Protocatechuic acid production from trans-ferulic acid by Pseudomonas sp. HF-1 mutants defective in protocatechuic acid catabolism

Summary A trans-ferulic acid-utilizing Pseudomonas sp. HF-1 was isolated from soil samples. Mutant HF-1124, capable of growing on trans-ferulic acid but not on protocatechuic acid, was isolated from HF-1 after mutagenesis with nitrosoguanidine. The optimum temperature was 30°C and the optimum pH was 7.0–8.0 for protocatechuic acid production from trans-ferulic acid by mutant HF-1124. Protocatechuic acid production reached 4 g/l from a concentration of 8 g/l trans-ferulic acid. As a result of co-oxidation of methoxy aromatic compounds by strain HF-1124 grown on acetic acid, protocatechuic acid was formed from vanillin and vanillic acid, and vanillic acid and isovanillic acid were formed from veratric acid. By the co-oxidative demethylation of substituted monomethoxybenzene, m- and p-hydroxybenzoic acids were accumulated from m-and p-anisic acid, respectively, while no products were detected from anisole, o-anisic acid, nitroanisole, methylanisole, methoxyphenol and dimethoxybenzene.     

Host selection by Ibalia leucospoides based on temporal variations of volatiles from the hosts’ fungal symbiont

Insect parasitoids locate hosts via reliable and predictable cues such as volatile emissions from hosts and/or host plants. For insects that depend on mutualistic organisms, such as many wood‐boring insects, symbiont‐derived semiochemicals may represent a source of such cues to be exploited by natural enemies. Ultimately, exploitation of these signals may increase fitness by optimizing foraging efficiency. Female parasitoids of Ibalia leucospoides use volatiles from the fungal symbiont Amylostereum areolatum of their host Sirex noctlio to find concealed host eggs and young larvae within the xylem. We hypothesize that the temporal pattern of fungal emissions may indicate not only the presence of host larvae but also be used as a cue that indicates host suitability and age. Such information would allow female parasitoids to discern more efficiently between hosts within ovipositor reach from those already buried too deep into the xylem and out of reach. In this context, we assessed the behaviour of I. leucospoides females to volatiles of A. areolatum in a ‘Y’‐tube olfactometer at regular intervals over 30 days. We concurrently examined the fungal volatiles by headspace sampling through solid‐phase microextraction (SPME) followed by gas chromatography mass spectrometry (GC‐MS). We observed that females were attracted to volatiles produced by two‐week‐old fungal cultures, a period that matches when older larvae are still within ovipositor reach. Four chemical compounds were detected: ethanol, acetone, acetaldehyde and the sesquiterpene 2,2,8‐trimethyltricyclo[6.2.2.01,6]dodec‐5‐ene, with each compounds’ relative abundance changing over time. Results are discussed in the context of parasitoids fitness. Future studies involving electrophysiology, different collection techniques and further behavioural assays will help in identifying the compounds that convey temporal information to female parasitoids and have the potential for being used in integrated pest management programmes.     

Inhibition of light-induced stomatal opening and of guard-cell-protoplast swelling in Vicia faba L. by tentoxin,an inhibitor of photophosphorylation

The fungal phytotoxin tentoxin and its natural derivative dihydrotentoxin impair light-induced stomatal opening in epidermal strips of broad bean (Vicia faba L.) incubated in a potassium-rich medium. Swelling of guard-cell protoplasts (GCPs) of the same species is inhibited in the presence of both substances. Swollen GCPs shrink after tentoxin or dihydrotentoxin treatment and these effects cannot be fully compensated by the phytoeffector fusicoccin. A comparison with the potassium carrier valinomycin shows that tentoxin acts in a different manner, because it is effective in the light only, whereas valinomycin causes shrinkage of GCPs also in the dark. Determination of adenine nucleotides in GCPs indicates a reduced ATP content and an enhanced ADP level after addition of tentoxin. At the same time, tentoxintreated GCPs contain more NADPH and less NAD⁺ than the control (NADP⁺ and NADH content does not differ). The results presented are consistent with the hypothesis that tentoxin closes stomata as a consequence of its inhibitory action on photophosphorylation.Abbreviations FC fusicoccin - GCP guard-cell protoplast - KIDA potassium iminodiacetate     

