* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Constitutive expression of Vitis vinifera thaumatin
Point mutation wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Plant virus wikipedia , lookup
Metalloprotein wikipedia , lookup
Plant breeding wikipedia , lookup
Paracrine signalling wikipedia , lookup
Biochemistry wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Signal transduction wikipedia , lookup
Gene expression wikipedia , lookup
Magnesium transporter wikipedia , lookup
Homology modeling wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein structure prediction wikipedia , lookup
Interactome wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein purification wikipedia , lookup
Expression vector wikipedia , lookup
Western blot wikipedia , lookup
Two-hybrid screening wikipedia , lookup
CSIRO PUBLISHING Functional Plant Biology, 2003, 30, 1105–1115 www.publish.csiro.au/journals/fpb Constitutive expression of Vitis vinifera thaumatin-like protein after in vitro selection and its role in anthracnose resistance Subramanian JayasankarA,B, Zhijian LiA and Dennis J. GrayA,C AMid-Florida Research and Education Center, Institute of Food Agricultural Sciences University of Florida, 2725 Binion Road, Apopka, FL 32703, USA. BCurrent address: Department of Plant Agriculture — Vineland Campus, University of Guelph, 4890 Victoria Avenue N, PO Box 7000, Vineland Station, Ontario, Canada L0R 2E0. CCorresponding author; email: [email protected] Abstract. Anthracnose-resistant grapevine (Vitis vinifera L. cv. Chardonnay) plants were regenerated from embryogenic cultures that had been subjected to in vitro selection with culture filtrate of Elsinoe ampelina (de Bary) Shear. Three secreted proteins differentially expressed by in vitro-selected embryogenic cultures and regenerated plants were identified. An 8-kDa protein was identified as a lipid-transfer protein (LTP) by N-terminal amino acid sequence comparison. Two other differentially expressed proteins, with estimated molecular weights of 21.6 and 22 kDa, immuno-reacted with antiserum raised against a thaumatin-like protein (TLP) protein from pinto bean (Phaseolus vulgaris L.). N-terminal amino acid sequencing of the 21.6-kDa protein showed a high degree of homology to V. vinifera thaumatin-like protein 2 (VVTL-2 = grapevine osmotin; Acc no. CAA71883), and that of the 22-kDa protein was homologous to V. vinifera thaumatin-like protein 1 (VVTL-1; AAB61590). Interestingly, both VVTL-1 and VVTL-2 are pathogenesis-related (PR) proteins, belonging to the PR-5 group. Protein produced from the cloned grapevine VVTL-1 gene significantly inhibited E. ampelina spore germination and hyphal growth in vitro. Plants regenerated from in vitro-selected cultures similarly inhibited fungal growth in vivo. Enhanced expression of antifungal VVTL-1 in anthracnose resistant grapevine strongly suggests that it plays an important role, either alone or in conjunction with other PR proteins, by suppressing pathogen growth. Keywords: Chardonnay, disease resistance, Elsinoe ampelina, lipid-transfer protein. Introduction The capacity of plants to counter the attack of pathogenic fungi depends, in part, on their ability to trigger defence mechanisms. Such defence mechanisms include elicitation of active oxygen species, biosynthesis of phytoalexins, strengthening of barriers to invasion, hypersensitive responses, and induction of pathogenesis-related (PR) genes (Fritig et al. 1998; Dempsey et al. 1999). Induction of PR genes has been associated with incompatibility and overexpression of one or more PR proteins can delay disease development (Hammond-Kosack and Jones 1996). PR proteins are induced intra- and extracellularly by pathogens, chemical elicitors or, in some instances, environmental stresses. PR proteins are separated into several groups, based primarily upon sequence homology, but electrophoretic mobility and functional characteristics also are considered (van Loon et al. 1994). Proteins with close sequence homologies to PR proteins have been added to respective PR groups without proof of inducibility; these are sometimes referred to as PR-like until functionality is determined (van Loon et al. 1994). The PR-5 protein group was established based upon sequence homology to thaumatin. Several PR-5 proteins have antifungal properties (Roberts and Selitrennikoff 1990; Vigers et al. 1992; Cheong et al. 1997). For example, osmotin, a tobacco (Nicotiana tabacum L.) PR-5 protein has antifungal activity in vitro and in vivo (Abad et al. 1996). Because some PR-5 proteins were discovered to be basic, the group was subdivided into the acidic thaumatin-like proteins (TLPs) and basic osmotin-like proteins (Kombrink and Somssich 1997). Thaumatin-like proteins are expressed in different tissues, such as pistils, fruits and seeds (Neale et al. 1990), associated with fruit ripening in banana, cherry and tomato (Vu and Huynh 1994; Fils-Lycaon et al. 1996; Barre et al. 2000). In addition, TLPs are induced by viral, bacterial and fungal infection. For example, in tobacco, infection with Abbreviations used: ECP, extracellular protein; ICF, intercellular fluid; LTP, lipid transfer protein; PEM, proembryogenic mass; PR, pathogenesis related; SEM, scanning electron microscopy; TLP, thaumatin-like protein; VVTL, Vitis vinifera thaumatin-like protein. © CSIRO 2003 10.1071/FP03066 1445-4408/03/111105 1106 Functional Plant Biology tobacco mosaic virus resulted in increased accumulation of TLP within 6–8 d (Pierpoint 1986; Pierpoint et al. 1987). The timing, localization and specificity of expression strongly suggest that TLPs function in microbial defence (Vigers et al. 1992; Velazhahan et al. 1999). Two TLPs (VVTL-1 and VVTL-2) were described from grapevine (Tattersall et al. 1997; Jacobs et al. 1999). VVTL-1 was identified as a developmentally-regulated, fruit-specific protein (Tattersall et al. 1997). VVTL-2, expressed in leaves and fruits, previously was named grapevine osmotin (Loulakakis 1997; Salzman et al. 1998), but was renamed when it was determined to be acidic. VVTL-2 shows considerable