Mechanisms in Growth-Promoting of Cucumber by the Endophytic Fungus Chaetomium globosum Strain ND35.

Endophytic fungi are effective in plant growth and development by secreting various kinds of plant hormones and nutrients. However, the cellular and molecular interactions between the endophytic fungi and plant growth-promoting have remained less explored. The present study was designed to explore the effects of the infection and colonization events of Chaetomium globosum strain ND35 on cucumber growth and the expression pattern of some metabolically important genes in development of the cucumber radicle. The results demonstrated that strain ND35 can infect and colonize the outer layers (cortical cells) of cucumber root and form a symbiotic structure with the host cell, similar to a periarbuscular membrane and establish chemical communication with the plant. Through transcriptome analysis, we found the differentially expressed genes (DEGs) caused by strain ND35 were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, plant-pathogen interaction and photosynthesis. Correspondingly, the contents of reactive oxygen species (ROS), hydrogen peroxide (H2O2), indole-3-acetic acid (IAA), gibberellin (GA), zeatin (ZT), salicylic acid (SA), jasmonic acid (JA) and the activity of phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) in ND35-colonized seedlings were generally higher than those of non-inoculated seedlings. Overall, the infection and colonization events of C. globosum strain ND35 increased cucumber growth through complex regulation of plant hormones biosynthesis and metabolism. Furthermore, although the endophytic fungus strain ND35 produced IAA, GA, ZT, and ergosterol in the fermentation broth, and there are enabled to promote growth of cucumber, it is uncertain whether there are ND35-derived microbial hormones in plants. This study of the interaction between cucumber and strain ND35 contributes to a better understanding of the plant-endophytic fungi interactions, and may help to develop new strategies for crop production.

Small One-Helix Proteins Are Essential for Photosynthesis in Arabidopsis.

The extended superfamily of chlorophyll a/b binding proteins comprises the Light-Harvesting Complex Proteins (LHCs), the Early Light-Induced Proteins (ELIPs) and the Photosystem II Subunit S (PSBS). The proteins of the ELIP family were proposed to function in photoprotection or assembly of thylakoid pigment-protein complexes and are further divided into subgroups with one to three transmembrane helices. Two small One-Helix Proteins (OHPs) are expressed constitutively in green plant tissues and their levels increase in response to light stress. In this study, we show that OHP1 and OHP2 are highly conserved in photosynthetic eukaryotes, but have probably evolved independently and have distinct functions in Arabidopsis. Mutations in OHP1 or OHP2 caused severe growth deficits, reduced pigmentation and disturbed thylakoid architecture. Surprisingly, the expression of OHP2 was severely reduced in ohp1 T-DNA insertion mutants and vice versa. In both ohp1 and ohp2 mutants, the levels of numerous photosystem components were strongly reduced and photosynthetic electron transport was almost undetectable. Accordingly, ohp1 and ohp2 mutants were dependent on external organic carbon sources for growth and did not produce seeds. Interestingly, the induction of ELIP1 expression and Cu/Zn superoxide dismutase activity in low light conditions indicated that ohp1 mutants constantly suffer from photo-oxidative stress. Based on these data, we propose that OHP1 and OHP2 play an essential role in the assembly or stabilization of photosynthetic pigment-protein complexes, especially photosystem reaction centers, in the thylakoid membrane.

Altered levels of LIL3 isoforms in Arabidopsis lead to disturbed pigment-protein assembly and chlorophyll synthesis, chlorotic phenotype and impaired photosynthetic performance.

Light-harvesting complex (LHC)-like (LIL) proteins contain two transmembrane helices of which the first bears a chlorophyll (Chl)-binding motif. They are widespread in photosynthetic organisms, but almost nothing is known about their expression and physiological functions. We show that two LIL3 paralogues (LIL3:1 and LIL3:2) in Arabidopsis thaliana are expressed in photosynthetically active tissues and their expression is differentially influenced by light stress. Localization studies demonstrate that both isoforms are associated with subcomplexes of LHC antenna of photosystem II. Transgenic plants with reduced amounts of LIL3:1 exhibited a slightly impaired growth and have reduced Chl and carotenoid contents as compared to wild-type plants. Ectopic overexpression of either paralogue led to a developmentally regulated switch to co-suppression of both LIL3 isoforms, resulting in a circular chlorosis of the leaf rosettes. Chlorotic sectors show severely diminished levels of LIL3 isoforms and other proteins, and thylakoid morphology was changed. Additionally, the levels of enzymes involved in Chl biosynthesis are altered in lil3 mutant plants. Our data support a role of LIL3 paralogues in the regulation of Chl biosynthesis under light stress and under standard growth conditions as well as in a coordinated ligation of newly synthesized and/or rescued Chl molecules to their target apoproteins.

