The recombinant pea defensin Drr230a is active against impacting soybean and cotton pathogenic fungi from the genera Fusarium, Colletotrichum and Phakopsora

Abstract

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.

Keywords: Colletotrichum gossypii var. cephalosporioides; Defensin; Fusarium tucumaniae; Phakopsora pachyrhizi; Pichia pastoris; Pisum sativum.

Fig. 1

Schematic description of the expression cassette construct designed to express Histidine-tagged recombinant Drr230a in yeast (P. pastoris). The drr230a gene was subcloned into the yeast expression vector pPICZαA (Invitrogen Co.) under the control of the alcohol oxidase 1 promoter (pAOX1) inducible by methanol. The orientations of the primers DRR230a01For and DRR230a02Rev, including the respective restriction EcoRI (resulting in addition of EF residues at the recombinant protein N-terminus) and NotI cloning sites (not expressed), are indicated by arrows. The recombinant protein (sequence depicted above) is expressed in frame with the Saccharomyces cerevisiae secretion mating α-factor signal peptide (α-Factor), which is cleaved before protein export at the Kex2 signal cleavage site (Kex2), resulting in the addition of four residues (EAEA) at the N-terminal end of the recombinant protein. A Histidine tag (6xHis; HHHHHH), followed by a stop codon (asterisk) was included at the C-terminal end of the sequence to facilitate recombinant protein purification by Immobilized metal affinity chromatography

Fig. 2

Expression of rDrr230a in P. pastoris X-33 cells. a SDS-TRICINE-PAGE analysis of the expression of the His-tagged rDrr230a protein from 24 to 96 h after methanol-induction. b Western blot analysis of expressed His-tagged rDrr230a protein probed with anti-His antibody (Invitrogen). Molecular mass marker See Blue^®Plus 2 Prestained Standard, Invitrogen (lane M). Supernatant from P. pastoris X-33 culture expressing rDrr230a 24 h (lane 1), 48 h (lane 2), 72 h (lane 3) and 96 h (lane 4) post-induction. Supernatant from P. pastoris X-33 untransformed culture 24 h (lane 5), 48 h (lane 6), 72 h (lane 7) and 96 h (lane 8) post-induction. Arrows indicate bands corresponding to the expressed rDrr230a (6.52 kDa)

Fig. 3

Purification of rDrr230a expressed in P. pastoris X-33 cells. rDrr230a was purified by immobilized-metal affinity chromatography (IMAC). a SDS-TRICINE-PAGE analysis of the purification of rDrr230a. b Western blot analysis of purification of rDrr230a, probed with anti-His antibody (Invitrogen). Molecular mass marker See Blue^®Plus 2 Prestained Standard, Invitrogen (lane M). Supernatant from P. pastoris X-33 culture expressing rDrr230a 96 h after methanol-induction (lane 1). Eluate fraction from IMAC, corresponding to P. pastoris-expressed rDrr230a 96 h after methanol-induction (lane 2). Arrows indicate bands corresponding to the purified rDrr230a (6.52 kDa)

Fig. 4

Inhibition of fungal growth of the non-biotrophic phytopathogenic fungi F. tucumaniae and C. gossypii var. cephalosporioides by rDrr230a. Bioassays were performed in microplate wells in liquid medium for 48 h. The data are mean ± standard deviation (n = 3). a Curve of inhibition of F. tucumaniae growth upon incubation with rDrr230a in a concentration range varying from 0.2 ng/µl up to 60 ng/µl (0.03 µM up to 9.20 µM). Calculated IC₅₀ against F. tucumaniae corresponds to 7.67 µM (50 ng/µl). b Curve of inhibition of C. gossypii var. cephalosporioides growth upon incubation with rDrr230a in a concentration range varying from 5 up to 50 ng/µl (0.77 µM up to 7.67 µM). Calculated IC₅₀ against C. gossypii var. cephalosporioides corresponds to 0.84 µM (5.5 ng/µl)

Fig. 5

Inhibition of spore germination of the non-biotrophic phytopathogenic fungi F. tucumaniae and C. gossypii var. cephalosporioides by rDrr230a. Bioassays were performed in microplate wells in liquid medium for 12 h. a Percentage of the inhibition of spore germination of F. tucumaniae and C. gossypii var. cephalosporioides upon incubation with rDrr230a. Fungal spores were incubated either with rDrr230a at 25 ng/µl, water or hydrogen peroxide at 70 µM. The data in a are mean ± standard deviation (n = 3). b Representative image of F. tucumaniae germinating spores (closed arrows) under water incubation. c Representative image of F. tucumaniae non-germinating spores (open arrows) under rDrr230a incubation. d Representative image of C. gossypii var. cephalosporioides germinating spores (closed arrows) under water incubation. e Representative image of C. gossypii var. cephalosporioides non-germinating spores (open arrows) under rDrr230a incubation

Fig. 6

Plant disease severity (Asian soybean rust) caused by the obligatory biotrophic phytopathogenic fungus P. pachyrhizi upon incubation with rDrr230a. a, c Representative half detached soybean leaflets inoculated with P. pachyrhizi uredospores either in presence rDrr230a at 3 µg/µl (a) or absence of rDrr230a (c) 14 days post inoculation. b, d Sampled areas from inoculated detached leaflets fixed at 1 day post inoculation and analyzed by scanning electron microscopy. Representative micrographs depict no uredospore (S) germination onto well preserved plant epidermal cells when rDrr230a is present (b), whereas there is normal uredospore (S) germination onto damaged epidermal plant cells (yellow arrows) in absence of rDrr230a (d)

Fig. 7

Inhibition of spore germination of the obligatory biotrophic phytopathogenic fungus P. pachyrhizi by rDrr230a. Bioassays were performed in microplate wells in liquid medium for 12 h. a Representative image of P. pachyrhizi germinating spores (arrows) under water incubation. b Representative image of P. pachyrhizi non-germinating spores under incubation with rDrr230a at 3 µg/µl