Variation in Capsidiol Sensitivity between Phytophthora infestans and Phytophthora capsici Is Consistent with Their Host Range

Plants protect themselves against a variety of invading pathogenic organisms via sophisticated defence mechanisms. These responses include deployment of specialized antimicrobial compounds, such as phytoalexins, that rapidly accumulate at pathogen infection sites. However, the extent to which these compounds contribute to species-level resistance and their spectrum of action remain poorly understood. Capsidiol, a defense related phytoalexin, is produced by several solanaceous plants including pepper and tobacco during microbial attack. Interestingly, capsidiol differentially affects growth and germination of the oomycete pathogens Phytophthora infestans and Phytophthora capsici, although the underlying molecular mechanisms remain unknown. In this study we revisited the differential effect of capsidiol on P. infestans and P. capsici, using highly pure capsidiol preparations obtained from yeast engineered to express the capsidiol biosynthetic pathway. Taking advantage of transgenic Phytophthora strains expressing fluorescent markers, we developed a fluorescence-based method to determine the differential effect of capsidiol on Phytophtora growth. Using these assays, we confirm major differences in capsidiol sensitivity between P. infestans and P. capsici and demonstrate that capsidiol alters the growth behaviour of both Phytophthora species. Finally, we report intraspecific variation within P. infestans isolates towards capsidiol tolerance pointing to an arms race between the plant and the pathogens in deployment of defence related phytoalexins.     

Interaction of a hydroxyproline-rich glycoprotein from tobacco callus with potential pathogens

A hydroxyproline-rich glycoprotein was isolated from tobacco (Nicotiana tabacum L.) callus tissue cultures by an acidic-ethanol extraction procedure and purified to about 95% homogeneity by ion exchange chromatography on carboxymethyl cellulose. This glycoprotein agglutinated cells of an avirulent strain (B-1) of the bacterial pathogen Pseudomonas solanacearum but not its parental, virulent isolate (K-60). Bacterial lipopolysaccharide (from K-60 strain) inhibited this agglutination. The tobacco glycoprotein also agglutinated zoospores of both compatible and incompatible races of Phytophthora parasitica var. nicotianae. Although 34 potential haptens were tested, no low-molecular-weight carbohydrate that inhibited bacterial or fungal agglutination was found. The agglutination activity of the tobacco glycoprotein was sensitive to pronase and sodium periodate. The apparent molecular weight of the glycoprotein was 120,000. The protein moiety was basic (12% lysine and 5% histidine) and contained 38% hydroxyproline. The carbohydrate moiety comprised 26% (by weight) of the glycoprotein, and contained 87% arabinose, 8% galactose, and 5% glucose. The glycoprotein labeled with fluorescein isothiocyanate bound significantly better to the avirulent isolate (B-1) of P. solanacearum than to the virulent strain (K-60). Binding to the avirulent cells was inhibited by incubation in a higher ionic strength medium (e.g. 0.2 m NaCl). The labeled glycoprotein also bound to cystospores and mycelia of both races of P. parasitica var. nicotianae. This fungal-glycoprotein interaction was inhibited by the lipopolysaccharide from strain K-60 and by higher ionic strength conditions.     

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

Migration of the Fungal Protein Cryptogein within Tobacco Plants

Cryptogein (CRY), a protein secreted by Phytophthora cryptogea, causes necrosis on tobacco (Nicotiana tabacum) plants at the site of application (the stem or the roots) and also on distant leaves. Autoradiography of plantlets after root absorption of radioiodinated CRY demonstrated a rapid migration of the label to the leaf lamina via the veins. Using an anti-CRY antiserum, a CRY-related antigen was detected in the stem and leaves of CRY-treated plants at a distance from the site of application. This antigen had the same molecular weight as CRY and was detected in the leaves as early as 1 hour after stem treatment, i.e. long before necrosis was detectable. The antigen was also detected in plants inoculated with P. cryptogea. The distant location of the necrosis induced by the fungus or by CRY can be ascribed to the migration of this protein, which is toxic to tobacco cells. It is proposed that CRY, which also elicits defense reactions in tobacco, might contribute to the hypersensitive response of tobacco to P. cryptogea.     

Biosynthesis and Secretion of Cryptogein, a Protein Elicitor Secreted by Phytophthora cryptogea

The phytopathogenic fungi Phytophthora subspecies elicit hypersensitive-like necroses on their nonhost tobacco (Nicotiana tabacum), with the exception of the tobacco pathogen Phytophthora nicotianæ. In culture, these fungi—except P. nicotianæ—secrete proteins, called elicitins, that cause these remote leaf necroses and are responsible for the incompatible reaction. These proteins protect tobacco against invasion by the agent of the tobacco black shank, P. nicotianæ, which is unable to produce such an elicitor. Cryptogein, secreted by Phytophthora cryptogea, has been purified, sequenced, and characterized as an elicitin, a novel family of 10 kilodalton holoproteins. In the present paper, we examined the secretion and biosynthesis of this protein elicitor from P. cryptogea culture. Results showed that the secretion of cryptogein began later than its synthesis and stopped earlier, simultaneously with mycelium growth, when the nitrogen source in the culture medium was nearly exhausted. Electrophoretic patterns of total protein from mycelium extracts and N-terminal sequence analysis showed that cryptogein accumulated in the mycelium in its mature form. The comparison of the immunoselected in vitro translation products with ³⁵S in vivo-labeled cryptogein showed that cryptogein was synthesized as a preprotein with a signal peptide removed cotranslationally before the secretion into the culture medium. Immunoselected in vitro-synthesized products were subjected to radiosequencing to clearly determine the N-terminal position and the size (20 amino acids) of the signal peptide. Cryptogein did not undergo any other posttranslational modification.     

