Elicitation of the Hypersensitive Responses in Tabacco by a10.6 kD Proteinaceous Elicitor from Phytophthora palmi

A 10.6 kD heat resistant, proteinacious elicitor was purified from the culture filtrate of Phytophthora palmivora Butler but not from P. melonis Katsura. The 10.6 kD elicitor is a holoprotein devoid of glycoside. It can cause hypersensitive necrosis of the detached tobacco ( Nicotiatna tabacum L. ) leaves 48 h post-inoculation with dosages of above 40 μg. Four cell types were investigated by using Confocal microscopy, and 2' ,7'-dichlorodihydrofluorescein diacetate (DCFDA) as a probe of H2O2 production. It was showed that oxidative burst occurred in cultured suspension cells as well as in mesophyll cells, epidermal cells and guard cells within 10 min upon the elicitor treatment. The hypersensitive cell death appeared 6 h after the treatment when inoculated with fluorescein diacetate (FDA) as indicator of cell viability. These results suggest that H2O2 accumulation was the main cause of the hypersensitive cell death in tobacco induced by the 10.6 kD elicitor. This 10.6 kD elicitor may belong to the family of elicitins.     

Phytophthora palmi分泌的10.6kD蛋白激发烟草的过敏反应

从疫霉菌Ｐｈｙｔｏｐｈｔｈｏｒａ ｐａｌｍｉｖｏｒａ Ｂｕｔｌｅｒ的培养滤液中分离出分子量为１０．６ｋＤ的不含糖基的耐热蛋白．这种１０．６ｋ蛋白能诱导烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）叶片发生过敏性坏死反应。而疫霉菌另一种Ｐ．ｍｅｌｏｎｉｓ Ｋａｔｓｕｒａ的培养滤液中不含这种类似蛋白,不能诱导烟草叶片发生过敏反应。利用共聚焦激光扫描显微镜,以荧光探剂ＦＤＡ（ｆｌｕｏｒｅｓｃｅｉｎ ｄ     

Amino acid sequence of cinnamomin, a new member of the elicitin family, and its comparison to cryptogein and capsicein

The phytopathogenic fungi Phytophthora cinnamomi cause systemic leaf necrosis on its non-host tobacco; in culture, it secretes a protein, called cinnamomin, which elicits leaf necrosis and protects tobacco against the pathogen Phytophthora nicotianoe, in a way similar to cryptogein and different from capsicein, elicitins of known amino acid sequences. The cinnamomin sequence has been determined and compared to other elicitins. The differences in the 3 elicitin sequences were correlated to the biological activities: 2 lysines were ascribed as the key amino acids involved in the differential control of protection with respect to necrosis.     

Relationship between Active Oxygen Species,Lipid Peroxidation,Necrosis, and Phytoalexin Production Induced by Elicitins in Nicotiana

Excised leaves of Nicotiana tabacum var Xanthi and Nicotiana rustica were treated with cryptogein and capsicein, basic and acidic elicitins, respectively. Both compounds induced leaf necrosis, the intensity of which depended on concentration and duration of treatment. N. tabacum var Xanthi was the most sensitive species and cryptogein was the most active elicitin. Lipid peroxidation in elicitin-treated Nicotiana leaves was closely correlated with the appearance of necrosis. Elicitin treatments induced a rapid and transient burst of active oxygen species (AOS) in cell cultures of both Nicotiana species, with the production by Xanthi cells being 6-fold greater than that by N. rustica. Similar maximum AOS production levels were observed with both elicitins, but capsicein required 10-fold higher concentrations than those of cryptogein. Phytoalexin production was lower in response to both elicitins in N. tabacum var Xanthi cells than in N. rustica cells, and capsicein was the most efficient elicitor of this response. In cryptogein-treated cell suspensions, phytoalexin synthesis was unaffected by diphenyleneiodonium, which inhibited AOS generation, nor was it affected by tiron or catalase, which suppressed AOS accumulation in the extracellular medium. These results suggest that AOS production, lipid peroxidation, and necrosis are directly related, whereas phytoalexin production depends on neither the presence nor the intensity of these responses.     

