Ectopic expression of Dahlia merckii defensin ...

Planta DOI 10.1007/s00425-006-0471-1

O RI G I NAL ART I C LE

Ectopic expression of Dahlia merckii defensin DmAMP1 improves papaya resistance to Phytophthora palmivora by reducing pathogen vigor Yun J. Zhu · Ricelle Agbayani · Paul H. Moore

Received: 15 December 2006 / Accepted: 19 December 2006 © Springer-Verlag 2007

Abstract Phytophthora spp., some of the more important casual agents of plant diseases, are responsible for heavy economic losses worldwide. Plant defensins have been introduced as transgenes into a range of species to increase host resistance to pathogens to which they were originally susceptible. However, the eVectiveness and mechanism of interaction of the defensins with Phytophthora spp. have not been clearly characterized in planta. In this study, we expressed the Dahlia merckii defensin, DmAMP1, in papaya (Carica papaya L.), a plant highly susceptible to a root, stem, and fruit rot disease caused by Phytophthora palmivora. Extracts of total leaf proteins from transformed plants inhibited growth of Phytophthora in vitro and discs cut from the leaves of transformed plants inhibited growth of Phytophthora in a bioassay. Results from our greenhouse inoculation experiments demonstrate that expressing the DmAMP1 gene in papaya plants increased resistance against P. palmivora and that this increased resistance was associated with reduced hyphae growth of P. palmivora at the infection sites. The inhibitory eVects of DmAMP1 expression in papaya suggest this approach has good potential to impart transgenic resistance against Phytophthora in papaya. Y. J. Zhu (&) · R. Agbayani Hawaii Agriculture Research Center, 99-193 Aiea Heights Drive, Aiea, HI 96701, USA e-mail: [email protected] R. Agbayani e-mail: [email protected] P. H. Moore Agriculture Research Service, USDA, 99-193 Aiea Heights Drive, Aiea, HI 96701, USA e-mail: [email protected]

Keywords Carica papaya · Genetic transformation · Root rot · Plant microbe interaction Abbreviations NPT II Neomycin phosphotransferase ELISA Enzyme linked immunosorbent assay PCR Polymerase chain reaction DmAMP1 Dahlia merckii defensin MS Murashige and Skoog plant culture medium CaMV35S CauliXower mosaic virus promoter NOS Nopaline synthase promoter SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis NBT Nitroblue tetrazolium GLM General linear model LSD Least signiWcant diVerence

Introduction Phytophthora spp. comprise at least 58 oomycete species infecting more than 1,000 plant species and causing billons of dollars in economic losses each year worldwide (Erwin and Ribeiro 1996). Papaya (Carica papaya L.), an important fruit crop of the tropics, is highly susceptible to Phytophthora palmivora (Nishijima 1994). Heavy yield losses associated with P. palmivora fruit and root rot can cause severe decline or death of papaya trees, particularly in poorly drained areas, during the cool and rainy season of winter (Nishijima 1994). Control of this pathogen is needed to decrease dependence on fungicides, increase crop productivity, and improve pre- and post-harvest fruit quality.

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Plant defensins are small (45–54 amino acids), highly basic cysteine-rich peptides that are apparently ubiquitous throughout the plant kingdom (Broekaert et al. 1997; Lay and Anderson 2005; Thomma et al. 2002). Although plant defensins share common chemical elements and three-dimensional structures, the plant defensin family is quite diverse in amino acid composition and biological activity (Lay and Anderson 2005; Thomma et al. 2002). Some defensins fail to show any antimicrobial activity, while others have been found to have antifungal and occasionally antibacterial activity in vitro (Broekaert et al. 1995, 1997; Osborn et al. 1995; Roy-Barman et al. 2006; Segura et al. 1998, 1999). Among defensins that have antifungal eVects, some cause hyperbranching of Wlamentous fungi while others do not produce morphological changes in the pathogens (Broekaert et al. 1997, 1995; Osborn et al. 1995; Segura et al. 1998, 1999). The plant defensins DmAMP1 from Dahlia merckii and RsAFP2 from Raphinus sativus have been shown to induce an array of relatively rapid responses, including increased K+ eZux and Ca2+ uptake, membrane potential changes, and membrane-permeabilization in cells of yeast (Saccharomyces cerevisiae) and black mold (Neurospora crassa) (Thevissen et al. 1996, 1999). In addition, plant defensins were shown to interact directly with plasma membrane components such as sphingolipids from mutants of S. cerevisiae (Thevissen et al. 2003) and glucosylceramindes from yeast species (Thevissen et al. 2004). These results demonstrate that structurally homologous antifungal peptides produced by species from diVerent eukaryotic kingdoms interact with the same target in fungal plasma membranes. Antifungal activity of these particular defensins is directly associated with deleterious alteration of the pathogen membrane system. Transgenic expression of plant defensins has been reported to increase protection of vegetative tissues against pathogen attack. Constitutive expression of the radish defensin clearly enhanced resistance of tobacco plants to the fungal leaf pathogen Alternaria longipes (Terras et al. 1995) and of tomato to Alternaria solani (Parashina et al. 2000). Transgenic canola (Brassica napus) constitutively expressing a pea defensin showed

