Reversible cytoplasmic rearrangements precede wall apposition,hypersensitive cell death and defense-related gene activation in potato/Phytophthora infestans interactions

We used video microscopy techniques as a tool for live examination of the dynamic aspects of plant/fungus interactions. Early, dynamic responses of epidermal midrib cells of leaves from a potato cultivar (Solanum tuberosum L. cv. Datura) carrying resistance gene R1 to Phytophthora infestans (race 1: compatible interaction, race 4: incompatible interaction) were monitored. Similar responses were observed in both types of interaction, ranging from no visible reaction of invaded plant cells to hypersensitive cell death. The overall defense response of each individual cell exhibited a highly dynamic behavior that appeared to be tightly coordinated with the growth of the fungus. Initial localized reactions, including major rearrangements within the cytoplasm, occurred directly at the fungal penetration site, where rapid apposition of autofluorescent material and callose took place. If fungal invasion stopped at this stage, the host cell restored its normal cytoplasmic activity and survived. Hypersensitive cell death occurred only when fungal growth had proceeded to the formation of a clearly identifiable haustorium. In such cases, cytoplasm and nucleus conglomerated around the intracellular fungal structure, followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium. Only small quantitative differences between the compatible and incompatible interactions of the two fungal races were observed for these early responses of epidermal cells. In the incompatible interaction, a slightly larger number of epidermal cells responded to fungal attack. More pronounced quantitative differences between compatible and incompatible interactions occurred upon fungal invasion of the mesophyll. These differences in the number of responding cells were not reflected at the level of gene expression: the spatial and temporal activation patterns of two defense-related genes, encoding phenylalanine ammonia-lyase and pathogenesis-related protein 1, were similar in both types of interaction.Dedicated to Professor Peter Sitte, Freiburg, Germany, on the occasion of his 65th birthday     

Induction of Resistance or Susceptibility in Pea to Races of Pseudomonas syringae pv. pisi

The phenomena of induced resistance and induced susceptibility were investigated in the pea-Pseudomonas syringae pv. pisi system, using two pea cultivars, Early Onward and Hurst Green Shaft, and races 1 and 2 of the pathogen. Preliminary treatment with heat-killed bacteria induced resistance in peas to infection by P. s. pv. pisi; the resistance induced was dependent on the time interval between the preliminary and challenge inoculations. The mechanism of induced resistance appears to vary between the cultivars. Similarly, both races of the pathogen appear to have different resistance-inducing efficiencies. Resistance in cvs Early Onward and Hurst Green Shaft to the compatible bacterium could not be induced by preliminary inoculation with live cells of an incompatible race. Heat-killed cells of the races failed to induce the hypersensitive reaction in cultivars that normally show this response when challenged with live avirulent bacteria. Preliminary inoculation with live race 1 cells failed to induce susceptibility in cv. Early Onward to live race 2 cells, irrespective of the challenge inoculation interval. On cv. Hurst Green Shaft, however, preliminary inoculation with live race 2 cells induced limited susceptibility to live race 1 cells. Preliminary treatment with sterile distilled water followed by challenge of the same leaves 24 h later with a compatible race induced a moderate resistance response in both cultivars.     

Induction of Resistance or Susceptibility in Pea to Races of Pseudomonas syringae pv. pisi

The phenomena of induced resistance and induced susceptibility were investigated in the pea-Pseudomonas syringae pv. pisi system, using two pea cultivgars, Early Onward and Hurst Green Shaft, and races 1 and 2 of the pathogen. Preliminary treatment with heat-killed bacteria induced resistance in peas to infection by P. s. pv. pisi; the resistance induced was dependent on the time interval between the preliminary and challenge inoculations. The mechanism of induced resistance appears to vary between the cultivars. Similarly, both races of the pathogen appear to have different resistance-inducing efficiencies. Resistance in cvs Early Onward and Hurst Green Shaft to the compatible bacterium could not be induced by preliminary inoculation with live cells of an incompatible race. Heat-killed cells of the races failed to induce the hypersensitive reaction in cultivars that normally show this response when challenged with live avirulent bacteria. Preliminary inoculation with live race 1 cells failed to induce susceptibility in cv. Early Onward to live race 2 cells, irrespective of the challenge inoculation interval. On cv. Hurst Green Shaft, however, preliminary inoculation with live race 2 cells induced limited susceptibility to live race 1 cells. Preliminary treatment with sterile distilled water followed by challenge of the same leaves 24 h later with a compatible race induced a moderate resistance response in both cultivars.     