Elicitin genes expressed in vitro by certain tobacco isolates of Phytophthora parasitica are down regulated during compatible interactions

Phytophthora spp. secrete proteins called elicitins in vitro that can specifically induce hypersensitive response and systemic acquired resistance in tobacco. In Phytophthora parasitica, the causal agent of black shank, most isolates virulent on tobacco are unable to produce elicitins in vitro. Recently, however, a few elicitin-producing P. parasitica strains virulent on tobacco have been isolated. We investigated the potential diversity of elicitin genes in P. parasitica isolates belonging to different genotypes and with various virulence levels toward tobacco as well as elicitin expression pattern in vitro and in planta. Although elicitins are encoded by a multigene family, parAl is the main elicitin gene expressed. This gene is highly conserved among isolates, regardless of the elicitin production and virulence levels toward tobacco. Moreover, we show that elicitin-producing P. parasitica isolates virulent on tobacco down regulate parAl expression during compatible interactions, whichever host plant is tested. Conversely, one elicitin-producing P. parasitica isolate that is pathogenic on tomato and avirulent on tobacco still expresses parAl in the compatible interaction. Therefore, some P. parasitica isolates may evade tobacco recognition by down regulating parA1 in planta. The in planta down regulation of parA1 may constitute a suitable mechanism for P. parasitica to infect tobacco without deleterious consequences for the pathogen.     

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca²⁺ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca²⁺ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca²⁺ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.     

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.     

Elicitin 172 from an isolate of Phytophthora nicotianae pathogenic to tomato

Elicitin 172, an acid protein with elicitor activity, has been isolated in true form from culture filtrates of Phytophthora nicotianae, the causal agent of crown and root rot of tomato (Lycopersicon esculentum). The M(r) (10,349 +/- 1) of the purified protein, determined by ES-MS, is identical to that calculated for parasiticein using the mean isotopic composition and assuming the occurrence of three disulfide bridges. The primary structure of elicitin 172, determined using also MALDI-MS experiments, shows complete identity with parasiticein, with elicitin 310 and a cloned elicitin gene from P. parasitica (= P. nicotianae), confirming conservation of the elicitin sequence within a single species. The protein induces necrosis (hypersensitive reaction) on tobacco, but no symptoms on tomato, when applied on the leaves. Tomato pretreated with elicitin 172 was affected by P. nicotianae, as well as by the phytotoxic aggregates, naturally occurring with the elicitin in the non permeated dialysis fraction of culture filtrates. Finally, the elicitin induce protection of capsicum (Capsicum annuum) and vegetable marrow (Cucurbita pepo) from P. capsici.     

Structures of elicitin isoforms secreted by Phytophthora drechsleri.

Most of the phytopathogenic fungi Phytophthora secrete holoproteins (elicitins) responsible for the incompatible reaction and systemic leaf necroses on tobacco. We found that Phytophthora drechsleri produces several elicitin isoforms of various toxicity on tobacco. The CD spectra showed that their secondary structure was largely conserved, exhibiting ca 50% alpha-helix and little or no beta-structure. These 98 residue proteins were sequenced and compared with other known elicitins. Only one point mutation correlated with the differences in necrotic activities. This residue could be either an active or a regulatory site, involved in the interaction with a receptor responsible for necrosis induction.     

