Nucellar callus of ‘Femminello’ lemon, selected for tolerance to Phoma tracheiphila toxin, shows enhanced release of chitinase and glucanase into the culture medium

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  Phoma tracheiphila is the causative agent of the disease mal secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogenP. tracheiphila and its toxin. Some cultivars (e.g., ‘Femminello’ lemon) are inherently sensitive
  Theor Appl Genet (1993) 86:527-532 9 Springer-Verlag 1993 Nucellar callus of Femminello lemon, selected for tolerance to homa tvacheiphila toxin, shows enhanced release of chitinase and glucanase into the culture medium A. Gentile 1, E. Tribulato 1, Z. N. Deng 2, E. Galun 3, R. Fluhr 3, A. Vardi 4 1 Istituto di Coltivazioni Arboree, University of Catania, Catania 95123, Italy 2 Hunan Horticultural Institute, Changsha, P.R. China 3 Department of Plant Genetics, The Weizmann Institute of Science, P. O. Box 26, Rehovot 76J00, Israel 4 Institute of Horticulture, Agricultural Research Organization, The Volcani Center, P. O. Box 6, Bet Dagan 50250, Israel Received: 30 September 1992/Accepted: 9 December 1992 Abstract. Phoma tracheiphila is the causative agent of the disease real secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogen P. tracheiphila and its toxin. Some cultivars (e.g., 'Femminello' lemon) are inherently sensitive while others (e.g., 'Tarocco' or- ange) are tolerant. Cell lines derived from nucellar tissue of 'Femminello', 'Tarocco' and a cell line selected for tolerance to the fungal toxin ('Femminello-S') were used to study host-pathogen interaction. Our results showed that calli or conditioned media of 'Tarocco' and 'Fem- minello-S' inhibited the size of co-cultivated fungal colonies when compared to 'Femminello'. In addition, conditioned medium of'Tarocco' as well as 'Femminello- S', but not 'Femminello', promoted bursting of hyphal tips. A ten-fold increase in chitinase and glucanase enzy- matic activity, as evaluated by radiometric assay and laminarin hydrolysis respectively, was detected in 'Fem- minello-S' extracellular extracts as compared to 'Fem- minello'. An increase in chitinase was also shown by immunoblot analysis. Our findings suggest a positive correlation between the presence of chitinase and glu- canase in the conditioned media of the cultured cells and the tolerance of those cells to P. tracheiphila toxin. Key words: Pathogenesis-related proteins (PR) - Glu- canase - Chitinase - Plant cell culture - Disease tolerance Introduction Phoma tracheiphila (Petri) Kanc. et Ghik is a pathogen that causes a serious tracheomycotic disease of citrus trees known as mal secco. So-far no genetic source of Communicated by G. Wenzel Correspondence to: A. Vardi tolerance to mal secco has been found among commer- cial lemon varieties (Perrotta and Tribulato 1977). How- ever, some other citrus species are tolerant e.g., orange, mandarin, grapefruit. The use of classical genetic breed- ing to transfer this trait is difficult, due to the high polyembryonic characteristic of lemon and the other spe- cies, which leads to a high percentage of parental types in the F 1 population. In addition, the zygotes from such crosses will not easily produce hybrids of high commer- cial values. An alternative approach is in-vitro selection for resistance at the cellular level. Recovery of disease-re- sistant plants by in-vitro selection for resistance against toxic chemicals, and toxins or culture filtrates produced by plant pathogens, has been reported for various cell cultures (for review see Daub 1986; Galun and Breiman 1992). Nachmias et al. (1977) have isolated a partially- purified toxin (PPT) from P. tracheiphila. Using tissue culture techniques, i.e., exposure of the sensitive 'Femminello' lemon cells to PPT, a cell line was established that was tolerant to the P. tracheiphila toxin and labelled 'Femminello-S' (Gentile et al. 1992b). The aim of the present work was to attain a better under- standing of the mechanism involved in in-vitro selection by testing the two 'Femminello' cell lines (non-selected versus selected) as compared to cell lines derived from cultivars with known in-vivo