Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

Abstract

Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination.

Regeneration of Transgenic Oil Palm Carrying GFP Gene Used as a Visual Selectable Marker

“…Furthermore, the gfp gene is useful to study functionality of candidate genes related to drought stress as demonstrated in barley (Nagy et al, 2011). The gfp expressing Aspergillus flavus fungus was also used to study fungal infection in cottonseed (Rajasekaran et al, 2008).…”

Antifungal Activity in Transgenic Peanut (Arachis hypogaeaL.) Conferred by a Nonheme Chloroperoxidase Gene

“…The stable transmission of the cpo-p gene was further confirmed by Southern blot and expression analysis of progeny from one transgenic line. To our knowledge, the bacterial cpo-p gene has been thoroughly studied in only two transgenic plant species, tobacco and cotton, with a vector carrying cpo-p and kanamycin resistance genes (Jacks et al , 2004Rajasekaran et al 2000Rajasekaran et al , 2008. We have previously shown that a codonmodified Bt cryIA(c) gene, which was introduced into three peanut cultivars (Singsit et al 1997), has potential to provide a barrier to fungal entry by reducing insect damage to peanut tissue.…”

“…All of these elements were subcloned into the plasmid vector pBlueScript-SK (Stratagene) to produce the vector gpd-EGFP (Rajasekaran et al 2008). Plasmid gpd-EGFP was cotransformed with the vector pSL82 harboring the A. parasiticus nitrate reductase (niaD) gene into the niaD mutant of A. flavus 70.…”

“…Mature transgenic peanut cotyledons and controls were inoculated with A. flavus 70-GFP to evaluate fungal spread in situ (Rajasekaran et al 2005(Rajasekaran et al , 2008. Cotyledons were soaked overnight in water and sliced (10 mm 3 6 mm 3 6 mm) to keep the explants uniform.…”

Development of Maize Host Resistance to Aflatoxigenic Fungi

“…A more recent use of reporter genes was performed on cotton using a green fluorescent protein reporter; a GFP-expressing A. flavus strain to successfully monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins [38]. This strain provides for an easy, potentially non-destructive, rapid and economical assay which can be done in real time, and may constitute an advance over GUS transformants.…”