Agroinfection-based high-throughput screening reveals specific recognition of INF elicitins in Solanum

We adapted and optimized the use of the Agrobacterium tumefaciens binary PVX expression system (PVX agroinfection) to screen Solanum plants for response to pathogen elicitors and applied the assay to identify a total of 11 clones of Solanum huancabambense and Solanum microdontum , out of 31 species tested, that respond to the elicitins INF1, INF2A and INF2B of Phytophthora infestans . Prior to this study, response to INF elicitins was only known in Nicotiana spp. within the Solanaceae. The identified S. huancabambense and S. microdontum clones also exhibited hypersensitivity-like cell death following infiltration with purified recombinant INF1, INF2A and INF2B, thereby validating the screening protocol. Comparison of INF elicitin activity revealed that Nicotiana plants responded to significantly lower concentrations than Solanum , suggesting variable levels of sensitivity to INF elicitins. We exploited natural variation in response to INF elicitins in the identified Solanum accessions to evaluate the relationship between INF recognition and late blight resistance. Interestingly, several INF-responsive Solanum plants were susceptible to P. infestans . Also, an S. microdontum  × Solanum tuberosum (potato) population that segregates for INF response was generated but failed to identify a measurable contribution of INF response to resistance. These results suggest that in Solanum , INF elicitins are recognized as general elicitors and do not have a measurable contribution to disease resistance.     

巴西橡胶树43 kD橡胶粒子膜蛋白基因的cDNA克隆及表达

对43 kD的橡胶粒子膜蛋白进行了分离纯化和其N端氨基酸序列分析,根据N端氨基酸序列,设计一简并引物,通过3'RACE(Rapid Amplification ofcDNA Ends)的方法,获得了43 kD的橡胶粒子膜蛋白的cDNA.该cDNA含有1 385个核苷酸,含有完整的阅读框架,编码381个氨基酸.在终止密码子下游,包含有一个239bp的3'非编码区.该cDNA由5个首尾相连的重复单元组成,每个单元编码76个氨基酸组成的泛素(ubiquitin)单体.编码43 kD橡胶粒子蛋白的基因具有多个拷贝,在胶乳、叶片和树皮都表达.     

Molecular cloning and biological activity of alpha-, beta-, and gamma-megaspermin,three elicitins secreted by Phytophthora megasperma H20

We report on the molecular cloning of the Phytophthora megasperma H20 (PmH20) glycoprotein shown previously as an inducer of the hypersensitive response, of localized acquired resistance and of systemic acquired resistance in tobacco (Nicotiana tabacum), and of the PmH20 alpha- and beta-megaspermin, two elicitins of class I-A and I-B, respectively. The structure of the glycoprotein shows a signal peptide of 20 amino acids followed by the typical elicitin 98-amino acid-long domain and a 77-amino acid-long C-terminal domain carrying an O-glycosylated moiety. The molecular mass deduced from the translated cDNA sequence is 14,920 and 18,676 D as determined by mass spectrometry. This structure together with multiple sequence alignments and phylogenetic analyses indicate that the glycoprotein belongs to class III elicitins. It is the first class III elicitin protein characterized, which we named gamma-megaspermin. We compared the biological activity of the three PmH20 elicitins when applied to tobacco cv Samsun NN plants. Although alpha- and gamma-megaspermin were similarly active, beta-megaspermin was the most active in inducing the hypersensitive response and localized acquired resistance, which was assessed by measuring the levels of acidic and basic pathogenesis-related proteins and of the antioxidant phytoalexin scopoletin. The three elicitins induced similar levels of systemic acquired resistance measured as the expression of acidic PR proteins and is increased resistance to challenge tobacco mosaic virus infection.     

Physiological changes of Fagus sylvatica seedlings infected with Phytophthora citricola and the contribution of its elicitin "citricolin" to pathogenesis

Beech seedlings were infected with the root rot pathogen Phytophthora citricola to study its impact on leaf physiology and water status. Net photosynthesis rate decreased two days after inoculation in infected seedlings. In contrast, electron quantum yield of photosystem II, leaf water potential, and total water consumption were only slightly impaired until 6 dpi. At the same time, wilt symptoms occurred on leaves. These results indicate the involvement of a mobile signal triggering the early changes in leaf physiology by root infection. As the elicitin gene of P. citricola was induced during root infection, we purified and characterised the elicitin protein and tested its ability to change leaf physiological parameters of beech and tobacco plants. P. citricola produced a single acidic elicitin (citricolin), which caused necrosis and decreased gas exchange of tobacco leaves. Furthermore, it induced an oxidative burst in tobacco cell suspension culture. However, none of these effects were observed in beech.     