tolerance to the real secco disease. Unexpectedly, our preliminary experiments on co-cultivated fungal and plant cells showed that cells derived from the tolerant lines actually inhibited fungal growth. We therefore used three approaches to study the plant-pathogen interaction. In the first approach differ- ent combinations of co-cultivated lines were examined similarly to the dual culture experiments performed by Storti et al. (1988) in tomato cell lines resistant to Fusar- ium oxysporum f. sp. lycopersici. Secondly, we examined fungal growth in the presence of conditioned media ob-  528 tained from cultured citrus cells. Thirdly, we examined extracellular proteins from the conditioned media of the cell-lines by immunoblot analysis using antisera specific for acidic endochitinase and for (1-3)-fl-glucanase. Our findings suggest a positive correlation between the extru- sion of chitinase and glucanase from the cultured cells into their respective media and the response of those cells to the P. tracheiphila toxin. Materials and methods Origin and maintenance of nucellar calli Embryogenic calli of nucellar srcin were derived from orange, Citrus sinensis (L.) Osb. ('Tarocco'), and from lemon, Citrus limon (L.) Burro. f. cv 'Femminello Continella' ('Femminello'), as described in Vardi et al. (1982). The nucellar calli were subcul- tured every 4 weeks on Murashige and Tucker (1969) medium containing 4% sucrose, solidified with 1% Difco agar (MT). The in-vitro selection of the 'Femminello-S' line has been de- scribed elsewhere (Gentile et al. 1992 b). Dual culture Dual culture experiments were carried out according to Storti et al. (1988) with some modifications. Two equal pieces of calli were placed oppositely and close to the periphery of a 5-cm Petri dish containing solidified MT medium. After 10, 20 and 30 days of culture in a growth chamber (27 ~ 1 ~ with a 16 h dim-light photoperiod) a small disk of P. tracheiphila mycelium [isolate "80(+)"; Pennisi et al. 1988] was placed in the center of each plate between the two callus pieces. Ten days later the fungal growth was monitored. Five plates were employed for each time and cell line replication. Conditioned media Suspension cultures were obtained by subculturing 200 mg of callus in 100 ml flasks containing 20 ml of liquid MT medium. The flasks were then horizontally shaken at 100 rpm. Thirty days from the beginning of culture, the liquid from individual flasks was centrifuged (1000 g, 5 min), the supernatants were collected and mixed with equal volumes of MT containing 1.8 % agar. The mixed media from each of the flasks were then plated: 5 ml per 5-cm Petri dish. A small disk ofP. tracheiphila myceli- um was placed at the center of each dish. Five Petri dishes were employed for each cell-line. The growth of fungal colonies (cm 2) was evaluated after 10 days of incubation. Extraction of proteins, polyacrylamide-gel electrophoresis and immunoblotting Conditioned media were separated from suspension cultures as described by Gavish et al. (1992). Immunoblots were prepared essentially as described by Gavish et al. (1991) and modified by the "Enhanced chemiluminescence detection system" (Amer- sham) and autoradiographed. Assay for chitinase and glucanase Chitinase activity was measured by using the radiometric assay described by Boller et al. (1983). The activity of (1-3)-•-glu- canase was measured according to Abeles et al. (1970). Assay of antifungal activity Microscope glass-slides were coated with a thin layer of carrot agar medium. A small disk (2 mm) of fungal mycelium was placed on the center of each slide. The slides were then placed above wet filter paper in 9-cm Petri dishes and the dishes were sealed with 'Parafilm'. The dishes were incubated (27~ 1 ~ 16 h dim light) until the fungal colony reached a diameter of about 2 cm. Antifungal activity was tested by applying the protein-extract solution from the cell-suspension culture at the edge of the growing fungal colony and the hyphae were observed microscopically. Results Dual-culture and conditioned-medium experiments Three callus lines were selected for dual-culture and con- ditioned-media experiments. Two of them were derived