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Plant Physiol. Jun 1998; 117(2): 465–472.
PMCID: PMC34966

Coordinate Accumulation of Antifungal Proteins and Hexoses Constitutes a Developmentally Controlled Defense Response during Fruit Ripening in Grape1

Abstract

During ripening of grape (Vitis labruscana L. cv Concord) berries, abundance of several proteins increased, coordinately with hexoses, to the extent that these became the predominant proteins in the ovary. These proteins have been identified by N-terminal amino acid-sequence analysis and/or function to be a thaumatin-like protein (grape osmotin), a lipid-transfer protein, and a basic and an acidic chitinase. The basic chitinase and grape osmotin exhibited activities against the principal grape fungal pathogens Guignardia bidwellii and Botrytis cinerea based on in vitro growth assays. The growth-inhibiting activity of the antifungal proteins was substantial at levels comparable to those that accumulate in the ripening fruit, and these activities were enhanced by as much as 70% in the presence of 1 m glucose, a physiological hexose concentration in berries. The simultaneous accumulation of the antifungal proteins and sugars during berry ripening was correlated with the characteristic development of pathogen resistance that occurs in fruits during ripening. Taken together, accumulation of these proteins, in combination with sugars, appears to constitute a novel, developmentally regulated defense mechanism against phytopathogens in the maturing fruit.

Plants have evolved a number of strategies to resist fungal infection. One strategy involves the accumulation of defense proteins that have direct inhibitory activity against the hyphae and/or germinating spores of the pathogen. Among these are PR proteins including chitinases (PR-3 family), thaumatin-like proteins (PR-5 family), and nsLTPs. Typically, these antifungal proteins are expressed constitutively at low levels in cells and accumulate in response to fungal attack or in response to other inducers of acquired resistance (Uknes et al., 1992). Reproductive organs are apparently an exception to induced acquired resistance. Presumably, the importance of flowers and ovaries to the maintenance of the species has mandated that reproductive organs acquire pathogen resistance during development. Developmentally regulated expression of PR proteins has been observed in floral organs (Lotan et al., 1989; Neal et al., 1990), including numerous examples of defensive gene mRNA accumulation in fruits. However, comparatively few data are available that interrelate developmental accumulation of antifungal proteins and the acquisition of resistance against fruit pathogens (Fils-Lycaon et al., 1996; Meyer et al., 1996).

Another physiological adaptation of plants that affects fungal pathogenesis, but one that has received considerably less attention, is the accumulation of sugars. Results from studies of several host/pathogen systems have implicated accumulation or depletion of sugars in resistance to fungal infection (VanderPlank, 1984). Increased susceptibility to fungi in sugar-depleted vegetative tissues, a phenomenon termed “sink-induced loss of resistance,” has been documented in tomato (Horsfall, 1975), cotton (Eaton and Rigler, 1946), and maize (Holbert et al., 1935). Conversely, moderate levels of sugars can enhance colonization rates of some fungal pathogens, presumably because these are important sources of C for the microbes (Mains, 1917). However, higher levels of sugars can reverse this effect, leading to decreased susceptibility, a phenomenon termed “high-sugar resistance” (Horsfall and Dimond, 1957). This reversal of susceptibility has been suggested to occur as a result of high sugar levels furnishing an osmotic challenge to the fungi and suppressing their colonization of the plant (VanderPlank, 1984).

We report here that accumulation of hexoses in grape (Vitis labruscana L. cv Concord) berries is accompanied by a developmental-stage-specific increase in a suite of proteins that are homologous to proteins known to be antifungal determinants. These proteins have been identified as a thaumatin-like protein (Salzman et al., 1994) (here we have named it GO), chitinases (both CBC and AC forms), and a nsLTP. Physiological levels of GO or CBC assayed in vitro alone or in combination with physiological levels of Glc exhibited individual and/or combinatorial activities against the important grape pathogens Guignardia bidwellii, the causal agent of black rot, and Botrytis cinerea. The interaction of the antifungal proteins and hexoses appears to constitute a developmentally regulated defense mechanism to restrict fungal pathogen infection as seeds are maturing in ripening berries.

MATERIALS AND METHODS

Unless otherwise stated, all plant material was obtained from field-grown vines of grape (Vitis labruscana L. cv Concord).

