Effect of formulated root endophytic fungus Piriformospora indica and plant growth promoting rhizobacteria fluorescent pseudomonads R62 and R81 on Vigna mungo

In the present investigation, the effect of three beneficial organisms (root endophytic fungus Piriformospora indica (Pi) and pseudomonads strains R62 and R81) and their four different consortia (Pi+R62, Pi+R81, R62+R81, Pi+R62+R81) was investigated on the plant Vigna mungo through their inorganic carrier-based (talcum powder and vermiculite) formulations. All the treatments resulted in significant increase in growth parameters under glasshouse as well as field conditions and showed a consistency in their performance on moving from glasshouse to field conditions. In glasshouse conditions, a maximum increase of 4.5-fold in dry root weight and 3.9-fold in dry shoot weight compared to control was obtained with vermiculite-based consortium formulation of Pi+R81. In field studies using vermiculite as carrier, a maximum enhancement of 3.2-fold in dry root weight, 3.0-fold in dry shoot weight, 8.4-fold in number of nodules and 4.0-fold in number of pods in comparison to control was obtained with the bio-inoculant formulation containing consortium of Pi+R81. The same treatment also caused the highest improvement of 1.9-fold in nitrogen content and 1.7-fold in phosphorus content, while the highest increase of 1.4-fold in potassium content was obtained with Pi alone.     

A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots

Calcium (Ca²⁺), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca²⁺] elevations are early events in the interaction between the plant growth‐promoting fungus Piriformospora indica and Arabidopsis thaliana. A cell wall extract (CWE) from the fungus promotes the growth of wild‐type seedlings but not of seedlings from P. indica‐insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca²⁺ signalling‐related functions. The CWE induces a transient cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum) plants, as well as in BY‐2 suspension cultures expressing the Ca²⁺ bioluminescent indicator aequorin. Nuclear Ca²⁺ transients were also observed in tobacco BY‐2 cells. The Ca²⁺ response was more pronounced in roots than in shoots and involved Ca²⁺ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca²⁺ response by staurosporine and the refractory nature of the Ca²⁺ elevation suggest that a receptor may be involved. The CWE does not stimulate H₂O₂ production and the activation of defence gene expression, although it led to phosphorylation of mitogen‐activated protein kinases (MAPKs) in a Ca²⁺‐dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica. Thus, Ca²⁺ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.     

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica.     

Culture filtrate of root endophytic fungus Piriformospora indica promotes the growth and lignan production of Linum album hairy root cultures

The effect of addition of autoclaved and filter-sterilized culture filtrate of Piriformospora indica (a root endophytic fungus) to the growing Linum album hairy root cultures on growth and lignan production was investigated. The addition resulted in a significant enhancement in lignan production and growth. The podophyllotoxin and 6-methoxypodophyllotoxin (the lignans) concentrations were maximally improved by 3.8 times (233.8 mg/L) and 4.4 times (131.9 mg/L) in comparison to control cultures, respectively, upon addition of 3.0% (v/v) filter-sterilized culture filtrate of P. indica to the hairy root cultures of L. album for exposure time of 48 h. This increase in the lignan content also coincided with the increase in phenylalanine ammonia lyase activity, which was 3.1-fold (371.4 μkat/kg protein) higher compared to control cultures under the same conditions. The maximal increase in hairy root biomass was, however, obtained under different conditions; it was enhanced by 1.4 times (21.8 g/L) in comparison to control cultures, when 2% (v/v) filter-sterilized culture filtrate was in contact with L. album cultures for 96 h.     

A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana

Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica-insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica-induced growth promotion and enhanced seed production in Arabidopsis thaliana.     

In vitro plant development and root colonization of Coleus forskohlii by Piriformospora indica

The present study was conducted for optimization of in vitro substrates under aseptic conditions for interaction of Piriformospora indica with the medicinal plant Coleus forskohlii. It aims to test the effects of different substrates on P. indica colonization as well as growth parameters of the in vitro raised C. forskohlii. Interaction of in vitro C. forskohlii with root endophyte P. indica under aseptic condition resulted in increase in growth parameters in fungus colonized plants. It was observed that P. indica promoted the plant’s growth in all irrespective of substrates used for co-culture study. The growth was found inferior in liquid compared to semisolid medium as well as there was problem of hyperhydricity in liquid medium. P. indica treated in vitro plantlets were better adapted for establishment under green house compared to the non treated plants due to fungal intervention.     

Piriformospora indica,a plant-root-colonising fungus enhances growth and tolerance of wheat to seedlings damping-off caused by Fusarium oxysporum

In greenhouse experiments, we investigated the potentials of Piriformospora indica (Pi) to penetrate and colonise roots of wheat and to induce beneficial effects on growth as well as to reduce seedlings damping-off disease of wheat caused by the soil-borne fungus Fusarium oxysporum (Fo). By microscopy we observed the intracellular hyphae within root cortex cells of 7–14 day-old plants and chlamydospores within root cortex cells of 14–28 day-old plants and within root hair cells of 21–28 day-old plants. Moreover, diagnostic PCR based on the β-tubulin gene marker confirmed the presence of Pi in roots of inoculated plants. Also, we found that plants inoculated with Pi exhibited a better growth of roots and shoots as well as early flowering as compared with non-treated plants. Moreover, Pi conferred tolerance of wheat to seedlings damping-off i.e. by reducing the harmful effects of Fo on infected plants.     