from citrus cultivars having known responses to the real secco disease; the tolerant cultivar 'Tarocco' orange and the sensitive 'Femminello' lemon. The third line was 'Femminello-S' that resulted from in-vitro selection of 'Femminello' nucellar calli resistant to the partially puri- fied toxin (Gentile et al. 1992 b). P. tracheiphila did not grow on unconditioned MT medium, while growth was normal in the presence of calli on MT medium or on carrot medium (Fig. 1 and Table 1). During dual-culture experiments we noted that fungal growth was stimulated by the presence of all citrus calli (Fig. 1 B). However, fungal growth was inhibited by 'Tarocco' and 'Femminello-S' when inoculating plugs were transferred after at least 20 days of callus growth (Fig. 1 C). If calli-containing plates were inoculated earli- er, no inhibition was obtained and near-centrifugal growth of fungal hyphae was observed (Fig. 1 B). These results suggest that extracellular metabolites may be involved in the inhibition of fungal growth. We therefore examined the conditioned medium of each cell line. Cells were grown for 30 days as suspension cultures and the respective conditioned media were then used for fungal culture as described in Materials and methods. After 10 days of inoculation the fungal colonies reached sizes of 7.0, 4.8 and 5.2 cmz when plated in conditioned medium prepared from 'Femminello', 'Femminello-S' and 'Tarocco' respectively (Table 1). Statistical analysis showed that the fungal colony sizes on 'Femminello'- conditioned medium was significantly larger then on 'Tarocco'- and 'Femminello-S'-conditioned media. In comparison, during 10 days incubation on uncondi- tioned MT medium there was no fungal growth (0.9 cm 2) while the size of fungal colonies on carrot medium was 9.8 cm 2. Analysis of extracellular protein After 20 days of growth, proteins were detected in the conditioned medium of all cultures. The 'Femminello' cell culture contained 15 pg/ml of protein while the medi- um of 'Femminello-S' or the medium of 'Tarocco' con- tained 47 btg/ml of protein. Total soluble extracellular  529 Fig. 1A-C. Fungal growth in citrus calli/fungal mycelium combination. The dishes were photographed 10 days after fungus inoculation. A Growth on defined media. Left plate shows normal average size of fungal colony on control carrot medium and right plate shows near absence of fungal growth on unconditioned MT medium. B Fungat growth on MT medium by 10-day-old citrus calli. The calli were cultured for 10 days prior to fungal inoculation and co-cultured with fungus for an additional 10 days. Left plate 'Femminello', middle plate 'Femminello-S' right plate 'Tarocco'. C Fungal growth on MT medium in the presence of 30-day-old citrus calli. The calli were cultured for 30 days prior to fungal inoculation and co-cultured with fungus for an additional 10 days. Left plate 'Femminello', middle plate 'Femminello-S', right plate ~Tarocco' proteins were analyzed by SDS-PAGE. A polypeptide of 30 kDa size was common to ~ and 'Fem- minello-S'; however in 'Femminello-S' two additional polypeptides with apparent molecular masses of 25 kDa and 37 kDa were evident. The respective profile from the ~Tarocco' cell culture was basically similar to that of Temminello-S' but the former also contained a number of bands with apparent masses of 45-50 kDa (Fig. 2). Fractionated polypeptides (Fig. 2 A) were transferred to nitrocellulose sheets and reacted with antiserum specific for the acidic endochitinase of tobacco (PR-3; Fig. 2 B). A 25 kDa polypeptide was detected in TMV- treated tobacco plants which is the expected size of the acidic endochitinase (lane 1, Fig. 2). Two immunoreac- tive polypeptides of apparent molecular masses of 24 and 37 kDa were detected in the extracellular proteins of non- selected lemon ('Femminello') cells (lane 2, Fig. 2B). In the selected lemon 'Femminello-S' cell line, the 37 kDa polypeptide was considerably enhanced (lane 3, Fig. 2 B) and two additional highly-immunoreactive polypeptides  530 Fig. 2A, B. SDS-PAGE fractionation of extracellular protein. A Coomassie-stained fractionated protein extracts from total tobacco leaves and from extracellular