Protein Purification

For antifungal protein purification, grape berries were destemmed, crushed, and squeezed in a horizontal press (Vaslin Bucher, Chalonnes-sur-Loire, France) to obtain juice. The juice was clarified by centrifugation at 10,000g for 15 min at 4°C. The supernatant was adjusted to pH 3.7 with malic acid, and passed through a column of Amberlite CG 50 cation-exchange resin (Rohm and Haas, Philadelphia, PA) that was preequilibrated with 10 mm KH2PO4 buffer, pH 3.7. The column was washed with buffer until A280 was undetectable. The proteins were eluted with 1.5% NH4OH (adjusted to pH 9.5 with malic acid). The eluate from the ion-exchange column was dialyzed against 100 volumes of distilled water, with one change, for 48 h. The dialyzed ion-exchange fraction was adjusted to 20 mm KH2PO4, pH 6.0, and applied to an S-300 cation-exchange HPLC column (Synchropak, Lafayette, IN). The flow-through fraction was collected for subsequent chromatographic separation. The column was then eluted using NaCl and pH-gradient fractionation (0–0.5 m NaCl in 20 mm KH2PO4, pH 6.0–8.0, for a period of 60 min) to isolate the CBC and nsLTP. After elution, the column was equilibrated with 10 mm KH2PO4, pH 3.5. The flow-through fraction was adjusted to pH 3.5 with malic acid and then applied to the column. GO and AC were eluted using a gradient of 0 to 0.5 m NaCl in 10 mm KH2PO4, pH 3.5, during a period of 60 min. The fractions were dialyzed as described above, and lyophilized to dryness. The proteins were resolubilized to a concentration of 2 mg mL−1 in distilled water. Chitin-binding capacity was assessed using a colloidal chitin column (Pierpoint, 1983) and elution with 10 mm NaOH.

Electrophoresis and Detection Methods and Antibody Production

For determination of protein levels in fruit, fresh berries were deseeded and ground in a mortar and pestle with 5× sample buffer (Laemmli, 1970), 250 μL g−1 fresh weight of fruit. The homogenate was boiled for 3 min and centrifuged at 13,000g for 10 min, and the supernatant was applied to SDS-PAGE 15% gels, 25 μL of extract per lane. For immunodetection, proteins were electroblotted onto nitrocellulose according to the method of Zhu et al. (1996) and detected by reaction with IgY polyclonal antibodies (Salzman et al., 1996) raised against tobacco (Nicotiana tabacum L.) osmotin, tobacco chitinase T2 (Yun et al., 1996), or grape LTP. After reaction with the primary antibody, blots were incubated with alkaline phosphatase-linked rabbit anti-chicken IgY secondary antibodies (Jackson Immunoresearch, West Grove, PA), and the color product was developed according to standard protocols (Bio-Rad).

Amino Acid Sequence Determination

Purified proteins were subjected to SDS-PAGE and then electroblotted to a PVDF (Bio-Rad) membrane (Zhu et al., 1996). Protein bands were visualized with Coomassie blue R and excised from the membrane. The N-terminal amino acid sequence determination was performed by the Purdue Laboratory for Molecular Structure (West Lafayette, IN) on a sequencer (model 470A, Applied Biosystems) using the Edman degradation method.

Measurement of Total Sugars

Total soluble sugars in grape berries were determined from measurements of the refractive index, which accurately reflects total soluble solids. In grape berries Glc and Fru typically represent 95 to 99% of soluble solids (Zoeklein et al., 1996). Molarity (as Glc equivalents) of total sugars in freshly expressed juice was calculated from refractive index measurements using a Glc standard curve.

Chitinase and Antifungal Assays

Chitinase activity was determined colorimetrically as described previously (Yun et al., 1996). Hyphal growth measurements were determined on one-half-strength potato dextrose-broth (Sigma) medium (Abad et al., 1996). Autoclaved potato dextrose-broth medium was cooled to 40°C, followed by inclusion of purified antifungal proteins, individually or in combination, and without or with Glc or Xyl. The medium was distributed into 2.5-mm (width) by 6-mm (depth) slots in assay plates and allowed to solidify. The medium in each slot was then inoculated at one end with a small block of potato dextrose-broth medium containing fungal hyphae excised from the edge of an actively growing culture (Abad et al., 1996). Hyphal elongation was quantified with the aid of a stereomicroscope equipped with a micrometer (Olympus Optical Co., Ltd., Tokyo, Japan).

Each treatment contained six replicates per experiment, and each experiment was conducted at least twice. Dose-response data from one representative experiment are presented for each fungus/protein/solute combination.

