Prezygotic mate selection is only partially correlated with the expression of NaS-like RNases and affects offspring phenotypes.

Nicotiana attenuata styles preferentially select pollen from among accessions with corresponding expression patterns of NaS-like-RNases (SLRs), and the postpollination ethylene burst (PPEB) is an accurate predictor of seed siring success. However, the ecological consequences of mate selection, its effect on the progeny, and the role of SLRs in the control of ethylene signaling remain unknown. We explored the link between the magnitude of the ethylene burst and expression of the SLRs in a set of recombinant inbred lines (RILs), dissected the genetic underpinnings of mate selection through genome-wide association study (GWAS), and examined its outcome for phenotypes in the next generation. We found that high levels of PPEB are associated with the absence of SLR2 in most of the tested RILs. We identified candidate genes potentially involved in the control of mate selection and showed that pollination of maternal genotypes with their favored pollen donors produces offspring with longer roots. When the maternal genotypes are only able to select against nonfavored pollen donors, the selection for such positive traits is abolished. We conclude that plants' ability of mate choice contributes to measurable changes in progeny phenotypes and is thus likely a target of selection.

Arbuscular mycorrhizal fungi influence the intraspecific competitive ability of plants under field and glasshouse conditions.

MAIN CONCLUSION: Nicotiana attenuata's capacity to interact with arbuscular mycorrhizal fungi influences its intraspecific competitive ability under field and glasshouse conditions, but not its overall community productivity. Arbuscular mycorrhizal (AM) fungi can alter the nutrient status and growth of plants, and they can also affect plant-plant, plant-herbivore, and plant-pathogen interactions. These AM effects are rarely studied in populations under natural conditions due to the limitation of non-mycorrhizal controls. Here we used a genetic approach, establishing field and glasshouse communities of AM-harboring Nicotiana attenuata empty vector (EV) plants and isogenic plants silenced in calcium- and calmodulin-dependent protein kinase expression (irCCaMK), and unable to establish AM symbioses. Performance and growth were quantified in communities of the same (monocultures) or different genotypes (mixed cultures) and both field and glasshouse experiments returned similar responses. In mixed cultures, AM-harboring EV plants attained greater stalk lengths, shoot and root biomasses, clearly out-competing the AM fungal-deficient irCCaMK plants, while in monocultures, both genotypes grew similarly. Competitive ability was also reflected in reproductive traits: EV plants in mixed cultures outperformed irCCaMK plants. When grown in monocultures, the two genotypes did not differ in reproductive performance, though total leaf N and P contents were significantly lower independent of the community type. Plant productivity in terms of growth and seed production at the community level did not differ, while leaf nutrient content of phosphorus and nitrogen depended on the community type. We infer that AM symbioses drastically increase N. attenuata's competitive ability in mixed communities resulting in increased fitness for the individuals harboring AM without a net gain for the community.

A suite of complementary biocontrol traits allows a native consortium of root-associated bacteria to protect their host plant from a fungal sudden-wilt disease.

The beneficial effects of plant--bacterial interactions in controlling plant pests have been extensively studied with single bacterial isolates. However, in nature, bacteria interact with plants in multitaxa consortia, systems which remain poorly understood. Previously, we demonstrated that a consortium of five native bacterial isolates protected their host plant Nicotiana attenuata from a sudden wilt disease. Here we explore the mechanisms behind the protection effect against the native pathosystem. Three members of the consortium, Pseudomonas azotoformans A70, P. frederiksbergensis A176 and Arthrobacter nitroguajacolicus E46, form biofilms when grown individually in vitro, and the amount of biofilm increased synergistically in the five-membered consortium, including two Bacillus species, B. megaterium and B. mojavensis. Fluorescence in situ hybridization and scanning electron microscopy in planta imaging techniques confirmed biofilm formation and revealed locally distinct distributions of the five bacterial strains colonizing different areas on the plant-root surface. One of the five isolates, K1 B. mojavensis produces the antifungal compound surfactin, under in vitro and in vivo conditions, clearly inhibiting fungal growth. Furthermore, isolates A70 and A176 produce siderophores under in vitro conditions. Based on these results we infer that the consortium of five bacterial isolates protects its host against fungal phytopathogens via complementary traits. The study should encourage researchers to create synthetic communities from native strains of different genera to improve bioprotection against wilting diseases.

