The oxidative processes induced in cell suspensions of Solanum species by culture filtrate of Phytophthora infestans

Solanum genotypes that differ in the level of polygenic resistance to the oomycete plant pathogen Phytophthora infestans were studied for their oxidative response to culture filtrate (CF) of the pathogen. Reactive oxygen species (ROS) production, peroxidase activity and lipid peroxidation have been studied in the CF-treated cell suspensions derived from leaves of the resistant S. nigrum (nonhost) and S. tuberosum cv. Bzura as well as from the susceptible S. tuberosum cv. Tarpan and clone H-8105. In both the resistant and susceptible cells the CF induced similar processes, but these varied with respect to the kinetics and intensity. In all cells probably the membrane-bound NADPH oxidase, was responsible for the ROS production. This process was more intensive and prolonged in the susceptible cells than in the resistant ones. The CF treatment slightly affected peroxidase activity in all cells studied. Lipid peroxidation that occurred as a consequence of the ROS accumulation was pronounced mainly in the susceptible cells. We suggest that lack of stringent control of the oxidative processes and sensitivity to the pathogen toxins may be decisive for limited polygenic resistance in potato.     

马铃薯非共生血红蛋白基因StHb1的克隆及表达

采用RT-PCR技术成功分离了马铃薯StHb1基因序列.经半定量RT-PCR分析表明,StHb1基因的表达在抗性品种(陇薯三号)和感性品种(荷兰十五)块茎中均受致病疫霉的侵染所抑制;StHb1基因在正常生长的马铃薯块茎组织中表达量最高;外源NO和H2O2的作用可明显地抑制StHb1基因的表达,但在抗性品种中该基因受抑制的程度低于感性品种.上述试验结果暗示了StHb1基因与马铃薯对致病疫霉侵染的抗性应答具有一定的相关性.     

Activation of elicitor defensive properties by systemic signal molecules during the interaction between potato and Phytophthora

The elicitor arachidonic acid in combination with jasmonic acid (JA) induced a higher level of defense against the late blight agent in potato (Solanum tuberosum L.) tissues than in combination with salicylic acid (SA). On the contrary, the elicitor chitosan displayed a higher inductive effect in combination with SA as compared with JA. The optimal concentrations of tested compounds were selected for designing the compositions activating wound repair, induction of proteinase inhibitors, and resistance to the biotrophic pathogen Phytophthora infestans (Mont.) de Bary. It was demonstrated that the compositions of elicitor and systemic signal molecules provided a faster spreading of an inducing effect in the potato tissues.     

Race nonspecific resistance for potato late blight

The late blight fungus (Phytophthora infestans) rots susceptible species of potato plants. None of the major varieties of potato (Solanum tuberosum) grown in the USA is resistant to US-8, the most prevalent genotype of the fungus. Now, Junqi Song, James Bradeen and colleagues have cloned the RB gene from the wild diploid potato species, Solanum bulbocastanum, using a map-based approach in combination with long-range PCR. Transgenic plants containing the gene, normally fully susceptible, displayed broad-spectrum late blight resistance.     

The elicitor-induced oxidative processes in leaves of Solanum species with differential polygenic resistance to Phytophthora infestans

Previous studies have shown that suspension-cultured cells of Solanum genotypes with various polygenic resistances to Phytophthora infestans differed in activities of early oxidative processes in response to culture filtrate (CF) from this pathogen. These studies have now been extended by analysing production of reactive oxygen species (ROS), lipid peroxidation and Lipoxygenase (LOX, E.C.1.13.11.12) activity induced by CF in detached leaves of S. tuberosum cv Bzura and clone H-8105, polygenically resistant and susceptible, respectively, as well as S. nigrum, nonhost, completely resistant. The relative increase in the ROS production was higher in the susceptible clone H-8105 than in both resistant genotypes. Lipid peroxidation increased only in the nonhost S. nigrum. An increase in lipid peroxidation in S. nigrum leaves coincided with enhanced LOX activity. In both S. tuberosum genotypes, significant increases in LOX activity were delayed and unaccompanied by changes in the level of lipid peroxidation. LOX activity attained a higher level in both of the resistant genotypes than in the susceptible one. The present results suggest that the involvement of both ROS production and LOX activity in the defense strategy in Solanum species/P. infestans interactions.     