Chitinases of <Emphasis Type="Italic">Coffea arabica</Emphasis> genotypes resistant to orange rust <Emphasis Type="Italic">Hemileia vastatrix</Emphasis>

Two Coffea arabica — Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra — rust race VI and I₂: coffee cv S4 Agaro — rust race II) and a compatible interaction (coffee cv. Caturra — rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.     

Structures of Conversion Products Formed from 18β-Glycyrrhetinic Acid by Streptomyces sp. G-20

Dynamic profiles of the rate of O₂ generation from press-injured and inoculated rice leaf slices, versus the time after inoculation, discriminated between the incompatible and compatible combination of blast fungus races with a cultivar. The application of sodium saccharin to rice seedlings via the root system for 6 days changed the compatible to incompatible profile. Even after press- injury and inoculation with the compatible conidia, the leaf application of sodium saccharin enhanced superoxide generation. The application of N-methylsaccharin in a similar manner, however, did not enhance the superoxide generation. Inoculation of press-injured leaves with incompatible conidia in the presence of an aqueous diffusate of the germinating compatible conidia changed the incompatible to compatible profile. The application to press-injured of concanavalin A or a lyophylized preparation from 5 m ammonia extracts of rice leaf homogenate prior to stimulating with a resistance-inducing factor (RIF) from the fungus also enhanced the superoxide generation. The RIF, either from the incompatible or compatible race, gave a quite similar profile of activation upon the generation of the superoxide anion.     

Investigation of the mechanism of glyceollin accumulation in soybean infected by Phytophthora megasperma f. sp. glycinea

Soybean seedlings (Glycine max, cv. Harosoy 63) which had been inoculated in the hypocotyls with mycelium from either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma f. sp. glycinea were pulse labeled with ¹⁴CO₂. The time course of accumulation of glyceollin and daidzein and of ¹⁴C incorporation into these compounds was determined. Metabolic rates of glyceollin were measured by pulse-chase experiments. Differences in glyceollin accumulation between the incompatible and compatible interaction were not apparent before about 14 h after inoculation. Subsequently glyceollin accumulated to a higher level in the incompatible interaction. This difference is also reflected in the rate of ¹⁴C incorporation, which declines more rapidly in the compatible interaction. The apparent half-life of glyceollin metabolism was 28 ± 7 h for inoculation with race 1, while no metabolism was observed with race 3. In contrast to a previous report (M. Yoshikawa, K. Yamauchi, and H. Masago (1979)Physiol. Plant Pathol.14, 157–169), our data prove that the higher accumulation of glyceollin in the incompatible interaction is due to a longer duration of synthetic activity and that the level of glyceollin in both the incompatible and compatible interaction is determined predominantly by its rate of synthesis.     

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.     

Effects of changing temperature regimes on resistance to races of Melampsora medusae in a cultivar of poplar

Leaf discs of Populus deltoides cv. W-79/307 inoculated with race 4–C of Melampsora medusae, give a compatible reaction when incubated at 16°C (LT), but an incompatible reaction at 26°C (HT). When, over a 12–day period, sets of inoculated leaf discs were reciprocally transferred between the temperature regimes (LT to HT, or HT to LT), incubation for as short as 15 h at HT resulted in incompatibility which was not reversed by subsequent incubation at LT. In contrast, incubation of the inoculated discs at LT for at least 4 days was necessary for the development of a compatible reaction following transfer to HT. Further, incompatibility induced in discs by inoculation with race 4–C and incubation at HT is epistatic to expected compatibility following subsequent inoculation with race 4–M, a temperature non-sensitive biotype. The rapidity, irreversibility and epistatic nature of the temperature-induced incompatibility suggests that initial recognition in this pathosystem may be for incompatibility. The significance of these results in this host/pathogen system is discussed.     