RESULTS

Identification of Major Proteins Accumulating in Grape Berries

Polypeptides with molecular masses of 32, 29, 27, and 9 kD were the predominant proteins recovered from the juice of grape berries at the fully ripe stage (Fig. (Fig.1).1). These proteins were purified to apparent homogeneity based on Coomassie blue staining of SDS-PAGE gels (Fig. (Fig.2).2). The 27-kD protein (GO) has an N-terminal amino acid sequence that is 72% identical to VVTL1 from Vitis vinifera (Tattersall et al., 1997), and is 68% identical to TPM-1, an osmotin-like protein from tomato (Fig. (Fig.3).3). GO has a sequence divergent from that of VVTL1, possibly because the proteins were obtained from different genotypes. GO reacted strongly with tobacco anti-osmotin. The 9-kD protein was identified as nsLTP based on the N-terminal amino acid sequence identity (79%) with NLT4 from V. vinifera × Vitis berlanchen (Coutos-Thevenot et al., 1993) and conservation of motifs prevalent in other LTPs (Fig. (Fig.3).3). The molecular mass determined by SDS-PAGE and heat stability are also characteristics of plant LTPs. Amino acid sequence information was not obtained for the 29- and 32-kD proteins because these were N-terminally blocked. However, both of these proteins had potent endochitinase activity but not exochitinase activity (Table (TableI). I). These proteins reacted strongly with tobacco anti-chitinase (Yun et al., 1996) and comigrated with the tobacco homologs on SDS-PAGE gels (data not shown). Increased accumulation of these two proteins was also correlated with increases in chitinase activity levels shown to occur in ripening grapes (Robinson et al., 1997). The 29-kD chitinase (AC) was more acidic than the 32-kD form based on elution from a cation-exchange column, and lacked chitin-binding activity, analogous to class II chitinases. The chitin-binding 32-kD protein (CBC) was more basic and presumably contained a chitin-binding domain (Fig. (Fig.4),4), both characteristics of class I chitinases.

Figure 1
Pattern of protein accumulation in grape berries during ripening. A, Total protein accumulation profile in cv Concord. Total protein from fresh berries was extracted, separated by SDS-PAGE (15% gel), and visualized after staining with Coomassie ...
Figure 2
Purification of proteins that accumulate in grape berries during ripening. Proteins extracted from berries at the fully ripe stage were fractionated and separated by SDS-PAGE. Molecular mass standards (STD) are indicated on the left. TP, Total fruit ...
Figure 3
N-terminal amino acid sequence comparison identifies GO and LTP. GO had high sequence homology with thaumatin-like proteins: V. vinifera (Vvtl1) (GenBank [G] accession no. ...
Table I
Grape chitinases CBC and AC have high endochitinase activity
Figure 4
Ripe grape berries accumulate abundant quantities of CBC. Proteins from different fractions of colloidal chitin chromatography were separated by SDS-PAGE (15% gel) and stained with Coomassie blue R. Molecular mass standards (STD) are indicated ...

Sugar and Antifungal Protein Accumulation Occur Coordinately during Fruit Ripening

Total sugar levels in berries increased substantially during the onset of ripening (termed “veraison”), coincident with major accumulation of the antifungal proteins (Fig. (Fig.5).5). During this period the molarity of total sugars, which are composed primarily of Glc and Fru (Zoeklein et al., 1996), increased from 0.58 to 1.1 m Glc equivalents. The antifungal proteins represented the predominant proteins in the berries during this period, in part because of reduced abundance of photosynthetic enzymes. The absolute levels of GO and CBC increased to as much as 200 μg g−1 fresh weight of the berry, estimated by comparing total protein extracts with known amounts of purified proteins on SDS-PAGE (data not shown), although accumulation levels varied among different genotypes (Fig. (Fig.1B).1B). High levels of the proteins also persisted during senescence.

Figure 5
Antifungal proteins coordinately accumulate with sugars during grape berry ripening. A, Sugar accumulation during ripening. Grape berry sugar concentration was measured by refractometry, and effective molarity of total sugars was derived from a Glc ...

