Role of Phenylalanine Ammonia Lyase and Polyphenol Oxidase in Host Resistance to Bacterial Wilt of Tomato

Plants respond to bacterial pathogen attack by activating various defence responses, which are associated with the accumulation of several factors like defence-related enzymes and inhibitors which serve to prevent pathogen infection. The present study focused on the role of the defence-related enzymes phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in imparting resistance to tomato against bacterial wilt pathogen Ralstonia solanacearum . The temporal pattern of induction of these enzymes showed maximum activity at 12 h and 15 h for PAL and PPO, respectively, after the pathogen inoculation (hpi) in resistant cultivars. Twenty different tomato cultivars were analyzed for PAL, PPO and total phenol content following pathogen inoculation. The enzyme activities and total phenol content increased significantly (P < 0.05) in resistant cultivars upon pathogen inoculation. The increase in enzyme activities and total phenol content were not significant in susceptible and highly susceptible cultivars. The role of PAL and PPO in imparting resistance to tomato against bacterial wilt disease is discussed.     

Induction of early blight resistance in tomato by <Emphasis Type="Italic">Quercus infectoria</Emphasis> gall extract in association with accumulation of phenolics and defense-related enzymes

Treatment of tomato leaves with aqueous extract (0.5%) of the galls of Quercus infectoria significantly reduced infection from subsequent inoculation with Alternaria solani, the tomato early blight pathogen. When the leaves were challenge-inoculated with A. solani 3 d after application of Q. infectoria gall extract (QIGE), the percent defoliation decreased from 33.6 to 7.3. Two to three day pre-treatment with QIGE reduced the percent defoliation by 77 percent. The biochemical responses of tomato plants to QIGE were also studied. In tomato plants treated with QIGE, phenolic content increased rapidly, reached the maximum at 2 d after treatment. Phenylalanine ammonia-lyase (PAL) activity increased significantly from 1 d after treatment and the maximum enzyme activity was recorded 2 d after treatment at which period a 3-fold increase in PAL activity was observed when compared to the control. Peroxidase (PO) activity was also significantly increased 1 d after treatment and the maximum activity was reached 2 d after treatment. Peroxidase isozyme analysis indicated that PO-1 was increased dramatically in tomato leaves 1 d after treatment and maintained at the same level throughout the experimental period of 6 d. When tomato leaves were treated with QIGE, a two-fold increase in chitinase and β-1,3-glucanase activities was recorded 2 and 3 d respectively, after treatment. The enhanced activities of defense-related enzymes and elevated levels of phenolics in QIGE-treated tomato plants between 1 and 3 d after treatment suggest that these induced biochemical defenses may be involved in the suppression of early blight by QIGE.     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

Tomato resistance to Alternaria stem canker: localization in host genotypes and functional expression compared to non-host resistance

Summary The Alternaria stem canker resistance locus (Asc-locus), involved in resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici and in insensitivity to host-specific toxins (AAL-toxins) produced by the pathogen, was genetically mapped on the tomato genome. Susceptibility and resistance were assayed by testing a segregating F₂ population for sensitivity to AAL-toxins in leaf bioassays. Linkage was observed to phenotypic markers solanifolium and sunny, both on chromosome 3. For the Asc-locus, a distance of 18 centiMorgan to solanifolium was calculated, corresponding to position 93 on chromosome 3. This map position of the resistance locus turned out to be the same in three different resistant tomato accessions, one Dutch and two American, that are at least 40 years apart. AAL-toxin sensitivity in susceptible and resistant tomato genotypes was compared with AAL-toxin sensitivity in a non-host Nicotiana tabacum during different levels of plant cell development. In susceptible and resistant tomato genotypes, inhibitory effects were demonstrated at all levels, except for leaves of resistant genotypes. However, during pollen and root development, inhibitory effects on susceptible genotypes were larger than on resistant genotypes. In the non-host Nicotiana tabacum, hardly any effects of AAL-toxins were demonstrated. Apparently, a cellular target site is present in tomato, but not in Nicotiana tabacum. It was concluded that three levels of AAL-toxin sensitivity exist: (1) a susceptible host sensitivity, (2) a resistant host sensitivity, (3) a non-host sensitivity, and that the resistance mechanism operating in tomato is different from that operating in Nicotiana tabacum.     

