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Resistance to Fusarium oxysporum f.sp. gladioli in transgenic Gladiolus plants expressing either a bacterial chloroperoxidase or fungal chitinase genes

Kathryn Kamo, Dilip Lakshman, Rudi Pandey, Mary Ann Guaragna, Patricia Okubara, Kanniah Rajasekaran, Jeffrey Cary, Ramon Jordan
Plant cell tissue and organ culture 2015 pp. 1-13
Burkholderia pyrrocinia, Fusarium oxysporum f. sp. gladioli, Gladiolus, RNA, Southern blotting, Western blotting, bacterial proteins, chitinase, disease resistance, enzyme activity, fungi, gene expression, growth retardation, hyphae, leaves, necrosis, organ culture, peroxidases, promoter regions, reverse transcriptase polymerase chain reaction, roots, shoots, transgenes, transgenic plants
Three antifungal genes, a non-heme chloroperoxidase from Pseudomonas pyrrocinia, and an exochitinase and endochitinase from Fusarium venetanum under regulation by the CaMV 35S promoter, were used to transform Gladiolus for resistance to Fusarium oxysporum f. sp. gladioli. Gladiolus plants were confirmed to be transgenic by Southern hybridization. Semi quantitative RT-PCR of RNA isolated from leaves and roots demonstrated expression of the Fusarium exochitinase and endochitinase genes in transgenic plants compared to controls. All transgenic plants expressing the Fusarium exochitinase or endochitinase gene had chitinase activity higher than that of the control plants. Semi-quantitative RT-PCR verified that three of the four plant lines with the chloroperoxidase gene expressed the transgene in leaves and roots while no expression was detected in control plants. Western hybridization confirmed the presence of the chloroperoxidase protein in both leaves and roots of transgenic plants. Cell extracts from one endochitinase plant line inhibited growth of germinated F. oxysporum spores more consistently than extracts from the four chloroperoxidase and three endochitinase plant lines. Three chloroperoxidase, two exochitinase, and three endochitinase transgenic plant lines sustained a significantly (P\0.05) lower density of hyphae on roots compared to roots of nontransformed Gladiolus plants three to four days following exposure of the roots to Fusarium. Shoots from two plant lines, one containing a chloroperoxidase and the other an endochitinase gene, had less necrosis when rated on a scale of 1–3 and appeared visually to be healthier and without obvious Fusarium infection than non-transformed, regenerated Gladiolus plants 17–21 days following exposure to F. oxysporum.