Date Published: February 13, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Ariane Ferreira Lacerda, Rafael Perseghini Del Sarto, Marilia Santos Silva, Erico Augusto Rosas de Vasconcelos, Roberta Ramos Coelho, Vanessa Olinto dos Santos, Claudia Vieira Godoy, Claudine Dinali Santos Seixas, Maria Cristina Mattar da Silva, Maria Fatima Grossi-de-Sa.
Plant defensins are antifungal peptides produced by the innate immune system plants developed to circumvent fungal infection. The defensin Drr230a, originally isolated from pea, has been previously shown to be active against various entomopathogenic and phytopathogenic fungi. In the present study, the activity of a yeast-expressed recombinant Drr230a protein (rDrr230a) was tested against impacting soybean and cotton fungi. First, the gene was subcloned into the yeast expression vector pPICZαA and expressed in Pichia pastoris. Resulting rDrr230a exhibited in vitro activity against fungal growth and spore germination of Fusarium tucumaniae, which causes soybean sudden death syndrome, and against Colletotrichum gossypii var. cephalosporioides, which causes cotton ramulosis. The rDrr230a IC50 corresponding to inhibition of fungal growth of F. tucumaniae and C. gossypii var. cephalosporioides was 7.67 and 0.84 µM, respectively, demonstrating moderate activity against F. tucumaniae and high potency against C. gossypii var. cephalosporioides. Additionally, rDrr230a at 25 ng/µl (3.83 µM) resulted in 100 % inhibition of spore germination of both fungi, demonstrating that rDrr230a affects fungal development since spore germination. Moreover, rDrr230a at 3 µg/µl (460.12 µM) inhibited 100 % of in vitro spore germination of the obligatory biotrophic fungus Phakopsora pachyrhizi, which causes Asian soybean rust. Interestingly, rDrr230a substantially decreased the severity of Asian rust, as demonstrated by in planta assay. To our knowledge, this is the first report of a plant defensin active against an obligatory biotrophic phytopathogenic fungus. Results revealed the potential of rDrr230a as a candidate to be used in plant genetic engineering to control relevant cotton and soybean fungal diseases.
Plant diseases caused by fungi affect a broad range of crops worldwide, being responsible for significant losses and for the decrease in quality and safety of agricultural products. In crop plants, fungi cause more economic damage than any other group of microorganisms, with annual losses estimated at more than US$ 200 billion (Horbach et al. 2011). There are several fungal diseases impacting in tropical areas, which include cotton ramulosis, Asian soybean rust and soybean sudden death syndrome caused by Colletotrichum gossypii (South) var. cephalosporioides A. S. Costa, Phakopsora pachyrhizi H. & P. Syd., and Fusarium solani (Mart.) Sacc. f. sp. glycines (= Fusarium tucumaniae T.) (Aoki et al. 2003), respectively. Currently, there are no cotton or soybean varieties with good agronomic traits, which at the same time present high resistance against these important diseases. In addition, none of these diseases are satisfactorily controlled by fungicides.
The successful expression and purification of the defensin rDrr230a by using P. pastoris yeast system, as well as the antimicrobial activity of rDrr230a against impacting cotton and soybean pathogenic fungi was demonstrated in this article. Notably it was demonstrated the inhibition of fungal growth and spore germination of the economically important phytopathogenic fungi F. tucumaniae, P. pachyrhizi and C. gossypii var. cephalosporioides, which cause soybean sudden death syndrome, Asian soybean rust and cotton ramulosis, respectively. The data herein presented indicate moderate activity of rDrr230a against F. tucumaniae and high potency against C. gossypii var. cephalosporioides. rDrr230a also drastically decreased the severity of Asian soybean rust. Moreover, rDrr230a affects the development of the three target fungi tested since spore germination. These results reveal the potential of rDrr230a for plant genetic engineering to control relevant tropical fungal diseases of cotton and soybean, for which there are neither satisfactory chemical control nor resistant varieties commercially available.