Deletion of Two <i>Endo</i>-β-1,4-Xylanase Genes Reveals Additional Isozymes Secreted by the Rice Blast Fungus

Wu, Sheng‐Cheng; Ham, Kyung-Sik; Darvill, Alan G.; Albersheim, Peter

doi:10.1094/mpmi.1997.10.6.700

Cited by 99 publications

(82 citation statements)

References 43 publications

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“…There are cases where single-gene disruption caused some decrease in virulence (43,50). In other cases, multiple-gene disruption failed to significantly decrease virulence (8,12,13). Therefore, conclusive evidence for or against a role for any particular enzyme activity in any aspect of pathogenesis has been difficult to obtain (51).…”

Section: Discussionmentioning

confidence: 99%

“…A variety of lines of evidence for their role in pathogenesis, including the correlation between the level of such enzymes and virulence, immunocytochemical evidence for the secretion of the enzyme during infection, protection of the host by inhibition of the enzymes with selective inhibitors or specific antibodies, and enhancement of virulence by gene transfer, have been challenged on the basis of the results of disruption experiments on genes that encode cutinase and cell wall-degrading enzymes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In the case of animal pathogens, single and double proteinase gene disruption failed to show a clear decrease in virulence (15)(16)(17).…”

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confidence: 99%

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Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

Rogers

Kim

Guo

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

127

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Fungal pathogens usually have multiple genes that encode extracellular hydrolytic enzymes that may degrade the physical barriers in their hosts during the invasion process. Nectria hematococca, a plant pathogen, has two inducible pectate lyase (PL) genes (pel) encoding PL that can help degrade the carbohydrate barrier in the host. pelA is induced by pectin, whereas pelD is induced only in planta. We show that the disruption of either the pelA or pelD genes alone causes no detectable decrease in virulence. Disruption of both pelA and pelD drastically reduces virulence. Complementation of the double disruptant with pelD gene, or supplementation of the infection droplets of the double disruptant with either purified enzyme, PLA, or PLD, caused a recovery in virulence. These results show that PL is a virulence factor. Thus, we demonstrate that disruption of all functionally redundant genes is required to demonstrate the role of host barrier-degrading enzymes in pathogenesis and that dismissal of the role of such enzymes based on the effects of single-gene disruption may be premature.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

Rogers

Kim

Guo

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

127

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“…Transformation of M. grisea and M. graminicola was performed essentially as described (33,34), but with ␤-glucanase (InterSpex Products, San Mateo, CA). Transformants were purified and DNA was extracted as described (33). PCR was used to identify homologous recombination events.…”

Section: Methodsmentioning

confidence: 99%

“…Targeted integration (TI) for creating gene-specific mutations is very efficient and requires only 50-bp fragments of target gene homology on either side of a selectable marker (11,12). In contrast, many filamentous fungi have genome sizes in the range of [30][31][32][33][34][35][36][37][38][39][40] Mb and are estimated to contain at least 10,000 genes (13). Genome studies using expressed sequence tag analysis suggest that more than half of these genes lack homologues in S. cerevisiae (14).…”

mentioning

confidence: 99%

Gene discovery and gene function assignment in filamentous fungi

Hamer

Adachi

Montenegro-Chamorro

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

104

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Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposonarrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.A powerful asset for functional genomic analysis is the ability to create large annotated single gene mutant collections. For model research organisms such as baker's yeast, Drosophila, Caenorhabditis, Arabidopsis, and mice, whole genome knockout collections (1), transposon lines (2), or insertional mutant collections (3, 4) are well developed. However, in addition to these model organisms there is a vast array of economically important organisms where genome sequences and functional genomic technologies are lacking. For example, filamentous fungi are causal agents of severe human (5, 6) and crop (7, 8) diseases, and many others are being exploited in the fermentation and food industries (9). Few of these fungal genomes have been analyzed, and large-scale approaches to functional analysis are needed.The model fungus, Saccharomyces cerevisiae, has Ϸ6000 genes in 12 Mb of DNA sequence (10). Targeted integration (TI) for creating gene-specific mutations is very efficient and requires only 50-bp fragments of target gene homology on either side of a selectable marker (11,12). In contrast, many filamentous fungi have genome sizes in the range of 30-40 Mb and are estimated to contain at least 10,000 genes (13). Genome studies using expressed sequence tag analysis suggest that more than half of these genes lack homologues in S. cerevisiae (14). TI occurs at very low frequencies (1-20%) for many filamentous fungi, and larger fragments of target gene homology must be used to obtain targeted insertion events (15).To initiate genome-wide mutagenesis studies in filamentous fungi we developed an approach we call transposon-arrayed gene knockouts (TAGKO) (Fig. 1). In vitro transposition (IVT) (16-19) into cosmid libraries is used to create gene sequencing templates. Subsequent sequencing and analysis from these templates creates an annotated collection of insertional gene disruption vectors. We demonstrate that IVT can...

