Plant defenses against parasitic plants show similarities to those induced by herbivores and pathogens

Herbivores and pathogens come quickly to mind when one thinks of the biotic challenges faced by plants. Important but less appreciated enemies are parasitic plants, which can have important consequences for the fitness and survival of their hosts. Our knowledge of plant perception, signaling and response to herbivores and pathogens has expanded rapidly in recent years, but information is generally lacking for parasitic species. In a recent paper we reported that some of the same defense responses induced by herbivores and pathogens—notably increases in jasmonic acid (JA), salicylic acid (SA), and a hypersensitive-like response (HLR)—also occur in tomato plants upon attack by the parasitic plant Cuscuta pentagona (field dodder). Parasitism induced a distinct pattern of JA and SA accumulation, and growth trials using genetically-altered tomato hosts suggested that both JA and SA govern effective defenses against the parasite, though the extent of the response varied with host plant age. Here we discuss similarities between the induced responses we observed in response to Cuscuta parasitism to those previously described for herbivores and pathogens and present new data showing that trichomes should be added to the list of plant defenses that act against multiple enemies and across kingdoms.Key words: Cuscuta, induced defenses, parasitic plant, jasmonic acid, salicylic acid, phytohormones, hypersensitive response, trichomes, defense signalingSeveral thousand species of plants are parasitic, stealing water and nutrients from other plants through a specialized feeding structure, the haustorium.¹ Haustoria are thought to be modified roots that grow into tissues and fuse with the vascular system of their photosynthetic hosts.¹ Considering that these parasites include some of the world''s most devastating agricultural pests² and are influential, fascinating components of natural communities,^1,3 surprisingly little is known about host defenses induced by parasitic plants. To address this shortcoming, we used a metabolomics approach to track biochemical changes induced in tomato shoots by invasion of C. pentagona haustoria.⁴We found that parasitism induced large increases in both JA and SA beginning about 24 hr after formation of haustoria began, but that production of JA and SA was largely separated in time. Host production of JA was transitory and reached a maximum at 36 hr, whereas SA peaked 12 hr later and remained elevated 5 d later. We also found that C. pentagona grew larger on mutant tomato plants in which the SA (NahG) or JA (jasmonic acid-insensitive1) pathways were disrupted, suggesting that these hormones can act independently to reduce parasite growth. Taken together, these findings suggest the staggered production of JA and SA may be an adaptive response to parasitism—by sequentially activating the JA and SA pathways, tomato plants may minimize the potential for cross-talk between these sometimes antagonistic pathways^5,6 and utilize both signaling molecules.^6,7 Thus, defenses against C. pentagona contain elements characteristic of responses to both herbivores (primarily JA-mediated⁸) and pathogens (primarily SA-mediated⁹)—though it should be noted that some herbivores induce SA¹⁰ and some pathogens JA.¹¹ It is worth noting that parasitism induced predominately cis-JA, the same jasmonate isomer induced by herbivore feeding.¹² Host responses to Cuscuta seem to most resemble that of known plant responses to some pathogens in which a similar sequence of JA and SA production is required to limit disease.¹³C. pentagona also triggered a hypersensitive-like response (HLR) localized around the points of parasite attachment. Using a trypan blue staining technique, we verified host cell death in these parasite-induced lesions. The deposition of eggs by some insect herbivores can elicit the formation of necrotic tissue,¹⁴ but localized cell death is most widely associated with the hypersensitive response (HR) of plants to pathogens. This complex early defense response can restrict the growth and spread of viruses, fungi and bacteria.⁹ Our work adds to existing evidence¹⁵ that the Cuscuta-induced HLR can play a similar role by preventing or limiting the growth of the parasite.An interesting discovery was that the first attachment by C. pentagona elicited almost no response from young 10-day-old hosts, whereas a subsequent attachment after 10 days induced the wholesale changes discussed above (we also found changes in abscisic acid and free fatty acids). Trials in which we varied the age of the host and parasite indicated that host age, rather than a priming effect on defenses, determined the magnitude of response. We have previously observed that Cuscuta spp. in natural populations germinate very early in the growing season, and hypothesized that this tactic promotes successful parasitism by ensuring the presence of young hosts; recent field work seems to corroborate this.¹⁶ As with the response to Cuscuta parasitism, levels of host plant defenses against insects¹⁷ and pathogens¹⁸ are known to be vary with host age.In an earlier paper we reported that tomato plants parasitized by C. pentagona released greater amounts of volatiles than did unparasitized control plants.¹⁹ The production and release of volatiles is a hallmark of plant responses to feeding by herbivores.²⁰ Herbivore-induced volatiles serve as an indirect plant defense by attracting herbivores'' natural enemies,²¹ repelling herbivores,²² or acting as intra-plant signals that prime systemic responses.²³ Although less well documented, pathogen attack can also induce emissions of volatile compounds,²⁴ some of which are antimicrobial and may serve as a direct defense against infection.²⁵ The same volatile compounds induced by Cuscuta (e.g., 2-carene, α-pinene, limonene, β-phellandrene) were also induced by caterpillar feeding and application of JA.¹⁹ Like herbivores, the JA induced by C. pentagona may regulate the emissions of plant volatiles. Whether or how parasitic plant-induced volatiles might function in defense is unknown, but they presumably could affect host plant choice by Cuscuta seedlings, which use plant volatiles to locate and select hosts.²⁶Following on from our previous studies we examined the potential role of host trichomes in resistance to parasitism by C. pentagona. Plant trichomes have been long appreciated as the first line of defense against insect herbivores^27,28 and more recently pathogens.²⁹ We hypothesized that trichomes could also defend against parasitic plants based on our observations that (1) tomato trichomes become denser with age (Fig. 1), notably on hypocotyls which is the first area contacted by Cuscuta seedlings, and (2) these trichomes can act as a physical barrier to C. pentagona seedlings. To test this hypothesis we allowed seedlings of C. pentagona to attach to 25-day-old tomato plants (Solanum lycopersicum ‘Halley 3155’) in a climate controlled growth chamber. Of 20 trials conducted, in six (30%) the parasite seedling was completely blocked by trichomes and was unable to reach the host stem—the parasite perished in each of these. Type I glandular trichomes, which are several millimeters long with a glandular tip,³⁰ were primarily responsible for the blocking effect. Thus, trichomes can defend against parasitic plants in a manner analogous to herbivores by physically obstructing their movement. Interestingly, the effectiveness of trichomes is also dependent on age of the host since those on younger tomato plants (<20 days old) are too sparse to impede Cuscuta seedlings (Fig. 1).Open in a separate windowFigure 1A newly germinated Cuscuta pentagona seedling encircles and attaches to the hypocotyl of a 10-day-old tomato seedling; the early development of haustoria are visible as nod-like swellings. The trichomes on hypocotyls of young tomato seedlings are not dense enough to affect C. pentagona seedlings, but the increased density of trichomes on 25-day-old plants can act as a physical barrier that blocks parasite seedlings (inset).Considering that the majority of plant defenses are mediated by only a small number of master regulators (e.g., JA, SA, ethylene),⁷ it is not surprising that plant responses to parasitic plants share commonalities with those induced by herbivores and pathogens. These few molecules mediate complex, interacting signaling networks that can be variously activated and modified by plants to tune defenses against a seemingly endless variety of attackers.⁷ Our finding that JA and SA act to defend plants from attack by other plants, further support these phytohormones as ‘global’ defense signals. It is also apparent that constitutive defenses, such as trichomes, can be effective against diverse antagonists (e.g., herbivores and parasitic plants). These new insights into host defenses against parasitic plants suggest many avenues of needed research including the molecular events induced by parasitic plant attack, the parasite-derived cues that elicit responses, and the ways in which JA and SA act to reduce parasite growth. Finally, our findings suggest it might be possible to manipulate induced responses or host plant age by varying planting date to control parasitic plants in agriculture.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Duration of Priming of Two Indirect Plant Defenses