homology to VVTL-1, but is more responsive to biotic and abiotic stresses (Jacobs et al. 1999). Antifungal properties of VVTL-1 have not been determined, whereas purified VVTL-2 inhibited fungal growth in the presence of sugars (Salzman et al. 1998). The phenomenon of phenotypic changes induced through the process of cell culture is well documented and termed ‘somaclonal variation’ (Larkin and Scowcroft 1981). Exposure of cell cultures to various toxins from disease-causing organisms led to disease-resistant plants (Daub 1986), presumably by the same mechanism. Induction of PR proteins, such as chitinases (PR-3) and glucanases (PR-2), has been demonstrated after challenge with fungal or bacterial culture filtrates in several cell culture systems including grapevine (Gentile et al. 1993; Busam et al. 1997; Jayasankar and Litz 1998; Jayasankar et al. 2000). However, PR-5 proteins induced by pathogen challenge have not been identified. Earlier we demonstrated that in vitro selection of grapevine embryogenic cultures with Elsinoe ampelina (de Bary) Shear (causal agent of grapevine anthracnose disease) culture filtrate resulted in resistant phenotypes that overexpressed chitinase (Jayasankar et al. 2000). Here we show that VVTL-1 and VVTL-2 are differentially expressed in these in vitro-selected grapevine embryogenic cultures as well as plants regenerated from them. We also demonstrate that purified, recombinant VVTL-1 protein inhibits spore germination and hyphal growth of E. ampelina. This is the first demonstration that grapevine TLPs possess antifungal activity, strengthening the evidence that they play a role in plant defence mechanisms. Materials and methods In vitro selection, culture establishment and plant regeneration Suspension culture initiation, somatic embryogenesis and plant regeneration of Vitis vinifera L. cv. Chardonnay were described previously (Jayasankar et al. 1999). In vitro selection was carried out as described by Jayasankar and Litz (1998). Log-phase proembryogenic masses (PEM) cultures were passed through a 960-µm sieve to generate a synchronized culture. Synchronized PEMs were selected in vitro against a 40% (v/v) culture filtrate-containing medium as described previously (Jayasankar et al. 2000). The culture filtrate was obtained by growing a virulent strain of Elsinoe ampelina (de Bary) Shear for 3 weeks in Czapek–Dox broth. S. Jayasankar et al. Cell-free extract was collected and stored at 20°C until further use. Selection was carried out for four or five cycles, each cycle lasting for 10 d. After the fourth and fifth cycles (40 or 50 d total exposure), putative resistant cultures were proliferated in suspension culture medium and established as ‘resistant culture 1’ (RC1) and ‘resistant culture 2’ (RC2), respectively. Somatic embryogenesis was achieved by culturing selected PEMs in auxin-free suspension culture medium and resulting somatic embryos were germinated on solid medium. Regenerated plants were acclimatized in potting mixture and established in a greenhouse. A set of non-selected PEMs were cultured in a similar way; the non-selected cultures and plants regenerated from them served as experimental controls. Extraction and quantification of extracellular proteins Extracellular proteins (ECP) were precipitated by the method of Gavish et al. (1992). Spent medium was collected in sterile flasks during subculture and filtered through a double layer KimwipeTM to eliminate any cellular debris. ECP was precipitated from the filtered medium by adding three volumes of ice-cold 95% ethanol and kept overnight at –20°C. Proteins were pelleted by centrifugation, concentrated by vacuum and re-suspended in sterile distilled water. Protein quantification was accomplished by the Bradford protein assay with bovine serum albumin as a standard. Protein samples were stored at –20°C until further use. Extraction of intercellular fluids and protein concentration Intercellular fluid (ICF) was extracted as described by Ignatius et al. (1994) with minor modifications. Fully expanded, flaccid leaves were collected from greenhouse-grown plants early in the morning. The leaves were washed thoroughly with distilled water and blot-dried. Lamina were cut into 2-cm-wide strips and vacuum infiltrated for 15 min in a buffer containing 100 mM TRIS–HCl, (pH 6.8), 2.0 mM CaCl2, 10 mM EDTA, 50 mM β–mercaptoethanol and 0.5 M sucrose, at a ratio of 10 mL per g of leaf tissue. After infiltration, leaf strips were gently blotted and rolled to fit into 0.5 mL centrifuge tubes (without caps) with a 1-mm-diameter hole at the bottom. Only two or three strips were placed in each microfuge tube. These tubes were then placed so that they fitted into 1.5-mL centrifuge tubes. The apparatus was centrifuged at 3140 g for 15 min at room temperature. ICF was collected as dense drops in the 1.5 mL centrifuge tubes. The ICF was diluted with four volumes of distilled water and proteins were precipitated with three volumes of ice-cold 95% ethanol overnight at 0°C. Protein pellets were collected by centrifugation, concentrated by vacuum and re-suspended in sterile distilled water. Protein quantification was accomplished as described above. Electrophoresis of proteins SDS–PAGE with 1-mm-thick mini gels was carried out as described by Laemmli (1970). Proteins were diluted with an equal volume of SDS–PAGE buffer (Sigma, St Louis, MO) and diluted samples were boiled for 5 min and then cooled. Samples were centrifuged at 5590 g for 5 min at room temperature to remove insoluble particles. Total protein (10 µg) was loaded onto each lane and electrophoresed for approximately 80 min at 200 V. The gels were then silver-stained using SilverSnapTM (Pierce, Rockford, IL). Immuno-detection of proteins After SDS–PAGE, proteins were transferred to ImmunoBlotTM PVDF membranes (Bio-Rad, Almeda, CA) in a mini trans-blotter according to manufacturer’s instructions. Transferred proteins were probed with a PR-5 antiserum raised against pinto bean thaumatin-like protein (kindly provided by Dr OP Sehgal, University of Missouri, Columbia, MO) at a