An Attempt to Predict Response of Sunflowers to Races of Plasmopara halstedii Using Cultured Sunflower Cells

Cultured sunflower cells reacted alike to inoculation in compatible and incompatible combinations with three races of Plasmopara halstedii, cause of downy mildew. Infection structures formed on the surfaces of cells derived from both resistant and susceptible sunflowers, but no intracellular fungal structures or other signs of penetration were evident in either case. There was no indication of a hypersensitive reaction (rapid death) in cells within 20 h of inoculation in incompatible combinations. The reaction of cells in culture was not like that of intact plants in either compatible or incompatible combinations.     

Seed treatment with salicylic acid invokes defence mechanism of Helianthus annuus against Orobanche cumana

The root holoparasitic angiosperm sunflower broomrape (Orobanche cumana) specifically affects sunflower (Helianthus annuus) growth and causes severe damage all over the world. This investigation was designed to examine the protective effects of salicylic acid (SA) treatment to the seeds of an O. cumana‐susceptible cultivar of sunflower (TK0409). Sunflower seeds were pretreated with different concentrations (0, 0.5 and 1 mM) of SA and inoculated with O. cumana for 4 weeks. O. cumana infection resulted in reduction in plant biomass, endogenous SA level, and the expression of SA‐related genes including pal, chs and NPR1. By contrast, O. cumana infection enhanced the production of reactive oxygen species, activities of antioxidant enzymes as well as contents of phenolics and lignin. Seed treatment with 1 mM SA increased sunflower biomass in terms of plant height, fresh weight and dry weight by 10%, 13% and 26%, respectively, via reducing the number and biomass of established O. cumana. The increase of hydrogen peroxide contents by 14% in the 1 mM SA treated sunflower plants appeared to be because of the inhibition of ascorbate peroxidase and catalase by exogenous SA. The enhanced expression of pathogenesis‐related genes (PR3 and PR12, encoding chitinase and defensin, respectively) after 4 weeks of inoculation indicated that systemic acquired resistance was induced in the SA treated sunflower in which the level of endogenous SA was also elevated in a dose‐dependent manner. The increased expression of a hypersensitive‐responsive (HR) gene hsr indicated that the resistance of sunflowers might be associated with a hypersensitive reaction which was activated by exogenous SA treatment.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Sugar-specific Attachment of Pseudomonas syringae pv. glycinea to Isolated Single Leaf Cells of Resistant Soybean Cultivars

Phase-contrast and scanning electron microscopy showed races of P. synngae pv. glycinea uniformly distributed over and attached to the whole surface of isolated single leaf cells of resistant soybean cultivars, as early as 30 to 180 min after inoculation. On the contrary, attachment in the compatible interaction did not occur within 10—15 h. In a later period, compatibility was characterized by the formation of adherent bacterial clusters. Early attachment of races 1 and 6 to cv. Harosoy and that of race 5 to cv. Flambeau leaf cells, each representing incompatible interaction, could be inhibited by L-rhamnose and D-glucose, respectively. Furthermore, the lack of Mn²⁺ and Fe²⁺ and heat-treatment of plant cells also affected the early attachment in incompatible combinations and resulted in cluster formation, suggesting incompatibility rather than compatibility to be the active phenomenon. Pre-inoculation of cells with an incompatible race induced changes that caused compatible bacteria also to distributively attach to plant cell surface indicating that a transfer of information or surface alterations occur upon attachment in incompatible interaction.     

Changes in the Hill Reaction and the Chlorophyll Fluorescence of Chloroplasts Isolated from Wheat Plants Infected with Stem Rust (Puccinia graminis f. sp. tritici)

Wheat plants with different resistance to stem rust were inoculated with urediospores of Puccinia graminis f. sp. tritici race 32. By means of isolated chloroplasts Hill activity and chlorophyll fluorescence were measured during the first 8–10 days of the infection. Highly resistant cultivars (infection type 0) showed no significant differences in Hill activity whereas resistant (infection type 1) and susceptible plants (infection type 4) exhibited a significant decrease in electron transport. This was associated with an apparent reduction of chlorophyll content. In both the incompatible as well as in the compatible interaction an additional factor had an effect on the inhibition of the Hill activity. Chlorophyll fluorescence was also affected in compatible and in incompatible interactions. However, the two interactions showed different patterns. Whereas the decline in variable fluorescence dominated in resistant varieties, susceptible cultivars showed an additional significant increase in basic fluorescence. The determination of Hill activity and chlorophyll fluorescence demonstrated that photosynthesis was inactivated in different ways in resistant and in susceptible cultivars.     