产紫杉醇内生真菌TMS-26的分离和鉴定

为丰富产紫杉醇植物内生真菌资源库,从曼地亚红豆杉Taxus media茎中分离得到一株产紫杉醇的内生真菌TMS-26。通过对TMS-26的发酵提取物进行高效液相色谱分析,发现其具有与紫杉醇标准品(4.545 min)相近的色谱特征峰。进一步通过液质联用仪检测发现,内生真菌TMS-26的发酵提取物中具有与紫杉醇标准品((M+Na)+=876)相近的质谱特征峰,表明内生真菌TMS-26能够产生紫杉醇。同时通过传统形态学分类鉴定方法和18S r DNA序列分析、Internal-transcribed spacer(ITS)序列分析等现代分子生物学分类鉴定方法,最终将内生真菌TMS-26鉴定为曲霉属烟曲霉Aspergillus fumigatus,并命名为"烟曲霉TMS-26"。     

Functional characterization of the ferroxidase, permease high-affinity iron transport complex from Candida albicans

Saccharomyces cerevisiae expresses two proteins that together support high‐affinity Fe‐uptake. These are a multicopper oxidase, Fet3p, with specificity towards Fe²⁺ and a ferric iron permease, Ftr1p, which supports Fe‐accumulation. Homologues of the genes encoding these two proteins are found in all fungal genomes including those for the pathogens, Candida albicans and Cryptococcus neoformans. At least one of these loci represents a virulence factor for each pathogen suggesting that this complex would be an appropriate pharmacologic target. However, the mechanism by which this protein pair supports Fe‐uptake in any fungal pathogen has not been elucidated. Taking advantage of the robust molecular genetics available in S. cerevisiae, we identify the two of five candidate ferroxidases likely involved in high‐affinity Fe‐uptake in C. albicans, Fet31 and Fet34. Both localize to the yeast plasma membrane and both support Fe‐uptake along with an Ftr1 protein, either from C. albicans or from S. cerevisiae. We express and characterize Fet34, demonstrating that it is functionally homologous to ScFet3p. Using S. cerevisiae as host for the functional expression of the C. albicans Fe‐uptake proteins, we demonstrate that they support a mechanism of Fe‐trafficking that involves channelling of the CaFet34‐generated Fe³⁺ directly to CaFtr1 for transport into the cytoplasm.     

Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.     

Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants.     

Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was approximately 10-fold lower than in the wild type (with 0.5 microM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 microM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.     

The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica

Arabidopsis growth and reproduction are stimulated by the endophytic fungus Piriformospora indica. The fungus produces low amounts of auxins, but the auxin levels and the expression of auxin-regulated genes are not altered in colonized roots. Also, mutants with reduced auxin levels (ilr1-1, nit1-3, tfl2, cyp79 b2b3) respond to P. indica. However, the fungus rescues the dwarf phenotype of the auxin overproducer sur1-1 by converting free auxin into conjugates, which also results in the downregulation of the auxin-induced IAA6 and the upregulation of the P. indica-induced LRR1 gene. The fungus produces relatively high levels of cytokinins, and the cytokinin levels are higher in colonized roots compared with the uncolonized controls. trans-Zeatin cytokinin biosynthesis and the CRE1/AHK2 receptor combination are crucial for P. indica-mediated growth stimulation, while mutants lacking cis-zeatin, impaired in other cytokinin receptor combinations, or containing reduced cytokinin levels respond to the fungus. Since root colonization is not affected in the cytokinin mutants, we propose that cytokinins are required for P. indica-induced growth promotion. Finally, a comparative analysis of the phytohormone mutants allows the conclusion that the response to P. indica is independent of the architecture and size of the roots.     

A mycovirus modulates the endophytic and pathogenic traits of a plant associated fungus

Fungi are generally thought to live in host plants with a single lifestyle, being parasitism, commensalism, or mutualism. The former, known as phytopathogenic fungi, cause various plant diseases that result in significant losses every year; while the latter, such as endophytic fungi, can confer fitness to the host plants. It is unclear whether biological factors can modulate the parasitic and mutualistic traits of a fungus. In this study, we isolated and characterized a mycovirus from an endophytic strain of the fungus Pestalotiopsis theae, a pathogen of tea (Camellia sinensis). Based on molecular analysis, we tentatively designated the mycovirus as Pestalotiopsis theae chrysovirus-1 (PtCV1), a novel member of the family Chrysoviridae, genus Alphachrysovirus. PtCV1 has four double-stranded (ds) RNAs as its genome, ranging from 0.9 to 3.4 kbp in size, encapsidated in isometric particles. PtCV1 significantly reduced the growth rates of its host fungus in vitro (ANOVA; P-value < 0.001) and abolished its virulence in planta (ANOVA; P-value < 0.001), converting its host fungus to a non-pathogenic endophyte on tea leaves, while PtCV1-free isolates were highly virulent. Moreover, the presence of PtCV1 conferred high resistance to the host plants against the virulent P. theae strains. Here we report a mycovirus that modulates endophytic and phytopathogenic fungal traits and provides an alternative approach to biological control of plant diseases caused by fungi.Subject terms: Fungal biology, Applied microbiology, Virus-host interactions     

Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.