Induction of defence-related enzymes in susceptible variety of banana: role of Fusarium-derived elicitors

Elicitor prepared from the Fusarium oxysporum f. sp cubense (Foc) isolated from infected banana rhizosphere induced the accumulation of resistance-associated enzymes in leaves of susceptible and resistant variety of banana. Roots of Grand Naine (susceptible) and robusta (resistant) variety were inoculated with 1 g/l Foc elicitors. Distinct difference in peroxidase, polyphenol oxidase, β-1,3-glucanase, chitinase and phenolics was observed in control plants of resistant and susceptible varieties. Induced defence-related enzymes in susceptible variety were increased tothe level of untreated resistant variety. This depicted that Fusarium-derived elicitor effectively induced defence in susceptible variety to the apparent level of untreated resistant variety.     

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.     

Tobacco rattle virus 29K movement protein is the elicitor of extreme and hypersensitive-like resistance in two cultivars of Solanum tuberosum

Leaf infection experiments were used to analyze the host responses of Solanum tuberosum cultivars known to be resistant or susceptible to natural, nematode-mediated infection of tubers and necrosis induction ("spraing") by Tobacco rattle virus (TRV) isolate PpK20 (TRV-PpK20). Extreme and hypersensitive-like resistance (ER and HR-like, respectively) as well as spreading veinal necrosis and systemic infection were observed. Agroinfection of leaves with a DsRed-expressing TRV cDNA clone revealed ER to function on the single-cell level, inhibiting virus replication and possessing the potential to initiate a cell death response. HR-like necrosis was characterized by initial virus replication and cell-to-cell movement before the onset of necrosis. Transient agroexpression and Potato virus X (PVX)-mediated expression assays demonstrated that the 29K-PpK20 movement protein (MP) can elicit ER and HR-like cell-death. A TRV isolate, PpO85M, known to overcome the resistance to spraing in plants that are resistant to TRV-PpK20 encoded a variant 29K protein which did not elicit HR in PpK20-HR plants. Our results show that the TRV MP is the elicitor of both ER and HR-like cell-death, that no other TRV-encoded proteins or RNA replication are required for its elicitor activity, and that the host reactions are likely to be controlled by single dominant resistance genes.     

Changes in the antioxidant status in leaves of Solanum species in response to elicitor from Phytophthora infestans

Three Solanum genotypes with various polygenic resistance levels to the oomycete pathogen Phytophthora infestans (Mont.) De Bary were studied for their antioxidant response to the pathogen culture filtrate (CF). Detached plant leaves were treated with CF for 6, 18 and 30 h, and assayed for changes in hydrogen peroxide content, total ascorbate and glutathione pools and redox ratios (reduced form to total pool), as well as for changes in activities of ascorbate peroxidase, glutathione reductase and glutathione-S-transferase. In CF treated leaves of non-host resistant S. nigrum var. gigantea and field resistant S. tuberosum cv Bzura, the H(2)O(2) content did not change in comparison to water treated control leaves, whereas in the susceptible S. tuberosum clone H-8105 it decreased below the control level. In CF treated leaves of all genotypes, the total ascorbate pools were relatively unaltered and their redox ratio changed only transiently. In Bzura leaves the total glutathione content increased earlier than in the two other genotypes. The glutathione redox ratio remained rather stable, except for the susceptible clone H-8105, where it decreased transiently by about 42%. The relative increases in activity of all the studied enzymes were the highest in the susceptible clone H-8105. The results are discussed in the light of oxidative processes occurring in CF treated leaves. We conclude that stringent control of pro- and anti-oxidant reactions bringing the H(2)O(2) and/or cellular redox state to the threshold level is decisive for deployment of an effective defense strategy.     

Modulation of plant resistance to diseases by water-soluble chitosan

Low-molecular-weight water-soluble chitosan with a molecular weight of 5 kDa obtained after enzymatic hydrolysis of native crab chitosan was shown to display an elicitor activity by inducing the local and systemic resistance of Solanumi tuberosum potato and Lycopesicon esculentum tomato to Phytophthora infestans and nematodes, respectively. Chitosan induced the accumulation of phytoalexins in tissues of host plants, decreased the total content and changed the composition of free sterols producing adverse effects on infesters, activated chitinases, beta-glucanases, and lipoxygenases, and stimulated the generation of reactive oxygen species. The activation of protective mechanisms in plant tissues inhibited the growth of taxonomically different pathogens (parasitic fungus Phytophthora infestans and root knot nematode Meloidogyne incognita).     