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“…High molecular mass and plasmid-free DNA was prepared according to Wu et al (1997) with minor modifications. PCR of pure culture was performed in a GeneAmp PCR system 7900 thermal cycler (Perkin-Elmer) using the general eubacterial primers, 27f (5-AGAGTTTGATCCTGGCTCAG-3) (Giovannoni, 1991) and 1492r (5-GGTTACCTTGTTAC-GACTT-3) (Lane, 1991).…”

Section: Dna Extraction Amplrfication and Sequencingmentioning

confidence: 99%

A Bacterial Strain with Antibacterial and Antioxidant Activities from Tasae, a Burmese Indigenous Alcohol Starter

Aung

Tanaka²,

Zheng

et al. 2004

FSTR

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A total of 85 microbial strains from indigenous alcohol starters of some Asian countries were screened for antibacterial and antioxidant activities. From the result of co-incubation, only one strain, M2-1 showed the antibacterial activity against all target bacteria and this strain was selected. Radical scavenging activity of ethyl acetate extract of M2-1 strain was compared with BHA using DPPH. The free radical scavenging activities were 18.0 and 29.5 % , respectively, at the concentration of 100 and 200 ppm. The antioxidant activity of extract was determined according to the thiocyanate method using AAPH. The inhibition of peroxidation of extract was 22.4% at 250 ppm concentration. Antibacterial activity of extract was assayed by paper disk method using 450 ug extract per disk. The extract has broad inhibition against all the target bacteria. M2-1 strain was identified as Bacillus sp. on the basis of 16S rDNA sequence.Keywords: tasae, an indigenous alcohol starter, antioxidant and antibacterial activities, 16S rDNA sequence.Burma (Myanmar) is a country in Southeast Asia. In making rice wines, Burmese people are still using indigenous alcohol starters in many parts of the country (Aye and Myint, 1978). An indigenous alcohol starter may contain different kinds of microorganisms such as yeasts, molds, and even bacteria (Steinkraus, 1996). The metabolites produced by microorganisms may serve as antioxidants (Hayashi et al., 1995;Ishikawa et al., 1992), scavengers (Kato et al., 1993), Iipoxygenase inhibitors (Nihei et al., 1993) and/or metal chelating agents (Kunze et al., 1992). Derivatives from food constituents produced by the action of microbial enzymes have antioxidant and/or synergistic activity (Murakami et al., 1984). Furthermore, microorganisms can decompose lipid peroxides and use them as nutrients (Rashid et al., 1992).Studies on the antibacterial compounds produced by bacterial flora in indigenous alcohol starter may contribute to:( 1) reduce microbial contamination during the fermentation and, (2) provide new bacteriocin -Iike inhibitory substances with activity on associated alcohol starters as well as on food pathogens or spoilage microorganisms.In our laboratory, we are studying antioxidant and antimicrobial activities of metabolites from yeasts isolated from indigenous alcohol starters of some Asian countries.Therefore, we collected such starters from Indonesia, China, Cambodia, Nepal, and Burma. From those source samples, Abbreviations: BHA. Butylated hydroxyanisole; DPPH, 1,1-Diphenyl-2-picrylhydrazyl; AAPH, 2,2-Azobis (2-amidinopropane) dihydrochloride *TO Whom correspondence should be addressed. E-mail : hasinaga @ chem. agri .kagoshima-u . ac . jp a total of 85 microbial strains were isolated. Fortunately, we found only one bacterial strain that possessed antibacterial activity against target bacteria when screening tests were carried out with the co-incubation method. That strain, isolated from tasae, a Burmese indigenous alcohol starter, was therefore selected to study the antioxidant and an...

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Deletion of Two Endo-β-1,4-Xylanase Genes Reveals Additional Isozymes Secreted by the Rice Blast Fungus

Cited by 99 publications

References 43 publications

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

Gene discovery and gene function assignment in filamentous fungi

A Bacterial Strain with Antibacterial and Antioxidant Activities from Tasae, a Burmese Indigenous Alcohol Starter

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