The effect of trans-ferulic acid and gamma-oryzanol on ethanol-induced liver injury in C57BL mouse

The effects of the oral administration of trans-ferulic acid and gamma-oryzanol (mixture of steryl ferulates) with ethanol (5.0 g per kg) for 30 days to c57BL mice on ethanol-induced liver injury were investigated. Preventions of ethanol-induced liver injury by trans-ferulic acid and gamma-oryzanol were reflected by markedly decreased serum activities of plasma aspartate aminotransferase, alanine aminotransferase and significant decreases in hepatic lipid hydroperoxide and TBARS levels. Furthermore, the trans-ferulic acid- and gamma-oryzanol-treated mice recovered ethanol-induced decrease in hepatic glutathione level together with enhancing superoxide dismutase activity. These results demonstrate that both trans-ferulic acid and gamma-oryzanol exert a protective action on liver injury induced by chronic ethanol ingestion.     

Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells

Summary. Programmed cell death plays a vital role in normal plant development, response to environmental stresses, and defense against pathogen attack. Different types of programmed cell death occur in plants and the involvement of mitochondria is still under investigation. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin induces cell death that shows apoptotic features, including chromatin condensation, DNA fragmentation, and release of cytochrome c from mitochondria. In this work, we show that cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria, inhibits the cell death, DNA fragmentation, and cytochrome c release induced by fusicoccin. In addition, we show that fusicoccin induces a change in the shape of mitochondria which is not prevented by cyclosporin A. These results suggest that the release of cytochrome c induced by fusicoccin occurs through a cyclosporin A-sensitive system that is similar to the permeability transition pore of animal mitochondria and they make it tempting to speculate that this release may be involved in the phytotoxin-induced programmed cell death of sycamore cells. Correspondence and reprints: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.     

Effect of fusicoccin on plant cell cultures and protoplasts

We have assayed the capacity of the fungal toxin fusicoccin to induce some of its characteristic effects (acidification of the medium, stimulation of K⁺, and of 3-O-methyl-D-glucose uptake) in cell suspensions of Parthenocissus tricuspidata (Siebold et Zucc.) Planchon, Acer pseudoplatanus L. and Oryza sativa L., and in protoplast suspensions prepared from leaves of Nicotiana tabacum L. and Spinacia oleracea L. or from cultures of P. tricuspidata. Evidence is presented showing that all tested biological materials respond to the addition of fusicoccin. The observation that the toxin is also active on protoplasts indicates that the cell wall is not involved in the mechanism of action of fusicoccin.Abbreviations FC Fusicoccin - 3-O-MG 3-O-Methyl-D-glucose     

Host plant selection behaviour of Chilo partellus and its implication for effectiveness of a trap crop

Female lepidopterans can display a hierarchy of preference among potential host species, a trait thought to arise from the balance between attractants and deterrents to which the insects respond. Host plant ranking by moths and larvae of Chilo partellus Swinhoe (Lepidoptera: Crambidae), an important pest of cereals in Africa, was investigated, and whether eggs deposited on specific host plants yield larvae of particular host preferences. Trap plants are used in management of this pest. However, any ‘disagreement’ in host ranking between moths and larvae could potentially reduce effectiveness of trap crops as larvae emigrate to the main crop from the parent’s preferred trap plant. We also investigated whether host plant preference is influenced by the diet upon which larvae fed as part of an integrated assessment of the relationship between host plant selection and learning in C. partellus. Five host plants (all Poaceae) were used: maize (Zea mays L.), sorghum (Sorghum bicolor Moench), Napier grass (Pennisetum purpureum Schumach), and two varieties of signal grass [Brachiaria brizantha (A. Rich.) Stapf], viz., local (henceforth signal grass) and improved (‘Mulato’). In multiple choice tests, C. partellus female moths preferentially oviposited on Napier grass, followed by sorghum, maize, and signal grass, and least preferred ‘Mulato’. Larvae however equally orientated and settled on leaf cuts of maize, sorghum, signal grass, and Napier grass, but least preferred ‘Mulato’. Moreover, eggs from specific host plants did not yield larvae of particular host preferences. Furthermore, oviposition preference was not altered by the larval food. These results imply only a slight ‘disagreement’ in host ranking behaviour between moths and larvae, which is beneficial for trap cropping as larvae would not ‘reject’ the trap plant and appreciably disperse to the neighboring plants. Moreover, absence of larval learning behaviour indicates that regardless of the larval food C. partellus moths would still be attracted to the selected trap plant.     