Priming and filtering of antiherbivore defences among Nicotiana attenuata plants connected by mycorrhizal networks.

Arbuscular mycorrhizal fungi (AMF) establish symbiotic associations with a majority of terrestrial plants to form underground common mycorrhizal networks (CMNs) that connect neighbouring plants. Because Nicotiana attenuata plants do not respond to herbivory-elicited volatiles from neighbours, we used this ecological model system to evaluate if CMNs function in interplant transmission of herbivory-elicited responses. A mesocosm system was designed to establish and remove CMNs linking N. attenuata plants to examine the herbivory-elicited metabolic and hormone responses in CMNs-connected "receiver" plants after the elicitation of "donor" plants by wounding (W) treated with Manduca sexta larval oral secretions (OS). AMF colonization increased constitutive jasmonate (JA and JA-Ile) levels in N. attenuata roots but did not affect well-characterized JAs-regulated defensive metabolites in systemic leaves. Interestingly, larger JAs bursts, and higher levels of several amino acids and particular sectors of hydroxygeranyllinalool diterpene glycoside metabolism were elevated in the leaves of W + OS-elicited "receivers" with CMN connections with "donors" that had been W + OS-elicited 6 hr previously. Our results demonstrate that AMF colonization alone does not enhance systemic defence responses but that sectors of systemic responses in leaves can be primed by CMNs, suggesting that CMNs can transmit and even filter defence signalling among connected plants.

Complex regulation of microRNAs in roots of competitively-grown isogenic Nicotiana attenuata plants with different capacities to interact with arbuscular mycorrhizal fungi.

BACKGROUND: Nicotiana attenuata is an ecological model plant whose 2.57 Gb genome has recently been sequenced and assembled and for which miRNAs and their genomic locations have been identified. To understand how this plant's miRNAs are reconfigured during plant-arbuscular mycorrhizal fungal (AMF) interactions and whether hostplant calcium- and calmodulin dependent protein kinase (CCaMK) expression which regulates the AMF interaction also modulates miRNAs levels and regulation, we performed a large-scale miRNA analysis of this plant-AMF interaction. RESULTS: Next generation sequencing of miRNAs in roots of empty vector (EV) N. attenuata plants and an isogenic line silenced in CCaMK expression (irCCaMK) impaired in AMF-interactions grown under competitive conditions with and without AMF inoculum revealed a total of 149 unique miRNAs: 67 conserved and 82 novel ones. The majority of the miRNAs had a length of 21 nucleotides. MiRNA abundances were highly variable ranging from 400 to more than 25,000 reads per million. The miRNA profile of irCCaMK plants impaired in AMF colonization was distinct from fully AMF-functional EV plants grown in the same pot. Six conserved miRNAs were present in all conditions and accumulated differentially depending on treatment and genotype; five (miR6153, miR403a-3p, miR7122a, miR167-5p and miR482d, but not miR399a-3p) showed the highest accumulation in AMF inoculated EV plants compared to inoculated irCCaMK plants. Furthermore, the accumulation patterns of sequence variants of selected conserved miRNAs showed a very distinct pattern related to AMF colonization - one variant of miR473-5p specifically accumulated in AMF-inoculated plants. Also abundances of miR403a-3p, miR171a-3p and one of the sequence variants of miR172a-3p increased in AMF-inoculated EV compared to inoculated irCCaMK plants and to non-inoculated EV plants, while miR399a-3p was most strongly enriched in AMF inoculated irCCaMK plants grown in competition with EV. The analysis of putative targets of selected miRNAs revealed an involvement in P starvation (miR399), phytohormone signaling (Nat-R-PN59, miR172, miR393) and defense (e.g. miR482, miR8667, Nat-R-PN-47). CONCLUSIONS: Our study demonstrates (1) a large-scale reprograming of miRNAs induced by AMF colonization and (2) that the impaired AMF signaling due to CCaMK silencing and the resulting reduced competitive ability of irCCaMK plants play a role in modulating signal-dependent miRNA accumulation.

Nicotiana attenuata's capacity to interact with arbuscular mycorrhiza alters its competitive ability and elicits major changes in the leaf transcriptome.