When plants are infested by herbivores, they emit herbivore-induced plant volatiles (HIPVs) that attract carnivorous natural enemies of herbivores. Furthermore, there are increasing evidences that defenses of intact plants against herbivores are primed when exposed to HIPVs. We previously reported that lima bean leaf volatiles induced by the herbivorous mites Tetranychus urticae primed two T. urtiae-induced indirect defenses in neighboring conspecific plants: HIPV emission and extrafloral nectar (EFN) secretion. An intriguing unanswered question is whether the durations of these two defenses are the same. Here, we show that the durations of the two defenses were the same for up to two days after the initiation of T. urticae damage. The two induced primed defense would act as a battery of defense in exposed plants.Key Words: herbivore-induced plant volatiles, indirect, defense, induced response, plant-plant interaction, primingWhen infested by herbivores, plants defend themselves indirectly by emitting herbivore-induced plant volatiles (HIPVs). One of the ecological functions of HIPVs is to attract carnivorous natural enemies of the herbivores.^1,2 Recently, it was reported that the emission of HIPVs primed defenses against herbivores in neighboring intact plants.^3–7 Thus, HIPVs also mediate interactions between infested and intact plants.⁸ The enhanced defense in response to HIPVs in intact plants is called ‘priming’, which has been studied intensively in plant-pathogen interactions,⁹ but not so in plant-insect interactions.We previously reported that exposure to HIPVs emitted from lima bean leaves infested by Tetranychus urticae primed HIPV production in detached intact conspecific leaves.³ We also reported that exposure to HIPVs, produced in response to T. urticae damage,⁴ primed the induced production of extrafloral nectar (EFN; an alternative food source for predators^10,11 in lima bean plants. An intriguing question is whether the two primed defenses work as a battery against T. urticae. To answer this, we examined the duration of primed HIPV production by lima bean plants using the same experimental set-up as our previous study of EFN priming by conspecific plants.⁴For exposure of plants to HIPVs, we used a 60 × 60 × 60 cm cage with two 30 × 30 cm windows on opposite sides of the cage.¹² As odor sources, we used eight plants that had been infested with 60 adult T. urticae females per plant for 1 day. Eight uninfested plants were used as control odor sources. Two uninfested plants were placed in a cage with the odor source plants and exposed to either HIPVs or uninfested plant volatiles (UPVs) for 10 days in a climate-controlled room (25 ± 2°C, 60–70% RH, 16:8; L:D).A Y-tube olfactometer¹³ was used to examine the response of the predators to HIPVs. Adult female P. persimilis were randomly selected from a colony and individually positioned at the beginning of the iron wire. When test mites reached the end of one arm of the olfactometer, their choice was recorded. We tested the olfactory responses of the predator toward (1) plants infested by T. urticae for two days after exposure to UPVs vs. plants infested by T. urticae for two days after exposure to HIPVs, and (2) plants infested by T. urticae for four days after exposure to UPVs vs. plants infested by T. urticae for four days after exposure to HIPVs.HIPV-exposed plants attracted more predators than UPV-exposed plants in a Y-tube olfactometer when infested by T. urticae for two days (Fig. 1A). By contrast, the predators did not distinguish between HIPV- and UPV-exposed plants when infested by T. urticae for four days (Fig. 1B). Our previous study showed that HIPV-exposed plants secreted significantly larger amounts of EFN secretion than UPV-exposed plants infested by T. urticae for two days under the same experimental condition as in this study.⁴ However, the difference was not significant when they were infested for four days.⁴Open in a separate windowFigure 1The olfactory response of P. persimilis females to volatiles from the odor-exposed plants, as determined in a Y-tube olfactometer: (A) plants infested by T. urticae for two days after exposure to UPVs (UPV-exposed—T. urticae 2d) vs. plants infested by T. urticae for two days after exposure to HIPVs (HIPV-exposed—T. urticae 2d), and (B) plants infested by T. urticae for four days after exposure to UPVs (UPV-exposed—T. urticae 4d) vs. plants infested by T. urticae for four days after the exposure to HIPVs (HIPV-exposed—T. urticae 4d). Asterisks beside each bar indicate a significant difference between the first trifoliate leaves and the primary leaves. Asterisks beside a bar indicate a significant difference (binomial test: p < 0.001).Lima bean plants increase the amount of endogenous jasmonic acid after exposure to HIPVs.¹⁴ Jasmonic acid, an important plant hormone regulating a defense signaling pathway against herbivores and pathogens,^15,16 is reported to be involved in the induction of both volatile emission^17,18 and EFN secretion¹⁹ in response to T. urticae damage in lima bean plants. The increase of endogenous jasmonic acid in HIPV-exposed plants may partly explain the simultaneous priming of the two defenses.In this study, we showed that the durations of priming of two indirect defenses were roughly the same for up to two days. Priming of these two indirect defenses would thus be a battery of defense at the outset of T. urticae damage. Further study is necessarily to test whether the primed battery of induced defense increases the fitness of the exposed plants.     