medium of citrus calli. Lanes 1, tobacco; 2, 'Femminello'; 3, 'Femminello-S' and 4, 'Tarocco'. B Immunoblot of fractionated proteins shown in A developed with chitinase antibodies 3000- 2000 1000 0 9 'Femminello' [] 'Femminello-S' [] 'Tarocco' A 6( 2O B Fig. 3 A, B. Endochitinase and glucanase activity in the medium of cultured citrus cells. Aliquots of extracellular medium from 20-day-old ceils were analyzed for A, endochitinase and B, (1-3)- fl-glucanase as described in Materials and methods Table 1. Growth of P. tracheiphila on defined and conditioned media. Values indicated by the same letters are not statistically different by Duncan's test at P= 0.05 Source of medium Growth Statistical (cm 2) grouping 'Femminello' ~ 7.0 c 'Femminello-S' a 4.8 b 'Tarocco' a 5.2 b MT medium 0.9 a Carrot agar medium 9.8 d a Filtered conditioned medium was prepared from 30-day-old citrus cell cultures grown in MT medium; the filtered medium was solidified with agar prior to fungal inoculation of 20 and 24 kDa were observed. In the 'Tarocco' culture medium four polypeptides reacted with the antiserum (lane 4, Fig. 2 B); two of them had apparent molecular masses similar to those of the selected lemon (24 and 37 kDa) and two had intermediate apparent masses of about 28 and 31 kDa (Fig. 2). Fig. 4A, B. Light micrographs of hyphae 5 min after applica- tion of 101~1 of approximately 30 gg of proteins extracted from the conditioned medium of 'Femminello-S' (A) and 'Femminel- lo' (B). Bar is equal to 10 microns Chitinase and 1-3)-~-glucanase activity Conditioned media from citrus cells cultured for 20 days was analyzed by a radiochemical assay for endochitinase. A ten-fold increase of specific chitinase activity in the culture medium from 'Femminello-S' as compared to 'Femminello' was detected. The value obtained from the culture medium of 'Tarocco' cells was half of that ob- tained from 'Femminello-S' (Fig. 3A). The activity of (1-3)-fl-glucanase was evaluated by using laminarin as a substrate. The amount of reducing groups released from the laminarin substrate indicated a ten-fold increase of glucanase activity in the medium of 'Femminello-S' cells as compared to 'Femminello'. The glucanase activity in the medium of 'Tarocco' orange cultures was similar to that of 'Femminello-S' (Fig. 3 B). Lysis of the hyphal tips Microscope slides coated with a thin layer of carrot agar and inoculated with P. tracheiphila (as described in Ma-  531 terials and methods) were used in the hyphal tip lysis tests. Small drops of protein extract (containing approx- imately 30 gg/ml protein) derived from the conditioned media of each of the three cell lines were applied at the edge of the growing myeelium. The extracellular proteins from 'Femminello-S' (Fig. 4) and 'Tarocco' (data not shown) caused abrupt swelling of the fungal tips. Some of the fungal tips started to burst within a few minutes after the application of the protein extract. Neither swelling nor bursting of hyphal tips were recorded after the application of the protein extract from 'Femminello' cultures. Discussion Nucellar-derived citrus cell lines have been shown to ac- cumulate stage-specific extracellular proteins. One group of extracellular proteins was found to play an important role in somatic embryogenesis (Gavish et al. 1991). Addi- tional enzymatic activities in the conditioned medium include peroxidases and polypeptides that cross-react to specific classes of tobacco pathogenesis-related proteins (Garish et al. 1991). Their constitutive expression has been detected in a few additional cell culture systems (Esaka et al. 1990). In contrast, in many reported cases the secretion of PR proteins in cell culture was condition- ally induced by pathogens, elicitors, hormones, or stress, as reported in tobacco (Antoniw et al. 1981; Ohashi and Matsuoka 1987), parsley (Kombrink and Hahlbrock 1986), and rice (Nishizawa and Hibi 1991). The exact function of these extracellular polypeptides is unknown though in one case it was shown to be involved in in-vitro carrot embryogenesis (De Jong et al. 1992). Here we compare two isogenic cell lines from one cultivar and another cell line from a related cultivar that differ in their ability to grow in the presence of a fungal toxin. We showed that both the selected line, 'Femminel- lo-S', and the line derived from a cultivar with intrinsic fungal tolerance, 'Tarocco' orange, repressed the growth of P. tracheiphila. In each case repression was correlated with an immunochemically-detected increase in the PR protein, chitinase, We also show a concomitant increase in the enzymatic activity of chitinase and of (l-3)-fl-glu- canase in the culture media of the tolerant cell lines. Furthermore, this increased activity was correlated with a direct repression of fungal growth as illustrated by lysis of the hyphal tips. Thus the observed repression of gross fungal growth is consistent with the increase in extracel- lular accumulation of cell wall hydrolytic enzymes. The results obtained are consistent with the observation that P. tracheiphila contains chitin as one of its major cell wall components (Bartnicki-Gracia 1968). Total soluble ex- tracellular protein from two additional callus lines, the sensitive sour orange and tolerant 'Murcott' tangor, was analyzed by SDS-PAGE and by immunobloting with acidic endo-chitinase of tobacco (data not shown). Inter- estingly, we found that the profile from the sensitive sour orange was similar to the profile of 'Femminelle' while the profile from the tolerant 'Murcott' tangor was similar to that of 'Tarocco' orange. The function of PR proteins in plants is obscure. They were srcinally detected in pathogenesis responses in a wide range of plants; however, recent evidence for their stage-specific accumulation may indicate an alter- native role for these genes in plant development (Eyal and Fluhr 1991). In only one case has the specific en- hancement of chitinase activity been shown to promote plant-resistance to a fungal disease (Broglie et al. 1991); therefore, it remains to be seen whether plants regenerat- ed from the resistant cell lines will show resistance at the whole plant level and whether this resistance will be cor- related with elevated levels of chitinase. Evidence shows that the partially-purified toxin produced from the cul- ture filtrate of P. tracheiphila is involved in its virulence (Nachmias et al. 1977; Pennisi et al. 1988; Sesto et al. 1990). It was also shown that the response of calli and calli-derived protoplasts (Gentile et al. 1992 a), as well as leaf protoplasts, to the P. tracheiphila toxin (Sesto et al. 1990) is in full agreement with what is known about the response of citrus cultivars to the mal secco disease. Re- cently, we reported that protoplasts isolated from leaves of regenerated 'Femminello-S' plants displayed a wide range of segregation with a noticeable proportion of tol- erant plants (Gentile et al. 1992b). However, it still re- mains enigmatic why selection for tolerance to a toxin, whose mode of action is presently unknown, would result in cell cultures that over-secrete hydrolases. Acknowledgements. This research was supported by the Assesso- rato Agricoltura e Foreste della Regione Siciliana and by an endowment Fund for Basic Research in Life Sciences (Charles H. Revson Foundation) administered by the Israeli Academy of Sciences and Humanities. R. Fluhr is a recipient of the Jack and Florence Goodman Career Development Chair. References Abeles FB, Bosshart RP, Forrence LE, Habig WH (1970) Prep- aration and purification of glucanase and chitinase from bean leaves. Plant Physiol 47:129-134 Antoniw JF, Kueh JSH, Walkey DGA, White RF (1981) The presence ofpathogenesis - related proteins in callus of Xanthi - nc tobacco. Phytopathology 101:179-184 Bartnicki-Gracia S (1968) Cell wall chemistry, morphogenesis and taxonomy of fungi. Annu Rev Microbiol 22: 87-107 Boller T, Gehri A, Mauch F, Vogeli U (1983) Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. Planta 157:22-31 Broglie K, Chet I, Hollidat M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R (1991) Transgenic plants with en- hanced resistance to the fungal pathogen Rhizoctonia solani. Science 254:1194-1197 Daub ME (1986) Tissue culture and the selection of resistance to pathogens. Annu Rev Phytopathol 24:159-186
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