dilution of 1 :500 for 2 h at room temperature with gentle Constitutive expression of Vitis vinifera thaumatin-like protein shaking. Colour development was carried out with a Opti-4Cn kit (Bio-Rad), according to manufacturer’s instructions. N-terminal amino acid sequencing For N-terminal amino acid sequencing, proteins were transferred to an ImmunoBlotTM PVDF membrane with a buffer lacking glycine. After transfer, proteins were stained with Coomassie blue (Sigma). Appropriate bands were identified on the basis of molecular weight and excised. N-terminal amino acid sequence determination was accomplished by the automated Edman degradation method in the Protein Chemistry Core Laboratory (University of Florida, Gainesville, FL) with a protein sequencer (Model 494HT, Applied Biosystems, Foster City, CA). Phenylthiohydantoin amino acid derivatives were detected by a 120A analyser used in conjunction with the sequencer. Amino acid sequences were compared with those in the database by BLAST (Altschul et al. 1997). Cloning and sequencing VVTL-1 Total genomic DNA was isolated from immature leaf tissues of both selected and non-selected plants of ‘Chardonnay’, according to a modified procedure of Lodhi et al. (1994). Two oligonucleotide primers were designed based on the sequence information from the database for VVTL-1 (AF003007, Tattersall et al. 1997). The forward primer GTL-51 contained 5′-ATGGATCCAACATCAAAATGCGCTTCACCACCA-3′ and the reverse primer GTL32 contained 5′-TGTCTAGAGTTCCAACTCTCAAGGGCAAAACGTGACCTTG-3′. Polymerase chain reactions (PCR) were performed using a reaction mixture containing 1 µg template DNA, 20 pmol of each primer, 200 µM dNTP, 2 mM MgCl2, 1× Mg-free reaction buffer and 2 units Taq polymerase in a total volume of 80 µL. Cycling conditions were as follows: 95°C for 3 min followed by 1 min each of 93°C, 55°C and 72°C for a total of 35 cycles. The resulting PCR products were separated by agarose gel electrophoresis. The amplified DNA fragments were isolated from gel blocks, purified and then cloned into pUC18 cloning vector (Amersham–Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. Cloned DNA fragments from both selected and non-selected plants were subsequently sequenced and analysed with the Vector NTI DNA analysis program (InforMax Inc., Frederick, MD). Construction of the VVTL-1 transfer vector and recombinant virus To generate the viral transfer plasmid, PCR was used to add BamHI (5′) and NotI (3′) restriction enzyme sites, a putative viral ribosomebinding site, and a C-terminal hexa-histidine sequence to the cloned VVTL-1 gene sequence. The PCR product was directly subcloned into the baculovirus transfer vector pVL1393 (PharMingen, San Diego, CA) and the sequence verified by double stranded sequencing using primers based on the polyhedrin promoter and 3′ flanking sequences. Liposome-mediated co-transfection (Invitrogen, Carlsbad, CA) of Spodoptera frugiperda J.E. Smith (Sf9) cells was performed with 2.5 µg of the recombinant transfer plasmid and 0.5 µg of linearized viral DNA. After 5 d the resultant virus was collected and a portion used to infect Sf9 cells to generate an amplified viral stock. The virus was titered by plaque assay (O'Reilly et al. 1994) and used for expression of recombinant VVTL-1. Expression of VVTL-1 in insect cells The Trichoplusia ni Hübner cell line BTI-TN-5B1-4 (Invitrogen) was grown in spinner flasks at 27°C in Ex-Cell 400 (JRH Biosciences, Lenexa, KS) to a density of 2 × 106 cells per mL. The cells were collected by centrifugation and infected with a multiplicity of infection (MOI) of five. The infected cells were tested at 24 h intervals for the expression of VVTL-1 by binding to Ni-NTA (Qiagen, Valencia, CA) and analysis by SDS–PAGE. The culture supernatants were collected by centrifugation when expression was determined to be maximal (72 h post-infection). Functional Plant Biology 1107 Purification of recombinant VVTL-1 The culture supernatants were concentrated by ultrafiltration and the buffer exchanged to 20 mM TRIS, 100 mM NaCl, 10 mM imidazole, 0.01% Nonidet P-40 (NP-40), pH 8.0. After binding to Ni-NTA for 2 h at 4°C, the matrix was collected by centrifugation, transferred to a column, and washed with 10 column volumes of the same buffer. The bound protein was eluted with six column volumes of 20 mM TRIS, 100 mM NaCl, 250 mM imidazole, pH 8.0. The eluted protein was concentrated by ultrafiltration. In vitro spore germination assay Spores were collected from a culture plate of E. ampelina and suspended in 1 mL Czapek-Dox broth. After estimating the spore density, a stock suspension of 500 000 spores per mL in sterile distilled water was made. Spore germination was tested in 96-well microwell plates by diluting 100 µL of spore suspension with 100 µL of sterile distilled water per well. To test for the effect of VVTL-1 on spore germination, recombinant protein was dissolved into the sterile distilled water before spore dilution to yield final amounts of 20, 40 and 80 µg per 200 µL. Each treatment and a no-protein control was replicated three times (i.e. three wells). The microwell plate was sealed with ParafilmTM and incubated at room temperature. Spores that exhibited a visible germ tube were counted as positive for germination. Spore germination was observed at 24, 48 and 72 h after inoculation and data on germination was collected and averaged from five distinct zones in each well during each count. The relative amount of hyphal growth was rated subjectively. In vitro leaf bioassay and scanning electron microscopy (SEM) Young, fully expanded leaves (typically the third or fourth leaf from the shoot tip) of selected and non-selected greenhouse-grown plants were excised carefully with a sterile scalpel and gently rinsed with distilled water. After blot-drying the excess water, leaves were placed in a sterile Petri dish (100 × 15 mm), with the cut end of the petiole resting on a sterile KimwipeTM (Fisher Scientific, Savannah, GA) saturated with distilled water. E. ampelina spores were collected from an actively growing culture and suspended at a