The haustorial transcriptomes of Uromyces appendiculatus and Phakopsora pachyrhizi and their candidate effector families.

Haustoria of biotrophic rust fungi are responsible for the uptake of nutrients from their hosts and for the production of secreted proteins, known as effectors, which modulate the host immune system. The identification of the transcriptome of haustoria and an understanding of the functions of expressed genes therefore hold essential keys for the elucidation of fungus-plant interactions and the development of novel fungal control strategies. Here, we purified haustoria from infected leaves and used 454 sequencing to examine the haustorial transcriptomes of Phakopsora pachyrhizi and Uromyces appendiculatus, the causal agents of soybean rust and common bean rust, respectively. These pathogens cause extensive yield losses in their respective legume crop hosts. A series of analyses were used to annotate expressed sequences, including transposable elements and viruses, to predict secreted proteins from the assembled sequences and to identify families of candidate effectors. This work provides a foundation for the comparative analysis of haustorial gene expression with further insights into physiology and effector evolution.

A novel structural effector from rust fungi is capable of fibril formation.

It has been reported that filament-forming surface proteins such as hydrophobins are important virulence determinants in fungi and are secreted during pathogenesis. Such proteins have not yet been identified in obligate biotrophic pathogens such as rust fungi. Rust transferred protein 1 (RTP1p), a rust protein that is transferred into the host cytoplasm, accumulates around the haustorial complex. To investigate RTP1p structure and function, we used immunocytological, biochemical and computational approaches. We found that RTP1p accumulates in protuberances of the extra-haustorial matrix, a compartment that surrounds the haustorium and is separated from the plant cytoplasm by a modified host plasma membrane. Our analyses show that RTP1p is capable of forming filamentous structures in vitro and in vivo. We present evidence that filament formation is due to ß-aggregation similar to what has been observed for amyloid-like proteins. Our findings reveal that RTP1p is a member of a new class of structural effectors. We hypothesize that RTP1p is transferred into the host to stabilize the host cell and protect the haustorium from degradation in later stages of the interaction. Thus, we provide evidence for transfer of an amyloid-like protein into the host cell, which has potential for the development of new resistance mechanisms against rust fungi.

The rust transferred proteins-a new family of effector proteins exhibiting protease inhibitor function.

Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N-terminus and a conserved and structured C-terminus. The characterization of Uf-RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C-terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf-RTP1p and Us-RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi-specific family of modular effector proteins comprising an unstructured N-terminal domain and a structured C-terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.

Niche differentiation of two sympatric species of Microdochium colonizing the roots of common reed.

BACKGROUND: Fungal endophyte communities are often comprised of many species colonizing the same host. However, little is known about the causes of this diversity. On the one hand, the apparent coexistence of closely related species may be explained by the traditional niche differentiation hypothesis, which suggests that abiotic and/or biotic factors mediate partitioning. For endophytes, such factors are difficult to identify, and are therefore in most cases unknown. On the other hand, there is the neutral hypothesis, which suggests that stochastic factors may explain high species diversity. There is a need to investigate to what extent each of these hypotheses may apply to endophytes. RESULTS: The niche partitioning of two closely related fungal endophytes, Microdochium bolleyi and M. phragmitis, colonizing Phragmites australis, was investigated. The occurrences of each species were assessed using specific nested-PCR assays for 251 field samples of common reed from Lake Constance, Germany. These analyses revealed niche preferences for both fungi. From three niche factors assessed, i.e. host habitat, host organ and season, host habitat significantly differentiated the two species. M. bolleyi preferred dry habitats, whereas M. phragmitis prevailed in flooded habitats. In contrast, both species exhibited a significant preference for the same host organ, i.e. roots. Likewise the third factor, season, did not significantly distinguish the two species. Differences in carbon utilization and growth temperature could not conclusively explain the niches. The inclusion of three unrelated species of Ascomycota, which also colonize P. australis at the same locations, indicated spatio-temporal niche partitioning between all fungi. None of the species exhibited the same preferences for all three factors, i.e. host habitat, host organ, and time of the season. CONCLUSIONS: The fungal species colonizing common reed investigated in this study seem to exploit niche differences leading to a separation in space and time, which may allow for their coexistence on the same host. A purely neutral model is unlikely to explain the coexistence of closely related endophytes on common reed.

