Valentina Fiorilli

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Global and cell-type gene expression profiles in tomato plants colonized by an arbuscular mycorrhizal fungus.

*Arbuscular mycorrhizal symbiosis develops in roots; extensive cellular reorganizations and specific metabolic changes occur, which are mirrored by local and systemic changes in the transcript profiles. *A TOM2 microarray (c. 12 000 probes) has been used to obtain an overview of the transcriptional changes that are triggered in Solanum lycopersicum roots and shoots, as a result of colonization by the arbuscular mycorrhizal fungus Glomus mosseae. The cell-type expression profile of a subset of genes was monitored, using laser microdissection, to identify possible plant determinants of arbuscule development,. *Microarrays revealed 362 up-regulated and 293 down-regulated genes in roots. Significant gene modulation was also observed in shoots: 85 up- and 337 down-regulated genes. The most responsive genes in both organs were ascribed to primary and secondary metabolism, defence and response to stimuli, cell organization and protein modification, and transcriptional regulation. Six genes, preferentially expressed in arbusculated cells, were identified. *A comparative analysis only showed a limited overlap with transcript profiles identified in mycorrhizal roots of Medicago truncatula, probably as a consequence of the largely nonoverlapping probe sets on the microarray tools used. The results suggest that auxin and abscisic acid metabolism are involved in arbuscule formation and/or functioning.

GintAMT2, a new member of the ammonium transporter family in the arbuscular mycorrhizal fungus Glomus intraradices

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungus delivers mineral nutrients, such as phosphate and nitrogen, to the plant while receiving carbon. Previously, we identified an NH(4)(+) transporter in the AM fungus Glomus intraradices (GintAMT1) involved in NH(4)(+) uptake from the soil when preset at low concentrations. Here, we report the isolation and characterization of a new G. intraradicesNH(4)(+) transporter gene (GintAMT2). Yeast mutant complementation assays showed that GintAMT2 encodes a functional NH(4)(+) transporter. The use of an anti-GintAMT2 polyclonal antibody revealed a plasma membrane location of GintAMT2. GintAMT1 and GintAMT2 were differentially expressed during the fungal life cycle and in response to N. In contrast to GintAMT1, GintAMT2 transcript levels were higher in the intraradical than in the extraradical fungal structures. However, transcripts of both genes were detected in arbuscule-colonized cortical cells. GintAMT1 expression was induced under low N conditions. Constitutive expression of GintAMT2 in N-limiting conditions and transitory induction after N re-supply suggests a role for GintAMT2 to retrieve NH(4)(+) leaked out during fungal metabolism.

Comparative Analysis of Expression Profiles in Shoots and Roots of Tomato Systemically Infected by Tomato spotted wilt virus Reveals Organ-Specific Transcriptional Responses

Tomato (Solanum lycopersicon), a model species for the family Solanaceae, is severely affected by Tomato spotted wilt virus (TSWV) worldwide. To elucidate the systemic transcriptional response of plants to TSWV infection, microarray experiments were performed on tomato. Parallel analysis of both shoots and roots revealed organ-specific responses, although the virus was present in similar concentration. In the shoots, genes related to defense and to signal transduction were induced, while there was general repression of genes related to primary and secondary metabolism as well as to amino acid metabolism. In roots, expression of genes involved in primary metabolism and signal transduction appear unaffected by TSWV infection, while those related to the response to biotic stimuli were induced and those associated to the response to abiotic stress were generally repressed or unaltered. Genes related to amino acid metabolism were unaffected, except for those involved in synthesis of secondary compounds, where induction was evident. Differential expression of genes involved in metabolism and response to ethylene and abscisic acid was observed in the two organs. Our results provide new insight into the biology of the economically important interaction between tomato and TSWV.

The phosphate transporters LjPT4 and MtPT4 mediate early root responses to phosphate status in non mycorrhizal roots

Arbuscular mycorrhizal (AM) symbiosis improves host plant phosphorous (P) status and elicits the expression of AM-inducible phosphate transporters (PTs) in arbuscule-containing cells, where they control arbuscule morphogenesis and P release. We confirmed such functions for LjPT4 in mycorrhizal Lotus japonicus. Promoter-GUS experiments showed LjPT4 transcription not only in arbusculated cells but also in root tips, in the absence of the fungus: here LjPT4 transcription profile depended on the phosphate level. In addition, quantitative RT-PCR confirmed the expression of Lotus and Medicago truncatula PT4 in the tips of non-mycorrhizal roots. Starting from these observations, we hypothesized that AM-inducible PTs may have a regulatory role in plant development, irrespective of the fungal presence. Firstly, we focused on root development responses to different phosphate treatments in both plants demonstrating that phosphate starvation induced a higher number of lateral roots. By contrast, Lotus PT4i plants and Medicago mtpt4 mutants did not show any differential response to phosphate levels, suggesting that PT4 genes affect early root branching. Phosphate starvation-induced genes and a key auxin receptor, MtTIR1, showed an impaired expression in mtpt4 plants. We suggest PT4 genes as novel components of the P-sensing machinery at the root tip level, independently of AM fungi.

Rice flooding negatively impacts root branching and arbuscular mycorrhizal colonization, but not fungal viability.

