Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

Abstract

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.

Modernization in plant breeding approaches for improvingbiotic stress resistance in crop plants

“…This gene showed stable integration and was inherited by the next generations. This provided resistance against Magnaporthe grisea fungus, which caused rice blast disease (Coca et al, 2004). Wheat transformed with a viral gene encoding KP4 protein conferred 10%-30% resistance against Ustilago maydis causing wheat smut (Schlaich et al, 2007).…”

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“…Oryzae, Rhizoctonia solani, Fusarium graminearum, Magnaporthe grisea, have been succeeded by genetically modifying crops to over express genes of Polygalacturonase-Inhibiting Proteins (PGIPs), Chitinase, 1, 3-Glucanase, OsNPR1, OsAOS2, OsWRKY31, Puroindolines, Bovine Lactoferrin cDNA and Antifungal Proteins (AFP) from Aspergillus giganteus (Nishizawa et al, 1999;Krishnamurthy et al, 2001;Itoh et al, 2003;Coca et al, 2004;Chern et al, 2005;Moreno et al, 2005;Mei et al, 2006;Yuan et al, 2007;Zhang et al, 2008;Ferrari et al, 2012;Han et al, 2012;Fujikawa 2012).…”

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

“…The Aspergillus cDNA sequence encoding the mature AFP protein (51 amino acids and the stop codon) was amplified by PCR using the pBSK(-)afp plasmid as template [17]. Two different afp plasmid constructs in which the mature AFP protein was translationally fused to the yeast α-factor signal sequence for secretion were prepared.…”

“…These fungi are responsible for important diseases in a large number of plants with agricultural and economical importance, such as rice, wheat, potato, or tomato. In addition, transgenic expression of the afp gene, either alone or in combination with other antimicrobial genes, has been shown to confer disease resistance [6,[16][17][18].…”