To study the local and systemic effects of arbuscular mycorrhizal fungal (AMF) colonization, Nicotiana attenuata plants impaired in their interactions with AMF due to silencing of a calcium- and calmodulin dependent protein kinase (inverted repreat (ir)CCaMK) were grown competitively in pairs with empty vector (EV) plants, with and without two different types of inoculum. When inoculated, EV plants strongly outperformed irCCaMK plants. Foliar transcript profiling revealed that AMF colonization significantly changed gene expression of P-starvation and -transporter genes in irCCaMK plants. The Pht1 family phosphate transporter NaPT5 was not only specifically induced in roots after AMF colonization, but also in leaves of AMF-colonized irCCaMK plants, and in plants grown under low Pi conditions in the absence of AMF. The P-starvation signature of inoculated irCCaMK plants corresponded with increases in selected amino acids and phenolic compounds in leaves. We also found a strong AMF-induced increase in amino acids and phenolic metabolites in roots. Plants impaired in their interactions with AMF clearly have a fitness disadvantage when competing for limited soil nutrients with a fully functional isogenic line. The additional role of the AMF-induced Pht1 family transporter NaPT5 in leaves under P-starvation conditions will require further experiments to fully resolve.

Catechol, a major component of smoke, influences primary root growth and root hair elongation through reactive oxygen species-mediated redox signaling.

Nicotiana attenuata germinates from long-lived seedbanks in native soils after fires. Although smoke signals have been known to break seed dormancy, whether they also affect seedling establishment and root development remains unclear. In order to test this, seedlings were treated with smoke solutions. Seedlings responded in a dose-dependent manner with significantly increased primary root lengths, due mainly to longitudinal cell elongation, increased numbers of lateral roots and impaired root hair development. Bioassay-driven fractionations and NMR were used to identify catechol as the main active compound for the smoke-induced root phenotype. The transcriptome analysis revealed that mainly genes related to auxin biosynthesis and redox homeostasis were altered after catechol treatment. However, histochemical analyses of reactive oxygen species (ROS) and the inability of auxin applications to rescue the phenotype clearly indicated that highly localized changes in the root's redox-status, rather than in levels of auxin, are the primary effector. Moreover, H2 O2 application rescued the phenotype in a dose-dependent manner. Chemical cues in smoke not only initiate seed germination, but also influence seedling root growth; understanding how these cues work provides new insights into the molecular mechanisms by which plants adapt to post-fire environments.

Specificity of root microbiomes in native-grown Nicotiana attenuata and plant responses to UVB increase Deinococcus colonization.

Plants recruit microbial communities from the soil in which they germinate. Our understanding of the recruitment process and the factors affecting it is still limited for most microbial taxa. We analysed several factors potentially affecting root microbiome structure - the importance of geographic location of natural populations, the microbiome of native seeds as putative source of colonization and the effect of a plant's response to UVB exposure on root colonization of highly abundant species. The microbiome of Nicotiana attenuata seeds was determined by a culture-dependent and culture-independent approach, and the root microbiome of natural N. attenuata populations from five different locations was analysed using 454-pyrosequencing. To specifically address the influence of UVB light on root colonization by Deinococcus, a genus abundant and consistently present in N. attenuata roots, transgenic lines impaired in UVB perception (irUVR8) and response (irCHAL) were investigated in a microcosm experiment with/without UVB supplementation using a synthetic bacterial community. The seed microbiome analysis indicated that N. attenuata seeds are sterile. Alpha and beta diversities of native root bacterial communities differed significantly between soil and root, while location had only a significant effect on the fungal but not the bacterial root communities. With UVB supplementation, root colonization of Deinococcus increased in wild type, but decreased in irUVR8 and irCHAL plants compared to nontreated plants. Our results suggest that N. attenuata recruits a core root microbiome exclusively from soil, with fungal root colonization being less selective than bacterial colonization. Root colonization by Deinococcus depends on the plant's response to UVB.

Silencing a key gene of the common symbiosis pathway in Nicotiana attenuata specifically impairs arbuscular mycorrhizal infection without influencing the root-associated microbiome or plant growth.

