Ubiquitous urease affects soybean susceptibility to fungi
Beatriz Wiebke-Strohm
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Giancarlo Pasquali
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Marcia Margis-Pinheiro
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Marta Bencke
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Lauro Bu cker-Neto
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Arlete B. Becker-Ritt
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Anne H. S. Martinelli
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Ciliana Rechenmacher
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Joseph C. Polacco
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Renata Stolf
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Francismar C. Marcelino
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Ricardo V. Abdelnoor
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Milena S. Homrich
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Emerson M. Del Ponte
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Celia R. Carlini
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Mayra C. C. G. De Carvalho
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Maria Helena Bodanese-Zanettini
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J. C. Polacco Biochemistry Department, University of Missouri
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Columbia, MO, USA
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G. Pasquali A. B. Becker-Ritt A. H. S. Martinelli C. R. Carlini Programa de Pos-Graduacao em Biologia Celular e Molecular, Centro de Biotecnologia
, UFRGS, Porto Alegre,
Brazil
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B. Wiebke-Strohm M. Margis-Pinheiro M. Bencke L. Bucker-Neto C. Rechenmacher M. S. Homrich M. H. Bodanese-Zanettini (&) Programa de Pos-Graduacao em Genetica e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS)
, Porto Alegre,
Brazil
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E. M. Del Ponte Departamento de Fitossanidade
, Faculdade de Agronomia, UFRGS, Porto Alegre,
Brazil
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R. Stolf F. C. Marcelino R. V. Abdelnoor M. C. C. G. De Carvalho Empresa Brasileira de Pesquisa Agropecuaria, Embrapa Soja, Londrina,
Brazil
The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from nontransgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.
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The soybean plant (Glycine max) is affected by several
diseases that negatively affect plant yield, eventually
resulting in significant crop losses (Sinclair and Hartman
1999). Host genetic resistance is the most desirable and
efficient control measure when resistant genotypes are
available. However, for some fungal diseases, such as
Asian soybean rust, which is caused by Phakopsora
pachyrhizi, fungicides are the only efficient measure to
avoid crop losses. This measure often results in increasing
economic and environmental costs (Miles et al. 2007).
Understanding the molecular basis of the soybean plant
defence against fungal infection and growth, identifying
genes involved in hypersensitive or immune response, and
characterising their individual roles are key steps for
engineering durable and quantitative disease resistance.
Ureases (EC 3.5.1.5) are nickel-dependent
metalloenzymes that catalyse the conversion of urea to ammonia and
carbon dioxide, thus allowing organisms to use exogenous and
internally generated urea as a nitrogen source (Dixon et al.
1975; Krajewska 2009). These enzymes are synthesised by
numerous organisms, including plants, fungi and bacteria
(Follmer 2008; Krajewska 2009). Two isozymes, which share
87% amino acid identity, have been described for the soybean
plant (Goldraij et al. 2003). The embryo-specific urease,
encoded by the GmEu1 gene (GenBank accession AY230157,
Phytozome accession Glyma05g27840.1), is synthesised in
the developing embryo and accumulates in mature seeds
(Polacco and Havir 1979; Polacco and Winkler 1984; Polacco
and Holland 1993), while the ubiquitous urease, encoded by
the GmEu4 gene (GenBank accession AY230156, Phytozome
accession Glyma11g37250.1), is found in lower amounts in all
plant tissues (Torisky et al. 1994). The ubiquitous urease is
involved in recycling metabolically derived urea (Polacco
et al. 1985; Stebbins and Polacco 1995; Witte et al. 2002), but
an assimilatory role for the abundant seed urease has not been
demonstrated thus far (Carlini and Polac (...truncated)