Piriformospora indica Reprograms Gene Expression in Arabidopsis Phosphate Metabolism Mutants But Does Not Compensate for Phosphate Limitation.

Piriformospora indica is an endophytic fungus of Sebacinaceae which colonizes the roots of many plant species and confers benefits to the hosts. We demonstrate that approximately 75% of the genes, which respond to P. indica in Arabidopsis roots, differ among seedlings grown on normal phosphate (Pi) or Pi limitation conditions, and among wild-type and the wrky6 mutant impaired in the regulation of the Pi metabolism. Mapman analyses suggest that the fungus activates different signaling, transport, metabolic and developmental programs in the roots of wild-type and wrky6 seedlings under normal and low Pi conditions. Under low Pi, P. indica promotes growth and Pi uptake of wild-type seedlings, and the stimulatory effects are identical for mutants impaired in the PHOSPHATE TRANSPORTERS1;1, -1;2 and -1;4. The data suggest that the fungus does not stimulate Pi uptake, but adapts the expression profiles to Pi limitation in Pi metabolism mutants.

Functional Characterization of a Hexose Transporter from Root Endophyte Piriformospora indica.

Understanding the mechanism of photosynthate transfer at symbiotic interface by fungal monosaccharide transporter is of substantial importance. The carbohydrate uptake at the apoplast by the fungus is facilitated by PiHXT5 hexose transporter in root endophytic fungus Piriformospora indica. The putative PiHXT5 belongs to MFS superfamily with 12 predicted transmembrane helices. It possess sugar transporter PFAM motif (PF0083) and MFS superfamily domain (PS50850). It contains the signature tags related to glucose transporter GLUT1 of human erythrocyte. PiHXT5 is regulated in response to mutualism as well as glucose concentration. We have functionally characterized PiHXT5 by complementation of hxt-null mutant of Saccharomyces cerevisiae EBY.VW4000. It is involved in transport of multiple sugars ranging from D-glucose, D-fructose, D-xylose, D-mannose, D-galactose with decreasing affinity. The uncoupling experiments indicate that it functions as H(+)/glucose co-transporter. Further, pH dependence analysis suggests that it functions maximum between pH 5 and 6. The expression of PiHXT5 is dependent on glucose concentration and was found to be expressed at low glucose levels (1 mM) which indicate its role as a high affinity glucose transporter. Our study on this sugar transporter will help in better understanding of carbon metabolism and flow in this agro-friendly fungus.

Piriformospora indica: Potential and Significance in Plant Stress Tolerance.

Owing to its exceptional ability to efficiently promote plant growth, protection and stress tolerance, a mycorrhiza like endophytic Agaricomycetes fungus Piriformospora indica has received a great attention over the last few decades. P. indica is an axenically cultiviable fungus which exhibits its versatility for colonizing/hosting a broad range of plant species through directly manipulating plant hormone-signaling pathway during the course of mutualism. P. indica-root colonization leads to a better plant performance in all respect, including enhanced root proliferation by indole-3-acetic acid production which in turn results into better nutrient-acquisition and subsequently to improved crop growth and productivity. Additionally, P. indica can induce both local and systemic resistance to fungal and viral plant diseases through signal transduction. P. indica-mediated stimulation in antioxidant defense system components and expressing stress-related genes can confer crop/plant stress tolerance. Therefore, P. indica can biotize micropropagated plantlets and also help these plants to overcome transplantation shock. Nevertheless, it can also be involved in a more complex symbiotic relationship, such as tripartite symbiosis and can enhance population dynamic of plant growth promoting rhizobacteria. In brief, P. indica can be utilized as a plant promoter, bio-fertilizer, bioprotector, bioregulator, and biotization agent. The outcome of the recent literature appraised herein will help us to understand the physiological and molecular bases of mechanisms underlying P. indica-crop plant mutual relationship. Together, the discussion will be functional to comprehend the usefulness of crop plant-P. indica association in both achieving new insights into crop protection/improvement as well as in sustainable agriculture production.

Fungal association and utilization of phosphate by plants: success, limitations, and future prospects.

Phosphorus (P) is a major macronutrient for plant health and development. The available form of P is generally low in the rhizosphere even in fertile soils. A major proportion of applied phosphate (Pi) fertilizers in the soil become fixed into insoluble, unavailable forms, which restricts crop production throughout the world. Roots possess two distinct modes of P uptake from the soil, direct and indirect uptake. The direct uptake of P is facilitated by the plant's own Pi transporters while indirect uptake occurs via mycorrhizal symbiosis, where the host plant obtains P primarily from the fungal partner, while the fungus benefits from plant-derived reduced carbon. So far, only one Pi transporter has been characterized from the mycorrhizal fungus Glomus versiforme. As arbuscular mycorrhizal fungi cannot be cultured axenically, their Pi transporter network is difficult to exploite for large scale sustainable agriculture. Alternatively, the root-colonizing endophytic fungus Piriformospora indica can grow axenically and provides strong growth-promoting activity during its symbiosis with a broad spectrum of plants. P. indica contains a high affinity Pi transporter (PiPT) involved in improving Pi nutrition levels in the host plant under P limiting conditions. As P. indica can be manipulated genetically, it opens new vistas to be used in P deficient fields.

The activity of RTA2, a downstream effector of the calcineurin pathway, is required during tunicamycin-induced ER stress response in Candida albicans.