Plant defense pathways subverted by Agrobacterium for genetic transformation

The soil phytopathogen Agrobacterium has the unique ability to introduce single-stranded transferred DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the host cell in a process known as horizontal gene transfer. Following its entry into the host cell cytoplasm, the T-DNA associates with the bacterial virulence (Vir) E2 protein, also exported from Agrobacterium, creating the T-DNA nucleoprotein complex (T-complex), which is then translocated into the nucleus where the DNA is integrated into the host chromatin. VirE2 protects the T-DNA from the host DNase activities, packages it into a helical filament and interacts with the host proteins, one of which, VIP1, facilitates nuclear import of the T-complex and its subsequent targeting to the host chromatin. Although the VirE2 and VIP1 protein components of the T-complex are vital for its intracellular transport, they must be removed to expose the T-DNA for integration. Our recent work demonstrated that this task is aided by an host defense-related F-box protein VBF that is induced by Agrobacterium infection and that recognizes and binds VIP1. VBF destabilizes VirE2 and VIP1 in yeast and plant cells, presumably via SCF-mediated proteasomal degradation. VBF expression in and export from the Agrobacterium cell lead to increased tumorigenesis. Here, we discuss these findings in the context of the “arms race” between Agrobacterium infectivity and plant defense.Key words: Arabidopsis, defense response, proteasomal degradation, bacterial infection, F-box proteinAgrobacterium infection of plants consists of a chain of events that usually starts in physically wounded tissue which produces Plant defense pathways subverted by Agrobacterium for genetic transformation small phenolic molecules, such as acetosyringone (AS).¹ These phenolics serve as chemotactic agents and activating signals for the virulence (vir) gene region of the Ti plasmid.^2,3 The vir gene products then process the T-DNA region of the Ti plasmid to a single-stranded DNA molecule that is exported with several Vir proteins into the host cell cytoplasm, in which it forms a the T-DNA nucleoprotein complex (T-complex).^4,5 The plant responds to the coming invasion by expressing and activating several defense-related proteins,⁵ such as VBF⁶ and VIP1,⁷ aimed at suppressing the pathogen. However, the Agrobacterium has evolved mechanisms to take advantage of these host defense proteins.⁸ Some of the unique strategies for achieving this goal include (1) the use of VIP1 to bind the T-complex—via the VIP1 interaction with the T-DNA packaging protein VirE2,^9,10—and assist its nuclear import⁷ and chromatin targeting,¹¹ and (2) the use of VBF to mark VIP1 and its associated VirE2 for proteasomal degradation, presumably for uncoating the T-complex prior to the T-DNA integration into the plant genome.^6,12 Here, we examine these subversion strategies in the context of “arms race” between Agrobacterium and plants.     

