Tomato growth promotion by the fungal endophytes Serendipita indica and Serendipita herbamans is associated with sucrose de-novo synthesis in roots and differential local and systemic effects on carbohydrate metabolisms and gene expression.

Plant growth-promoting and stress resilience-inducing root endophytic fungi represent an additional carbohydrate sink. This study aims to test if such root endophytes affect the sugar metabolism of the host plant to divert the flow of resources for their purposes. Fresh and dry weights of roots and shoots of tomato (Solanum lycopersicum) colonised by the closely related Serendipita indica and Serendipita herbamans were recorded. Plant carbohydrate metabolism was analysed by measuring sugar levels, by determining activity signatures of key enzymes of carbohydrate metabolism, and by quantifying mRNA levels of genes involved in sugar transport and turnover. During the interaction with the tomato plants, both fungi promoted root growth and shifted shoot biomass from stem to leaf tissues, resulting in increased leaf size. A common effect induced by both fungi was the inhibition of phosphofructokinase (PFK) in roots and leaves. This glycolytic-pacing enzyme shows how the glycolysis rate is reduced in plants and, eventually, how sugars are allocated to different tissues. Sucrose phosphate synthase (SPS) activity was strongly induced in colonised roots. This was accompanied by increased SPS-A1 gene expression in S. herbamans-colonised roots and by increased sucrose amounts in roots colonised by S. indica. Other enzyme activities were barely affected by S. indica, but mainly induced in leaves of S. herbamans-colonised plants and decreased in roots. This study suggests that two closely related root endophytic fungi differentially influence plant carbohydrate metabolism locally and systemically, but both induce a similar increase in plant biomass. Notably, both fungal endophytes induce an increase in SPS activity and, in the case of S. indica, sucrose resynthesis in roots. In leaves of S. indica-colonised plants, SWEET11b expression was enhanced, thus we assume that excess sucrose was exported by this transporter to the roots. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬.

Salt Stress Tolerance of Dark Septate Endophytes Is Independent of Melanin Accumulation.

Dark septate endophytes (DSEs) represent a diverse group of root-endophytic fungi that have been isolated from plant roots in many different natural and anthropogenic ecosystems. Melanin is widespread in eukaryotic organisms and possesses various functions such as protecting human skin from UV radiation, affecting the virulence of pathogens, and playing a role in development and physiology of insects. Melanin is a distinctive feature of the cell walls of DSEs and has been thought to protect these fungi from abiotic stress. Melanin in DSEs is assumed to be synthesized via the 1,8-dihydroxynaphthalene (DHN) pathway. Its function in alleviation of salt stress is not yet known. The aims of this study were: (i) investigating the growth responses of three DSEs (Periconia macrospinosa, Cadophora sp., and Leptodontidium sp.) to salt stress, (ii) analyzing melanin production under salt stress and, (iii) testing the role of melanin in salt stress tolerance of DSEs. The study shows that the three DSE species can tolerate high salt concentrations. Melanin content increased in the hyphae of all DSEs at 100 mM salt, but decreased at 500 mM. This was not reflected in the RNA accumulation of the gene encoding scytalone dehydratase which is involved in melanin biosynthesis. The application of tricyclazole, a DHN-melanin biosynthesis inhibitor, did not affect either salt stress tolerance or the accumulation of sodium in the hyphae. In addition, melanin biosynthesis mutants of Leptodontidium sp. did not show decreased growth performance compared to the wild-type, especially not at high salt concentrations. This indicates that DSEs can live under salt stress and withstand these conditions regardless of melanin accumulation.

RAMOSA1 ENHANCER LOCUS2-Mediated Transcriptional Repression Regulates Vegetative and Reproductive Architecture.