Electrophysiological Responses to Fusaric Acid of Root Hairs from Seedlings of Date Palm-susceptible and -resistant to Fusarium oxysporum f. sp. albedinis

Bayoud, a vascular wilt of date palm caused by Fusarium oxysporum f. sp. albedinis (Foa), is the most devastating disease in palm groves of north Africa. Although Foa is able to induce resistance mechanisms in its host plant, no data are available on the early responses of the root cells. Fusaric acid (FA) is the main toxin found in culture filtrates from Foa aggressive strains. This phytotoxin induces modifications of membrane permeability or membrane potential in various cell types and could thus be involved in the early steps of signal exchange between the pathogen and the plant. We showed an early differential behaviour of the disease‐resistant and ‐susceptible cultivars from date palm when challenged by FA. This response could be due to a differential sensitivity of H⁺‐ATPases to FA.     

Distribution of fungal endophyte genotypes in doubly infected host grasses

Fungal endophytes of the genus Epichloë live intercellulary in above ground plant parts of many pooid ‘grasses of the temperate regions. The associations are characterized by single genotype entities since a given host individual normally contains a single endophyte genotype. They can persist over the life span of the hosts. This study examines whether two fungal genotypes can co-exist within a host plant, and how fungal genotypes are distributed within a host in the case of double infections. We selected four Epichloë bromicola strains that we identified as unique genotypes through RAPD‘ analysis. Young Bromus erectus plants, derived from callus cultures, were artificially inoculated with all possible double-strain mixtures of these fungal genotypes. For identification of fungal genotypes in planta, we designed genotype-specific primer pairs that flanked size-variable loci in the fungal genomes. Diagnostic PCR revealed that only one fungal genotype was present in most inoculated plants, but double infections were also observed with a frequency of 8% of all infected plants. Subsequent analyses of individual tillers of doubly infected plants revealed that, in a given tiller, both the leaf-blade and the leaf-sheath were colonized with only one endophyte genotype. Tillers without any detectable fungal DNA were also observed. Thus, co-existence of multiple endophyte genotypes within a single host plant is governed by mutual exclusion at the tiller level.     

Responses of embryogenic mango cultures and seedling bioassays to a partially purified phytotoxin produced by a mango leaf isolate of Colletotrichum gloeosporioides penz

Summary Colletotrichum gloeosporioides Penz., the causal agent of mango anthracnose, produces a phytotoxin in vitro. The partially purified phytotoxin, presumably colletotrichin, caused anthracnose-like symptoms on young mango leaves, was toxic to embryogenic suspension cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ and inhibited in vitro seed germination of two nonhosts, lettuce and tobacco. There were linear relationships between concentration of the partially purified phytotoxin and mortality of mango embryogenic cultures. Embryogenic cultures grown in the presence of the partially purified phytotoxin showed significantly lower growth rates than the controls. Similarly, embryogenic cultures grown in the presence of 40% (vol/vol) fungal culture filtrate showed significantly lower growth rates than unchallenged controls. Medium containing 40% (vol/vol) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures, indicating the production of phytotoxin in vitro. The results suggest that either fungal culture filtrate or purified phytotoxin can be used as in vitro selection agents to screen for resistance to this fungus.     

Host‐ and stage‐dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal fungi form the most wide‐spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host‐dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host‐ and stage‐dependent manner to contribute to their extremely wide host range. We investigated the expression of SP‐encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA‐seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host‐dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.     