Piriformospora indica mycorrhization increases grain yield by accelerating early development of barley plants

Root colonization by the basidiomycete fungus Piriformospora indica induces host plant tolerance against abiotic and biotic stress, and enhances growth and yield. As P. indica has a broad host range, it has been established as a model system to study beneficial plant-microbe interactions. Moreover, its properties led to the assumption that P. indica shows potential for application in crop plant production. Therefore, possible mechanisms of P. indica improving host plant yield were tested in outdoor experiments: Induction of higher grain yield in barley was independent of elevated pathogen levels and independent of different phosphate fertilization levels. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. Analysis of plant development and yield parameters indicated that positive effects of P. indica on grain yield are due to accelerated growth of barley plants early in development.Key words: mycorrhiza, barley development, Piriformospora indica, phosphate uptake, grain yield, pathogen resistanceThe wide majority of plant roots in natural ecosystems is associated with fungi, which very often play an important role for the host plants'' fitness.¹ The widespread arbuscular mycorrhizal (AM) symbiosis formed by fungi of the phylum Glomeromycota is mainly characterized by providing phosphate to their host plant in exchange for carbohydrates.^2,3 Fungi of the order Sebacinales also form beneficial interactions with plant roots and Piriformospora indica is the best-studied example of this group.⁴ This endophyte was originally identified in the rhizosphere of shrubs in the Indian Thar desert,⁵ but it turned out that the fungus colonizes roots of a very broad range of mono- and dicotyledonous plants,⁶ including major crop plants.^7–9 Like other mutualistic endophytes, P. indica colonizes roots in an asymptomatic manner¹⁰ and promotes growth in several tested plant species.^6,11,12 The root endophyte, moreover, enhances yield in barley and tomato and increases in both plants resistance against biotic stresses,^7,9 suggesting that application in agri- and horticulture could be successful.     

Plant Parasitic Oomycetes Such as Phytophthora Species Contain Genes Derived from Three Eukaryotic Lineages