density of 10 6 spores per mL (adjusted using a hemacytometer) in sterile water. Spore suspension (100 µL) was gently placed on three spots per leaf. Five leaves each from selected and non-selected plants were tested. The Petri dishes with inoculated leaves were carefully sealed with ParafilmTM and incubated at 25 ± 2°C with a 16 h photoperiod. For SEM, spore suspensions were placed on leaves of selected and non-selected plants for 48 h. Leaf discs from inoculated spots were processed and examined with a scanning electron microscope as described previously (Jayasankar et al. 2002). Results Grapevine embryogenic cultures were subjected to recurrent in vitro selection with E. ampelina culture filtrate and surviving embryogenic cultures were used to regenerate plants. These selected embryogenic cultures and plants were compared with a set of non-selected cultures and plants regenerated at the same time. Embryogenic cultures that survived in vitro selection and plants regenerated from them are hereafter referred to as ‘resistant cultures’ and ‘resistant plants’, respectively. In order to understand the molecular basis of the resistance, we profiled proteins secreted from resistant cultures and compared them with non-selected control. Polypeptides with molecular masses of 8, 21.6 and 22 kDa 1108 Functional Plant Biology S. Jayasankar et al. were predominant proteins differentially expressed by resistant cultures. In order to ascertain whether these elicited proteins also expressed in planta, similar profiling of proteins in the ICF of leaves from resistant plants was performed. All three proteins were differentially expressed in the ICF of resistant plants as well (Fig. 1). Molecular identity of differentially expressed proteins using N-terminal amino acid sequencing A BLAST search of the 21 amino acid residues from the 8-kDa protein N-terminus revealed that this sequence exhibits a high homology to non-specific lipid transfer proteins (nsLTP). Among the nsLTPs that showed high similarity was a 9-kDa protein identified from grapevine somatic embryos previously as LTP P4 (Coutos-Thevenot et al. 1993). The 8-kDa protein exhibited 75% sequence similarity with a 9-kDa nsLTP from grapevine berries (Salzman et al. 1998). The molecular mass and sequence identifies the 8-kDa protein as a nsLTP (Fig. 2A). The 21.6-kDa protein secreted by resistant cultures and regenerated plants had identical N-terminal sequences. This sequence had a high homology with grapevine osmotin from ‘Sultania’ (Loulakakis 1997) and ‘Concord’ (Salzman et al. 1998), which is very similar, but not identical, to V. vinifera M 1 * ** 2 3 4 5 6 7 22 21.6 8 Fig. 1. Extracellular protein profile of intercellular fluid (ICF) from regenerated plants and in vitro-selected embryogenic cultures. Proteins were separated by SDS–PAGE on a 12% mini-gel. (Lanes: M, marker; 1, ICF from non-selected plants; 2 and 3, ICF of regenerated plants from in vitro selected line RC1; 4 and 5, ICF of regenerated plants from in vitro-selected line RC2; 6, secreted proteins from embryogenic cultures RC1; 7, secreted proteins from embryogenic cultures RC2). Asterisks indicate a lightly-stained 22-kDa band in the control and two distinct 21.6- and 22-kDa bands in the selected plants and embryogenic cultures. thaumatin-like protein (VVTL-1) (Fig. 2B). The amino acid sequences of the 22-kDa protein from resistant cultures and regenerated plants were identical and also exhibited a very high similarity to that of VVTL-1 from grapevine berries (Tattersall et al. 1997). In addition, the 22-kDa protein exhibited a very high N-terminal sequence homology with several other TLPs including two from tobacco, E22 and E2 (Fig. 2C). The sequence homology implies that the 22-kDa protein is VVTL-1. To further verify the molecular identity of these two proteins, we carried out an immunoblot assay of secreted proteins from the spent medium of embryogenic cultures and ICF of leaves, using pinto bean PR-5 antibody as a probe. The antibody reacted with both 21.6- and 22-kDa proteins (Figs 3A, B). Therefore, our results show that both 21.6- and 22-kDa peptides are PR-5 proteins that are differentially and constitutively expressed by resistant cultures and plants as secreted proteins. Effect of recombinant VVTL-1 on E. ampelina spore germination The entire VVTL-1 gene was cloned from genomic DNA of resistant plants by PCR. The recombinant protein was produced in a baculovirus expression system that allowed recovery of 2.0 mg of purified VVTL-1 from 4 L of transfected cultures. The purified protein was subsequently tested for antifungal activity. Spore germination of E. ampelina was significantly inhibited at three VVTL-1 concentrations and two exposure periods, except for the lowest concentration (20 µg) at 48 h (Table 1). For example, more spores had germinated at 24 h in untreated control compared with spores treated with 20, 40 or 80 µg of VVTL-1 (Table 1). Even after 48 h, the single dose of VVTL-1 at 40 or 80 µg significantly inhibited spore germination. Similarly, spores kept for 24 or 48 h in untreated control conditions had longer germ tubes than those incubated for the same periods with 40 or 80 µg of VVTL-1 (Fig. 4). Among treatments, there was a gradual increase in spore germination and growth over time; this may have been due to protein degradation occurring over the course of the incubation period. This is illustrated in Table 1 and Fig. 4, which shows that the percentage of germination increased from 7.9 to 41.2% from 24 to 48 h in spores treated with 80 µg VVTL-1, and germ tubes became longer; however, germination and growth still was less than in the untreated control. At 72 h, germ tube length in the 40 µg treatment was similar to that of the control. The shorter germ tubes in VVTL-1 treatments correlated with respective germination data; this indicated that the protein had the effect of delaying germination and/or retarding growth. Because the germination percentage did not increase appreciably in the untreated control after the first count, it appears that 24 h is sufficient for