Resistance to broomrape (Orobanche crenata) in faba bean (Vicia faba): cell wall changes associated with prehaustorial defensive mechanisms

Broomrapes (Orobanche spp.) are parasitic angiosperms, which attach to the roots of the hosts to take water and nutrients from them. No complete control measures are available to date, but breeding for resistance remains as one of the most feasible and environmentally friendly methods. However, the mechanisms governing the interaction between these parasites and the host are not yet well understood. We studied the cellular changes associated with the resistance to Orobanche crenata in faba bean as mechanisms involved or responsible for resistance. Two cultivars of faba bean, resistant and susceptible to O. crenata infection, were used. The evolution of the infection and the changes in the cell and tissue organisation and wall components of the host cells were followed and evaluated in both genotypes. Samples of compatible and incompatible interactions were fixed and sectioned, and specific cytochemical methods for different cell components were applied, results being analysed under light and epifluorescence microscopy. A higher proportion of O. crenata seedlings unable to penetrate the root was found on the resistant genotype. Reinforcement of cell walls by callose deposition hampers parasite penetration through the cortex. Lignification of endodermal cells prevents further penetration of the parasite into the central cylinder.     

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea

Primary roots of soybean (Glycine max (L.), Merrill, cv. Harosoy 63) seedlings were inoculated with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea and total callose was determined at various times after inoculation. From 4 h onward, total callose was significantly higher in roots showing the resistant rather than the susceptible response. Local callose deposition in relation to location of fungal hyphae was determined in microtome sections by its specific fluorescence with sirofluor and was quantified on paper prints with an image-analysis system. Callose deposition, which occurs adjacent to hyphae, was found soon after inoculation (2, 3 and 4 h post inoculation) only in roots displaying the resistant response, and was also higher at 5 and 6 h after inoculation in these resistant roots than in susceptible roots. Early callose deposition in the incompatible root-fungus reaction could be a factor in resistance of soybean against P. megasperma.Abbreviation pi post inoculation     

Comparative proteomic analysis of bacterial wilt susceptible and resistant tomato cultivars

To investigate the molecular mechanisms of bacterial resistance in susceptible and resistant cultivars of tomato, a proteomic approach was adopted. Four cultivars of tomato were selected on the basis of their response to bacterial (Pseudomonas solanacearum) inoculation wherein cultivar Roma and Riogarande, and cultivar Pusa Ruby and Pant Bahr were considered as resistant and susceptible cultivars, respectively. Proteins were extracted from leaves of 3-week-old seedlings of the four cultivars and separated by 2-DE. A total of nine proteins were found to be differentially expressed between the susceptible and resistant cultivars. Amino acid sequences of these proteins were determined with a protein sequencer. The identified proteins belongs to the categories of energy, protein destination and storage, and defense. Of these proteins, a 60 kDa chaperonin and an apical membrane antigen were significantly upregulated in resistant cultivars compared with susceptible cultivars. Application of jasmonic acid and salicylic acid resulted in significant changes in levels of apical membrane antigen and protein disulfide-isomerase. Taken together, these results suggest that apical membrane antigen might be involved in bacterial resistance process through salicylic acid induced defense mechanism signaling in tomato plants.     

Defense responses in tomato landrace and wild genotypes to early blight pathogen Alternaria solani infection and accumulation of pathogenesis-related proteins