In this study, we demonstrate a novel function of a downstream effector molecule of the calcineurin pathway, RTA2 (Resistance To Aminocholesterol), in ER stress response. The deletion of RTA2 increases susceptibility to the ER stressor tunicamycin and morpholine-like drug, 7-aminocholesterol. Additionally, the expression of RTA2 is also transcriptionally induced by ergosterol biosynthesis inhibitors and cell-wall-damaging agents. As tunicamycin induces the unfolded protein response pathway (UPR) via the transcription factor, HAC1, we monitored the expression of a subset of HAC1-dependent UPR target genes in rta2Δ/Δ cells. Upon tunicamycin exposure, rta2Δ/Δ cells displayed a significantly reduced expression of UPR genes, in spite of only a moderate decrease in the HAC1 spliced mRNA levels and no change in Hac1 protein levels. Furthermore, hac1Δ/Δrta2Δ/Δ cells display an exacerbated sensitivity to tunicamycin compared to the single mutants. We propose that functional RTA2 is requisite for the regulation of Hac1p-dependent UPR target genes to maximal levels, thereby assisting survival during ER stress. Collectively, this study proposes, for the first time, existence of an interplay between the Hac1p- and calcineurin- controlled networks via a downstream effector molecule of the latter, RTA2, to facilitate survival during ER stress in Candida albicans.

Crystal structure of a eukaryotic phosphate transporter.

Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chemical energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane-buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asymmetrical 'rocker-switch' mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the organic cation and anion transporters of the solute carrier family (SLC22), which are implicated in cancer-drug resistance. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.

Transcriptional and proteomic profiles of group B Streptococcus type V reveal potential adherence proteins associated with high-level invasion.

Group B Streptococcus (GBS) is an opportunistic organism that can harmlessly colonize the human gut, vagina, and rectum but can also cause pneumonia, sepsis, and meningitis in neonates born to colonized mothers. We have shown previously that growth rate and oxygen level regulate the ability of GBS to invade eukaryotic cells in vitro. Herein we extend and expand on these observations to show that GBS type V, an emergent serotype, grown in a chemostat at a cell mass-doubling time (t(d)) of 1.8 h with oxygen invaded human ME-180 cervical epithelial cells in large numbers compared with those grown at the same t(d) without oxygen or at a slower t(d) of 11.0 h. The fact that several GBS type V cell wall-associated and membrane proteins were expressed exclusively under the invasive growth condition prompted an investigation, using genomics and proteomics, of all upregulated genes and proteins. Several proteins with potential roles in adherence were identified, including an undefined surface antigen (SAG1350), a lipoprotein (SAG0971), penicillin-binding protein 2b (SAG0765), glyceraldehyde-3-phosphate dehydrogenase (SAG0823), and an iron-binding protein (SAG1007). Mouse antisera to these five proteins inhibited binding of GBS type V to ME-180 cells by > or =85%. Recombinant undefined surface antigen (SAG1350), lipoprotein (SAG0971), and penicillin-binding protein 2b (SAG0765) each bound to ME-180 cells in a dose-dependent fashion, confirming their ability to act as ligands. Collectively, these data increase the number of potential GBS adherence factors and also suggest a role for these surface-associated proteins in initial pathogenic events.

Growth rate and oxygen regulate the interactions of group B Streptococcus with polarized respiratory epithelial cells.

We investigated growth conditions that regulate the ability of group B Streptococcus (GBS) to attach to, invade, and translocate through polarized human respiratory epithelial cells (RECs). GBS grown in a chemostat at a fast cell mass doubling time (t(d) = 1.8 h), invaded RECs from both the apical and basolateral surfaces in higher numbers compared with those held at a t(d) = 11 h. With the exception of adherence from the basolateral surface, GBS reached peak adherence to, invasion of, and translocation through RECs when held at the fast t(d) with 15% dissolved oxygen compared with 0% dissolved oxygen. Growth rate and oxygen level strongly influence the interaction of GBS with polarized RECs and likely GBS pathogenicity.

Adherence to, invasion by, and cytokine production in response to serotype VIII group B Streptococci.

The adherence to and invasion of the human epithelial cell line A549 by group B streptococcus (GBS) serotype VIII strains were compared with those of serotype III strains by a conventional method and the dynamic in vitro attachment and invasion system. Twenty GBS strains, including nine vaginal isolates and one invasive isolate each of serotypes III and VIII, were used in the conventional attachment and invasion assay. Adherence to and invasion of A549 cells by serotype VIII GBS strains were significantly greater (P < 0.0001) than those by serotype III strains for both the invasive strain and vaginal isolates. Cytokine production by A549 cells following stimulation with GBS serotypes III and VIII or their purified capsular polysaccharides (CPS) was measured. Serotype III strains stimulated significantly greater tumor necrosis factor alpha (TNF-alpha) (P < 0.0001) and interleukin-10 (IL-10) (P < 0.05) production than did serotype VIII strains. IL-8 production in response to serotype VIII was significantly higher (P < 0.001) than that in response to serotype III. TNF-alpha, IL-8, and IL-10 production was greater in A549 cells infected with GBS than in the untreated control cells. TNF-alpha production was significantly greater (P < 0.005) after stimulation with purified GBS serotype III CPS than after stimulation with serotype VIII CPS, a result similar to that after stimulation with whole GBS. IL-12 production by A549 cells was observed only in response to infection with GBS serotype III, resulting in the possibility of a greater TH1 response in serotype III GBS. These results suggest differences in immune responses to infection with GBS serotypes III and VIII.

Oxygen regulates invasiveness and virulence of group B streptococcus.

The facultative anaerobe group B Streptococcus (GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5- to 400-fold higher numbers when cultured at a cell mass doubling time (t(d)) of 1.8 h than at a slower t(d) of 11 h. Invasion was optimal when GBS was cultured at a t(d) of 1.8 h in the presence of >or=5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (t(d) of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (t(d) of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.