Immunolocalization of pathogen effectors.

The use of polyclonal antibodies enables the detection of proteins on a cellular and even subcellular level. Immunolocalization can be used on all pathosystems even if one or both partners of the interaction are unamenable to molecular tools like transformation. This chapter provides detailed information about how to obtain high quality antibodies, how to prepare samples, and finally how to detect the proteins. Methods for light and electron microscopy are presented.

Lack of evidence for a role of hydrophobins in conferring surface hydrophobicity to conidia and hyphae of Botrytis cinerea.

BACKGROUND: Hydrophobins are small, cysteine rich, surface active proteins secreted by filamentous fungi, forming hydrophobic layers on the walls of aerial mycelia and spores. Hydrophobin mutants in a variety of fungi have been described to show 'easily wettable' phenotypes, indicating that hydrophobins play a general role in conferring surface hydrophobicity to aerial hyphae and spores. RESULTS: In the genome of the grey mould fungus Botrytis cinerea, genes encoding three hydrophobins and six hydrophobin-like proteins were identified. Expression analyses revealed low or no expression of these genes in conidia, while some of them showed increased or specific expression in other stages, such as sclerotia or fruiting bodies. Bhp1 belongs to the class I hydrophobins, whereas Bhp2 and Bhp3 are members of hydrophobin class II. Single, double and triple hydrophobin knock-out mutants were constructed by consecutively deleting bhp1, bhp2 and bhp3. In addition, a mutant in the hydrophobin-like gene bhl1 was generated. The mutants were tested for germination and growth under different conditions, formation of sclerotia, ability to penetrate and infect host tissue, and for spore and mycelium surface properties. Surprisingly, none of the B. cinerea hydrophobin mutants showed obvious phenotypic defects in any of these characters. Scanning electron microscopy of the hydrophobic conidial surfaces did not reveal evidence for the presence of typical hydrophobin 'rodlet' layers. CONCLUSIONS: These data provide evidence that in B. cinerea, hydrophobins are not involved in conferring surface hydrophobicity to conidia and aerial hyphae, and challenge their universal role in filamentous fungi. The function of some of these proteins in sclerotia and fruiting bodies remains to be investigated.

The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea.

In all fungi studied so far, mitogen-activated protein (MAP) kinase cascades serve as central signalling complexes that are involved in various aspects of growth, stress response and infection. In this work, putative components of the yeast Fus3/Kss1-type MAP kinase cascade and the putative downstream transcription factor Ste12 were analysed in the grey mould fungus Botrytis cinerea. Deletion mutants of the MAP triple kinase Ste11, the MAP kinase kinase Ste7 and the MAP kinase adaptor protein Ste50 all resulted in phenotypes similar to that of the previously described BMP1 MAP kinase mutant, namely defects in germination, delayed vegetative growth, reduced size of conidia, lack of sclerotia formation and loss of pathogenicity. Mutants lacking Ste12 showed normal germination, but delayed infection as a result of low penetration efficiency. Two differently spliced ste12 transcripts were detected, and both were able to complement the ste12 mutant, except for a defect in sclerotium formation, which was only corrected by the full-sized transcript. Overexpression of the smaller ste12 transcript resulted in delayed germination and strongly reduced infection. Bc-Gas2, a homologue of Magnaporthe grisea Gas2 that is required for appressorial function, was found to be non-essential for growth and infection, but its expression was under the control of both Bmp1 and Ste12. In summary, the role and regulatory connections of the Fus3/Kss1-type MAP kinase cascade in B. cinerea revealed both common and unique properties compared with those of other plant pathogenic fungi, and provide evidence for a regulatory link between the BMP1 MAP kinase cascade and Ste12.