Response of Solanum tuberosum to Myzus persicae infestation at different stages of foliage maturity

Young leaves of the potato Solanum tuberosum L. cultivar Kardal contain resistance factors to the green peach aphid Myzuspersicae （Sulzer） （Hemiptera： Aphididae） and normal probing behavior is impeded. However, M. persicae can survive and reproduce on mature and senescent leaves of the cv. Kardal plant without problems. We compared the settling ofM. persicae on young and old leaves and analyzed the impact of aphids settling on the plant in terms of gene expression. Settling, as measured by aphid numbers staying on young or old leaves, showed that after 21 h significantly fewer aphids were found on the young leaves. At earlier time points there were no difference between young and old leaves, suggesting that the young leaf resistance factors are not located at the surface level but deeper in the tissue. Gene expression was measured in plants at 96 h postinfestation, which is at a late stage in the interaction and in compatible interactions this is long enough for host plant acceptance to occur. In old leaves of cv. Kardal （compatible interaction）, M. persicae infestation elicited a higher number of differentially regulated genes than in young leaves. The plant response to aphid infestation included a larger number of genes induced than repressed, and the proportion of induced versus repressed genes was larger in young than in old leaves. Several genes changing expression seem to be involved in changing the metabolic state of the leaf from source to sink.     

Interaction between Pseudomonas syringae pv. Pisi and Isolated Pea Mesophyll Protoplasts

The interaction between five races of Pseudomonas syringae pv. pisi (PSP) and isolated mesophyll protoplasts obtained from five pea cultivars was studied. There was no trend in the attachment of bacterial cells to surfaces of compatible and incompatible host protoplasts. The viability of protoplasts from compatible and incompatible host-pathogen interactions did not differ significantly; however, changes in the viability of cultivar Kelvedon Wonder protoplasts, compatible with all five races, was relatively more stable following inoculation with bacteria than those of cultivar Fortune, incompatible with all of the five races. Protoplast cell wall regeneration did not take place until 24–48 h after isolation. It is concluded that the use of pea protoplasts as a model system for studying the pea-PSP interaction appears to have considerable potential for the future, but more basic research is required.     

Increased Activity of a Cationic Peroxidase Associated with an Incompatible Interaction Between Xanthomonas oryzae pv oryzae and Rice (Oryza sativa)

Rice (Oryza sativa L.) cultivar Cas 209 carries the gene Xa-10 for resistance to race 2 of Xanthomonas oryzae pv oryzae, the bacterial blight pathogen. When seedling leaves of Cas 209 plants were infiltrated with bacterial cell suspensions of strain PXO86^Rif (race 2, incompatible), total peroxidase activity in extracts from extracellular spaces increased almost threefold between 16 and 24 hours after inoculation. The increase in total peroxidase activity in extracellular extracts was correlated with the appearance of a 43-kilodalton peroxidase isoenzyme with an isoelectric point of 8.6. Increases in the activities of two anionic peroxidase isoenzymes also were associated with the incompatible interaction. Later during the interactions, total peroxidase activities increased in both compatible (cv Cas 209 infiltrated with race 1, PXO61^Sm) and control (Cas 209 infiltrated with water) treatments, but final activity levels were less than that observed in the incompatible combination. Similarly, the cationic peroxidase was detected in all three treatments by 48 hours after infiltration, but at reduced levels in compatible and water-infiltrated control treatments relative to the incompatible combination. Accumulation of this peroxidase in extracellular spaces thus may play a role in the defense response in cultivar Cas 209.     

Response to Gamma Irradiation and Induced Virulent Mutation in Melampsora medusae of Poplars

When the urediniospores of three races of Melampsora medusae, the causal agent of leaf rust of poplars, were exposed to Co⁶⁰ gamma irradiation (range 100–1000 Gy), there were significant differences in the radiosensitivity of the races as assessed, in vitro, by incubation period to flecking and the uredinial survivability percentage. In race 5A the frequency of mutation to virulence on Populus deltoides cv. T-173, normally a resistant cultivar, was maximum (1.688 %) at an irradiation dose of 400 Gy. Importance of mutations as source of variability in pathosystems and usefullness of artificially induced mutations in the context of host-pathogen interactions are discussed.     