While the biochemical function of calcium and calmodulin-dependent protein kinase (CCaMK) is well studied, and plants impaired in the expression of CCaMK are known not to be infected by arbuscular mycorrhizal fungi (AMF) in glasshouse studies, the whole-plant and ecological consequences of CCaMK silencing are not well understood. Here we show that three independently transformed lines of Nicotiana attenuata plants silenced in CCaMK (irCCaMK) are neither infected by Rhizophagus irregularis in the glasshouse nor by native fungal inoculum in the field. The overall fungal community of field-grown roots did not differ significantly among empty vector (EV) and the transgenic lines, and the bacterial communities only showed minor differences, as revealed by the alpha-diversity parameters of bacterial OTUs, which were higher in EV plants compared with two of the three transformed lines, while beta-diversity parameters did not differ. Furthermore, growth and fitness parameters were similar in the glasshouse and field. Herbivory-inducible and basal levels of salicylic acid, jasmonic acid and abscisic acid did not differ among the genotypes, suggesting that activation of the classical defence pathways are not affected by CCaMK silencing. Based on these results, we conclude that silencing of CCaMK has few, if any, non-target effects.

Quantification of growth-defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover.

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for ¹5N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, ¹5N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover.

Determination of ¹5N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

The Costs and Benefits of Plant-Arbuscular Mycorrhizal Fungal Interactions.

The symbiotic interaction between plants and arbuscular mycorrhizal (AM) fungi is often perceived as beneficial for both partners, though a large ecological literature highlights the context dependency of this interaction. Changes in abiotic variables, such as nutrient availability, can drive the interaction along the mutualism-parasitism continuum with variable outcomes for plant growth and fitness. However, AM fungi can benefit plants in more ways than improved phosphorus nutrition and plant growth. For example, AM fungi can promote abiotic and biotic stress tolerance even when considered parasitic from a nutrient provision perspective. Other than being obligate biotrophs, very little is known about the benefits AM fungi gain from plants. In this review, we utilize both molecular biology and ecological approaches to expand our understanding of the plant-AM fungal interaction across disciplines.

Nicotiana tabacum agglutinin is active against Lepidopteran pest insects.

A jasmonate-inducible lectin called Nicotiana tabacum agglutinin or NICTABA was found in tobacco (Nicotiana tabacum cv Samsun) leaves. Since NICTABA expression is also induced after insect herbivory, a role in the defence response of tobacco was suggested. In this report, a detailed analysis was made of the entomotoxic properties of NICTABA using different transgenic approaches. First, purified NICTABA was shown to be strongly resistant to proteolytic degradation by enzymes present in the Lepidopteran midgut. To address the question of whether NICTABA is also active against Lepidopteran larvae, transgenic N. tabacum plants that silence endogenous NICTABA expression were constructed using RNA interference. Feeding experiments with these transgenic N. tabacum plants demonstrated that silencing of NICTABA expression enhances the larval performance of the generalist pest insect Spodoptera littoralis. In a second transgenic approach, NICTABA was ectopically expressed in the wild diploid tobacco Nicotiana attenuata, a species that lacks a functional NICTABA gene. When these transgenic N. attenuata plants were used in feeding experiments with S. littoralis larvae, a clear reduction in mass gain and significantly slower development were observed. In addition, feeding experiments with the Solanaceae specialist, Manduca sexta, provided further evidence that NICTABA exerts clear entomotoxic effects on Lepidopteran larvae.

An Endophytic Trichoderma Strain Promotes Growth of Its Hosts and Defends Against Pathogen Attack.

Plants host numerous endophytic microbes which promote plant performance, in particular under stress. A new endophytic fungus was isolated from the leaves of a deciduous wood tree Leucas aspera. Morphological inspection and multilocus phylogeny identified the fungus as a new Trichoderma strain. If applied to Arabidopsis thaliana and Nicotiana attenuata, it mainly colonizes their roots and strongly promotes initial growth of the plants on soil. The fungus grows on high NaCl or mannitol concentrations, and shows predatory capability on the pathogenic fungus Alternaria brassicicola. Colonized Arabidopsis plants tolerate higher salt stress and show lower A. brassicicola spread in roots and shoots, while arbuscular mycorrhiza formation in N. attenuata is not affected by the Trichoderma strain. These beneficial features of the novel Trichoderma strain are important prerequisites for agricultural applications.

Mitochondrial respiratory pathways modulate nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris.

Mitochondrial electron transport pathways exert effects on carbon-nitrogen (C/N) relationships. To examine whether mitochondria-N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered 'nitrate-sensing' phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of <1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots.

Symbiosis between Nicotiana attenuata and Glomus intraradices: ethylene plays a role, jasmonic acid does not.