Transcriptional repression in multicellular organisms orchestrates dynamic and precise gene expression changes that enable complex developmental patterns. Here, we present phenotypic and molecular characterization of the maize (Zea mays) transcriptional corepressor RAMOSA1 ENHANCER LOCUS2 (REL2), a unique member of the highly conserved TOPLESS (TPL) family. Analysis of single recessive mutations in rel2 revealed an array of vegetative and reproductive phenotypes, many related to defects in meristem initiation and maintenance. To better understand how REL2-mediated transcriptional complexes relate to rel2 phenotypes, we performed protein interaction assays and transcriptional profiling of mutant inflorescences, leading to the identification of different maize transcription factors and regulatory pathways that employ REL2 repression to control traits directly impacting maize yields. In addition, we used our REL2 interaction data to catalog conserved repression motifs present on REL2 interactors and showed that two of these, RLFGV- and DLN-type motifs, interact with the C-terminal WD40 domain of REL2 rather than the N terminus, which is known to bind LxLxL EAR motifs. These findings establish that the WD40 domain of TPL family proteins is an independent protein interaction surface that may work together with the N-terminal domain to allow the formation of large macromolecular complexes of functionally related transcription factors.

Beneficial Root Endophytic Fungi Increase Growth and Quality Parameters of Sweet Basil in Heavy Metal Contaminated Soil.

How interactions between plants, the rhizosphere, and contaminated soil affect environmental sustainability is still under research. We tested the effects of two root endophytic fungi, the arbuscular mycorrhiza fungus (AMF) Rhizophagus irregularis and the beneficial endophyte Serendipita indica, on sweet basil (Ocimum basilicum) growing on soil contaminated with lead and copper in a pot experiment under defined greenhouse conditions. Both fungi caused an increase in shoot and root dry weight of sweet basil plants under all conditions and decreased the amount of lead in shoots. The amount of copper was reduced by S. indica, while the AM fungus showed this effect only when the soil is contaminated with both copper and lead. Furthermore the AMF, but not the endophyte S. indica caused a strong increase on the concentrations of the essential oils linalool and eucalyptol even on sweet basil growing on contaminated soils. Hence, cultivating sweet basil in combination with beneficial fungi in case of difficult environmental conditions could be of interest for industry located in countries with widespread land pollution, because quantity and quality of plants are increased while the amount of heavy metals is generally reduced.

Growth of Arabidopsis seedlings on high fungal doses of Piriformospora indica has little effect on plant performance, stress, and defense gene expression in spite of elevated jasmonic acid and jasmonic acid-isoleucine levels in the roots.

The endophytic fungus Piriformospora indica colonizes the roots of many plant species including Arabidopsis and promotes their performance, biomass, and seed production as well as resistance against biotic and abiotic stress. Imbalances in the symbiotic interaction such as uncontrolled fungal growth result in the loss of benefits for the plants and activation of defense responses against the microbe. We exposed Arabidopsis seedlings to a dense hyphal lawn of P. indica. The seedlings continue to grow, accumulate normal amounts of chlorophyll, and the photosynthetic parameters demonstrate that they perform well. In spite of high fungal doses around the roots, the fungal material inside the roots was not significantly higher when compared with roots that live in a beneficial symbiosis with P. indica. Fifteen defense- and stress-related genes including PR2, PR3, PAL2, and ERF1 are only moderately upregulated in the roots on the fungal lawn, and the seedlings did not accumulate H2O2/radical oxygen species. However, accumulation of anthocyanin in P. indica-exposed seedlings indicates stress symptoms. Furthermore, the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) levels were increased in the roots, and consequently PDF1.2 and a newly characterized gene for a 2-oxoglurate and Fe2+ -dependent oxygenase were upregulated more than 7-fold on the dense fungal lawn, in a JAR1- and EIN3-dependent manner. We conclude that growth of A. thaliana seedlings on high fungal doses of P. indica has little effect on the overall performance of the plants although elevated JA and JA-Ile levels in the roots induce a mild stress or defense response.

HSPRO controls early Nicotiana attenuata seedling growth during interaction with the fungus Piriformospora indica.