Fungi and the oomycetes include several groups of plant pathogenic microbes. Although these two eukaryotic groups are unrelated they have a number of phenotypic similarities suggested to have evolved convergently. We have recently shown that gene transfer events have occurred from fungi to the oomycetes. These gene transfer events appear to be only one part of a complex and chimeric ancestry for the oomycete genome, which has also received genes from a red algal endosymbiont.Key Words: horizontal gene transfer, osmotrophy, phototrophy, biotrophy, endosymbiosis, fungi, Magnaporthe griseaAs genomic sampling increases, a persistent pattern of horizontal gene transfer (HGT) between microbial lineages is becoming evident.^1,2 So far, patterns of horizontal gene transfer have been identified in four main forms: (A) gene transfer between prokaryote lineages, such that a large proportion of many prokaryote genomes are likely to be chimeric,^3,4 (B) gene transfer from the prokaryote progenitors of the mitochondrion and the plastid organelles to a host eukaryote nuclear genome (e.g., refs. ^5–7), (C) gene transfer from prokaryote genomes to eukaryote microbes, often involving phagocytic eukaryotes and microbes that share similar habitats⁸ and (D) gene transfer from a eukaryotic endosymbiont to their host eukaryotic genomes.^9,10 This fourth form of gene transfer includes secondary and tertiary endosymbiotic events and has so far provided our best examples of eukaryote-to-eukaryote gene transfer.^11,12 Secondary and tertiary endosymbiotic events are typified by the engulfment of a photosynthetic eukaryote by another eukaryote followed by the reduction of the consumed photosynthetic eukaryote and transfer of genes from the endosymbiont to the host nuclei with some retargeting of the transferred gene products back to the remnant organelle.^9,10Gene transfer events can be identified using phylogenetic analysis when an individual gene tree topology contradicts a known species relationship. HGT can only be seriously considered, however, if the gene phylogeny shows that the putative HGT is nested within a donor clade with strong bootstrap support.² Endosymbiosis typically leads to multiple cases of nuclear-encoded genes demonstrating endosymbiotic ancestry, with the candidate genes grouping within a clade representing the lineage that gave rise to the progenitor of the endosymbiont.⁵ There have been multiple cases of both secondary and tertiary endosymbiosis within the eukaryotes, making the evolutionary reconstruction of phototrophy in the eukaryotes highly complex.⁹ Secondary and tertiary endosymbiotic remnant organelles are often identified by the presence of three or more membranes surrounding the organelle body.¹⁰ However, secondary endosymbiotic events have led to a range of different combinations of cell apparatus, from the total loss of the endosymbiont-derived organelle^13,14 to the maintenance of the organelle compartment¹⁰ and the possession of a remnant nucleus as a nucleomorph.¹⁵The oomycetes include the plant pathogenic Phytophthora spp. and are heterokonts (sometimes called Stramenopiles).¹⁶ The heterokonts also encompass numerous groups of photosynthetic algae (e.g., Bolidomonas, Diatoms, Xanthophyceae, Phaeophyceae and Chrysophyceae) and are proposed to be derived from an ancestrally photosynthetic cell that obtained its plastid by engulfment of a red alga.¹⁶ Cytological studies of the oomycetes have so far failed to identify a relic plastid organelle but the recent publication of the Phytophthora sojae and Phytophthora ramorum genomes identified 855 genes putatively originating from the genome of a photosynthetic microbe consistent with a phototrophic ancestry for the oomycetes.¹³Phytophthora plant pathogens include the causal agents of sudden oak death (P. ramorum), potato blight (P. infestans) and, P. sojae which causes serious root and stem rot of soybean plants. Initially, P. infestans was identified as a fungal pathogen and the causal agent of the great 1845 Irish potato famine by Rev. Miles J. Berkeley,¹⁷ due to life cycle similarities and an apparently homologous mode of plant infection to ascomycete plant pathogens. It was only with the use of molecular phylogenetic methods starting with small subunit rDNA analysis¹⁶ followed by multiple concatenated gene phylogenies¹⁸ that the oomycetes were demonstrated to group within the heterokont radiation. With the apparent phylogenetic origins of the oomycetes pinpointed it left the apparent similarities in pathogenic mechanism and infective lifecycle between the filamentous ascomycetes and the oomycetes a mysterious case of convergent evolution.¹⁹During the evolutionary analyses of the predicted proteome of the filamentous plant pathogenic ascomycete Magnaporthe grisea²⁰ we detected a series of unexpected similarities in the genomes of plant pathogenic ascomycetes and the oomycete genomes.¹³ We followed up this observation by further investigation using phylogenetic methods combined with comparative genomic analysis, which revealed a series of HGT events. We subjected our datasets to a range of tests: (A) to test that the level of support for the tree topology seen was robust given random resampling of the sequence alignments used to reconstruct the gene phylogenies; (B) to ensure that the possibility that similar topologies with the oomycete/filamentous ascomycete relationship removed could be rejected at the 0.05 confidence level and; (C) to test for alternative patterns of gene evolution including hidden paralogy (duplication with differential patterns of gene loss) were unlikely. Four of the datasets tested in this way held up to our scrutiny and were thus proposed as fungi-to-oomycete horizontal gene transfers.²¹ The predicted function of three of the four genes (CodB, a purine permease, AraJ, a sugar transporter and a PcaH an extracellular dioxygenase) could conceivably be useful for an osmotrophic microbe living in a plant associated habitat (biotrophy), suggesting that these HGT events could in-part explain the convergently evolved similarities in osmotrophy and filamentous growth habit seen in the oomycetes and fungi. Our analyses also suggested that three of these HGTs originated from a genome closely related to the last common ancestor of the Magnaporthe and Aspergillus evolutionary branches. Although the specific branching position of the transferred lineage could not be pinpointed in the fourth analysis, the same point of origin could not however be excluded. This suggests that the four HGTs we identified could be derived from the same source, a phenomenon similar in pattern (if not involving the same lineages) to that seen for phylogenetic tree topologies used to investigate the endosymbiotic events discussed above. Although these analyses do not shed any light on the circumstances in which these transfers occurred, it is possible that an intimate association between a fungus and a heterokont has led to genetic exchange and demonstrates that eukaryote-to-eukaryote gene transfers are not just associated with the acquisition of phototrophy by secondary/tertiary endosymbiosis.Our published study was conducted using only published genome sequences as a seed for comparative genomic analyses.²¹ However, with the very recent publication of two Phytophthora genomes¹³ it is possible that further analyses will identify additional candidate Phytophthora-Fungi HGT events when they are carried out. These tests may determine how pervasive the pattern of HGT is within the oomycetes.The oomycetes have been classified within the phylum Pseudofungi¹⁶ which comprises a number of microbial lineages with phenotypic similarities to true fungi, including hyphae-like structures and osmotrophy. Originally, the term Pseudofungi was used to group together ‘water-moulds’ possessing mastigonemes (tubular tri-partite hairs) on one flagellum. Currently the phylum Pseudofungi comprises the biotrophic oomycetes including parasites of plants and brown algae, the phagotrophic Developayella and the biotrophic hyphochytrids, including the diatom ectoparasite Pirsonia.¹⁶ It will be interesting to ascertain at what point within the diversification of the Pseudofungi the HGTs that are identified²¹ became fixed and how the acquisition of these phenotypes relates to the evolution of Pseudofungi phenotypes within the heterokonts. Independent of the specific ancestry of the gene transfer events within the Pseudofungi it is clear that P. sojae and P. ramorum have chimeric genomes, originating from three separate eukaryotic lineages, the ancestral heterokont nuclear genome, the red algal endosymbiont and at least four genes of fungal ancestry donated to an oomycete nuclear genome.     