Structure-function relationships of α and β elicitins,signal proteins involved in the plant-Phytophthora interaction

Elicitins form a family of 10-kDa holoproteins secreted by various Phytophthora species. The large-scale purification of parasiticein, a novel elicitin secreted by P. parasitica, led to the determination of its sequence. We have compared the necrotic activities and the primary and secondary structures (determined through circular dichroism) of four elicitins. On tobacco plants, they could be classified into two classes: a, comprising capsicein and parasiticein (less necrotic), and , comprising cryptogein and cinnamomin (very toxic with a necrosis threshold of 0.1 g per leaf). The features of elicitin structure which might be involved in the interaction of elicitins with the leaf target cells and that could explain the different necrosis-inducing properties of the two proteins are investigated. About 75% sequence identity was observed between the four elicitins: only two short terminal regions are heterologous, while the central core is mainly conserved. The circular-dichroism spectra showed that the secondary structure of the elicitins was largely conserved. All of them consisted of approx. 50% -helix with little or no -structure. Comparisons of the complete sequences, amino-acid compositions, isoelectric points, hydropathy indices and the secondary-structure predictions correlated with the necrotic classification. Alpha elicitins corresponded to acidic molecules with a valine residue at position 13, while elicitins were basic with a lysine at this position, which appeared to be a putative active site responsible for necrosis induction.Abbreviations CD circular dichroism - RPLC reversed-phase liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The authors are indebted to Dr. A. Van Dorrselaer (Laboratoire de Chimie Organique des Substances Naturelles, Strasbourg, France) for mass-spectrometry measurements. They are grateful to their staff in Versailles, more particularly to Marc Sallantin for electrophoreses, to Françoise Beauvais for biological-activity determinations and to Monique Mansion and Christian Ouali for their skilful technical assistance.     

Initiator-independent and initiator-dependent rubber biosynthesis in Ficus elastica

The rubber-producing tree, Ficus elastica (the Indian rubber tree), requires the same substrates for rubber production as other rubber-producing plants, such as Hevea brasiliensis (the Brazilian or Para rubber tree), the major source of commercial natural rubber in the world, and Parthenium argentatum (guayule), a widely studied alternative for natural rubber production currently under commercial development. Rubber biosynthesis can be studied, in vitro, using purified, enzymatically active rubber particles, an initiator such as FPP, IPP as the source of monomer, and a metal cofactor such as Mg2+. However, unlike H. brasiliensis and P. argentatum, we show that enzymatically active rubber particles purified from F. elastica are able to synthesize rubber, in vitro, in the absence of added initiator. In this paper, we characterize, for the first time, the kinetic differences between initiator-dependent rubber biosynthesis, and initiator-independent rubber biosynthesis, and the effect of cofactor concentration on both of these processes.     

Nondestructive FTIR monitoring of leaf senescence and elicitin-induced changes in plant leaves

The applicability of the FTIR attenuated total reflectance technique for in situ monitoring of plant physiological processes such as leaf senescence and aging has been examined. Difference spectra obtained by subtracting the spectrum of the young plant leaf from that of the older one revealed positive bands at 1650-1500 cm(-1), indicating a higher relative concentration of phenolics in the older leaves of both black cherry and sweet pepper bush leaves. Prolonged physiological stress of tobacco leaves exhibited a progressive time-dependent increase of the absorbance at around 3475 cm(-1), corresponding to hydroxyl functional groups. Absorption changes were also observed between 1650 and 1500 cm(-1), which are likely to correspond to phenolics. The characteristic changes of the FTIR absorbance spectra resulting from physiological and induced aging were detected also as a response to treatment with a recombinant alpha-elicitin, cinnamomin. This allowed the first quantification of the biological activity of a recombinant elicitin using a spectroscopic method. We suggest that FTIR spectroscopy provides important information about physiological events occurring in plant tissue in vivo, and it could be useful for the in situ characterization of the plant responsiveness to fungal toxins such as elicitins.     

