The Isolation, Cloning and Characterisation of Novel Antifungal Genes and Their Encoded Products for use in Resistance Strategies Against Fungal Pathogens of Vitis Vinifera


Project Number
IWBT 05-08A

Project title
The isolation, cloning and characterisation of novel antifungal genes and their encoded products for use in resistance strategies against fungal pathogens of Vitis vinifera

Project leader
Vivier, M A

Institution
University of Stellenbosch. Faculty of AgriSciences. Institute for Wine Biotechnology

Team members
Vivier, M A

Project description
Manipulated disease resistance has been linked to plant improvement since it became possible to add genes of interest to economically important plant species. To reach this goal it is imperative to have available genes and promoters that are known to confer resistance to the pathogens targeted in the approach; these should preferably not be protected by patents and licenses. From previous work done in our laboratory it became clear that polygalacturonase inhibiting proteins, cell wall proteins that specifically inhibit various fungal polygalacturonases and that are involved in plant defense responses, as well as antifungal peptides are excellent targets in manipulated disease resistance.

The expression of PGIPs is regulated by various stimuli, including physiological, environmental and pathogen related factors. Recently a PGIP encoding gene from grapevine (PGIP1) has been cloned in our laboratory. Here we show that Vitis vinifera PGIP expression is berry specific, developmentally regulated and induced by a number of external stimuli including wounding, osmotic stresses, Botrytis cinerea infection, indole acetic acid (IAA) and salicylic acid (SA). Tissue specificity is abolished in response to all induction stimuli. Berry ripening is furthermore accompanied by significant increases in osmotic stress as well as changes in hormonal composition. Together these results indicate that temporal physiological conditions within tissues are in part responsible for the tissue specific regulation of grapevine PGIP. Grapevine PGIP is also up-regulated in response to the inhibition of serine/threonine kinases, indicating a role for protein phosphorylation in grapevine PGIP regulation. The core promoter of the pgip1 gene is situated within the first 100 bp upstream of the ATG. Transcription starts only 17 bp upstream from the putative ATG. Putative regulatory sequences will be identified using transient assays; this will hopefully enable us to identify specific pathways involved in grapevine stress responses.

Thirty-four additional PGIP encoding genes have also been isolated and are currently evaluated for specificity towards fungal pathogens. Similarly, six novel antifungal peptide encoding genes have also been isolated and are currently being evaluated. These results significantly strengthen our position and resources to enhance the fungal resistance of grapevine species.

Presentation(s)
Joubert, D A, De Lorenzo, G, Pretorius, I S and Vivier, M A. 2004. The transcriptional regulation of a polygalacturonase inhibiting protein from grapevine (Vitis vinifera L).Paper presented at the 7th International Symposium for Grapevine Physiology and Biotechnology. Davis, California, United States

Vivier, M A and Pretorius, I S. 2004. Innovation in the grapevine industry: The use of biotechnology in cultivar improvement. Paper presented at the 4th World Wine Forum. La Rioja, Spain.

Joubert, D A, De Lorenzo, G, Pretorius, I S, Becker, G and Vivier, M A. 2004. Vitis vinifera L. polygalacturonase inhibiting proteins and their involvement in plant defence response. Paper presented at the 29th International Congress of Biochemistry and Molecular Biology. Toronto, Canada

Joubert, D A, De Lorenzo, G, Pretorius, I S and Vivier, M A. 2004. Regulation of Vitis vinifera polygalacturonase inhibiting protein (PGIP) encoding genes. Paper presented at the Joint Congress of the South African Genetics and Microbiology Societies. Stellenbosch, South Africa.

De Beer, A and Vivier, M A. 2004. Isolation and characterisation of a defence-related gene, Vv-AMP1, from grapevine. Paper presented at the 13th Congress of the South African Society for Microbiology. Stellenbosch, South Africa.

De Beer, A and Vivier, M A. 2004. Over expression of an antimicrobial peptide from Heuchera Sanguinea in tobacco for inhibition of fungal pathogens. Paper presented at the 13th Congress of the South African Society for Microbiology. Stellenbosch, South Africa.

Vivier, M A, Carstens, M, Basson, E, De Beer, A and Becker, J. 2004. Isolation and evaluation of various antifungal genes for use against Botrytis cinerea in biotechnology approaches. Paper presented at the 13th International Botrytis Symposium. Antalya, Turkey

Joubert, D A and Vivier, M A. 2004. The activity of endo-polygalacturonase II from Botrytis cinerea is adversely affected in vivo, but not in vitro by the Vitis vinifera polygalacturonase inhibiting protein (VvPGIP1). Paper presented at the 13th International Botrytis Symposium. Panatela, Turkey

Joubert, D A, De Lorenzo, G, Pretorius, I S and Vivier, M A. 2004. Regulation of Vitis vinifera polygalacturonase inhibiting protein (PGIP) encoding genes. Paper presented at the 13th Congress of the South African Society for Microbiology. Stellenbosch, South Africa.