Emerging roles in plant defense for cis-jasmone-induced cytochrome P450 CYP81D11

Cis-jasmone is a volatile organic compound emitted constitutively by flowers or leaves of several plant species where it acts as an attractant for pollinators and as a chemical cue for host localization (or avoidance) for insects.^1–3 It is also released by some plant species after feeding damage inflicted by herbivorous insects and in this case might serve as a chemical cue for parasitoids to guide them to their prey (so called “indirect defense”).^4,5 Moreover, we have recently shown that plants can perceive cis-jasmone and that it acts as a signaling molecule in A. thaliana, inducing a discrete and distinctive suite of genes, of which a large subset is putatively involved in metabolism and defense responses.⁶ Cytochrome P450s feature prominently in these functional subsets and of these the highest fold change upon cis-jasmone treatment occurred with the cytochrome CYP81D11 (At3g28740).⁶ Hence this gene was chosen for a more thorough analysis of the potential biological relevance of the cis-jasmone induced defense response. Although the precise function of CYP81D11 remains to be determined, we could previously demonstrate its involvement in the indirect defense response in Arabidopsis, as plants exposed to cis-jasmone ceased to be attractive to the aphid parasitoid Aphidius ervi when this P450 was inactivated by T-DNA insertion mutagenesis.⁶ Here we report additional experiments which give further support to a role of CYP81D11 in the direct or indirect defense response of A. thaliana.Key words: cis-Jasmone, Cytochrome P450, indirect defense, tritrophic interactions, volatile signaling     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Piriformospora indica enhances plant growth by transferring phosphate

Piriformospora indica is an endophytic fungus that colonized monocot as well as dicot. P. indica has been termed as plant probiotic because of its plant growth promoting activity and its role in enhancement of the tolerance of the host plants against abiotic and biotic stresses. In our recent study, we have characterized a high affinity phosphate transporter (PiPT) and by using RNAi approach, we have demonstrated the involvement of PiPT in P transfer to the host plant. When knockdown strains of PiPT-P. indica was colonized with the host plant, it resulted in the impaired growth of the host plants. Here we have analyzed and discussed whether the growth promoting activity of P. indica is its intrinsic property or it is dependent on P availability. Our data explain the correlation between the availability of P and growth-promoting activity of P. indica.Key words: Piriformospora indica, phosphate transport, plant growth promotionPhosphorous (P) is one of the most essential mineral nutrients for plant growth and development. In the soil P is present mainly in the form of sparingly soluble complexes that are not directly accessible to plants. Thus, it is the nutrient that limits crop production throughout the world.¹ Plants have therefore evolved a range of strategies to increase the availability of soil P, which include both morphological and biochemical changes at the soil-root interface. For example, increased root growth and branching, proliferation of root hairs, and release of root exudates can increase plant access to inorganic phosphate (Pi) from otherwise poorly available sources.^2,3 Plant root possess two distinct modes of phosphate uptake, direct uptake by its own transporters and indirect uptake through mycorrhizal associations. In plants several high affinity P transporters specifically associated with the uptake of Pi from soil solution. Expression of these transporters is induced in response to P deficiency and enables Pi to be effectively taken up against the large concentration gradient that occurs between the soil solution and internal plant tissues.⁴ However, in arbuscular mycorrhizal associations (indirect uptake), plants acquire Pi from the extensive network of fine extra radical hyphae of fungus, that extend beyond root depletion zones to mine new regions of the soil.⁵ In the case of arbuscular mycorrhizal fungi (AMF), including Glomus versiforme and G. intraradices, the regulation of phosphate transporters that are expressed, typically upregulated under P deficiency but their role in P transfer to the host plant have not been characterized.^5,6P. indica was reported to be involved in high salt tolerance, disease resistance and strong growth-promoting activities leading to enhancement of host plant yield.^7–9 Recently, we have shown the role of PiPT in the P transport to the host plant.¹⁰ Here we discuss the performance of P. indica (grown under P-rich and -deprived conditions and colonized with the host plant) and its involvement in the P transportation to, and the growth of the host plant.     

Filamentous brown algae infected by the marine,holocarpic oomycete Eurychasma dicksonii: first results on the organization and the role of cytoskeleton in both host and parasite

