertig et al 1993, Weerakoon et al 1998. Numerous species of Oomyceres are important pathogens of plants, including many in the genus Phytophthora such as P. infestans, the late blight of potato (Erwin and Ribeiro 1996).…”

Section: Introductionmentioning

confidence: 99%

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Mitchell

Hardham

1999

Protoplasma

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Summary. The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment in P. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 +0.71 s. The WEV continues to pulse at this rate until approximately 4 rain after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgot pressure in the cyst. As in other organisms, the WEV in P. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H+-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H+-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions from P. nieotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H+-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H+-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists. Abbreviations: BMM butyl methylmethacrylate; F fix 4% formaldehyde fixation; GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation; V-ATPase vacuolar H+-ATPase; WEV water expulsion vacuole,

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Isolation and Characterization of the Single beta-tubulin Gene in Phytophthora cinnamomi

Cited by 17 publications

References 0 publications

Analysis of β-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

Analysis of β-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

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