Joubert, D A, Vivier, M A, De Lorenzo, G and Pretorius, I S. 2004. The regulation of polygalacturonase inhibiting proteins in grapevine (Vitis vinifera). Paper presented at the 7th International Symposium on Grapevine Physiology and Biotechnology. California, United States

Joubert, D A, Kemp, G, Bergmann, C, Van Kan, J A L and Vivier, M A. 2005. Characterisation of the interactions between endopolygalacturonases (PGs) from Botrytis cinerea and a polygalacturonase-inhibiting protein from grapevine (VvPGIP1) with in vitro and in vivo methodologies. Paper presented at the 1st Botrytis Cinerea Genome Workshop. Kaiserslautern, Germany

Vivier, M A, Joubert, D A, Becker, JvW, Wentzel, L, Taylor, K and Young, P R. 2005. Functional analysis of grapevine genes. Paper presented at the International Grape Genomics Symposium. 12-14 July, St Louis, United States

Joubert, D A, Kemp, G, Bergmann, C, Van Kan, J A L and Vivier, M A. 2005. Characterisation of the interactions between endopolygalacturonases (PGs) from Botrytis cinerea and a polygalacturonase-inhibiting protein from grapevine (VvPGIP1) with in vitro and in vivo methodologies. Paper presented at the 1st Botrytis Cinerea Genome Workshop, Kaiserslautern, Germany

De Beer, A and Vivier, M A. 2006. Isolation and characterisation of a fruit-specific defence from Vitis vinifera. Paper presented Plant Dynamic: from Molecular to Ecosystems: 3rd EPSO Conference. 28 May – 1 June, Visegrad, Hungary.

De Beer, A and Vivier, M A. 2006. Isolation and characterisation of a fruit-specific defences in from Vitis vinifera. Paper presented Plant Dynamic: from Molecular to Ecosystems: 3rd EPSO Conference. 28 May- 1 June, Visegrad, Hungary.

Becker, J v W, Joubert, D A and Vivier, M A. 2006. Transcriptome and phytohormone analysis of the resistance phenotype against Botrytis cinerea in transgenic tobacco overexpressing the Vitis vinifera polygalacturonase-inhibiting protein. Paper presented at the 2nd Botrytis Genome Workshop, Versailles, France.

De Beer, A and Vivier, M A. 2005. A small peptide from Vitis vinifera shows strong antifungal activity against the necrotrophic plant pathogen Botrytis cinerea. Paper presented at the 3rd International Congress of the South African Society for Enology and Viticulture. 28-30 July, Cape Town, South Africa

Vivier, M A. 2005. Grapevine biotechnology: Scope and value for viticulture. Paper presented at the 3rd International Congress of the South African Society for Enology and Viticulture. 28-30 July, Cape Town, South Africa.

De Beer, A and Vivier, M A. 2007. A small peptide from Vitis vinifera shows strong anti0fungal activity against the necrotrophic plant pathogen Botrytis cinerea. 14th International Botrytis Symposium, Cape Town, South Africa.

De Beer, A & Vivier, M A. 2007. A ripening induced defending gene from Vitis vinifera shows anti-fungal activity against a broad spectrum of fungal pathogens. Paper presented at the 5th International Table Grape Symposium. Somerset West, South Africa.

Tredoux, M M, de Beer, A, Mostert, L and Vivier, M A. 2007. The evaluation of the anti-fungal activity of a Vitis vinifera anti-fungal peptide. Paper presented at the 14th International Botrytis Symposium. Cape Town, South Africa

Venter, A, Mostert, D A and Vivier, M A. 2007. The evaluation of a range of Vitis polygalacturonase-inhibiting proteins (PGIPs) for their antifungal activity against Botrytis cinerea. Paper presented at the 14th International Botrytis Symposium. Cape Town, South Africa.

Vivier, M A. 2007. Grapevine biotechnology: Scope and value for viticulture and table grape product development. Paper presented at 5th International Table Grape Symposium. Somerset West, South Africa.

Article
Joubert, D A, Slaughter, A R, Kemp, G, Becker, J v W, Krooshof, G H, Bergmann, C, Benen, J, Pretorius, I S, Vivier, M A. 2006. The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases, Transgenic Research, v. 15 (6) (p. 687-702)

Dissertation
Joubert, D A. 2004. Regulation of the Vitis vinifera PGIP1 gene encoding a polygalacturonase-inhibiting protein. PhD. University of Stellenbosch, Stellenbosch, South Africa.

De Beer, A. 2008. Isolation and characterization of an antifungal peptide from grapevine. PhD. University of Stellenbosch, Stellenbosch, South Africa.

Thesis
Wentzel, L. 2005. Specificity analysis of the interaction between isozymes of the Vitis vinifera polygalacturonase inhibiting protein (PGIP) and endo-polygalacturonases (PGs) from Botrytis cinerea. MSc. University of Stellenbosch, Stellenbosch.

Venter, A. 2006. The effect of amino acid changes on the interaction specificity of various grapevine polygalacturonase-inhibiting proteins. MSc. University of Stellenbosch, Stellenbosch.

Tredoux, M M. 2007. Evaluation of transgenic tobacco and grapevine plant lines overexpressing antifungal peptides. MSc. University of Stellenbosch, Stellenbosch.

FinalReport.pdf

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