Induction of a proteinase K inhibitor in a potato cultivar with a high degree of field resistance against Phytophthora infestans

A proteinase K inhibitor (PLPKI) was isolated from a potato cultivar with a high level of field resistance ( Solanum tuberosum L. cv. Pampeana INTA), after 24 h of infection with Phytophthora infestans , when inhibitory activity was markedly increased. Purification was performed by heat treatment, gel filtration chromatography and affinity chromatography. A size of 60 kDa was estimated by SDS-PAGE in partially denaturing conditions and by gel filtration. It is multimeric and the monomer has a molecular mass of 8.5–9.0 kDa. PLPKI is highly active against proteinase K (EC 3.4.21.14) but poorly inhibits two serine proteinases of animal origin, trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). A differential expression (determined by activity and immunoblotting assays) of PLPKI was observed between two potato cultivars with different degrees of field resistance to P. infestans . In the resistant cultivar (cv. Pampeana INTA) PLPKI induction (19-fold with respect to healthy leaves) occurred 24 h after infection and remained over basal levels after 48 h infection. By contrast, in the susceptible cultivar (cv. Bintje), no induction was observed.     

马铃薯种质资源的晚疫病抗性鉴定

采用离体叶片接种法,对43份马铃薯种质资源进行晚疫病抗性鉴定、比较和评价。以接种5 d后的感病品种‘Désirée’和抗病品种‘加湘1号’叶片症状为对照,鉴定出5份表现为高抗的种质资源,其中包含3个Solanum phurejia和2个S. tuberosum ssp. andigena材料。另外,还鉴定出14份中抗材料(No. 7～20)。结果表明,野生种和安第斯山栽培亚种马铃薯资源的抗性材料较为丰富,可作为晚疫病抗病育种的亲本。     

Comparative analysis of Phytophthora infestans induced gene expression in potato cultivars with different levels of resistance

Differential gene expression was analyzed after infection with Phytophthora infestans in six potato cultivars with different levels of resistance to late blight. To verify the infection of the potato leaflets, the amount of phytopathogen mRNA within the plant material was quantified by real-time quantitative PCR. The expression of 182 genes selected from two subtracted cDNA libraries was studied with cDNA array hybridization using RNA from non-infected and infected potato leaflets. Gene up- and down-regulation were clearly detectable in all cultivars 72 h post inoculation. Gene expression patterns in susceptible cultivars differed from those in potato varieties with a higher level of resistance. In general, a stronger gene induction was observed in the susceptible cultivars compared to the moderately to highly resistant potato varieties. Five genes with the highest homology to stress and/or defence-related genes were induced specifically in the susceptible cultivars. Four genes responded to pathogen attack independently of the level of resistance of the cultivar used, and three genes were repressed in infected tissue of most cultivars. Even in the absence of P. infestans infection, six genes showed higher expression levels in the somewhat resistant cultivars Bettina and Matilda. Possible reasons for the different levels of gene expression are discussed.     

Changes in the initial phase of lipid peroxidation induced by elicitor from Phytophthora infestans in Solanum species

The initial phase of the lipid peroxidation process in leaves of Solanum nigrum var. gigantea, Solanum tuberosum cv Bzura and clone H-8105, which represent non-host resistance, field resistance and susceptibility, respectively, against Phytophthora infestans, was investigated. Based on quantitative and qualitative high-performance liquid chromatography (HPLC) analyses of free and esterified fatty acid hydroperoxides (FAHs), we characterized the lipid peroxidation process induced by the pathogen-derived elicitor, culture filtrate (CF), in leaves of the studied genotypes. In all plants, FAHs generated due to 13-lipoxygenase (LOX) action dominated over those from the non-enzymatic pathway. The FAHs derived from 9-LOX activity were found only in CF-treated leaves of the non-host resistant S. nigrum. However, experiments in vitro and in planta with exogenous linoleic acid (LA) as a substrate for LOX revealed high constitutive activity of 9-LOX in all genotypes, which increased in response to CF treatment. The time course changes in polyunsaturated fatty acid (PUFA) pools in the total lipid fractions as well as the degree of their oxidation suggested that CF-induced PUFA peroxidation was enhanced mostly in S. nigrum, less so in Bzura and least in the susceptible clone H-8105. The obtained results are discussed in light of the overall biochemical cell status of plants in the studied interactions.     