In a previous study aimed at identifying regulators of Nicotiana attenuata responses against chewing insects, a 26-nucleotide tag matching the HSPRO (ORTHOLOG OF SUGAR BEET Hs1(pro)(-)(1)) gene was found to be strongly induced after simulated herbivory (Gilardoni et al., 2010). Here we characterized the function of HSPRO during biotic interactions in transgenic N. attenuata plants silenced in its expression (ir-hspro). In wild-type plants, HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv tomato DC3000 and roots with the growth-promoting fungus Piriformospora indica. Reduced HSPRO expression did not affect the regulation of direct defenses against Manduca sexta herbivory or P. syringae pv tomato DC3000 infection rates. However, reduced HSPRO expression positively influenced early seedling growth during interaction with P. indica; fungus-colonized ir-hspro seedlings increased their fresh biomass by 30% compared with the wild type. Grafting experiments demonstrated that reduced HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots. This effect was accompanied by changes in the expression of 417 genes in colonized roots, most of which were metabolic genes. The lack of major differences in the metabolic profiles of ir-hspro and wild-type colonized roots (as analyzed by liquid chromatography time-of-flight mass spectrometry) suggested that accelerated metabolic rates were involved. We conclude that HSPRO participates in a whole-plant change in growth physiology when seedlings interact with P. indica.

The OXI1 kinase pathway mediates Piriformospora indica-induced growth promotion in Arabidopsis.

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H2O2 formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

Do ethylene response factorS9 and -14 repress PR gene expression in the interaction between Piriformospora indica and Arabidopsis?

The plant hormone ethylene (ET) plays a crucial role in the signalling network when plants have to respond to biotic stresses. We investigate the beneficial interaction between the model plant Arabidopsis thaliana and the endophytic fungus Piriformospora indica. Recently, we showed that ET signalling and ETHYLENE RESPONSE FACTOR (ERF)1 are important to balance beneficial and nonbeneficial traits in this symbiosis. 147 ERF genes in Arabidopsis encode transcriptional regulators with a variety of functions involved in development, physiological processes as well as plant/microbe interactions. In the beneficial symbiosis between Arabidopsis and P. indica, overexpression of ERF1 activates defence responses, strongly reduces root colonization and thus abolishes the benefits for the plants. Here we show that additional transcription factors of the ERF family, the ERF DOMAIN PROTEIN9 (ERF9) and the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR14 (ERF14) are involved in the interaction between the two symbionts and are required for growth promotion of the host plant. Expression of these genes is upregulated in colonized wild-type roots. Insertional inactivation of ERF9 and ERF14 diminishes the P. indica-induced growth promotion and activates the expression of the PATHOGENESIS-RELATED (PR)-1 and PR-2 genes. We propose that ERF9 and ERF14 repress PR gene expression in colonized Arabidopsis roots and thus contribute to the establishment of a beneficial interaction.

Ethylene signalling and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis between the endophytic fungus Piriformospora indica and Arabidopsis thaliana.

*The endophytic fungus Piriformospora indica colonizes the roots of the model plant Arabidopsis thaliana and promotes its growth and seed production. The fungus can be cultivated in axenic culture without a host, and therefore this is an excellent system to investigate plant-fungus symbiosis. *The growth of etr1, ein2 and ein3/eil1 mutant plants was not promoted or even inhibited by the fungus; the plants produced less seeds and the roots were more colonized compared with the wild-type. This correlates with a mild activation of defence responses. The overexpression of ETHYLENE RESPONSE FACTOR1 constitutively activated defence responses, strongly reduced root colonization and abolished the benefits for the plants. *Piriformospora indica-mediated stimulation of growth and seed yield was not affected by jasmonic acid, and jasmonic acid-responsive promoter beta-glucuronidase gene constructs did not respond to the fungus in Arabidopsis roots. *We propose that ethylene signalling components and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis. The results show that the restriction of fungal growth by ethylene signalling components is required for the beneficial interaction between the two symbionts.

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.