Proteins That Accumulate in Fruits during Ripening Inhibit Growth of Grape-Pathogenic Fungi and This Activity Is Enhanced by Sugars

Hyphal growth of grape isolates of Guignardia bidwellii and Botrytis cinerea was inhibited by GO and CBC (Figs. (Figs.66 and and7). 7). Virtually no chitinase activity was detected in the GO protein fraction (Table (TableII),II), indicating that antifungal activity was not attributable to contaminating chitinases. Petri-dish and quantitative assays established that the effects of GO and CBC against B. cinerea and G. bidwellii were enhanced substantially by inclusion of 1 m Glc in the medium (Figs. (Figs.66 and and7).7). This effect was more evident with GO, which had substantially less antifungal activity in the absence of solute (Fig. (Fig.7). 7). Fungal-growth inhibition was additive when GO and CBC were assayed in combination; however, no synergism was evident between the two proteins, either in the presence or absence of Glc (Fig. (Fig.6).6). The sugar modulation of antifungal protein activity seemed to be mediated through a nutrition-independent mechanism, because Xyl (a nonmetabolizable osmolyte) also enhanced the activity of the proteins (Table III). The LTP exhibited no antifungal activity against either G. bidwellii or B. cinerea at doses up to 200 μg mL−1 (data not shown).

Figure 6
Hyphal growth inhibition of G. bidwellii and B. cinerea by GO and CBC is enhanced by Glc, although the sugar does not have antifungal activity. Hyphal plugs were inoculated onto medium without (w/o) or with 1 m Glc. After the fungal colony diameter ...
Figure 7
GO and CBC antifungal activities are facilitated by Glc. Hyphal growth of G. bidwellii (G.bidw) and B. cinerea (B.cin) was measured at time intervals on medium containing GO or CBC without (glc−) or with 1 m Glc (1Mglc). Values represent mean ...
Table II
Purified GO and LTP fractions are devoid of chitinase activity
Table III
Glc or Xyl are equivalent in their capacity to facilitate antifungal activity of GO and CBC against G. bidwellii

DISCUSSION

Antifungal Proteins Are Components of Fungal Resistance Developed during Grape Berry Ripening

The demonstration that the grape berry proteins have antifungal activity, and their abundant and developmentally controlled accumulation in berries implicate a potential role for these proteins in host-plant defense against fruit pathogens. Ripe fruits typically are considered vulnerable to pathogen attack because of their enriched levels of sugars and other nutrients, as well as physical changes that facilitate infection (Swinburn, 1983). Plants with climacteric fruits, such as avocado, tomato, and banana, evolved ethylene during ripening, which fostered infection by Colletotrichum spp. (Flaishman and Kolattukudy, 1994). However, for some years it has been known that grape berries acquire resistance to several pathogens during ripening (Delp 1954; Lafon and Clerjeau 1988; Pearson, 1988), and it has been suggested that an osmotin-like protein may be involved in this acquisition of resistance (Tattersall et al., 1997).

Here we establish that GO and CBC, which are effective antifungal proteins against isolates of the grape berry pathogens, accumulate synchronously at stages of ripening when the fruits become resistant to black rot (G. bidwellii), powdery mildew (Uncinula necator), and downy mildew (Plasmopara viticola) (Delp, 1954; Lafon and Clerjeau, 1988; Pearson, 1988; Ramsdell and Milholland, 1988). The levels of GO and CBC in fruits that acquired resistance were consistent with those that substantially inhibited fungal growth in bioassays. Accumulation of thaumatin in fruits of Thaumatococcus daniellii (van der Wel and Loeve, 1972) and thaumatin-like proteins in those of cherry and V. vinifera (Fils-Lycaon et al., 1996; Tattersall et al., 1997) have been reported; however, thaumatin essentially lacks antifungal activity (Vigers et al., 1991) and antifungal capacity has yet to be established for the cherry and the V. vinifera fruit-expressed PR-5 proteins. Typically, grape berries are susceptible to postharvest infection by B. cinerea, so the effective antifungal activity of the fruit GO and CBC was unpredicted. However, the cv Concord used in this study has been classified as B. cinerea resistant (Reich et al., 1993). Perhaps the proposed defense mechanism is unique to this genotype, or it may mitigate colonization rather than mediate immunity. Supporting this, another grape cultivar (SV 5276, Seyval Blanc), classified as highly susceptible to B. cinerea (Reich et al., 1993), accumulated distinctly lower levels of the antifungal proteins than the cv Concord, at the same fully ripe stage (Fig. (Fig.11B).