Rice is mostly cultivated in wetlands, where arbuscular mycorrhization (AM) is reported to decrease. The mechanisms regulating such events are largely unknown. Rice uninoculated and inoculated with Rhizophagus irregularis were grown in dry and flooded conditions, allowing also for the transfer of plants from one water regime to the other. Roots were sampled at different times, from 7 to 35 d post-inoculation (dpi). The morphological and molecular parameters (root branching, aerenchyma formation, mycorrhizal colonization, AM marker gene expression) were evaluated. Root branching was more pronounced in dry conditions, and such phenotype was enhanced by the fungus. In wetlands, the colonization level was comparable till 21 dpi, when the mycorrhization then decreased, paralleled by an increase in aerenchyma. Expression of the fungal transporters was comparable under the two conditions. The root apparatus, when shifted from one water regime to the other, rapidly adapted to the new condition, revealing a marked plasticity. The reversibility of the AM rice symbiosis was also mirrored by expression changes of plant marker genes. The results demonstrate that the water regime is the driving force that regulates AM colonization under flooding conditions, by directly influencing root architecture and anatomy, but without impacting the basic AM functionality.

Host and non-host roots in rice: cellular and molecular approaches reveal differential responses to arbuscular mycorrhizal fungi

Oryza sativa, a model plant for Arbuscular Mycorrhizal (AM) symbiosis, has both host and non-host roots. Large lateral (LLR) and fine lateral (FLR) roots display opposite responses: LLR support AM colonization, but FLR do not. Our research aimed to study the molecular, morphological and physiological aspects related to the non-host behavior of FLR. RNA-seq analysis revealed that LLR and FLR displayed divergent expression profiles, including changes in many metabolic pathways. Compared with LLR, FLR showed down-regulation of genes instrumental for AM establishment and gibberellin signaling, and a higher expression of nutrient transporters. Consistent with the transcriptomic data, FLR had higher phosphorus content. Light and electron microscopy demonstrated that, surprisingly, in the Selenio cultivar, FLR have a two-layered cortex, which is theoretically compatible with AM colonization. According to RNA-seq, a gibberellin inhibitor treatment increased anticlinal divisions leading to a higher number of cortex cells in FLR. We propose that some of the differentially regulated genes that lead to the anatomical and physiological properties of the two root types also function as genetic factors regulating fungal colonization. The rice root apparatus offers a unique tool to study AM symbiosis, allowing direct comparisons of host and non-host roots in the same individual plant.

A dipeptide transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in the intraradical phase

Arbuscular mycorrhizal fungi (AMF), which form an ancient and widespread mutualistic symbiosis with plants, are a crucial but still enigmatic component of the plant micro biome. Nutrient exchange has probably been at the heart of the success of this plant-fungus interaction since the earliest days of plants on land. To characterize genes from the fungal partner involved in nutrient exchange, and presumably important for the functioning of the AM symbiosis, genome-wide transcriptomic data obtained from the AMF Rhizophagus irregularis were exploited. A gene sequence, showing amino acid sequence and transmembrane domains profile similar to members of the PTR2 family of fungal oligopeptide transporters, was identified and called RiPTR2. The functional properties of RiPTR2 were investigated by means of heterologous expression in Saccharomyces cerevisiae mutants defective in either one or both of its di/tripeptide transporter genes PTR2 and DAL5. These assays showed that RiPTR2 can transport dipeptides such as Ala-Leu, Ala-Tyr or Tyr-Ala. From the gene expression analyses it seems that RiPTR2 responds to different environmental clues when the fungus grows inside the root and in the extraradical phase.

THE ARBUSCULAR MYCORRHIZAL SYMBIOSIS REDUCES DISEASE SEVERITY IN TOMATO PLANTS INFECTED BY BOTRYTIS CINEREA

SUMMARY The majority of plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. The symbiosis provides the plants with an improved mineral nutrition and, to some extent, higher tolerance to biotic and abiotic stresses. In this work we have evaluated whether AM symbiosis modifies the response of tomato plants to the attack of the necrotrophic pathogen Botrytis cinerea. Leaves of tomato plants, colonized or not by the AM fungus Glomus mosseae, were infected with B. cinerea. A higher disease index in control plants (60.3%) compared to mycorrhizal plants (37.5%) was observed. To assess the potential involvement of salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) in this response, the levels of these hormones were also measured in the leaves of mycorrhizal and non mycorrhizal plants. While JA was not detected and no differences were observed in the SA content between the two biological conditions, a statistically significant lower content of ABA was detected in mycorrhizal vs control plants. Our results show that AM symbiosis reduces disease severity in tomato plants infected by B. cinerea and suggest that ABA is one component of the AM-induced lower susceptibility to B. cinerea.

The arbuscular mycorrhizal symbiosis attenuates symptom severity and reduces virus concentration in tomato infected by Tomato yellow leaf curl Sardinia virus (TYLCSV).

The arbuscular mycorrhizal (AM) symbiosis is considered a natural instrument to improve plant health and productivity since mycorrhizal plants often show higher tolerance to abiotic and biotic stresses. However, the impact of the AM symbiosis on infection by viral pathogens is still largely uncertain and little explored. In the present study, tomato plants were grown under controlled conditions and inoculated with the AM fungus Funneliformis mosseae. Once the mycorrhizal colonization had developed, plants were inoculated with the Tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus causing one of the most serious viral diseases of tomatoes in Mediterranean areas. Biological conditions consisted of control plants (C), TYLCSV-infected plants (V), mycorrhizal plants (M), and TYLCSV-infected mycorrhizal plants (MV). At the time of analysis, the level of mycorrhiza development and the expression profiles of mycorrhiza-responsive selected genes were not significantly modified by virus infection, thus indicating that the AM symbiosis was unaffected by the presence and spread of the virus. Viral symptoms were milder, and both shoot and root concentrations of viral DNA were lower in MV plants than in V plants. Overall F. mosseae colonization appears to exert a beneficial effect on tomato plants in attenuating the disease caused by TYLCSV.