E. ampelina spores to germinate under favourable conditions. Thus, the significant reduction in spore germination and germ tube length at 24 h and later Constitutive expression of Vitis vinifera thaumatin-like protein Functional Plant Biology 1109 A Present study 8 kDa protein nsLTP - P4 nsLTP Sorghum LTP - 2 Rice LTP - 1 T V T X G O V A S A V G P X I S Y L O T V T C T I T C X V T C X I T C G O V A S A L S P C I D Y L O G O V A S A L S P I I N Y L O G O V S S A I G P C L S Y X X G O V N S A V G P C L T Y A R B Present study 21.6 kDa protein VVOsm A T F N I Q N K G G Y T V A T F N I Q N H G G G Y T V A T F N I Q N H C P Y T V A T F D I L N K X T Y T V X VVTL1 A T F D I L N K C T Y T V W A Tobacco PR5 E22 A T F D I V N K C T Y T V W A Tobacco PR5 E2 A T F D I V N O C T Y T V W A GO C Present study 22 kDa protein VVOsm GO A A T F N I Q N H G G Y T V W A A T F N I Q N H C P Y T V W A Fig. 2. N-terminal amino acid sequence comparison of differentially expressed extracellular proteins. (A) 8-kDa protein from embryogenic cultures. This protein had high sequence homology with non-specific lipid transfer proteins (nsLTP): Vitis nsLTP P4 (SwissProt [SP] accession number P 80274), Vitis nsLTP (Salzman et al. 1998), Sorghum nsLTP (SP accession number Q 43194), rice nsLTP (SP accession number P 23096). (B) 21.6-kDa protein from in vitro-selected embryogenic cultures and plants had high homology with grapevine osmotins: VVOsm (SP accession number Y 10992), and grape osmotin (GO; Salzman et al. 1998). (C) 22-kDa ICF protein from in vitro-selected embryogenic cultures and plants. This protein had a high homology with several thaumatin-like proteins (TLPs): VVTL-1 (GenBank accession number AF 003007), Tobacco TLP-E22 (SP accession number P 13046), Tobacco TLP-E2 (SP accession number P 07052), VVOsm and GO. Identical amino acids are shaded dark and similar amino acids are shaded light. X denotes unidentified amino acid residues. 1110 Functional Plant Biology S. Jayasankar et al. suggests that VVTL-1 plays a pivotal role in inhibiting E. ampelina. Plants regenerated from in vitro selected cultures show enhanced resistance to E. ampelina The antifungal activity of VVTL-1 prompted us to explore whether plants regenerated from in vitro selected lines also exhibited resistance to E. ampelina. Inoculation with E. ampelina spores resulted in diagnostic necrotic spots with greyish centers on leaves from non-selected control plants within 5 d (Fig. 5A, left). However, plants regenerated from in vitro-selected lines had fewer symptoms at 5 d postinoculation (Fig. 5A, right). 1 2 Discussion 3 A 22 21.6 1 2 To further analyse the nature of observed resistance we performed scanning electron microscopy (SEM) on inoculated leaves from resistant and control plants. SEM analysis showed widespread spore germination and hyphal growth on leaves from non-selected control plants (Fig. 5B). In these leaves, most spores formed germ tubes, which developed into hyphae. At infection sites, the surface wax layer was degraded, suggesting that hyphae had the ability to freely penetrate the outer epidermis. In contrast, spore germination was greatly reduced on leaves of resistant plants (Fig. 5C) and very few spores had germ tubes. The majority of spores remained ungerminated and tumid, indicating that hyphal growth from germ tubes was arrested. In addition, the wax layer of resistant plant leaves remained intact. 3 B 22 21.6 Fig. 3. (A) Immuno-detection with PR-5 antiserum of secreted proteins from embryogenic cultures. (Lanes: 1, non-selected control; 2, secreted proteins from RC1; 3, secreted proteins from RC2). (B) Immuno-detection with PR-5 antiserum of secreted proteins in ICF of regenerated plants (Lanes: 1, non-selected control; 2, in vitroselected line RC1; 3, in vitro-selected line RC2). We reported previously that grapevine plants regenerated after in vitro selection with E. ampelina culture filtrate had a high degree of anthracnose resistance and that a chitinase (PR-1 or PR-2) was constitutively expressed (Jayasankar et al. 2000). We now demonstrate that resistance is accompanied by expression of three additional proteins, including an nsLTP and two PR-5 proteins. One PR-5 protein, VVTL-1, is shown here to have antifungal properties. Lipid-transfer proteins belong to a multigene family of cysteine-rich antimicrobial peptides that are present in many species (Garcia-Olmedo et al. 1995; Broekaert et al. 1997). Recently, nsLTPs were grouped into the PR-14 family of proteins (Van Loon and Van Strien 1999). In grapevine, LTPs occurred in somatic embryos as secreted proteins, and in fruits (Coutos-Thevenot et al. 1993; Salzman et al. 1998). Based upon deduced amino acid sequence, it is likely that the LTP expressed after in vitro selection is the same as previously reported. In contrast to other plant nsLTPs, grapevine LTP was not considered to be antifungal per se (Salzman et al. 1998). However, it is possible that nsLTP may have a synergistic effect with PR proteins in anthracnose resistance, since all have membrane permeating capacity. Originally, VVTL-2 was identified as grapevine osmotin based on its nucleotide sequence (Loulakakis 1997; Table 1. Effect of VVTL-1 on germination of E. ampelina spores Means were separated by Duncan’s multiple range test. Means within each column followed by the same letter are not significantly different at P=0.05 VVTL-1 Treatments (Fg/200:l) Control 20 40 80 Spore germination after 24h (%) Spore germination after 48h (%) 90.7 ± 1.94a 62.0 ± 3.68b 41.5 ± 3.75c 7.9 ± 3.21d 91.4 ± 3.58a 83.9 ± 5.50a 64.8 ± 6.54b 41.2 ± 4.77c Constitutive expression of Vitis vinifera thaumatin-like protein Functional Plant Biology Salzman et al. 1998). Osmotins are basic proteins having a C-terminal extension that targets them to the vacuole (Kombrink and Somssich 1997). However, Jacobs et al. (1999) placed it into the TLP subgroup of PR-5 and renamed it VVTL-2 because, although the predicted protein product of the cloned gene corresponded to osmotin, it had an acidic pI with no vacuole-targeting C-terminal extension. In the present