Subversion of host kinases: a key network in cellular signaling hijacked by Helicobacter pylori CagA

Helicobacter pylori is a paradigm of persistent pathogens and major risk factor for developing severe diseases including adenocarcinoma in the human stomach. An important bacterial factor linked to gastric disease progression is the cag pathogenicity island‐encoded type‐IV secretion system (T4SS) effector protein CagA. Translocated CagA undergoes tyrosine phosphorylation at EPIYA‐motifs and then activates or inactivates multiple host signaling proteins in a phosphorylation‐dependent and phosphorylation‐independent fashion. In this way, intracellular CagA acts as a ‘masterkey’ or ‘picklock’, which evolved during evolution to hijack key host cell signal transduction functions. Crucial targets of CagA represent a variety of serine/threonine and tyrosine kinases, which control major checkpoints of eukaryotic signaling. Here we review the signal transmission by translocated CagA on multiple receptor kinases (c‐Met and EGFR) and non‐receptor kinases (Src, Abl, Csk, aPKC, Par1, PI3K, Akt, FAK, GSK‐3, JAK, PAK1, PAK2 and MAP kinases), manipulating a selection of fundamental processes in the human gastric epithelium such as cell adhesion, polarity, proliferation, motility, receptor endocytosis, cytoskeletal rearrangements, apoptosis, inflammation and cell cycle progression. This enormous complexity generates a highly remarkable and puzzling scenario during H. pylori infection. The contribution of these signaling pathways to bacterial survival, persistence and gastric pathogenesis is discussed.     

Une phytotoxine de Xanthomonas campestris pv. vasculorum

A phytotoxin from Xanthomonas campestris pv. vasculorum I. Purification and partial characterization Xanthomonas campestris pv. vasculorum, the causal agent of sugarcane disease produces, when maintained in pure culture, a host selective phytotoxin. The molecule obtained at a high purity level is a strongly acid polyalcohol.     

PR-1 gene family of grapevine: a uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco

A functional contribution of pathogenesis-related 1 (PR-1) proteins to host defense has been established. However, systematic investigation of the PR-1 gene family in grapevine (Vitis spp.) has not been conducted previously. Through mining genomic databases, we identified 21 PR-1 genes from the Vitis vinifera genome. Polypeptides encoded by putative PR-1 genes had a signal sequence of about 25 residues and a mature protein of 10.9–29 kDa in size. PR-1 mature proteins contained a highly conserved six-cysteine motif and pI values ranging from 4.6 to 9. A major cluster with 14 PR-1 genes was mapped to a 280-kb region on chromosome 3. One particular PR-1 gene within the cluster encoding a basic-type isoform (pI 7.77), herein named VvPR1b1, was isolated from various genotypes of grapevine (Vitis spp.) for functional studies. Sequence analysis of PCR-amplified DNA revealed that all genotypes contained a single VvPR1b1 gene except for a broad-spectrum bacterial and fungal disease resistant Florida bunch grape hybrid, ‘BN5-4’, from which seven different homologues were identified. Duplication of VvPR1b1-related genes encoding acidic-type PR-1 isoforms was also observed among several genotypes. However, transgenic expression analysis of grapevine PR-1 genes under strong constitutive promoters in transgenic tobacco revealed that only the basic-type VvPR1b1 gene duplicated in ‘BN5-4’ was capable of conferring high level resistance to bacterial disease caused by Pseudomonas syringae pv. tabaci.     

Logistic growth of the host‐specific obligate insect pathogenic fungus Entomophthora muscae in house flies (Musca domestica)

Insect pathogenic fungi (IPF) need to overcome the host immune system in order to sporulate and ensure transmission to new hosts. Some IPF produce immunosuppressive toxins, whereas others rely on rapid fungal proliferation to kill the host by sheer fungal mass, resulting in a trade‐off between allocating resources to toxin production and fungal proliferation. The obligate entomopathogenic fungus, Entomophthora muscae sensu stricto, is host specific to the common house fly, Musca domestica. E. muscae grows as protoplast cells without cell walls and is not known to produce toxins. Here, we assessed the growth of E. muscae, in vivo, using real‐time PCR to measure the amount of a single‐copy actin gene. We find that E. muscae exhibits S‐shaped logistic growth between time post‐exposure and the number of fungal nuclei. The results show that E. muscae initially grows exponentially inside the host until depletion of available nutrient sources signifies the ‘limiting capacity’ where after the host is killed. This growth pattern differs markedly from toxin‐producing IPF species of Metarhizium and Beauveria in which maximal (plateau) growth and sporulation do not occur until well after the death of the host.     