Differential Activation of Expression of a Suberization-Associated Anionic Peroxidase Gene in Near-Isogenic Resistant and Susceptible Tomato Lines by Elicitors of Verticillium albo-atratrum

We tested whether the expression of the suberization-associated anionic peroxidase gene is involved in the timely appearance of the vascular suberized coating involved in the resistance of a tomato line to Verticillium albo-atrum. The mRNA for this peroxidase appeared at a higher level one day earlier in wound-healing fruits of the resistant tomato line than in a near-isogenic susceptible line. Cell cultures from the resistant line, when treated with low levels (nanograms per milliliter) of fungal elicitor, generated the peroxidase mRNA and this apparent activation of the peroxidase gene expression could be detected in minutes, whereas the cells from the susceptible line hardly responded.     

Phenylalanine Ammonia Lyase Activity in Pearl Millet Seedlings and its Relation to Downy Mildew Disease Resistance

Phenylalanine ammonia lyase (PAL) activity was studied in differentgenotypes of pearl millet with varying degrees of susceptibilityto downy mildew disease, after inoculating with Pathotype 1of Sclerospora graminicola. In resistant genotypes, the enzymeactivity significantly increased 24 h after fungal inoculationwhile in the susceptible genotypes, the activity decreased.The increase or decrease in enzyme activity was well-correlatedwith the degree of host resistance to the pathogen. A time-courseof change in activity of PAL after inoculation showed a considerabledifference between resistant and susceptible genotypes. Studieson the activity of PAL in different parts of pearl millet seedlingsrevealed that in the resistant genotype, enzyme activity significantlyincreased at 24 h post-inoculation only in the shoot portion,whereas in mesocotyl and root the activity decreased. In susceptibleseedlings, enzyme activity decreased at 24 h post-inoculationin shoot, mesocotyl and root. The activity of PAL was also foundto be pathotype-specific. Histochemical tests for lignin werepositive in infected cells in the resistant genotypes. The roleof PAL in imparting resistance to pearl millet against downymildew disease is discussed. Key words: Sclerospora graminicola, resistance screening, enzyme activity     

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.     

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean

A new disease resistance locus in Arabidopsis, RPS3 , was identified using a previously cloned avirulence gene from a non- Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis , and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB , and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F₂ plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3 . Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1 . In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1 . Thus, RPG1 and RPS3 both confer avrB -specific disease resistance, suggesting that these genes may be homologs.     

Differential expression of tomato proteinase inhibitor I and II genes during bacterial pathogen invasion and wounding

Expression of proteinase inhibitor I and II genes was investigated during infection by Pseudomonas syringae pv. tomato, the causal agent of bacterial speck disease in tomato. Inoculation of leaves with P. s. pv. tomato of two inbred tomato lines that are resistant and susceptible to the pathogen resulted in the accumulation of proteinase inhibitor I and II mRNAs in this organ. Our data showed that in the lines used in this study, proteinase inhibitor II mRNAs accumulated in leaves to higher levels than proteinase inhibitor I mRNA in response to P. s. pv. tomato infection and wounding. Proteinase inhibitor II mRNAs accumulated more rapidly in disease-resistant than in disease-susceptible plants. Proteinase inhibitor I mRNAs were first detected in the disease-susceptible line during infection and wounding. In contrast to wounding, the systemic induction of these genes during pathogen ingression was limited. These data show that the plant proteinase inhibitors constitute one of the components of the plant defense system that are induced in response to bacterial pathogen invasion.     

Suppression of phenylalanine ammonia-lyase activity elicited in date palm by Fusarium oxysporum f. sp. albedinis hyphal wall elicitor

The inoculation of the seedling roots of the resistant (Bousthami Noir) and susceptible (Jihel) date palm (Phoenix dactylifera) cultivars by Fusarium oxysporum f. sp. albedinis induced an increase in phenylalanine ammonia-lyase (PAL) activity. The response of the PAL activity in the resistant cultivar was faster and higher than in the susceptible one. However, the elicitation of the seedlings with the hyphal wall elicitor (HWE) of the pathogen induced identical PAL activity in both cultivars. In the resistant cultivar, the the PAL activity elicited with the HWE was not influenced by the addition of the fungal culture filtrate (FCF) whereas it was suppressed in the susceptible cultivar. This FCF suppressor effect was dose-dependent, not influenced by sodium periodate, whereas it was strongly reduced by the heat (121 °C for 45 min) and pronase E. These results show that differential induction of the defence mechanisms in both cultivars was not related to differences in the induction of the PAL activity, but to the suppression of its elicitation in the susceptible cultivar.     