I. Pathogenic variation in fungi and bacteria and mycorrhizal compatibility: Variation in Pseudomonas morsprunorum and the selection of cherry cultivars resistant to bacterial canker

During 1971 and 1972 leaf spot infection caused by Pseudomonas morsprunorum was far more severe on the cv. Roundel than on the normally more susceptible cv. Napoleon, and Roundel supported higher leaf surface populations of the pathogen. This unprecedented reversal in the established field performance of the two cultivars was associated with the presence on the trees of a colony variant (race 2) of P. morsprunorum that differed from the forms of the organism previously described (race 1) in physiological, pathological and phage sensitivity characteristics. In inoculation experiments race 2 isolates showed some specificity for Roundel and race 1 isolates for Napoleon. This difference was reflected in the distribution of the races on trees of the two cultivars growing in experimental plots and commercial orchards. Similar interactions between race and host genotype were observed amongst progeny from breeding programmes and material originally selected for resistance to bacterial canker after inoculation with race 1 proved susceptible to race 2. Three distinct groups of isolates with the colony characteristics of race 2 have been identified and one of these has affinities with Pseudomonas syringae. Group 1 isolates, corresponding to P. morsprunorum and comprising non-fluorescent forms, were the most virulent in pathogenicity tests and accounted for the majority of race 2 infections in the field.     

Quantitative and Microscopic Assessment of Compatible and Incompatible Interactions between Chickpea Cultivars and Fusarium oxysporum f. sp. ciceris Races

Background

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris, a main threat to global chickpea production, is managed mainly by resistant cultivars whose efficiency is curtailed by Fusarium oxysporum f. sp. ciceris races.

Methodology

We characterized compatible and incompatible interactions by assessing the spatial-temporal pattern of infection and colonization of chickpea cvs. P-2245, JG-62 and WR-315 by Fusarium oxysporum f. sp. ciceris races 0 and 5 labeled with ZsGreen fluorescent protein using confocal laser scanning microscopy.

Findings

The two races colonized the host root surface in both interactions with preferential colonization of the root apex and subapical root zone. In compatible interactions, the pathogen grew intercellularly in the root cortex, reached the xylem, and progressed upwards in the stem xylem, being the rate and intensity of stem colonization directly related with the degree of compatibility among Fusarium oxysporum f. sp. ciceris races and chickpea cultivars. In incompatible interactions, race 0 invaded and colonized ‘JG-62’ xylem vessels of root and stem but in ‘WR-315’, it remained in the intercellular spaces of the root cortex failing to reach the xylem, whereas race 5 progressed up to the hypocotyl. However, all incompatible interactions were asymptomatic.

Conclusions

The differential patterns of colonization of chickpea cultivars by Fusarium oxysporum f. sp. ciceris races may be related to the operation of multiple resistance mechanisms.     

Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of Puccinia striiformis f. sp. tritici,the causal agent of yellow rust disease

Chitinase specific activity was measured spectrophotometrically in wheat leaf tissues during the compatible and incompatible interactions with Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. The wheat cultivar, Federation^* 4/Kavkaz, was inoculated with virulent (134E134A+) or avirulent (4EOA+) races of P. striiformis f. sp. tritici in the first leaf stage. The results showed that chitinase activity pattern was similar in both compatible and incompatible interactions up to 72 hrs after inoculation. However, the specific activity increased rapidly in the incompatible reaction thereafter. In susceptible reaction, chitinase activity gradually declined after 72 hrs post-inoculation reaching a level similar to that in the control plants two weeks after inoculation. Chitinase specific activity in resistance response was at least three times greater than that in the susceptible reaction two weeks following the inoculation. Electrophoresis of native polyacrylamide gel impregnated with 0.1% (w/v) glycol chitinas the substrate revealed the presence of eight chitinase isoforms with relative electrophoretic mobility (R_m) values ranging from 0.11 to 0.64 in the resolving gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Host-Pathogen Interactions : XX. BIOLOGICAL VARIATION IN THE PROTECTION OF SOYBEANS FROM INFECTION BY PHYTOPHTHORA MEGASPERMA F. SP. GLYCINEA

Phytophthora megasperma f.sp. glycinea, which causes soybean (Glycine max) root and stem rot, exists as several races which differ in their ability to infect a range of soybean cultivars. A glycoprotein-rich fraction (Fraction I) isolated from fungal culture fluid protects soybean seedlings from infection with compatible races. In an early study (13), seedlings were protected only by Fraction I purified from incompatible races. In 1979, seedlings were better protected by Fraction I isolated from incompatible races than by Fraction I isolated from compatible races. In 1980, seedlings were protected equally well by Fraction I from incompatible and compatible races. Materials similar in composition to Fraction I did not protect seedlings from infection. No cause could be identified for the apparent change, during the 3-year period, in the race specificity of the protection assay. Variability in the bioassay prohibited further purification or characterization of Fraction I components that protect seedlings from infection.