slightly enhanced resistance against blackleg (Leptosphaeria maculans) disease (Wang et al. 1999). Transgenic potato expressing an alfalfa defensin exhibited robust resistance against Verticillium dahliae under Weld conditions (Gao et al. 2000). Recently, transgenic wheat expressing a lipid transfer protein, Ace-AMP1, increased resistance to Neovossia indica (Roy-Barman et al. 2006). These studies indicate that engineering disease resistance in crops with a range of plant defensins has potential to provide protection against various diseases. The dahlia DmAMP1 defensin inhibits the in vitro growth of a broad range of fungi at micromolar concentration (Osborn et al. 1995; Thevissen et al. 1996). We hypothesized therefore, that resistance of papaya to Phytophthora spp. might be increased by transformation with the DmAMP1 gene. In this paper we report transformation of papaya plants with the dahlia defensin gene, expressed with a constitutive promoter, showed inhibitory eVects on growth of Phytophthora in bioassays in vitro and in situ. The inhibition on Phytophthora growth is associated with the reduced thickness of hyphae cell wall at the infection sites. These results suggest this approach has good potential to improve plant resistance to Phytophthora.

Materials and methods Plant materials and transformation procedure Previously published procedures (Fitch et al. 1990) were used for producing and multiplying somatic embryogenic cultures from seedling hypocotyls of papaya cv. Kapoho. The embryogenic cultures were grown in Petri plates on agar solidiWed MS nutrient media. Twenty plates of embryogenic callus derived from papaya cv. Kapoho were used for transformation with the plasmid construct DmAMP1 (Fig. 1) containing the antifungal plant defensin gene DmAMP1 from Dahlia merckii (Osborn et al. 1995) under the control of the constitutive promoter cauliXower mosaic virus promoter (CaMV35S). The transformation plasmid also contained a selectable marker construct consisting

Fig. 1 Diagram showing core features and restriction sites of the DmAMP1 construct that was used for transformation of papaya. Size of elements not drawn to scale

TMV omega HindIII

Nos 3''

NOS prmtr NPTII

13

SP

EcoRI XhoI NcoI NcoI SacI

2 x 35S prom

Nos 3''

DmAMP1

Planta

of the kanamycin or geneticin resistance gene neomycin phosphotransferase (NPT II) Xanked by the nopaline synthase promoter (NOS) promoter and the NOS terminator. As a negative control, a vector containing only the NPT II selectable marker genes was transformed into calli. Gene insertion was by particle bombardment with conditions optimized for papaya embryogenic callus (Fitch et al. 1990). Bombarded callus was given a 10-day recovery period by transfer to fresh callus induction medium without antibiotic selection. After the recovery period, the growing callus tissues were transferred to a selection medium containing 100 mg l¡1 geneticin. Bombarded cultures were grown on the selection medium by subculturing onto fresh medium at 3–4-week intervals. After 3 months, selectively growing geneticin-resistant calli were selected, placed on regeneration medium, and regenerated into plants using published methods (Fitch 1993). Transgenic plant multiplication and growth conditions Putatively transformed plants were multiplied by using the micropropagation methods of Fitch (1993). When roots appeared, plants were transplanted to pots containing plant growth medium for greenhouse growth. The transgenic clonal lines were grown in pots in the greenhouse at 25 § 4°C and relative humidity ranging from 30 to 60% under natural daylight for about 3 months at which time the plants were suYciently large to be harvested for leaves that were used for molecular and pathology evaluations. Protein extract and ELISA assay Protein extraction of calli and plant leaves was performed as previously described (Zhu et al. 1997). Tissues (0.1 g) were homogenized under liquid nitrogen and extracted with 50 mM MES (pH 6.0) containing a mixture of protease inhibitors (1 mM phenylmethylsulfonylXuoride, 1 mM N-ethylmaleimide, 5 mM EDTA, and 0.02 mM pepstatin A). Crude extracts were separated by centrifugation at 15,000 g for 10 m at 4°C. Total protein content of the supernatant was determined using the Bradford protein assay (Bradford 1976) with BSA as a standard. Enzyme linked immunosorbent assay (ELISA) assays were conducted as competitive assays essentially as previously described (Francois et al. 2002; Penninckx et al. 1996). The ELISA microtiter plates were coated with 50 ng ml¡1 puriWed DmAMP1 protein (provided by Dr. Cammue) dissolved in coating buVer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6). Primary antisera were used as a 1,000-fold diluted solu-