Elicitation of capsidiol accumulation in suspended callus cultures of capsicum annuum

Capsidiol was elicited in suspended callus cultures of Capsicum annuum in response to commercial cellulase (ex Trichoderma viride), or pectinase (ex Aspergiltus niger), or a sterile extract from Gliocladium deliquescens. Amounts of capsidiol up to 2.9 mg per 100 ml of culture were accumulated in response to the G. deliquescens extract. Capsidiol was the preponderant phytoalexin produced in the cultures: minor congeners were present at levels below 0.1% of the amounts of capsidiol.     

Defence response of pepper (Capsicum annuum) suspension cells to Phytophthora capsici

Cell suspension cultures of three cultivars of Capsicum annuum L., with different degrees of sensibility to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate. They showed conductivity changes, browning, production of the phytoalexin capsidiol and synthesis or accumulation of pathogenesis-related (PR) proteins with glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) activities. The cultivation medium was optimised for growth of both the plant and the fungus in order to avoid any stress during their combination. The resistant cv. Smith-5, showed a more rapid and intense response to the elicitor preparations than the sensitive cvs Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate, when differences became clearly visible after only 6 h incubation. The greatest rate of capsidiol accumulation occurred after 18 h in the mycelium-elicited cells and after 12 h in those elicited with the filtrate. These times are the optimal for capsidiol accumulation, and the phytoalexin is produced much more rapidly than it can be excreted into the extracellular medium. The inhibition threshold of fungal growth (300 µg capsidiol [g dry weight]^?1) was reached only in the resistant cultivar. The induction of an intracellular glucanase (pI 8.9 and R_f 0.18) and an extracellular chitinase (pI 5.4 and R_f 0.70) only in the resistant cultivar 24 h after elicitation suggests that these enzymes are involved in the resistance to Phytophthora capsici, while other hydrolases common to all three cultivars form part of a more general defence. The results indicate that elicitation of pepper cell suspension cultures by signal molecules from P. capsici exhibits properties of a multicomponent dynamic system in which different protective mechanisms play complementary roles in the overall expression of the defence reaction. We confirm that the differential responses of resistant and susceptible pepper cultivars to P. capsici previously seen in plant stem sections are retained in suspension culture.     

The effect of size and acetylation degree of chitosan derivatives on tobacco plant protection against Phytophthora parasitica nicotianae

Enzymatic degradation of chitosan polymer with Pectinex Ultra SPL was used to obtain derivatives with biological potential as protective agents against Phytophthora parasitica nicotianae (Ppn) in tobacco plants. The 24 h hydrolysate showed the highest Ppn antipathogenic activity and the chitosan native polymer the lowest. The in vitro growth inhibition of several Phytophthora parasitica strains by two chitosans of different DA was compared. While less acetylated chitosan (DA 1%) fully inhibited three P. parasitica strains at the doses 500 and 1000 mg/l the second polymer (DA 36.5%) never completely inhibited such strains. When comparing two polymers of similar molecular weight and different DA, again the highest antipathogenic activity was for the less acetylated polymer. However, degraded chitosan always showed the highest pathogen growth inhibition. Additionally, a bioassay in tobacco seedlings to test plant protection against Ppn by foliar application demonstrated that partially acetylated chitosan and its hydrolysate induced systemic resistance and higher levels of glucanase activity than less acetylated chitosan. Similarly, when treatments were applied as seeds coating before planting, about 46% of plant protection was obtained using chitosan hydrolysate. It was concluded that, while less acetylated and degraded chitosan are better for direct inhibition of pathogen growth, partially acetylated and degraded chitosan are suitable to protect tobacco against P. parasitica by systemic induction of plant resistance.     

Incorporation of mevalonic Acid into ribosylzeatin in tobacco callus ribonucleic Acid preparations

The incorporation of ¹⁴C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis-ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of ¹⁴C-2-mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the ¹⁴C-2-mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue.     