The important role of the cytoskeletal scaffold is increasingly recognized in host-pathogen interactions. The cytoskeleton potentially functions as a weapon for both the plants defending themselves against fungal or oomycete parasites, and for the pathogens trying to overcome the resisting barrier of the plants. This concept, however, had not been investigated in marine algae so far. We are opening this scientific chapter with our study on the functional implications of the cytoskeleton in 3 filamentous brown algal species infected by the marine oomycete Eurychasma dicksonii. Our observations suggest that the cytoskeleton is involved in host defense responses and in fundamental developmental stages of E. dicksonii in its algal host.Oomycetes are important plant and animal pathogens and are the cause of significant crop losses every year. Hence, a plethora of studies with different cultivated and model plant species investigate the diversity of parasite infection pathways and host defense responses.¹ However, little information is available on the interactions between algae and marine oomycetes, despite the epidemic outbreaks reported² and the huge impact on intensive algal aquaculture.³Eurychasma dicksonii is a biotrophic, intracellular marine oomycete, capable to infect at least 45 species of brown seaweeds in laboratory cultures.⁴ Molecular data reveal that E. dicksonii has a basal phylogenetic position in the oomycete lineage.^5,6 The basic stages of the infection are known: the attachment of the parasite spore to the host cell wall, the penetration of its cytoplasm into the host cell, the formation of a multinucleated, unwalled thallus, and zoosporogenesis.⁶ Hitherto, though, there was no knowledge about the role of cytoskeleton in the context of infection, which stimulated our research.In land plants, reorganization of the cytoskeleton is part of the reaction to infection by fungal pathogens. The rearrangement of the cytoplasm and the relocation of the nuclei and other organelles are accompanied by rapid rearrangements of the cytoskeletal elements.⁷ The plant cytoskeleton shows an extreme plasticity in order to serve the intracellular realignment.At the same time, this indicates that the plant cytoskeleton could be the parasite’s target by producing anti-cytoskeletal compounds in an effort to overcome plant resistance, a mechanism known in several fungal and oomycete pathogens of higher plants.^8,9Consequently, the changes in microtubule (MT) organization are associated with both the plant defense and/or susceptibility toward oomycetes, respectively.¹⁰ Therefore, our research on the organization and role of cytoskeleton in the host and the parasite sheds some light into the enormous variability in the specificity of the recognition, defense, and infection mechanisms.     