tion in 3% (w/v) gelatin (Sigma, St. Louis, MO, USA) in phosphate-buVered saline (140 mM NaCl, 3 mM KCl, 2 mM KH2PO4, and 8 mM Na2HPO4, pH 7.4) containing 0.05% (v/v) Tween 20. The quantity of DmAMP1 protein equivalents in plant extracts was expressed as percent total soluble protein (TSP). Western-blot analysis Crude proteins were extracted from callus (0.1 g each line) homogenized in 2.5% SDS, 10% sucrose and 50 mM CaCl2, heated at 60°C for 5 m. Protein concentration was determined using the Bradford assay (Bradford 1976). Twenty
g total proteins were loaded onto each lane and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) using a Bio-Rad Mini Electrophoresis system per the manufacturer’s instructions. The positive control consisted of loading 100 ng of puriWed DmAMP1 protein. After electrophoresis, the separated proteins were transferred onto nitrocellulose membranes (BioRad, Hercules, CA, USA) using an Electro Transblot apparatus (Bio-Rad). Molecular masses of the proteins were determined on the SDS-PAGE gel by comparison to prestained Kaleidoscope peptide standards in the range of 3.5–31 kDa (Bio-Rad). Immunodetection of transgenically produced DmAMP1 was done on nitrocellulose membranes washed with a polyclonal antibody prepared against puriWed DmAMP1 peptide (provided by Dr. Cammue). The puriWed antibody was used at a dilution of 1:5,000. The nitrocellulose membranes were blocked for 1 h in 5% BSA in TBS (20 mM Tris–HCl, pH 7.5, 150 mM NaCl). The blots were incubated for 1 h against primary antibody in TBS containing 5% BSA then subsequently with alkaline-phosphatase-conjugated goat anti-rabbit IgG antibody (Sigma) for 1 h. The color reaction was performed on the blots using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3indolyl phosphate in the buVer containing 0.1 M NaHCO3 and 1.0 mM MgCl2, pH 9.8. Phytophthora palmivora zoospore extraction and in vitro pathogen growth in the presence of the crude protein extract Zoospore suspensions were prepared by combining two or three Petri plates of P. palmivora cultures grown on 8% V8 juice agar at 25°C for 7–8 days. Sterilized water (5–10 ml) was placed on the surface of cultures to be harvested and a spatula was used to gently rub the agar surface to dislodge the sporangia. The suspension solutions from all plates were pooled. An

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aliquot of suspension was placed in an Eppendorf tube that was shaken vigorously on a vortex mixer to induce zoospore encystment. The non-mobile spores were counted on a haemocytometer. The suspension concentration was adjusted to 1 £ 104–1 £ 106 spores ml¡1 and used immediately for inoculation. Antimicrobial activity of plant protein was evaluated as previously described (Broekaert et al. 1997; Cammue et al. 1992) with a slight modiWcation. Tests were performed with 20
g total crude proteins (0.22
M Wlter-sterilized) in 20
l solution to which had been added 80
l of a zoospore suspension (2 £ 104 spores ml¡1) in 8% V8 juice broth in each well of a microtiter plate. The control consisted of 20
g of crude protein extract from non-transformed plants. The solutions were incubated in a shaker (100 rpm) at 25°C for 24–48 h. Growth of P. palmivora cultures was observed on an inverted microscope. Quantitative measures of pathogen density were taken after 48 h by staining cultures in microtiter plate wells with 50
l of trypan blue solution (10 g phenol, 10 ml glycerol, 10 ml lactic acid, 10 ml distilled water and 0.02 g of trypan blue) and reading the absorbance at 595 nm in a microplate reader (MRX, Dynatech Laboratories, Alexandria, VA, USA). Pathogen growth is presented as growth relative to that of the control (mean of absorbance at 595 nm of control were normalized as 100%). The data are the means of the three independent experiments consisting of six replicates each. Leaf-disk challenge assay The leaf-disk challenge assays were performed according to previously published methods (Zhu et al. 2004). The youngest fully expanded mature leaves from seven transgenic papaya lines (four having the DmAMP1 gene and three transformed controls with the vector having only the NPT II selectable marker) plus leaves from non-transformed control plants were harvested, washed, and dried on a paper tower. Leaf-disks (20 mm in diameter) were excised with a cork borer, taking care to avoid major veins. The leaf-disks were immediately placed, with adaxial side up, in Petri plates containing water agar. Twenty
l of spore suspension (1 £ 106 spores ml¡1) were pipetted onto the center of each leaf-disk and the Petri plates were placed in a growth chamber maintained at 24°C, 12 h light, and 100% rh. Water soaked spots on the leaf discs could be observed after 24 h. A day later, necrotic lesions appeared that over time expanded as more tissue was damaged by the pathogen. Diameters of lesions on the leaf-disks were measured 3 days after inoculation. For each transgenic line, six disks from the

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youngest fully expanded leaf from three individual plants were pooled to make 18 leaf-disks per line for each assay. The data presented are the means of the three independent experiments that were conducted on diVerent dates. Greenhouse plant assay Greenhouse evaluations of transgenic plants in response to root drench inoculations with spores of P. palmivora were conducted on two diVerent occasions testing diVerent lines of transformed papaya. The initial experiment was conducted with ten cloned plants per treatment of transgenic line DMA6. The second experiment on transgenic lines DMA24 and DMA32 consisted of four plants per treatment. Both experiments were designed as completely randomized blocks. Inoculation treatment consisted of a 10 ml inoculation adjacent to the stem of 3-month-old plants with a zoospore suspension (1 £ 104 spore ml¡1) of P. palmivora. Seedling responses were scored and root weighs were taken 14 days after inoculation. A visual disease rating of 0–4 was given to each plant based on the following symptoms: 0 = healthy plant with no visual symptom, 1 = showing slight leaf wilt or stress, 2 = severe leaf wilt, 3 = leaf abscission plus stem wilt, and 4 = dead plant. Roots of transformed and non-transformed plants were carefully collected, washed to remove the potting mix, blotted on paper towels, and weighed. Root tissues were collected from inoculated pots of non-transformed control, DMA24, and DMA32 plants at 0, 48, 96, and 240 h after inoculation in the second experiment for an ELISA assay for pathogen quantiWcation, using a commercial DAS ELISA kit for Phytophthora (Agdia, Elkhart, IN, USA) according to the manufacturer’s instruction. Approximately 0.1 g root tissues were used for each extraction with non-inoculated healthy root as a blank check. The absorbance at 405 nm of each sample well was measured on a microplate reader (MRX, Dynatech Laboratories). The data presented are means of four biological replicates with three technical replicates. The absorbance was normalized to the mean value of the control at 96 h (mean of control at 96 h was calculated as 100%). Microscopic analysis The microscopic analysis of hyphal growth was carried out on leaf-disks 72-h post-inoculation with P. palmivora as described above for the leaf-disk challenge assay. Images of infecting hyphae were taken using a Zeiss inverted microscope at 200£ magniWcation

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equipped with a Kodak CCD camera. The hyphae relative thickness was obtained by using the Image-Pro Plus analysis program (Media Cybernetics, Silver Springs, MD, USA) to measure the diameters of hyphae growing on leaf discs. Diameters of the hyphae on transformed plants are expressed relative to those of hyphae growing on leaf discs from a nontransformed control plant. Six disks from the youngest fully expanded leaf from three individual plants were pooled to make 18 leaf-disks per line for each assay of four transgenic lines and one non-transformed control. The data presented are the means of the three independent experiments conducted on diVerent dates. Data analysis Data of mycelia growth, lesion size, disease rating and fresh weight of roots were analyzed using the general linear model (GLM) procedure of the SAS (Statistical Analysis System Inc., Cary, NC, USA). When treatment eVects were signiWcant (P < 0.05), means were separated using the least signiWcant diVerence (LSD) (P = 0.05).

Results Over-expression of DmAMP1 defensin in transgenic papaya Twenty-one independent transgenic calli, based on their resistance to geneticin, were selected from 20 bombarded Petri plate cultures. Transformation events were conWrmed by an ELISA assay for the presence of NPT II selectable marker, and by polymerase chain reaction (PCR) using primers speciWc for the DmAMP1 gene. ELISA assay has conWrmed that all 21 transgenic calli were positive for the NPT II selectable marker protein and the PCR assay ampliWed the DmAMP1 gene in all lines that were selected as geneticin resistant (data not shown). The transformation frequency was about two independent transformation events per gram bombarded callus. This frequency is consistent with that previously reported for papaya using the biolistics delivery system with NPT II selection on tissue culture media containing the antibiotic geneticin (Fitch et al. 1990). Integration of the coding region of the DmAMP1 transgene into the papaya genome was conWrmed by Southern blots (Southern 1975) showing a DNA band of 1.2 kb in all four of the transgenic lines tested (data not shown). This is the same sized DNA fragment as

seen in the plasmid (positive control) but not seen in the non-transformed plant (negative control). Western-blot analyses of a random set of seven PCR positive transgenic lines showed the presence of a 5 kDa protein that was not seen in the non-transformed negative control (Fig. 2). This protein from all seven transgenic lines co-migrated with puriWed DmAMP1. The quantity of the transgenic protein was visibly less than the 100 ng of puriWed DmAMP1 protein run on the gel. Based on the relative intensities of the 5 kDa bands of plant extracts and the DmAMP1 standard, the plant extracts of, DmAMP1 protein were estimated to be 10–40 ng in 20
g total proteins. The Western blot showed a second band »16 kDa in size that crossreacted reacting with the Dm-AMP1 antibodies. Since this band was also present in the non-transformed control, we consider the 16 kDa band as an unknown protein with properties allowing non-speciWc hybridization against the DmAMP1 polyclonal antibody. On the basis of the ELISA assay, the levels of DmAMP1 protein in individuals from diVerent transgenic events ranged from 0.07 to 0.14% TSP in callus and from 0.05 to 0.08% TSP in leaves of young plants (Fig. 3). At these concentrations, the amount of DmAMP1 protein visualized in the gel (Fig. 2) as produced by the seven transformed callus lines calculated to be 12–28 ng, quantities that seem reasonable when compared to the puriWed DmAMP1 in the lane labeled Pep. The non-transformed callus also gave a low ELISA reading for DmAMP1 (less than 0.01% TSP), possibly due to the non-speciWc band that was seen in the Western blot. Transformed lines Nontr DMA6

DMA7

DMA9 DMA10 DMAC28 DMA24 DMA32 Pep

MW Std 31000

21500

14400

6500 3496

Fig. 2 Immunoblot of proteins extracted from papaya transgenic callus lines transformed with DmAMP1 gene. Twenty
g total protein was loaded in each lane and the protein was visualized in the nitrocellulose membrane blot by treating it with polyclonal antibodies raised against the DmAMP1 peptide followed by staining with nitroblue tetrazolium. Non-transformed callus (Nontr) is a negative control and puriWed DmAMP1 peptide (Pep) is a positive control. DMA6, DMA7, DMA9, DMA10, DMAC28, DMA24, DMA32 are independent transgenic calli. Molecular sizes are indicated on the right in daltons (Da)

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DmAMP1, % TSP

Planta 0.18 0.16

Callus

0.14 0.12

Leaf

0.1 0.08 0.06 0.04 0.02 0 Nontransform

DMA6

DMA24

DMA32

DMAC28

Fig. 3 DmAMP1 defensin levels in papaya callus and leaves. ELISA was used to determine the concentration of DmAMP1 expressed as a percent of total soluble protein. The data shown are means and standard error of the mean of three independent experiments. Non-transform: non-transformed papaya control, DMA6, DMA24, DMA32 and DMAC28: independent transgenic lines transformed with DmAMP1 gene

In vitro and in planta inhibition of P. palmivora by transgenically expressed DmAMP1 The inhibitory eVect of leaf crude-protein extracts (20
g total protein) from DmAMP1-transformed or control papaya plants on P. palmivora growth was readily seen by trypan blue staining of hyphae cultures grown in microtiter plates (Fig 4a). QuantiWcation of hyphae growth in the presence of the protein extracts showed that lines transformed with the DmAMP1 gene had 35–50% less growth than in the presence of protein extracts from the non-transformed, or the empty vector transformed, plant line DMSCH14 (Fig. 4b).

b

120

Relative growth (%)

a

100

a

a

b

80

b

b

60 40 20 0

Nontransform

DMSCH14

DMA6

DMA24

DMA32

Fig. 4 Growth of P. palmivora in crude protein extracts from papaya leaves. a Microscopic observation of mycelium growth in a microtiter well 48 h after incubation. b Absorbance at 595 nm was measured on each reaction mixture that had been stained with trypan blue. Non-transform non-transformed control; DMSCH14 plant transformed with empty vector containing NPT II selectable marker only; DMA6, DMA24, and DMA32 transgenic papaya lines transformed with DmAMP1 gene

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Inoculation of the non-transformed, or empty vector-transformed, leaf discs with zoospores of P. palmivora showed water-soaked necrotic spots within 24 h after inoculation (data not shown). The water-soaked area increased over time to cause necrosis of the entire leaf disc of non-transformed plants within 3 days (Fig. 5a). Lesion diameters measured 3 days after inoculation were signiWcantly smaller (P < 0.05) in all four DmAMP1 transformed lines than in the non-transformed and empty vector control lines (Fig. 5a, b). On average, the diameter of lesions occurring on leaves of the transgenic plants was about 25–30% smaller so that the infected area was about 40–50% less than on the control leaves. Trypan blue dye, used to stain the lesions followed by hand sectioning and microscopic viewing of the leaf discs, showed the presence of P. palmivora mycelia in every lesion. Every plant of the transformed lines DMA6, DMA24, and DMA32 appeared healthier than did non-transformed control plants when challenged with P. palmivora zoospores applied as pot drenches (Fig. 6a, line DMA6 is not included in the photograph). Disease ratings of the transformed lines were less than half those of the non-transformed lines, ranging from 1.0 for DMA32, 0.9 for DMA6, to 1.6 for DMA24, compared to 2.85 and 3.4 for non-transformed lines (Table 1). Evaluation of transformation event DMA6 in Experiment 1 showed that 10/20 of the control plants developed root rot symptoms and died within 14 days, while none of the transgenic DMA6 plants died during the same period (Table 1). At the other extreme, 11/20 of the transgenic plants maintained a healthy appearance without any obvious root rot symptoms, while only 2/20 of the control plants remained healthy. The average disease rating, on a scale of 0–4, of the DMA6 transgenic line was 0.9, compared to a rating of 2.85 for the non-transformed control plants. The level of disease in line DMA6 was about one-third of that of the controls in all disease ratings. Similar results were observed in the second experiment where the totals of two replicates of the control plants showed 4/8 developed severe root rot leading to plant death while none of the transformed lines developed such severe symptoms. Only 1/8 and 0/8 of the DMA24 and DMA32 plants showed stem wilt symptoms and half of the plants, 1/8 of DMA24 and 3/8 of DMA32, showed no symptoms. The average disease ratings of DMA24 and DMA32 were 1.6 and 1.0, compared to a rating of 3.4 for the non-transformed control. Fresh weights of roots, as a quantitative measure of the disease severity, conWrmed that the transformed lines DMA24 and DMA32 were more resistant to P. palmivora than were the non-transformed controls

Planta

a

b

Non-transfm

14

a

Lesion (mm)

12

DMAC28

a

a

DMA32

a b

b

10

b

b

8 6 4 2

Empty vector

DMA28

DMA32

DMA24

DMA6

DMSCH24

DMSCH18

DMSCH14

Nontransform

0

DmAMP1 transgenic plants

Fig. 5 Disease responses of papaya leaves to P. palmivora. a Petri plates containing papaya leaf disks challenged 3 days earlier by inoculation with P. palmivora zoospores suspensions (20
l £ 106 zoospores ml¡1). Non-transf non-transformed control; DMA C28 and DMA32 transgenic plants transformed with DmAMP1 gene. b Lesion diameter (mm) of P. palmivora inoculated leaf disks. Transgenic lines (DMA6, DMA24, DMA32, and DMAC28), along with empty vector transgenic lines (DMSCH14,

DMSCH18, and DMSCH24) and a non-transformed control were tested for tolerance to P. palmivora inoculation. Diameters of necrotic lesions were measured 3 days after inoculation. Data are means from three independent experiments. Values noted with diVerent letters (a, b) are signiWcantly diVerent (P · 0.05) by the least signiWcant diVerence (LSD) test of SAS (Statistical Analysis System Inc)

(Fig. 6b). The fresh weight of control plants averaged 0.7 g per plant, signiWcantly less than the averages of DMA24 (1.5 g) and DMA32 (1.8 g). ELISA assay also indicated the levels of resistance in transgenic plants (Fig. 7). Phytophthora was barely detected immediately after root drench inoculation (0 h time point for all three lines), but it increased rapidly in the non-transformed plants to about 25% of its maximum within 48 h while remaining below 10% of maximum in the transformed lines DMA24 and DMA32. At 96 h, Phytophthora in all three groups reached their peaks but average levels in of DMA24 and DMA32 were only 45% and 37% of the non-transformed control. Interestingly, the control at 240 h (10 days) after inoculation had a slightly lower Phytophthora level than at 96 h. This decline was, possibly due to a deWcient level of nutrients in susceptible roots to support strong Phytophthora growth. The Phytophthora levels in DMA24 and DMA32 at 240 h remained the same as at 96 h. Overall, the DMA32 plants appeared to be more resistant than the DMA24 plants in both disease rating and in root fresh weight, but these diVerences were not statistically signiWcant.

Fitness of P. palmivora at infection sites Microscopic observation of infection sites on the leaf sections revealed fewer number of Phytophthora hyphae present in the infection sites on the transgenic plants. The hyphae were also thinner and less robust than those growing on non-transformed plants (compare Fig. 8a, b). The diameter of hyphae growing in transformed plants was 40–50% of that in non-transformed plants. It appears that the smaller necrotic lesions seen on the inoculated transgenic plants may be the result of reduced hyphae growth in the presence of transgenic DmAMP1 defensin protein.

Discussion Our earlier work (Zhu et al. 2004) showed that papaya transformed to express a grapevine stilbene synthase gene exhibited increased resistance to P. palmivora. That report established that transgenic production of an antifungal protein appears to enhance papaya resistance to fungal diseases. In the present study, we report that the defensin gene, DmAMP1 from Dahlia merckii,

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Planta

a

DMA32

DMA24

Control

Root fresh weigh (g)

b

b

2

b 1.5

a

1 0.5 0

Control

DMA24

Fig. 6 Papaya plants challenged with root drenched P. palmivora. a Pictures were taken 14 days after P. palmivora challenge. b Root weighs were taken 14 days after P. palmivora inoculation. Controls are plants that went through the same tissue culture process as the transgenic plants but that were not bombarded for

DMA32

transformation. DMA24 and DMA32 are papaya lines transformed with the DmAMP1 gene. Inoculums consisted of P. palmivora zoospores suspensions (10 ml £ 104 zoospores ml¡1) as described in Materials and methods

Table 1 Disease rating of transgenic lines and non-transformed control papaya plants 14 days after P. palmivora inoculation Experiment

1

Treatment

Non-transformed DMA6

2

Non-transformed DMA24 DMA32

Trial number

Number of plants in each disease rating 4 dead

3 stem wilt

2 leaf wilt

1 slight wilt

0 healthy

1 (10) 2 (10) 1 (10) 2 (10) 1 (4) 2 (4) 1 (4) 2 (4) 1 (4) 2 (4)

6 4 0 0 2 2 0 0 0 0

2 1 2 1 2 1 1 0 0 0

2 1 2 1 0 1 1 2 0 1

0 2 1 2 0 0 2 1 2 2

0 2 5 6 0 0 0 1 2 1

Trial disease ratinga

Treatment averageb

3.4 2.3 1.1 0.7 3.5 3.3 1.8 1.3 1.0 1.0

2.85 0.9 3.4 1.6 1.0

Transgenic and non-transformed lines were clonally multiplied, rooted, and maintained in the greenhouse. Experiment 1 evaluations were based on ten individual plants per replicate trial and Experiment 2 evaluations were based on four individual plants per replicate trial in a completely randomized design for each treatment (control and transgenic lines). Inoculums consisted of P. palmivora zoospores suspensions (10 ml £ 104 zoospores ml¡1 ) applied to the plants as described in Materials and methods. Disease ratings, based on plant appearance (0 = no symptom, healthy plant, 1 = slight leaf wilt or stress, 2 = leaf wilt, 3 = leaf fall oV and stem wilt and 4 = dead), were assigned 14 days after P. palmivora challenge a Trial disease rating calculated as the number of plants at each rating times the rating number divided by the number of plants in that trial b Treatment average of disease incidence is the mean of two experimental trials

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Relative Absorbance (405 nm)

Planta 120 100 0 hrs

80

48 hrs

60

96 hrs

40

240 hrs

20 0 Control

DMA24

DMA32

Fig. 7 Detection of Phytophthora in papaya roots by microtiter plate ELISA assay at a time course of 0, 48, 96 and 240-h postdrench inoculation. Control plants are non-transformed plants, DMA24 and DMA32 are transgenic plants with DmAMP1 gene. Inoculums consisted of P. palmivora zoospores suspensions (10 ml £ 104 zoospores ml¡1) as described in materials and methods. The absorbance at 405 nm was normalized to the mean value of the control at 96 h

increased leaf resistance of papaya to P. palmivora. Taken together, these studies indicate that a transgenic approach may increase disease resistance in papaya. In vitro inhibition assays reported in the literature suggest that the DmAMP1 defensin might inhibit growth of a broad range of fungal pathogens (Osborn et al. 1995; Thomma et al. 2002). However, it was not known whether the expression level in papaya would be suYcient for inhibition of P. palmivora. Our study shows that the constitutive expression level of the DmAMP1 gene under the CaMV35S promoter is suYcient, as evaluated both by in vitro growth inhibition by

a

Nontransformed

c

140

Hyphal thickness (%)

Fig. 8 Hyphae of P. palmivora in the infection sites on papaya leaves. Microscopic view of hyphae growing on a leaf discs of non-transformed plants, b leaf discs of DMA6 transformed plant, Scale bar, 20
m, and c The relative hyphae thickness of P. palmivora. Non-transform non-transformed papaya control; DMA6, DMA24, DMA32, and DMAC28, transgenic papaya transformed with DmAMP1

leaf protein extracts and by in vivo whole plant inoculation, to inhibit P. palmivora development. The quantity of transgenically expressed DmAMP1 peptide, estimated by ELISA, ranged from 0.07 to 0.14 % TSP in callus and from 0.05 to 0.08% TSP in leaves. These levels are consistent with other reported values of transgenic protein production using the CaMV35S promoter. The in vitro growth inhibition assay carried out with 20
g of total protein in 100
l Wnal test volume corresponded to a DmAMP1 peptide concentration of 1.0–1.2
g ml¡1. The level of inhibition of P. palmivora growth by this dose was »50%. This is in line with a 50% growth inhibition (IC50) of the fungal pathogen Fusarium culmorum by puriWed DmAMP1 in the range of 1 to 2.8 § 0.5
g ml¡1 (Francois et al. 2002; Osborn et al. 1995). However, our data do not permit us to calculate a precise IC50 of DmAMP1 protein against P. palmivora since we did not have a suYcient amount of puriWed DmAMP1 to conduct an in vitro growth inhibition assay. Moreover, we cannot extrapolate an IC50 from the transgenic plant crude protein extract because the mixture of proteins could have multiple eVects on P. palmivora. Although root rot and stem rot are the more common forms of Phytophthora disease expression in nature, our leaf disk bioassay indicated that P. palmivora is capable of becoming suYciently establishment in papaya leaves to cause disease symptoms. The range in susceptibility revealed by this assay also indicated

120

b

DMA6

a

100

b

80

b

b

60

b

40 20 0

nontransform

DMA6

DMA24

DMA32

DMAC28

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Planta

that although the transgene product was not suYcient to prevent infection, it was suYcient to inhibit pathogen growth and reduce its elicitation of a toxic response. This conclusion is consistent with the in vitro pathogen inhibition assay in which the pathogen was not killed in the transgenic plant but its pathogenicity was restricted. Most plant defensins isolated in the early 1990s were seed-derived (Terras et al. 1995). The quantity of defensins released from a single seed was suYcient to inhibit fungal growth. Therefore, it has been proposed that plant defensins contribute to the protection of seeds or seedling against attack by soil-borne pathogens to enhance seedling survival (Terras et al. 1995). It was recognized that plant defensins are also expressed in peripheral cell layers (Penninckx et al. 1996; Thomma et al. 1998), the epidermal cell layer, and primordia (Moreno et al. 1994) of plant tissues, which is consistent with defensins having an important role in the Wrst line of defense against pathogens (Gu et al. 1992; Terras et al. 1995). Defensins are also found in stomatal cells and in the cell walls lining substomatal cavities of beet leaves (Kragh et al. 1995), which is interesting since stomata are well-known entry points for speciWc pathogens. Thus, cell walls lining substomatal cavities may still be the Wrst line of defense for stomatal penetrating pathogens. In the present study, we used the CaMV35S promoter so that the DmAMP1 peptide might be produced throughout the plant. Constitutive expression would be good for control of pathogens, such as P. palmivora, that attack multiple sites on the plant such as roots, stems, and fruits of papaya. The transgenic papaya plants produced in the present research appeared phenotypically normal, but Weld data will be needed to determine whether constitutive production of the defensin protein has an eVect on yield. PCR ampliWcation of the DmAMP1 gene in all 21 lines surviving selection on culture medium containing geneticin (data not shown) indicated a highly eVective selection screen since it allowed no selection of nontransformed lines. The Wdelity and copy number of transgenes were not determined, but rearranged sequences and more than one copy might be expected since the biolistic gene gun method is recognized as commonly scrambling non-contiguous transgene and genomic sequences (Svitashev et al. 2002) and inserting multiple copies of transgenes. Either type of faulty plasmid insertion might be associated with some level of transgene silencing (Kohli et al. 1998), but, if this were the case, the degree of silencing was not suYcient to hamper selection nor to negate increased resistance to Phytophthora.

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DmAMP1 has been reported to induce membrane permeabilization of susceptible fungi (Thevissen et al. 1999). When inhibited fungi were visualized under the microscope, the plant defensins were seen to cause distinct morphological changes (Osborn et al. 1995). DmAMP1 can inhibit the rate of elongation of fungal germ tubes but does not cause the swelling and budding seen with the other anti-fungal peptides (e.g., RsAFP2). Our results demonstrated that DmAMP1 caused a reduction in hyphae thickness of Phytophthora and an apparent collapse of the plasma membrane leading to fragmentation of the cytoplasm. The present research thus expands the phylogenetic range over which DmAMP1 has been shown to be eVective in inhibiting hyphae growth and development and in conferring disease resistance at the level of bioassays and greenhouse tests. The challenge is to translate these results into robust disease resistance at the Weld level. Acknowledgments We thank Dr. B. Cammue from University of Leuven, Belgium, for providing the DmAMP1 construct, protein, and antisera; Dr. M. Fitch, ARS/USDA, for advice on papaya transformation and clonal propagation; Dr. S. Schenck, Hawaii Agriculture Research Center, and Dr. W. Nishijima, University of Hawaii, for providing pathogen cultures and advice on pathogen studies; Ms. R. Tumpap, Charminade University, for helping with the leaf disk assay; Ms. N. Rosette, Hawaii Agriculture Research Center, for assisting with papaya tissue cultures and Ms. A. Aoki, Hawaii Agriculture Research Center, for assisting with PCR and ELISA assays. Finally, we thank Ms. M. Moore for her critical editing of this manuscript. This work was supported in part by a U.S. Department of Agriculture ARS cooperative agreement, CA 585320-3-460, with the Hawaii Agriculture Research Center.

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