Resistenzinduktion gegen systemische Virusinfektionen durch Kulturfiltrate von Stachybotrys chartarum (Ehrenb. ex Link) Hughes

Induced resistance in systemic host-virus combinations by culture filtrates of Stachybotrys chartarum (Ehrenb. ex Link) Hughes Culture filtrates of the fungus Stachybotrys chartarum sprayed on systemic hosts decreased the development of symptoms of different elongated and isometric viruses. The degree of induced resistance depended on the host-virus system. An interval of three or five days between application of the filtrate and virus inoculation was sufficient to induce resistance. High inoculum concentration reduced the efficiency of induced resistance in cucumber against CMV. The content of CMV in inoculated andsystemically infected, induced resistant cucumber leaves was decreased. TMV inoculated leaves of induced resistant tobacco plants contained higher, systemically infected leaves lower virus amounts as comparable untreated control leaves. Reduced virus content and distribution in induced resistant plants obviously resulted from inhibition of multiplication and spread of viruses.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Glycoproteins from Russian wheat aphid infested wheat induce defence responses

Elicitors are molecules which can induce the activation of plant defence responses. Elicitor activity of intercellular wash fluid from Russian wheat aphid, Diuraphis noxia (Mordvilko) infested resistant (cv Tugela DN), and susceptible (cv Tugela), wheat (Triticum aestivum L.), was investigated. Known Russian wheat aphid resistance related responses such as peroxidase and beta-1,3-glucanase activities were used as parameters of elicitor activity. The intercellular wash fluid from infested resistant plants contains high elicitor activity while that from infested susceptible plants contains no or very little elicitor activity. After applying C-18 reverse phase and concanavalin A Sepharose chromatography, elicitor active glycoproteins were isolated from the intercellular wash fluid of Russian wheat aphid infested resistant wheat. The elicitor-active glycoproteins separated into three polypeptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated glycoproteins elicited peroxidase activity to higher levels in resistant than in susceptible cultivars. It was evident that the glycoproteins were probably a general elicitor of plant origin. Information gained from these studies is valuable for the development of plant activators to enhance the defence responses of plants.     

Isolation and Characterization of an Elicitor of Necrosis Isolated from Intercellular Fluids of Compatible Interactions of Cladosporium fulvum (Syn. Fulvia fulva) and Tomato

Intercellular fluids of compatible race-cultivar interactions of Cladosporium fulvum and tomato contain specific elicitors of necrosis. These elicitors which are of fungal origin induce chlorosis and necrosis in resistant but not in susceptible plants. With the tomato cultivar Sonatine (carrying resistance gene Cf9, resistant to the fungal races 0, 4, 5, 2, 2.4, and 2.4.5 but susceptible to race 2.4.5.9) as the test plant for assaying necrosis-inducing activity, we isolated and partially characterized an elicitor of necrosis on this cultivar. The elicitor bound to CM-Sephadex but not to DEAE-Sephadex; it was stable to heat (10 minutes at 100°C), HCl (0.01 normal), NaOH (0.01 normal), and NaIO₄ (0.02 molar), sensitive to pronase and protease (from Bacillus polymyxa) but not to other proteases such as α-chymotrypsin and trypsin. After electrophoresis of partially purified elicitor preparations under low pH conditions, the necrosis-inducing activity was association with a peptide with an apparent molecular weight of 5500. Races 0, 4, 5, 2.4, and 2.4.5 but not race 2.4.5.9 produced this elicitor in high yields. The elicitor is probably a product of avirulence gene A9 which is present in all races except in race 2.4.5.9 and induces necrosis in cultivars carrying resistance gene Cf9.     

Effect of cadmium and Phytophthora infestans on polyamine levels in potato leaves

Changes in putrescine, spermidine and spermine concentrations in potato ( Solanum tuberosum L.) leaves stressed either with cadmium, with the fungal pathogen Phytophthora infestans Mont. (de Bary) or with both simultaneously were investigated. The leaves of two cultivars, Bintje and Bzura, respectively susceptible and resistant to P. infestans were examined. The level of of putrescine did not change under any stress conditions. Cadmium stress caused at least a 2-fold increase in spermidine and spermine concentrations in susceptible leaves. In resistant leaves there was a 4-fold increase in spermidine and a decrease in spermine. The pathogenic stress produced similar changes in polyamine concentrations, i. e. with differences between the cultivars. The double stress induced antagonistic alterations in the concentrations of spermidine and spermine.