The development of improved maize genotypes with increased resistance to fungal pathogens is one of the major objectives of breeding biotechnology strategies. F. verticillioides attacks maize, causing root, stem, and ear rot diseases, and produces mycotoxins (fumonisins), often associated with mycotoxicoses in livestock and also in humans. In maize endosperm a cytosolic albumin with a molecular weight of 32 kDa, termed b-32, has homology with several previously characterized Ribosome-Inactivating Proteins (RIPs). It was found that b-32 is a functional RIP and shows anti-fungal activity by in vitro and in vivo experiments. Research is in progress to verify if maize plants expressing b32 in various organs and tissues have an increased defence against fungal pathogens in comparison with plants expressing b32 only in the kerne. For these purpoes transgenic plants were obtained through genetic transformation using the vector pSC1b32 containing the b-32 coding seguence clone under the constitutive promoter 35SCaMV and the cassette ubil-bar for basta herbicide reisstance as selectable marker. A set of six homozygous progenies PCR-b32 and western b32 positive and western-b32 negative (as negative control) were raised to maturity into a containment-greenhouse and used, at flowering stage, for a detailed analyiss of b-32 expression in leaves and for pathogenicity tests.A differential b-32 expression in the lesves of various progenies was recorded. Proteomic experimentson protein leaf extracts have been set up. The 2DE map matching clearly showed the presence of additional spots in a progeny b-32 wesetrn positive, in comparison to a progeny basta-sensitive and B-32 western negative. These spots have been cut and digested with trypsin to achieve proetin identification by MALDI-TOF MS. Both induced b-32 and herbicide resistance in multiple spots have been successfully identified the identification of progenies with a differential b-32 expression in the leaves was useful for setting up pathogenicity experiments, in order to evaluete a possible differential response to Fusarium attack in leaf tissue colonization bioassay. Plants were raised to maturity into a containment-greenhouse. Preliminary experiments supported the choice of bioassay parameters (spore concentration, detection time) useful for a reliable evaluation of genotype. the negative control was the most susceptible to F.Verticilloides attack, in comparison to all the progenies expressing b-32. Experiments are in progress to extend pathogenicity tests to other palnt tissues and to evaluate the specificity of b-32 role in the defence against other fungal pathogens (i.e., Aspergillus, Penicillum).
The maize ribosome-inactivating protein b-32: genetic engineering for maize protection againstmycotoxigenic fungal pathogens
MG Giuffrida;
2006
Abstract
The development of improved maize genotypes with increased resistance to fungal pathogens is one of the major objectives of breeding biotechnology strategies. F. verticillioides attacks maize, causing root, stem, and ear rot diseases, and produces mycotoxins (fumonisins), often associated with mycotoxicoses in livestock and also in humans. In maize endosperm a cytosolic albumin with a molecular weight of 32 kDa, termed b-32, has homology with several previously characterized Ribosome-Inactivating Proteins (RIPs). It was found that b-32 is a functional RIP and shows anti-fungal activity by in vitro and in vivo experiments. Research is in progress to verify if maize plants expressing b32 in various organs and tissues have an increased defence against fungal pathogens in comparison with plants expressing b32 only in the kerne. For these purpoes transgenic plants were obtained through genetic transformation using the vector pSC1b32 containing the b-32 coding seguence clone under the constitutive promoter 35SCaMV and the cassette ubil-bar for basta herbicide reisstance as selectable marker. A set of six homozygous progenies PCR-b32 and western b32 positive and western-b32 negative (as negative control) were raised to maturity into a containment-greenhouse and used, at flowering stage, for a detailed analyiss of b-32 expression in leaves and for pathogenicity tests.A differential b-32 expression in the lesves of various progenies was recorded. Proteomic experimentson protein leaf extracts have been set up. The 2DE map matching clearly showed the presence of additional spots in a progeny b-32 wesetrn positive, in comparison to a progeny basta-sensitive and B-32 western negative. These spots have been cut and digested with trypsin to achieve proetin identification by MALDI-TOF MS. Both induced b-32 and herbicide resistance in multiple spots have been successfully identified the identification of progenies with a differential b-32 expression in the leaves was useful for setting up pathogenicity experiments, in order to evaluete a possible differential response to Fusarium attack in leaf tissue colonization bioassay. Plants were raised to maturity into a containment-greenhouse. Preliminary experiments supported the choice of bioassay parameters (spore concentration, detection time) useful for a reliable evaluation of genotype. the negative control was the most susceptible to F.Verticilloides attack, in comparison to all the progenies expressing b-32. Experiments are in progress to extend pathogenicity tests to other palnt tissues and to evaluate the specificity of b-32 role in the defence against other fungal pathogens (i.e., Aspergillus, Penicillum).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