Chemical characterization of stress-induced vascular coating in tomato

Indirect evidence suggests that vascular coatings formed by plants in response to stress consist of suberin-like substances containing lipid and phenolic compounds. To provide more direct chemical evidence that coatings are suberin, we used a natural pathogen, Verticillium albo-atrum, or a stress-responsive hormone, abscisic acid, to induce coating in two isolines of tomato (Lycopersicon esculentum L. cultivar Craigella) that are resistant or susceptible to the pathogen. Using treated petioles that had been monitored cytologically, chemical depolymerization followed by combined gas-liquid chromatography-mass spectrometry analysis of alkane-α,ω-diol levels confirmed the presence of suberin after induction of coating and showed quantitative differences between the isolines that correlated with cytological measurements of the coating response. Northern analysis of suberization-associated anionic peroxidase mRNA showed corresponding increases, and tissue blot analysis further indicated that induction of the mRNA was localized in the responding vascular bundles, as determined by suberin histochemistry. Taken together, these results provide chemical evidence that the coatings are mainly suberin.     

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M₂ mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.     

黄瓜褐斑病菌毒素对抗、感黄瓜品种的作用

在黄瓜褐斑病菌毒素的作用下,黄瓜品种的根数、根长、芽长受到了抑制,根的电导值及叶片的苯丙氨酸解氨酶(PAL)活性升高。抗病品种的根数、根长、芽长受毒素的影响较感病品种小,即感病品种对毒素敏感,且抗、感品种的电导值差异达显著水平。抗病品种的PAL活性增加幅度较感病品种大。     

籽用美洲南瓜叶片对白粉病菌的抗逆生理响应

以籽用美洲南瓜(Cucurbita pepo L.)白粉病抗病品系F2和感病品系M3为试材,在人工气候箱内接种白粉病生理小种2US孢子悬浮液,考察在接种白粉病菌后南瓜幼苗植株与白粉病菌的互作、叶片活性氧代谢及保护酶活性的变化,探讨南瓜抵御白粉病的生理机制。结果表明：(1)与感病品系M3相比,接种白粉病菌后,抗病品系F2叶片上病原菌发育缓慢,较难侵染叶片。(2)抗病品系F2在感病初期叶片H₂O₂、O₂^-·含量迅速升高后逐渐下降,而感病品系在感病初期H₂O₂、O₂^-·含量上升缓慢,在达最大值后始终保持较高水平,且感病品系叶片MDA含量始终高于抗病品系;组织化学染色分析发现,抗病品系叶片着色比感病品系快,之后着色面积有所减少并趋于较低水平。(3)抗病品系F2和感病品系M3叶片抗氧化酶CAT、SOD、POD活性及PAL、PPO活性在接种白粉病菌后均显著增加,但抗病品系的活性及其增幅均高于感病品系。研究发现,籽用美洲南瓜抗病品系叶片上白粉病菌发育缓慢,较难受到侵染,生成菌丝体后叶片上粉状斑点较小;抗病品系在被白粉病菌侵染初期依靠活性氧的增加抵御病原菌的入侵,随着活性氧含量增加抗病品系通过迅速增加自身抗氧化酶活性来防止氧化胁迫;与感病品系相比,抗病品系在受病原菌侵染后能迅速增加PAL、PPO活性以抵御病原菌侵染。     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum     

Differential induction of phenylalanine ammonia-lyase activity in date palm roots in response to inoculation with Fusarium oxysporum f. sp. albedinis and to elicitation with fungal wall elicitor

The inoculation of the roots of resistant (BSTN) and susceptible (JHL) cultivars of date palm seedlings byFusarium oxysporum f. sp.albedinis (Foa) induces an increase in activity of phenylalanine ammonia-lyase (E.C. 4. 3. 1. 5., PAL). The post-infectional response in the PAL activity in the resistant cultivar roots was faster and higher than that in the susceptible cultivar. However, the elicitation of the seedlings by the hyphal wall preparation (HWP) ofFoa induces an identical PAL response in the resistant and the susceptible cultivars. The elicitor activity of HWP was dose-dependent, the optimal concentration which induces a maximum PAL activity was 10 mg of mycelium per mL. The elicitor present in the HWP was thermostable since its elicitor activity was maintained after heat treatment (121 °C for 45 min). The treatment of the HWP with protease (Pronase E) does not have an effect on the HWP elicitor activity. However, the treatment of the HWP with sodium periodate inhibits its elicitor activity. This data suggests that the HWP elicitor is a carbohydrate compound. In addition, the HWP elicitor is non-specific since it induces identical responses of the PAL activity in two cultivars showing different behaviors to the pathogen. The absence of specificity of HWP elicitors and the differential response of the PAL activity to the infection byFoa and to the elicitation by the HWP are discussed. An explanation of the general interactions between plant and parasite is proposed.