Induction of Antifungal Proteins during Fruit Ripening

At least two potentially interrelated mechanisms may be involved in the regulation of antifungal protein accumulation during ripening: osmotic due to sugar accumulation, or sugar signaling. Osmotin accumulation was first shown to occur as a function of salt/osmotic adaptation of tobacco cells in culture (Singh et al., 1985). The osmotin gene is responsive to hyperosmolarity and pathogen infection, as well as many inducers that have been implicated as components of the signal cascades leading from perception of these stresses (Kononowicz et al., 1992). The osmolarity in grape berries (>1.5 m Glc equivalents) is far in excess of that required to induce accumulation of osmotin, chitinases, and nsLTPs (Singh et al., 1985; Torres-Schumann et al., 1992; Yun et al., 1996). In fact, potentially inductive osmotic levels (380–580 mm Glc equivalents) occur in fruits coincident with the acquisition of resistance (Fig. (Fig.55).

As an alternative, sugars may act as a signal molecule to regulate the expression of antifungal protein genes during fruit ripening. There are numerous examples of sugar-repressed genes, most notably those that encode major C-assimilatory enzymes, because these are not required when sugars are in excess (Sheen, 1990; Cheng et al., 1992; Krapp et al., 1993; Jang and Sheen, 1994). However, sugars also up-regulate many genes in a hexose-specific manner, including those that encode defensive proteins such as protease inhibitor II (Johnson and Ryan, 1990), chalcone synthase (Tsukaya et al., 1991), and PR proteins PR3 (Herbers et al., 1995), PR1b, and PR-Q (Herbers et al., 1996a). Both Glc and Fru (the two major sugars accumulating in grape berries) mediate defense gene induction in tobacco leaves (Herbers et al., 1996b) at levels (100–500 mm) that accumulate in ripening grape berries (Fig. (Fig.5).5). This response seems sugar specific because the osmotic agents sorbitol and PEG 8000 did not mediate gene induction.

Sugars and Antifungal Proteins Interact to Mediate Host Plant Defense against Phytopathogens

Our results indicate that the antifungal efficacy of GO and CBC is enhanced by concentrations of Glc that occur in fruits as resistance to phytopathogens is acquired. The antipathogenic effect of potential metabolic interactions between sugars and the pathogens apparently involves nutrition-independent mechanisms, since Glc and Xyl, at equivalent osmolarities have the same effect, and Xyl is not metabolized by either G. bidwellii or B. cinerea (neither of these organisms was able to grow on Xyl as a sole C source). The previous concept that sugars furnish an “osmotic challenge” also seems unlikely, since fungal growth rates on medium supplemented with either 1 m Glc or 1 m Xyl were as high or higher than those on basal medium (data not shown). Consequently, the fungi were able to adjust osmotically and generate sufficient turgor for hyphal growth.

A more likely mechanism for the observed enhancement of antifungal protein activity by sugars might be a disruption of fungal gene regulation caused by sugar repression (Ronne, 1995). The most notable examples of sugar-repressed genes, which are relevant in this context, are those that encode pectate lyase in Fusarium solani (Guo et al., 1995) and cellulase in Trichoderma reesei (Ilmen et al., 1996). These enzymes are involved in host colonization by the phytopathogens. Sugar repression of genes involved in pathogenesis or virulence might be facilitated by GO because members of the PR-5 family are presumed to cause formation of membrane pores and this would enhance intracellular uptake of sugars. The maize PR-5 zeamatin presumably facilitated uptake of nikkomycin Z into Candida albicans cells (Vigers et al., 1991).

Another potential mechanism by which hexoses could enhance antifungal activity of GO and CBC is based on solute-mediated preservation of native protein structure, since sugars are well known to stabilize proteins against denaturation (Lee and Timasheff, 1981; Timasheff and Arakawa, 1988). At physiological temperatures, proteins are preferentially hydrated. Glc and other sugars, and polyhydric alcohols, are preferentially excluded from the hydration surface, a process that requires free energy. Unfolding of proteins is energetically unfavorable because solute exclusion would be required for a larger surface area (Arakawa and Timasheff, 1982). Presumably, the hexoses in ripening grape berries would enhance the function of the antifungal proteins by facilitating active conformation in the physiological solution. A chemical basis for sugar enhancement of antifungal activity is very plausible because GO and CBC are proteins with very different structures and functions.

ACKNOWLEDGMENTS

We thank Dr. Meena Narasimhan and Dr. Keyan Zhu-Salzman for their critical review of the manuscript.

Abbreviations:

AC
acidic chitinase
CBC
chitin-binding chitinase (basic)
GO
grape osmotin
LTP
lipid-transfer protein
nsLTP
nonspecific LTP
PR
pathogenesis-related

Footnotes

1This research was partially supported by a grant from the Purdue Agricultural Research Program. This is publication no. 15,479 of the Purdue University Agricultural Experiment Station.

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