Plants dispense localised and systemic defense responses against biotic colonisers and plant resistance to pathogens depends upon timely recognition of pathogen infection and subsequent rapid activation of defense responses through signal transduction pathways. Induction of host defense responses involving compatible and incompatible interactions in tomato landrace, LE996, LE150 and LE1165 (Solanum lycopersicum), and wild relatives, Seijima Jeisei and I979 (S. hirsutum) genotypes, and early blight (EB) pathogen, Alternaria solani were studied. Accumulation patterns of different defense related proteins in resistant genotypes (LE996, Seijima jeisei and I979) and susceptible genotypes (LE150 and LE1165) are reported here. Challenge inoculation led to similar protein profiles in resistant genotypes yielding 11 proteins, unique are 14, 54, and 58 kD proteins that are absent with susceptible genotypes. Resistant genotypes accumulated more proteins well in advance, 6-h after challenge inoculation. Resistant genotype LE996 expressed 54%, 90%, and 52% enhanced enzymatic activity of peroxidase, polyphenol oxidase and phenylalanine ammonia lyase respectively than susceptible LE150 and correlated phenolics accumulation peaked 2-d after challenge inoculation. Activity gel assay indicated the unique expression of PO1 and PO2 in LE996 upon challenge inoculation. Significant increase in expression of chitinases (63%) and β-1,3-glucanase (71%) upon challenge inoculation than susceptible LE150 control was recorded. Western blotting indicated the unique presence of less than 30 kD chitinase in resistant LE996 and Seijima Jeisei and was absent in susceptible LE150. The results demonstrate the importance of pathogenesis-related (PR) proteins in EB resistance and their use as biochemical markers for genotype selection.     

Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75 kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17 kDa), at 12 to 24 h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24 h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins. Received October 9, 2001 Accepted January 18, 2002     

Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics

Molecular changes elicited by plants in response to fungal attack and how this affects plant–pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label‐free proteomics and NMR‐based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis‐related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea–Foc interactions.     

Temporal and spatial patterns of 1, 3-β-glucanase and chitinase induction in potato leaves infected by Phytophthora infestans

Infection of potato (Solarium tuberosum L.) leaves with the fungal pathogen Phytophthora infestans caused a similar, strong and coordinated induction of 1, 3-β-glucanases and chitinases in compatible (plant susceptible) and incompatible (plant resistant) interactions of two selected plant cultivars with appropriate races of the fungus. The temporal and spatial patterns of 1, 3-β-glucanase induction were studied in further detail by immunohistochemical and in-situ hybridization methods. Accumulation of the protein was preceded by progressive activation of the corresponding gene, commencing near infection sites and spreading rapidly throughout the whole infected leaf as well as to adjacent, non-infected leaves. Protein and mRNA distribution patterns were nearly identical in compatible and incompatible interactions. In comparison with 1, 3-β-glucanase mRNA, phenyl-alanine ammonia-lyase mRNA accumulated more rapidly and remained restricted to the vicinity of fungal infection sites, in addition to its constitutive occurrence in the vascular bundles. Even more rapid than any detectable mRNA induction was the accumulation of auto fluorescing material in plant cells immediately surrounding fungal structures, particularly and invariably in incompatible interactions and less frequently in compatible interactions. It is concluded that cultivar-race-specific resistance is established early in the interaction of potato leaves with P. infestans and hence the observed massive accumulation of 1, 3-β-glucanase and chitinase is presumably not involved in determining this specificity.     

Regulation of superoxide dismutase isoforms in resistant and susceptible strawberry cultivars subjected to leaf spot disease

Abstract

Macroscopic symptoms were observed in two strawberry cultivars, with the degree of symptom intensity varying depending on the susceptibility of the cultivars, i.e. resistant or susceptible. The symptoms presented as red spots and were observed 30 d following leaf tissue inoculation with the Mycosphaerella fragariae pathogen. A comparison of the superoxide dismutase isoform profiles obtained by gel electrophoresis in all samples extracted from both resistant and susceptible cultivars indicated one constant sharp band, identified as Mn[sbnd]SOD with a molecular mass of 19 kDa. The intensity of this band was higher in all samples derived from the resistant cultivar than in those from the susceptible cultivar. Another superoxide dismutase (SOD) isoform, identified as CuZn[sbnd]SOD with a molecular mass of 16 kDa, was detected in all soluble proteins derived from the resistant cultivar. This isoform was not observed in the susceptible cultivar; however, following an incremental increase in the amount of loaded protein, it was illuminated as a faint band in a sample collected 3 d after inoculation, indicating insufficient production of the CuZn[sbnd]SOD isoform in the susceptible cultivar during an oxidative burst induced by the M. fragaria pathogen. Several bands were also characterized in both cultivars containing Fe and Mn as their co-factors (Fe, Mn[sbnd]SOD). Unlike in the resistant cultivar, where the activity of Fe, Mn[sbnd]SOD isoforms gradually and regularly increased and reached its highest level on the third day after inoculation, the activity of the isoforms changed irregularly over 20 days of study in the susceptible cultivar.     