study, the 21.6-kDa protein expressed by resistant cultures and regenerated plants exhibited a high N-terminal sequence homology to VVTL-2 (Loulakakis 1997); it was extracellular and thus, presumably acidic. Hence, the 21.6-kDa protein was identified as VVTL-2 as reported earlier (Jacobs et al. 1999; Davies and Robinson 2000). Induction of PR proteins following challenge with fungal or bacterial culture filtrate was considered to be epigenetic because PR protein expression returned to low levels after the challenge was withdrawn (Punja and Zhang 1993). However, mango embryogenic cultures produced the PR proteins chitinase and glucanase several months after 1111 in vitro selection with fungal culture filtrate, indicating that a constitutive change in expression had occurred (Jayasankar and Litz 1998). Prior to Jayasankar et al. (2000) and the present report, only transient expression of PR proteins was identified in non-transgenic grapevine. For example PR-3 chitinase was transiently expressed in cell cultures and plants after treatment with various elicitors (Busam et al. 1997). Selection of embryogenic cells with toxic filtrates can cause somaclonal variation (Larkin and Scowcroft, 1981) and the plants regenerated from such resistant cells then express changed phenotype(s) as described by Daub (1986); this provides an explanation for the constitutive expression of PR-5 proteins and nsLTP for over 3 years to date. Most PR-5 proteins, including the original ‘thaumatin’, were identified only in reproductive organs, such as floral and fruit tissues and in seeds. In grapevine, expression of VVTL-1 (Tattersall et al. 1997) and VVTL-2 (Loulakakis 1997; Salzman et al. 1998), was reported to be fruit VVTL-1 (µg) 0 40 80 24 48 72 Incubation time (h) Fig. 4. Effect of 0, 40 and 80 µg of VVTL-1 protein (per 200 µL total volume) on germination of E. ampelina spores and subsequent germ tube growth over time (refer to Table 1 for numerical data on these treatments). At 24 h, germination is inhibited in both 40 and 80 µg treatments (note the longer germ tubes and greater number of germinants in the 0 µg VVTL-1 control). After 48 h, germ tube growth and number of germinants are less at 80 µg compared with 0 and 40 µg. After 72 h, germination increased at the 80 µg treatment (possibly due to protein degradation), but growth was less than at 40 µg treatment, which, in turn, was less than at 0 µg. 1112 Functional Plant Biology ripening-specific and developmentally controlled. VVTL-2 exhibited antifungal properties, especially in the presence of high concentrations of glucose, indicating it to be a defence protein, active in berries (Salzman et al. 1998). Based on its appearance in ripening berries, VVTL-1 was considered to function in disease resistance (Tattersall et al. 1997) but its antifungal properties remained unknown. In studies using other species, the actual induction of PR-5 proteins was thought to be caused by various elicitors and signal molecules (Xu et al. 1994) and enhanced expression occurred predominantly in leaves (Cornelissen et al. 1986; Vigers et al. 1991; Regalado and Ricardo 1996). In this study the antifungal nature of VVTL-1 and its enhanced expression in vegetative tissues of grapevine is S. Jayasankar et al. demonstrated, strongly suggesting the role of this PR-5 protein in anthracnose resistance. Programmed expression and accumulation of PR-5 proteins in ICF during fruit ripening was suggested to be a preemptive defence response to fungal pathogenesis, especially since fruit tissues accumulate high concentrations of sugars and hence are highly vulnerable to pathogen attack (Velazhahan et al. 1999). Correlation between accumulation of PR-5 proteins during a specific stage of fruit ripening and onset of resistance to powdery mildew was established (Tattersall et al. 1997; Salzman et al. 1998). Furthermore, Jacobs et al. (1999) showed that genes encoding two PR-5 proteins were induced upon infection by powdery mildew, both in leaves and berries of grapevine. However, actual A B C Fig. 5. Inoculation of in vitro-selected and non-selected V. vinifera cv. Chardonnay leaves with E. ampelina. (A) Non-selected (left) and in vitroselected (right) leaves 5 d after inoculation with spore suspension. Note severe and mild anthracnose symptoms on non-selected leaves v. in vitroselected, respectively. (B) SEM of a non-selected leaf inoculation site. Note that many spores have collapsed owing to successful germination and germ tube growth. More germ tubes are present; they have branched and appear to have attached to the leaf surface by development of appressoria. (C) SEM of an in vitro-selected leaf inoculation site. Note that E. ampelina spores remain tumid and most have not germinated. Germ tubes, when present, do not branch nor do they possess appressoria. Constitutive expression of Vitis vinifera thaumatin-like protein inhibition of powdery mildew by PR-5 proteins was not substantiated. Accumulation of sugars in concert with elevated levels of PR-5 proteins may provide resistance to powdery mildew in berries of resistant plants produced from in vitro selection, as suggested by Salzman et al. (1998). However, our resistant plants cannot yet be evaluated for differences in PR-5 protein content in berries because they require at least two more years to flower and fruit. Inability of E. ampelina spores to germinate and colonize leaves of resistant plants indicates the presence of an inhibitory substance. Restricted hyphal growth on the leaf surface is similar to that described for other examples of plant resistance (e.g. Hoch et al. 1987; Podila et al. 1993; Scholthof 2001) and has been attributed to several factors, including physical barriers (cuticular waxes and plant surface topography) and chemical signals (secondary metabolites and defence proteins). In the present study, the response of E. ampelina spores on leaves of plants from in vitro selection was strikingly similar to that exhibited when they were incubated in vitro with VVTL-1 protein (i.e. spores remained tumid, germination was reduced and germ tube growth was restricted — compare Fig. 5C with Fig. 4, VVTL-1 at 80 µg and 48 h), strongly suggesting that VVTL-1 is a key factor in providing resistance to E. ampelina, both in vitro and in vivo. In conclusion, we have demonstrated that recurrent in vitro selection of grapevine PEMs has resulted in the expression of several proteins. That these proteins were activated at the cellular level, but continued to express in regenerated resistant plants for three years to date suggests that the response is due to a stable genetic change. Molecular mechanisms such as demethylation and differential activation of promoter(s) leading to altered gene expression can occur as a result of selection pressure with fungal culture filtrate (Jayasankar and Litz 1998), resulting in somaclonal variants with enhanced protein levels as demonstrated here. In contrast to earlier studies, we have demonstrated that PR-5 proteins can be constitutively expressed throughout grapevine tissues after in vitro selection and that at least one of them (VVTL-1) is antifungal. Resistance to fungal diseases in transgenic grapevines expressing PR proteins is an encouraging step (Yamamoto et al. 2000; Gray et al. 2002). However, since in vitro selection is not encumbered by intellectual property issues or subject to societal concerns currently affecting transgenic plant development, this non-transgenic approach of constitutively expressing native defence proteins offers an attractive alternative method of producing genetically improved varieties. Acknowledgments We thank Dr OP Sehgal, Dept of Plant Pathology, University of Missouri, Columbia, MO, for the generous gift of antiserum; Dr S Muthukrishnan, Department of Bio- Functional Plant Biology 1113 chemistry, Kansas State University, Manhattan, KS, and Dr Senthil K Ramu, Department of Genetics, Harvard Medical School, Boston, MA, for critical reading of the manuscript; Dr Nancy Denslow and Scott McClung, PCCL, University of Florida, Gainesville performed N-terminal amino acid sequencing; Dr Peter Snow of Protein Expression Center, Beckman Institute, California Institute of Technology assisted in the expression and purification of VVTL-1. We acknowledge the excellent technical assistance of Marilyn Van Aman, Karen Kelley, Ajitharani Ravindran, Nancy Barnett and Sivagama Sundhari. This research was supported by the Florida Agricultural Experiment Station and a grant from The Florida Department of Agriculture and Consumer Services’ Viticulture Trust Fund and approved for publication as Journal Series No. R-08969. References Abad LR, Urzo D, Liu MP, Narasimhan ML, Reuveni M, Zhu JK, Niu X, Singh NK, Hasegawa PM, Bressan RA (1996) Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization. Plant Science 118, 11–23. doi:10.1016/0168-9452(96)04420-2 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402. doi:10.1093/NAR/25.17.3389 Barre A, Peumans WJ, Menu-Bouaouiche L, Van Damme EJM, May GD, Herrera AF, Van Leuven AF, Rouge P (2000) Purification and structural analysis of an abundant thaumatin-like protein from ripe banana fruit. Planta 211, 791–799. doi:10.1007/ S004250000354 Broekaert WF, Cammue BPA, De Bolle MFC, Thevissen K, De Samblanx GW, Osborn RW (1997) Antimicrobial peptides from plants. Critical Reviews in Plant Sciences 16, 297–323. Busam G, Kassemeyer H, Matern U (1997) Differential expression of chitinases in Vitis vinifera L. responding to systemic acquired resistance activators or fungal challenge. Plant Physiology 115, 1029–1038. doi:10.1104/PP.115.3.1029 Cheong NE, Choi YO, Kim WY, Chang S, Cho MJ, Lee SY (1997) Purification of an antifungal PR-5 protein from flower buds of Brassica campestris and cloning of its gene. Physiologia Plantarum 101, 583–590. doi:10.1034/J.1399-3054.1997. 1010319.X Cornelissen BJC, Hooft Van Huijsduijnen RAM, Bol JF (1986) A tobacco mosaic virus-induced tobacco protein is homologous to the sweet tasting protein, thaumatin. Nature 231, 531–532. Coutos-Thevenot P, Jouenne T, Maes O, Guerbette F, Grosbois M, La Caer JP, Boulay M, Deloire A, Kader JC, Guern J (1993) Four 9 kDa proteins excreted by somatic embryos of grapevine are isoforms of lipid-transfer proteins. European Journal of Biochemistry 217, 885–889. Daub ME (1986) Tissue culture and the selection of resistance to pathogens. Annual Review of Phytopathology 24, 159–186. doi:10.1146/ANNUREV.PY.24.090186.001111 Davies C, Robinson SP (2000) Differential screening indicates a dramatic change in mRNA profiles during grape berry ripening. Cloning and characterization of cDNAs encoding putative cell wall and stress response proteins. Plant Physiology 122, 803–812. doi:10.1104/PP.122.3.803 1114 Functional Plant Biology Dempsey DA, Shah J, Klessig DF (1999) Salicylic and disease resistance in plants. Critical Reviews in Plant Sciences 18, 547–575. doi:10.1016/S0735-2689(99)00391-3 Fils-Lycaon BR, Wiersma PA, Eastwell KC, Sautiere P (1996) A cherry protein and its gene, abundantly expressed in ripening fruit have been identified as thaumatin-like. Plant Physiology 111, 269–273. doi:10.1104/PP.111.1.269 Fritig B, Heitz T, Legrand M (1998) Antimicrobial proteins in induced plant defense. Current Opinions in Immunology 10, 16–22. doi:10.1016/S0952-7915(98)80025-3 Garcia-Olmedo F, Molina A, Segura A, Moreno M (1995) The defensive role of non-specific lipid-transfer proteins in plants. Trends in Microbiology 3, 72–74. doi:10.1016/S0966-842X(00) 88879-4 Gavish H, Vardi A, Fluhr R (1992) Suppression of somatic embryogenesis in citrus cell cultures by extracellular proteins. Planta 186, 511–517. Gentile A, Tribulato E, Deng ZN, Galun E, Fluhr R, Vardi A (1993) Nucellar callus of ‘Femminello’ lemon, selected for tolerance to Phoma tracheiphila toxin, shows enhanced release of chitinase and glucanase into culture medium. Theoretical and Applied Genetics 86, 527–532. Gray DJ, Jayasankar S, Li Z, Cordts J, Scorza R, Srinivasan C (2002) Transgenic grapevines. In ‘Transgenic plants and crops’. (Eds GG Khachatourians, A McHughen, R Scorza, W Nip and YH Hui) pp. 397–405. (Marcel Dekker, Inc.: New York) Hammond-Kosack