Interactions between elicitins and radish Raphanus sativus

Two responses to elicitins are described in cultivars of radish (Raphanus sativus L.). Type I, exhibited by the cultivar Daikon, is characterised by wilting and desiccation within 24 h of elicitin application and was previously reported as the sensitive response (S. Kamoun et al. 1993, Mol Plant-Microbe Interact 6: 15–25). At 1 μg elicitin · g⁻¹ FW radish tissue, symptoms appeared after 8 h, a sensitivity comparable to that shown by tobacco to β elicitins (J.-C. Pernollet et al., 1993, Physiol Mol Plant Pathol 42: 53–67; S. Kamoun et al., 1993, Mol Plant-Microbe Interact 6: 15–25). Elicitin failed to induce these symptoms in the cultivar White Icicle, even at 100 μg · g⁻¹ FW of tissue. However, a different response (Type II) with symptoms resembling senescence appeared in White Icicle after 48 h and were fully developed by 72 h. The Type II response was induced at levels of elicitin above 0.3 μg · g⁻¹ FW. Elicitin-treated Daikon leaves held at 100% relative humidity, rather than ambient (50–60%) did not wilt and by 72 h displayed Type II symptoms. When treated Daikon leaves were removed to ambient humidity at any time during the latent period, they developed Type I symptoms within 2 h. Although Type I symptoms were suppressed in Daikon at high humidity, there was no indication that leaf diffusion resistance or plant water conductance were affected. Protoplasts from the cultivar Daikon responded to elicitin by H⁺ uptake and K⁺ release, with maximal response at 300 pM. The response was eliminated by K252a or staurosporine. Daikon protoplasts also showed transient uptake/secretion of Ca²⁺ on elicitin addition. Protoplasts from White Icicle gave neither of these responses. Both Daikon and White Icicle phenotypes could be transferred to progeny of Daikon-White Icicle crosses and in the F2 generation three phenotypes, including a null, segregated. Only those F2 plants which exhibited the Daikon phenotype produced protoplasts which responded to elicitin. Received: 13 May 1997 / Accepted: 27 August 1997     

Activation of the tobacco SIP kinase by both a cell wall-derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp.

Two purified proteinaceous fungal elicitors, parasiticein (an alpha elicitin) and cryptogein (a beta elicitin), as well as a fungal cell wall-derived carbohydrate elicitor all rapidly activated a 48-kD kinase in tobacco suspension cells. The maximum activation of this kinase paralleled or preceded medium alkalization and activation of the defense gene phenylalanine ammonia-lyase (PAL). In addition, the two elicitins, which also induced hypersensitive cell death, activated a 44- and a 40-kD kinase with delayed kinetics. By contrast, the cell wall-derived elicitor only weakly activated the 44-kD kinase and failed to activate the 40-kD kinase. The size and substrate preference of the 48-kD kinase are reminiscent of the recently purified and cloned salicylic acid-induced protein (SIP) kinase, which is a member of the mitogen-activated protein kinase family. Antibodies raised against a peptide corresponding to the unique N terminus of SIP kinase immunoreacted with the 48-kD kinase activated by all three elicitors from Phytophthora spp. In addition, the cell wall elicitor and the salicylic acid-activated 48-kD kinase copurified through several chromatography steps and comigrated on two-dimensional gels. Based on these results, all three fungal elicitors appear to activate the SIP kinase. In addition, inhibition of SIP kinase activation by kinase inhibitors correlated with the suppression of cell wall elicitor-induced medium alkalization and PAL gene activation, suggesting a regulatory function for the SIP kinase in these defense responses.