Debneyol,a fungicidal sesquiterpene from tnv infected Nicotiana debneyi

An antifungal sesquiterpene diol, debneyol, has been isolated from tobacco necrosis virus-inoculated leaves of Nicotiana debneyi and a structure is proposed from chemical, spectroscopic and biogenetic evidence. In contrast to the related phytoalexin capsidiol which is fungistatic, debneyol appears to exhibit genuine fungicidal activity.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

Endogenous indole-3-acetic Acid in the stem of tobacco in relation to flower neoformation as measured by mass spectroscopic assay

The contents of free indole-3-acetic acid (IAA) and alkali-labile, conjugated IAA were measured in relation to a `floral gradient' present in epidermis and subepidermis tissues of flowering plants of Nicotiana tabacum by capillary gas-chromatographic spectrometric analysis by selected ion monitoring (GC-SIM-MS) using 2,4,5,6,7-penta deutero IA (²H₅-IAA) as an internal standard. In floral axes, floral branches and stems with floral branches, free IAA levels (dry weight) were 387, 253, and 417 nanograms, and bound IAA levels were 99, 1089, and 268 nanograms. In vegetative tissue of the first plus second internodes (measured from top), and of the 11th to 13th internodes, free IAA levels were 826 and 500 nanograms, and bound IAA levels were 1421 and 286 nanograms, respectively. Since flower-forming ability of excised cells from the epidermis and subepidermis shows a gradient in an in vitro system, but levels of IAA in these tissues do not, there thus appears to be no correlation between flower-forming ability (in vitro) and endogenous IAA levels (at the time of excision) in tobacco stem tissues.     

Constitutive expression of a fusion gene comprising Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) confers enhanced disease and insect resistance in transgenic tobacco

Plant defensins are small, basic cysteine-rich peptides that can inhibit the growth of a broad range of fungi or bacteria at micro-molar concentrations. They have been introduced as transgenes into different species to enhance host resistance to pathogens. In this study, a fusion gene of two defensins, Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) fused by a linker peptide of a polyprotein precursor from Impatiens balsamina was introduced into tobacco (Nicotiana tabacum var. Xanthi) via Agrobacterium-mediated leaf section transformation. Putative transgenic plants were confirmed by PCR analysis and integration of the fusion gene was confirmed by Southern blotting. RT-PCR analysis showed that the fusion gene was expressed in several confirmed transgenic plants. Western blotting analysis of crude protein extracts from leaves of the transgenic plants with anti-Tfgd2 and anti-RsAFP2 antibodies exhibited an 8 and 9 kDa bands corresponding to size of the fusion gene and confirmed the expression of fusion protein. When the leaves of transgenic plants were challenged with Rhizoctonia solani and Phytophthora parasitica var. nicotianae pathogens, they showed enhanced levels of disease resistance along with resistance to the generalist herbivore, Spodoptera litura larvae compared to control. Our results demonstrate that Tfgd2–RsAFP2 fusion protein is effective in protecting the transgenic plants against fungal and insect pathogens.     

Potential Role of Elicitins in the Interaction between Phytophthora Species and Tobacco

The potential role of extracellular elicitor proteins (elicitins) from Phytophthora species as avirulence factors in the interaction between Phytophthora and tobacco was examined. A survey of 85 Phytophthora isolates representing 14 species indicated that production of elicitin is almost ubiquitous except for isolates of Phytophthora parasitica from tobacco. The production of elicitins by isolates of P. parasitica correlated without exception with low or no virulence on tobacco. Genetic analysis was conducted by using a cross between two isolates of P. parasitica, segregating for production of elicitin and virulence on tobacco. Virulence assays of the progeny on tobacco confirmed the correlation between production of elicitin and low virulence.     

Purification and characterization of abundant secreted protein in suspension-cultured pumpkin cells : abundant secreted protein may be a chitinase

The abundant secreted protein with molecular weight of 32,000 was purified from the culture medium of suspension-cultured pumpkin (Cucurbita sp.) cells. Two steps, ammonium sulfate fractionation and Sepharose 6B column chromatography, were sufficient for purification to homogeneity. Antibodies against the pure protein were used to show that a protein of the same size is made by callus cells. There is considerable homology between the amino-terminal amino acid sequence of this secreted protein and chitinase isolated from tobacco (Nicotiana tabacum L.) or bean (Phaseolus vulgaris L.).     

Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein

The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti-RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.

The previously reported dissociation of the binding protein-large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP.

     

Responses of Cultured Tobacco Cells to Cryptogein, a Proteinaceous Elicitor from Phytophthora cryptogea: Possible Plasmalemma Involvement

In culture, the phytopathogenic fungus Phytophthora cryptogea secretes a protein which elicits hypersensitive-like necroses and protects tobacco plants against invasion by the pathogen Phytophthora parasitica var. nicotianae. This protein, named cryptogein, has been purified and its amino acid sequence determined. In this work, we studied the effect of cryptogein on tobacco cell suspension cultures. Cryptogein was lethal at about 0.10 micromolar. When added at sublethal doses, it elicited the production of ethylene and phytoalexins. It also induced a rapid increase in pH and conductivity of the extracellular medium without affecting the integrity of the plasma membrane. Cryptogein reduced the fusicoccin-induced acidification of the extracellular medium. The concentration which inhibited the fusicoccin response by 50% was 0.8 nanomolar, while 1 micromolar erythrosine B, an ATPase inhibitor, was needed to produce the same inhibition. However, cryptogein did not inhibit the activity of a purified plasma membrane ATPase. Results of binding studies with whole cells suggested the presence of elicitor-binding sites with a high affinity for cryptogein. The involvement of the plasma membrane during the initial interaction between elicitor and cells is discussed.