Mycoparasitism of endophytic fungi isolated from reed on soilborne phytopathogenic fungi and production of cell wall-degrading enzymes in vitro.

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of beta-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of beta-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.

Host plant development, water level and water parameters shape Phragmites australis-associated oomycete communities and determine reed pathogen dynamics in a large lake.

In a 3-year-study, we analysed the population dynamics of the reed pathogen Pythium phragmitis and other reed-associated oomycetes colonizing fresh and dried reed leaves in the littoral zone of a large lake. Oomycete communities derived from internal transcribed spacer clone libraries were clearly differentiated according to substrate and seasonal influences. In fresh leaves, diverse communities consisting of P. phragmitis and other reed-associated pathogens were generally dominant. Pythium phragmitis populations peaked in spring with the emergence of young reed shoots, and in autumn after extreme flooding events. In summer it decreased with falling water levels, changing water chemistry and rising temperatures. Another Pythium species was also highly abundant in fresh leaves throughout the year and might represent a new, as-yet uncultured reed pathogen. In dried leaves, reed pathogens were rarely detected, whereas saprophytic species occurred abundantly during all seasons. Saprophyte communities were less diverse, less temperature sensitive and independent of reed development. In general, our results provide evidence for the occurrence of highly specialized sets of reed-associated oomycetes in a natural reed ecosystem. Quantitative analyses (clone abundances and quantitative real-time PCR) revealed that the reed pathogen P. phragmitis is particularly affected by changing water levels, water chemistry and the stage of reed development.

Colonization of barley roots by endophytic fungi and their reduction of take-all caused by Gaeumannomyces graminis var. tritici.

Fungal root endophytes obtained from natural vegetation were tested for antifungal activity in dual culture tests against the root pathogen Gaeumannomyces graminis var. tritici. Fifteen isolates, including Acremonium blochii, Acremonium furcatum, Aspergillus fumigatus, Cylindrocarpon sp., Cylindrocarpon destructans, Dactylaria sp., Fusarium equiseti, Phoma herbarum, Phoma leveillei, and a sterile mycelium, selected based on the dual culture test, were inoculated on barley roots in growth tubes under axenic conditions, both in the absence and presence of G. graminis var. tritici. All isolates colonized the rhizosphere and very often the root cortex without causing disease symptoms and without affecting plant growth. Eight isolates significantly reduced the symptoms caused by G. graminis var. tritici, and 6 of them reduced its presence in the roots.

Diversity, host, and habitat specificity of oomycete communities in declining reed stands (Phragmites australis) of a large freshwater lake.

We studied the diversity of oomycetes in a declining reed belt (Phragmites australis) of Lake Constance, Germany, using conventional baiting with specific reed and standard oak baits, accompanied by molecular techniques. Apart from an Aphanomyces sp. and a Phytophthora sp., baiting from reed rhizosphere samples from flooded, as well as drier, littoral sites revealed only Pythium spp. A total of 67 oomycete isolates was classified according to PCR-RFLP banding patterns and ITS sequencing, and 18 different sequence types could be separated. The majority of these seemed previously unknown species, as indicated by the degree of similarity to those deposited in nucleotide databases. Species communities in both flooded and drier habitats or both reed and oak baits clearly differed from one another, and only few species occurred in both dry and flooded sites, or in both oak and reed baits. A frequently occurring group of related Pythium species appeared to be specifically associated with reed, and these were the only species that proved pathogenic towards this host in vitro. Our study proved that unexplored natural ecosystems harbour diverse communities of oomycete species with specific habitat and host preferences within close-by, but ecologically contrasting, sites. Among the species isolated, those associated with the predominating plant might accumulate and thus may be reed pathogens of considerable importance.

Volatiles modulate the development of plant pathogenic rust fungi.

Rust fungi are obligate biotrophic pathogens that differentiate a series of specialized cells to establish infection. One of these cells, the haustorium, which serves to absorb nutrients from living host cells, normally develops only in planta. Here, we show that the rust fungus Uromyces fabae (Pers.) Schroet. stimulates volatile emission of its host, broad bean (Vicia faba L.). Volatiles were identified and shown to be perceived by the fungus in in vitro assays that excluded the host. Three of them, nonanal, decanal, and hexenyl acetate promoted the development of haustoria on artificial membranes. In contrast, the terpenoid farnesyl acetate suppressed this differentiation. In assays using whole plants, farnesyl acetate reduced rust disease not only on broad bean but also on several cereals and legumes including soybean. This natural substance was effective against all rusts tested when directly applied to the host. This demonstrated that farnesyl acetate may serve as a powerful novel tool to combat rust fungi including Phakopsora pachyrhizi that currently threatens the production of soybeans world-wide.

Cloning and characterization of a novel invertase from the obligate biotroph Uromyces fabae and analysis of expression patterns of host and pathogen invertases in the course of infection.

Invertases are key enzymes in carbon partitioning in higher plants. They gain additional importance in the distribution of carbohydrates in the event of wounding or pathogen attack. Although many researchers have found an increase in invertase activity upon infection, only a few studies were able to determine whether the source of this activity was host or parasite. This article analyzes the role of invertases involved in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant, Vicia faba. We have identified a fungal gene, Uf-INV1, with homology to invertases and assessed its contribution to pathogenesis. Expression analysis indicated that transcription began upon penetration of the fungus into the leaf, with high expression levels in haustoria. Heterologous expression of Uf-INV1 in Saccharomyces cerevisiae and Pichia pastoris allowed a biochemical characterization of the enzymatic activity associated with the secreted gene product INV1p. Expression analysis of the known vacuolar and cell-wall-bound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cell-wall-associated invertase exhibited increased expression. These changes were not confined to the infected tissue, and effects also were observed in remote plant organs, such as roots. These findings hint at systemic effects of pathogen infection. Our results support the hypothesis that pathogen infection establishes new sinks which compete with physiological sink organs.

Only a few fungal species dominate highly diverse mycofloras associated with the common reed.

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.

Pythium litorale sp. nov., a new species from the littoral of Lake Constance, Germany.

A description is given of Pythium litorale sp. nov., a new species from reed stands in Germany. Pythium litorale was among the most abundant species when the oomycete community of littoral soils of Lake Constance was studied. It was consistently isolated from flooded as well as from drier reed sites. The species is characterized by subglobose, papillate and internally proliferating sporangia, globose hyphal swellings, the absence of oogonia in single culture and a high optimum growth temperature. It proved to be nonpathogenic to Phragmites australis, the predominating plant in the investigated sites. Molecular analysis of ribosomal DNA internal transcribed spacer regions placed Pythium litorale in a clade together with its closest relatives Pythium megacarpum, Pythium boreale, Pythium montanum and Pythium carbonicum. The generic status of this basal clade in Pythium is currently under discussion, as it possibly represents a separate genus that is distinct from Pythium, and shares several characteristics with Phytophthora.

Microarray analysis of expressed sequence tags from haustoria of the rust fungus Uromyces fabae.

Rust fungi are plant parasites which colonise host tissue with an intercellular mycelium that forms haustoria within living plant cells. To identify genes expressed during biotrophic growth, EST sequencing was performed with a haustorium-specific cDNA library from Uromyces fabae. One thousand seventeen ESTs were generated, which assembled into 530 contigs. Several of the most frequently represented sequences in the EST database were identical to the in planta induced genes (PIGs) identified previously (Hahn, M., Mendgen, K., 1997. Characterisation of in planta-induced rust genes isolated from a haustorium-specific cDNA library, Mol. Plant-Microbe Interact. 10, 427-437). Virus-encoded sequences were identified, providing evidence for two novel RNA mycoviruses in U. fabae. Microarray hybridisation revealed many cDNAs that were significantly activated in rust-infected leaves compared to germinated uredospores. Very strong in planta expression was found for two PIGs encoding putative metallothioneins. Furthermore, several genes involved in ribosome biogenesis and translation, glycolysis, amino acid metabolism, stress response, and detoxification showed an increased expression in the parasitic mycelium. These data indicate a strong shift in gene expression in rust fungi between germination and the biotrophic stage of development.