Historical Overview on Plant Neurobiology

The review tracks the history of electrical long-distance signals from the first recordings of action potentials (APs) in sensitive Dionea and Mimosa plants at the end of the 19^th century to their re-discovery in common plants in the 1950''s, from the first intracellular recordings of APs in giant algal cells to the identification of the ionic mechanisms by voltage-clamp experiments. An important aspect is the comparison of plant and animal signals and the resulting theoretical implications that accompany the field from the first assignment of the term “action potential” to plants to recent discussions of terms like plant neurobiology.Key Words: action potentials, slow wave potentials, plant nerves, plant neurobiology, electrical signaling in plants and animailsFor a long time plants were thought to be living organisms whose limited ability to move and respond was appropriately matched by limited abilities of sensing.¹ Exceptions were made for plants with rapid and purposeful movements such as Mimosa pudica (also called the sensitive plant), Drosera (sundews), Dionea muscipula (flytraps) and tendrils of climbing plants. These sensitive plants attracted the attention of outstanding pioneer researchers like Pfeffer,^2,3 Burdon-Sanderson,^4,5 Darwin,⁶ Haberlandt^7–9 and Bose.^10–13 They found them not only to be equipped with various mechanoreceptors exceeding the sensitivity of a human finger but also to trigger action potentials (APs) that implemented these movements.The larger field of experimental electrophysiology started with Luigi Galvani''s discovery of “animal electricity” or contractions of isolated frog legs suspended between copper hooks and the iron grit of his balcony.¹⁴ It soon became clear that the role of the electric current was not to provide the energy for the contraction but to simulate a stimulus that existed naturally in the form of directionally transmitted electrical potentials. Studies by both Matteucci and Du Bois-Reymond¹⁵ recognized that wounding of nerve strands generated the appearance of a large voltage difference between the wounded (internal) and intact (external) site of nerves. This wound or injury potential was the first, crude measurement of what later became known as membrane or resting potential of nerve cells. It was also found that various stimuli reduced the size of the potential (in modern terms: they caused a depolarization), and to describe the propagating phenomenon novel terms such as action potential (AP) and action current were created (reviewed in refs. ¹⁵ and ¹⁶). Rather than relying on such indirect methods, the membrane theory of exicitation proposed by Bernstein in 1912¹⁷ made it desirable to directly measure the value of cell membrane potentials. Such progress soon became possible by the introduction of microelectrodes (KCl-filled glass micropipettes with a tip diameter small enough to be inserted into living cells) to record intracellular, i.e., the real membrane potentials (V_m). The new technique was simultaneously adopted for giant cells (axons) of cephalopods such as Loligo and Sepia¹⁸ and giant internodal cells of Charophytic green algae. In the 1930s Umrath and Osterhout^19–21 not only made the first reliable, intracellular measurements of membrane potentials in plant cells (reporting V_m values between −100 to −170 mV) but the first intracellular recordings of plant APs as well. When this technique was complemented with precise electronic amplifiers and voltage clamp circuits in the 1940s, one could measure ion currents (instead of voltages) and so directly monitor the activity of ion channels. The smart application of these methods led to a new, highly detailed understanding of the ionic species and mechanisms involved in V_m changes, especially APs.^22–27 Whereas the depolarizing spike in animal nerve cells is driven by an increased influx of Na⁺ ions, plant APs were found to involve influx of Ca²⁺ and/or efflux of Cl⁻¹ ions.The first extracellular recording of a plant AP was initiated by Charles Darwin and performed on leaves of the Venus flytrap (Dionea muscipula Ellis) by the animal physiologist Burdon-Sanderson in 1873.^4–6 Ever since APs have often been considered to fulfil comparable roles in plants and nerve-muscle preparations of animals. However, this was never a generally accepted view. While it is commonly assumed that the AP causes the trap closure, this had not been definitely shown (see refs. ²⁸ and ²⁹). Kunkel (1878) and Bose (1907, 1926) measured action spikes also in Mimosa plants where they preceded the visible folding movements of the leaflets.^{12–13,30–31} Dutrochet and Pfeffer^2–3 had already found before that interrupting vascular bundles by incision prevented the excitation from propagating beyond the cut and concluded that the stimulus must move through the vascular bundles, in particular the woody or hadrome part (in modern terms the xylem). Haberlandt⁷ cut or steam-killed the external, nonwoody part of the vascular bundles and concluded that the phloem strands were the path for the excitation, a notion which is confirmed by a majority of recent studies in Mimosa and other plant species. APs have their largest amplitude near and in the phloem and there again in the sieve cells.^{23–24,32–35} Moreover, APs can be recorded through the excised stylets of aphids known to be inserted in sieve tube elements.^36–37 Other studies found that AP-like signals propagate with equal rate and amplitude through all cells of the vascular bundle.³⁸ Starting studies with isolated vascular bundles (e.g., from the fern Adiantum), Bose found increasing amplitudes of heat-induced spikes by repeated stimulation (tetanisation) and incubation in 0.5 % solution of sodium carbonate.^10–13 Since the electrical behavior of isolated vascular strands was comparable to that of isolated frog nerves, Bose felt justified to refer to them as plant nerves.Although at the time a hardly noticed event, the discovery that normal plants such as pumpkins had propagating APs just as the esoteric “sensitive” plants was a scientific breakthrough with important consequences.^39–40,32 First, it corrected the long-held belief that normal plants are simply less sensitive and responsive than the so-called “sensitive plants” from Mimosa to Venus flytraps. Second, it led to the stimulating belief that so widely distributed electric signals must carry important messages.⁴¹ The ensuing studies made considerable progress in linking electrical signals with respiration and photosynthesis,^40–42 pollination,^43–44 phloem transport^{33,36–37,45} and the rapid, plant-wide deployment of plant defenses.^46–53The detailed visualization of nerve cells with silver salts by the Spanish zoologist S. Ramon y Cajal, the demonstrated existence of APs in Dionea and Mimosa as well as the discovery of plant mechanoreceptors in these and other plants⁹ at the end of the century was sufficient stimulation to start a search for structures that could facilitate the rapid propagation of these and other excitation signals. Researchers began to investigate easily stainable intracellular plasma strands that run across the lumen of many plant cells, and sometimes even continue over several cells for their potential role as nerve-like, excitation-conducting structures. Such strands were shown to occur in traumatized areas of many roots⁵⁴ and in insectivorous butterworts where they connect the glue-containing hair tips with the basal peptidase-producing glands of the Pinguicula leaves.^55–56 However, after investigating these claims, Haberlandt came to the conclusion that the only nerve-like structures of plants were situated the long phloem cells of the vascular bundles.^7–8 From that time on papers, lectures and textbooks reiterated statements that “plants have no nerves”.This unproductive expression ignores the work of Darwin, Haberlandt, Pfeffer and Bose together with the fact that in spite of their anatomical differences, nerve cell networks and vascular bundles share the analog function of conducting electrical signals. Similar anatomical differences have not been an obstacle to stating that both plants and animals consist of cells. The mechanistic similarity of excitations (consisting of a transient decline in cell input resistance) in plant and nerve cells was later elegantly demonstrated by the direct comparison of action potentials in Nitella and the giant axon of squids.^57–58 Today, consideration of nerve-like structures in plants involves increasingly more aspects of comparison. We know that many plants can efficiently produce electric signals in the form of action potentials and slow wave potentials (= variation potentials) and that the long-distance propagation of these signals proceeds in the vascular bundles. We also know that plants like Dionea can propagate APs with high efficiency and speed without the use of vascular bundles, probably because their cells are electrically coupled through plasmodesmata. Other analogies with neurobiology include vesicle-operated intercellular clefts in axial root tissues (the so-called plant synapses)⁵⁹ as well as the certain existence and operation of substances like neurotransmitters and synaptotagmins in plant cells (e.g., refs. ⁶⁰ and ⁶¹). The identification of the role(s) of these substances in plants will have important implications. Altogether, modern plant neurobiology might emerge as a coherent science.⁶²Electrophysiological and other studies of long-distance signals in plants and animals greatly contributed to our knowledge of the living world by revealing important similarities and crucial differences between plants and animals in an area that might directly relate to their different capacities to respond to environmental signals. Even at this stage the results are surprising. Rather than lacking electric signals, higher plants have developed more than just one signal type that is able to cover large distances. In addition to APs that occur also in animals and lower plants,⁶³ higher plants feature an additional, unique, hydraulically propagated type of electric signals called slow wave potentials.⁶⁴