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

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Abstract

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 non-transformed 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.

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Acknowledgments

Mention of a trademark, proprietary product or vendor does not imply its approval to the exclusion of other products or vendors that may also be suitable. Siobhan O’Connor provided excellent technical assistance with the DNA blot and RNA analysis and Sarah Wantoch with the Western blot.

Author’s contributions

KK developed the study, wrote the manuscript and did the antifungal challenge experiments with DL and RP. MAG and RJ did the Western blot. PO provided the two chitinase genes. KR and JC provided the CPO gene that JC had subcloned for bombardment.

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Authors and Affiliations

  1. Floral and Nursery Plants Research Unit, USDA, National Arboretum, Beltsville, MD, USA

    Kathryn Kamo, Dilip Lakshman, Ruchi Pandey, Mary Ann Guaragna & Ramon Jordan

  2. Root Disease and Biological Control Research Unit, USDA, Pullman, WA, USA

    Patricia Okubara

  3. Food and Feed Safety Research Unit, Southern Regional Research Center, USDA, New Orleans, LA, USA

    Kanniah Rajasekaran & Jeffrey Cary

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  1. Kathryn Kamo
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  2. Dilip Lakshman
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Correspondence to Kathryn Kamo.

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Kamo, K., Lakshman, D., Pandey, R. et al. Resistance to Fusarium oxysporum f. sp. gladioli in transgenic Gladiolus plants expressing either a bacterial chloroperoxidase or fungal chitinase genes. Plant Cell Tiss Organ Cult 124, 541–553 (2016). https://doi.org/10.1007/s11240-015-0913-1

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  • DOI: https://doi.org/10.1007/s11240-015-0913-1

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