Phytohormones are thought to mediate plant-arbuscular mycorrhizal (AM) interactions. To explore the role of phytohormones in the interaction between Nicotiana attenuata and Glomus intraradices, we analysed levels of jasmonic acid (JA) and its amino acid conjugate JA-isoleucine/JA-leucine (JA-Ile), salicylic acid (SA) and ethylene in either infected or non-infected N. attenuata wild-type (WT) plants growing in soils that mimic the nutrient supply rates found in the plant's native environment. Under these conditions, the infection decreases plant growth and reproductive performance. Levels of JA, JA-Ile and SA did not change upon infection, but ethylene release was slightly decreased. Transgenic N. attenuata plants defective in JA signalling (aslox3 and ircoi1) did not differ significantly in growth or reproductive performance compared with infected WT. Furthermore, no difference in infection rates could be observed. Transgenic plants unable to produce (iraco) or perceive (etr1) ethylene showed significantly larger decreases in growth and number of seed capsules produced between infected and non-infected plants compared with WT plants. We conclude that ethylene, rather than JA, signalling plays a role in the interaction between N. attenuata and AM, from which the plant does not realize a fitness benefit.

Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi.

High-through-put (HTP) screening for functional arbuscular mycorrhizal fungi (AMF)-associations is challenging because roots must be excavated and colonization evaluated by transcript analysis or microscopy. Here we show that specific leaf-metabolites provide broadly applicable accurate proxies of these associations, suitable for HTP-screens. With a combination of untargeted and targeted metabolomics, we show that shoot accumulations of hydroxy- and carboxyblumenol C-glucosides mirror root AMF-colonization in Nicotiana attenuata plants. Genetic/pharmacologic manipulations indicate that these AMF-indicative foliar blumenols are synthesized and transported from roots to shoots. These blumenol-derived foliar markers, found in many di- and monocotyledonous crop and model plants (Solanum lycopersicum, Solanum tuberosum, Hordeum vulgare, Triticum aestivum, Medicago truncatula and Brachypodium distachyon), are not restricted to particular plant-AMF interactions, and are shown to be applicable for field-based QTL mapping of AMF-related genes.

Redox regulation of peroxiredoxin and proteinases by ascorbate and thiols during pea root nodule senescence.

Redox factors contributing to nodule senescence were studied in pea. The abundance of the nodule cytosolic peroxiredoxin but not the mitochondrial peroxiredoxin protein was modulated by ascorbate. In contrast to redox-active antioxidants such as ascorbate and cytosolic peroxiredoxin that decreased during nodule development, maximal extractable nodule proteinase activity increased progressively as the nodules aged. Cathepsin-like activities were constant throughout development but serine and cysteine proteinase activities increased during senescence. Senescence-induced cysteine proteinase activity was inhibited by cysteine, dithiotreitol, or E-64. Senescence-dependent decreases in redox-active factors, particularly ascorbate and peroxiredoxin favour decreased redox-mediated inactivation of cysteine proteinases.

In wild tobacco, Nicotiana attenuata, variation among bacterial communities of isogenic plants is mainly shaped by the local soil microbiota independently of the plants' capacity to produce jasmonic acid.

The phytohormone jasmonic acid (JA) plays a central role in defense against necrotrophic pathogens and herbivores in Nicotiana attenuata. Recently Santhanam et al.(1) showed that JA does not have a major role in shaping the root- and shoot associated bacterial communities, though a few taxa differed among control (empty vector, EV) plants and plants impaired in their capacity to produce JA (irAOC). In this addendum, we provide additional data showing that the composition of the plant bacterial communities is mainly shaped by tissue type. The qualitative data analysis revealed that at the order level, 5 bacterial OTUs formed a core community found in all tissues irrespective of genotypes, while 9 OTUs were different among roots and shoots. The heterogeneity among individual plants was high masking the potential genotype effect on bacterial communities. Using a culture-dependent approach, 3 of 18 bacterial taxa retrieved either only from one of the genotypes or from both had a growth promoting effect on EV and irAOC seedlings. The data suggest that the local soil niche in which the roots grows is a major driver of the variability in root bacterial communities recruited by different individuals, and the plant growth-promoting effects of some taxa are independent of the genotype.

Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large-scale gene expression studies across different species induce of a core set of genes of similar functions. However, additional factors seem to influence the overall pattern of gene expression, resulting in high variability among independent studies with different hosts. We conclude that VIGS is a powerful tool with which to investigate the function of genes involved in plant-AMF interactions but that inoculum strength can strongly influence the outcome of the interaction.