Cloning of Trypanosoma cruzi trans-Sialidase and Expression in Pichia pastoris

Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface. This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5′-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltransferase structure is not present. To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells. Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification. Using P. pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium.     

A nonclassically secreted effector of Magnaporthe oryzae targets host nuclei and plays important roles in fungal growth and plant infection

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N-terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice–M. oryzae interactions.     

Behind the curtain of the compartmentalization process: Exploring how xylem vessel diameter impacts vascular pathogen resistance

A key determinant of plant resistance to vascular infections lies in the ability of the host to successfully compartmentalize invaders at the xylem level. Growing evidence supports that the structural properties of the vascular system impact host vulnerability towards vascular pathogens. The aim of this study was to provide further insight into the impact of xylem vessel diameter on compartmentalization efficiency and thus vascular pathogen movement, using the interaction between Vitis and Phaeomoniella chlamydospora as a model system. We showed experimentally that an increased number of xylem vessels above 100 μm of diameter resulted in a higher mean infection level of host tissue. This benchmark was validated within and across Vitis genotypes. Although the ability of genotypes to restore vascular cambium integrity upon infection was highly variable, this trait did not correlate with their ability to impede pathogen movement at the xylem level. The distribution of infection severity of cuttings across the range of genotype's susceptibility suggests that a risk-based mechanism is involved. We used this experimental data to calibrate a mechanistic stochastic model of the pathogen spread and we provide evidence that the efficiency of the compartmentalization process within a given xylem vessel is a function of its diameter.     

Diferulic acid as a component of cell walls of Lolium multiflorum

Trans,trans-, cis,trans- and cis,cis-diferulic acids were released from cell walls of Lolium multiflorum by treatment with sodium hydroxide. The isomers were apparently bound via ester links to the structural carbohydrates of the cell walls. Sodium hydroxide treatment gave, per g of wall, 0.18 mg trans,trans-diferulic, 0.02 mg cis,trans-diferulic and a trace of cis,cis-diferulic acids compared with 5.3 mg trans-ferulic, 1.2 mg cis-ferulic, 0.78 mg trans-p-coumaric and 0.12 mg cis-p-coumaric acids. The significance of these acids in lignin biosynthesis is discussed. The effect of UV light on the trans,trans isomer and its fully silylated trimethylsilyl either derivative was also investigated.     

Does mass rearing of field collected Trichogramma brassicae wasps influence acceptance of European corn borer eggs?

The purpose of our study was to identify a trait that changes quickly during Trichogramma mass rearing, and that could therefore be used to monitor stock deterioration. Quality deterioration in mass reared Trichogramma wasps was evaluated by examining host acceptance behaviour of Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) on the target host Ostrinia nubilalis Hübner (Lepidoptera: Crambidae). We compared three replicate lines (designated ‘E’) reared in the laboratory on the factitious host Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) for 27 generations, a line ‘O’ reared in the laboratory on O. nubilalis for 24 generations, and a line ‘F’ reared in the laboratory on O. nubilalis for only two generations. All lines were initiated from field collections of O. nubilalis egg masses. We also evaluated natal host effects by rearing each line on E. kuehniella as well as on O. nubilalis for the last generation prior to testing. The percentage of wasps accepting the O. nubilalis egg mass was significantly higher for the E lines (69.6%) than for the F line (46.5%), while wasps of the O line showed intermediate (57.4%) acceptance. Thus, wasps laboratory reared on E. kuehniella performed better than wasps which had recently been collected in the field. Wasps of the O line showed extended probing behaviour compared to the other lines. Lines did not differ in the duration from the first host contact to the beginning of the drilling, probing or trembling behaviour. Natal host (E. kuehniella or O. nubilalis) did not affect acceptance of the target host O. nubilalis. Even though there is some evidence of adaptation to laboratory rearing conditions, we found no indication for quality deterioration in terms of acceptance behaviour of the target host O. nubilalis when T. brassicae was mass reared on the factitious host E. kuehniella.