KE, Jones JD (1996) Resistance gene-dependent plant defense responses. The Plant Cell 8, 1773–1791. doi:10.1105/TPC.8.10.1773 Hoch HC, Staples RC, Whitehead B, Comeau J, Wolf ED (1987) Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 234, 1659–1662. Ignatius SMJ, Chopra RK, Muthukrishnan S (1994) Effects of fungal infection and wounding on the expression of chitinases and β-1,3-glucanases in near-isogonic lines of barley. Physiologia Plantarum 90, 584–592. doi:10.1034/J.1399-3054.1994.900321.X Jacobs AK, Dry IB, Robinson SP (1999) Induction of different pathogenesis-related cDNAs in grapevine infected with powdery mildew and treated with ethephon. Plant Pathology 48, 325–336. doi:10.1046/J.1365-3059.1999.00343.X Jayasankar S, Litz RE (1998) Characterization of resistance in mango embryogenic cultures selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin. Theoretical and Applied Genetics 96, 823–831. doi:10.1007/S001220050808 Jayasankar S, Gray DJ, Litz RE (1999) High frequency somatic embryogenesis and plant regeneration from suspension cultures of grapevine. Plant Cell Reports 18, 533–537. doi:10.1007/ S002990050617 Jayasankar S, Li Z, Gray DJ (2000) In vitro selection of Vitis vinifera ‘Chardonnay’ with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase. Planta 211, 200–208. doi:10.1007/S004250000285 Jayasankar S, Bondada BR, Li Z, Gray DJ (2002) A unique morphotype of grapevine somatic embryo exhibits accelerated germination and early plant development. Plant Cell Reports 20, 907–911. doi:10.1007/S00299-001-0427-7 Kombrink E, Somssich IE (1997) Pathogenesis-related proteins and plant defense. In ‘The Mycota V, part A’. (Eds GC Carroll and PU Tudzynski) pp. 107–127. (Springer-Verlag: Berlin) Larkin PJ, Scowcroft SC (1981) Somaclonal variation — a novel source of variability from cell culture for plant improvement. Theoretical and Applied Genetics 60, 197–214. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. S. Jayasankar et al. Lodhi MA, Ye GN, Weeden NF, Reisch BJ (1994) A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Molecular Biology Reporter 12, 6–13. Loulakakis KA (1997) Nucleotide sequence of a Vitis vinifera L. cDNA (accession no.Y10992) encoding for osmotin-like protein (PGR97-064). Plant Physiology 113, 1464–1465. Neale AD, Wahleithner JA, Lund M, Bonnet HT, Kelly A, Meeks-Wagner DR, Peacock WJ, Dennis ES (1990) Chitinase, β-1,3-glucanase, osmotin and extensin are expressed in tobacco explants during flower formation. The Plant Cell 2, 673–684. doi:10.1105/TPC.2.7.673 O’Reilly DR, Miller LK, Luckow VA (1994) ‘Baculovirus expression vectors: a laboratory manual.’ pp. 124–127. (Oxford University Press: New York) Pierpoint WS (1986) The pathogenesis-related proteins of tobacco leaves. Phytochemistry 25, 1595–1601. doi:10.1016/S00319422(00)81215-7 Pierpoint WS, Thatham AS, Pappin DJC (1987) Identification of the virus-induced protein of tobacco leaves that resembles the sweet tasting protein thaumatin. Physiological and Molecular Plant Pathology 31, 291–298. Podila GK, Rogers LM, Kolattukudy PE (1993) Chemical signals from avocado surface wax trigger germination and appressorium formation in C. gloeosporioides. Plant Physiology 103, 267–272. Punja ZK, Zhang Y (1993) Plant chitinases and their roles in resistance to fungal diseases. Journal of Nematolology 25, 526–540. Regalado AP, Ricardo CPP (1996) Study of the intercellular fluid of healthy Lupinus albus organs. Presence of a chitinase and thaumatin-like protein. Plant Physiology 110, 227–232. doi:10.1104/PP.110.1.227 Roberts WK, Selitrennikoff CP (1990) Zeamatin, an antifungal protein from maize with membrane permeabilizing activity. Journal of General Microbiology 136, 1771–1778. Salzman RA, Tikhonova I, Bordelon BP, Hasegawa PM, Bressan RA (1998) Coordinate accumulation of antifungal proteins and hexoses constitutes a developmentally controlled defense response during fruit ripening in grape. Plant Physiology 117, 465–472. doi:10.1104/PP.117.2.465 Scholthof HB (2001) Molecular plant–microbe interactions that cut the mustard. Plant Physiology 127, 1476–1483. doi:10.1104/PP.127.4. 1476 Tattersall DB, van Heeswijck R, Hoj PB (1997) Identification and characterization of a fruit-specific, thaumatin-like protein that accumulates at very high levels in conjunction with the onset of sugar accumulation and berry softening in grapes. Plant Physiology 114, 759–769. doi:10.1104/PP.114.3.759 van Loon LC, Van Strien EA (1999) The families of pathogenesis related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology 55, 85–97. doi:10.1006/PMPP.1999.0213 van Loon LC, Pierpoint WS, Boller T, Conejero V (1994) Recommendations for naming plant pathogenesis-related proteins. Plant Molecular Biology Reporter 12, 245–264. Velazhahan R, Datta SK, Muthukrishnan S (1999) The PR-5 family: thaumatin-like proteins. In ‘Pathogenesis-related proteins in plants’. (Eds S Muthukrishnan and SK Datta) pp. 107–129. (CRC Press: Boca Raton) Vigers AJ, Roberts WK, Selitrennikaff CP (1991) A new family of plant antifungal proteins. Molecular Plant–Microbe Interactions 4, 315–323. Vigers AJ, Weidemann S, Roberts WK, Legrand M, Selitrennikaff CP, Fritig B (1992) Thaumatin-like pathogenesis-related proteins are antifungal. Plant Science 83, 153–161. Constitutive expression of Vitis vinifera thaumatin-like protein Vu L, Huynh QK (1994) Isolation and characterization of a 27 kDa antifungal protein from the fruits of Diospyros texana. Biochemical and Biophysical Research Communications 202, 666–672. doi:10.1006/BBRC.1994.1928 Xu Y, Chang PFL, Liu D, Narasimhan ML, Hasegawa PM, Bressan RA (1994) Plant defense genes are synergistically induced by ethylene and methyl jasmonate. The Plant Cell 6, 1077–1085. doi:10.1105/TPC.6.8.1077 Functional Plant Biology 1115 Yamamoto T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hibi T, Hayashi T, Matsuta N (2000) Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Reporter 19, 639–646. doi:10.1007/ S002999900174 Manuscript received 10 April 2003, accepted 11 September 2003 http://www.publish.csiro.au/journals/fpb