Changes in the redox status of chickpea roots in response to infection by Fusarium oxysporum f. sp. ciceris: apoplastic antioxidant enzyme activities and expression of oxidative stress-related genes

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas ( Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15–17 days after inoculation) and (iii) when all plants had developed disease symptoms (20–22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier ( i.e. GR and SOD) or to higher levels ( i.e. DAO) in the incompatible interaction. Expression of apx , cat , sod , lipoxygenase ( lox ) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier ( lox ) or higher ( actin ) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.     

Peroxidase Activity in Leaves of Wheat Cultivars Differing in Resistance to Erysiphe graminis DC.

The peroxidase activities in leaves from resistant and susceptible cultivars of wheat infected and non-infected by Erysiphe graminis DC were studied. In non-infected wheat, soluble and ionic bound peroxidase activity level was found to be higher in the resistant cultivar than that in the one susceptible to Erysiphe graminis DC. After infecting wheat leaves with Erysiphe graminis DC a remarkable increase in the activity of soluble and ionic bound peroxidases was detected 5 days after inoculation only in the resistant cultivar. In the susceptible cultivar a high increase in the activity of the soluble and ionic bound peroxidases occurred only 15 days after inoculation. Using ion exchange chromatography four peroxidase fractions were obtained from infected susceptible and resistant cultivars as from non-infected ones. The fraction II in non-inoculated resistant cultivars was much higher than that in the susceptible one. This fraction increased after inoculation in both cases reaching a higher level in resistant cultivars. Fraction I was higher in the susceptible cultivar. Electrofocusing profiles of peroxidase from the susceptible and resistant cultivar differed from one another. New peroxidase bands after inoculation appeared only in the resistant cultivar.     

Biochemical and molecular characterization of non-host resistance keys in food crops

Generally, under normal conditions plants are resistant to many of the incompatible pathogens (viral, fungal and bacterial), and this is named “non-host resistance phenomenon”. To understand this phenomenon, different types of food crops (faba bean, squash, barley and wheat) were inoculated with compatible and incompatible pathogens. Strong resistance symptoms were observed in the non-host/incompatible pathogen combinations as compared with host/compatible pathogen combinations, which showed severe infection (susceptibility). Reactive oxygen species (ROS) mostly hydrogen peroxide and superoxide were significantly increased early 24 and 48 h after inoculation (hai) in the non-host plants comparing to the host. Antioxidant enzymes activity (catalase, polyphenol oxidase and peroxidase) were not increased at the same early time 24, 48 hai in the non-host resistant and host resistant plants, however, it increased later at 72 and 168 hai. Electrolyte leakage decreased significantly in non-host resistant and host resistant/pathogen combinations. Catalase and peroxidase genes were significantly expressed in non-host resistant and in host resistant plants as compared to the host susceptible one, which did not show expression using RT-PCR technique. Furthermore, Yr5, Yr18 and Yr26 resistant genes were identified positively using PCR in all treatments either host susceptible or non-host resistant plants in which prove that no clear role of these resistant genes in resistance. Early accumulation of ROS could have a dual roles, first role is preventing the growth or killing the pathogens early in the non-host, second, stimulating the gene appearance of related genes in addition the activition of antioxidant enzymes later on which thereby, neutralize the harmful effect of ROS and consequently suppressing disease symptoms. The new finding from this study supporting the plant breeders with new source of resistance to develop new resistant cultivars and/or stop the breakdown of resistance in resistant cultivars.     

Influence of inoculation with Ascochyta rabiei on the mineral contents of resistant and susceptible cultivars of chickpea

The determination of mineral contents of healthy as well as Ascochyta rabiei inoculated resistant, moderately resistant, moderately susceptible and susceptible cultivars revealed that the amount of N, P, Zn and Fe did not vary much in healthy plants of the resistant and susceptible cultivars. The amount of K and S was greater in the susceptible cultivars compared to the resistant cultivars while the reverse was true for Cu and Mn. Barring the recovery of Cu and Fe, the amount of all other elements (N, P, K, S, Zn and Mn) was enhanced upon inoculation of resistant, moderately resistant, moderately susceptible and susceptible cultivars. There was a noticeable increase in the amount of K in the resistant cultivars and the reverse was true for P, S and Mg contents after inoculation.     

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus × domestica) with Erwinia amylovora

Background  

The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceaespecies, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora.