A tribute to Graziella Berta (1948-2024): research milestones and highlights.

Graziella Berta, a well-known mycorrhiza researcher, passed away in her home in Torino (Italy) on March 2nd, 2024, at the age of 75. We were both fortunate to know Graziella personally and to greatly appreciate her professionally, by working closely with her in the same research group in Alessandria (GL) or through many collaborative projects over the years (VGP). Here, we recall some of the milestones in her research and particularly the important contribution she has made to knowledge about plant interactions with mycorrhizal fungi and beneficial rhizosphere bacteria.

Life's rich tapestry : A tribute to Sally E. Smith (1941-2019).

With the recent passing away of Emeritus Professor Sally (Sarah) E. Smith, the mycorrhiza research community has lost one of its most outstanding members. Sally's contribution to knowledge in the fields of soil microbiology and root physiology will continue to be an inspiration to present and future scientists.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Local and systemic mycorrhiza-induced protection against the ectoparasitic nematode Xiphinema index involves priming of defence gene responses in grapevine.

The ectoparasitic dagger nematode (Xiphinema index), vector of Grapevine fanleaf virus (GFLV), provokes gall formation and can cause severe damage to the root system of grapevines. Mycorrhiza formation by Glomus (syn. Rhizophagus) intraradices BEG141 reduced both gall formation on roots of the grapevine rootstock SO4 (Vitis berlandieri×V. riparia) and nematode number in the surrounding soil. Suppressive effects increased with time and were greater when the nematode was post-inoculated rather than co-inoculated with the arbuscular mycorrhizal (AM) fungus. Using a split-root system, decreased X. index development was shown in mycorrhizal and non-mycorrhizal parts of mycorrhizal root systems, indicating that both local and systemic induced bioprotection mechanisms were active against the ectoparasitic nematode. Expression analyses of ESTs (expressed sequence tags) generated in an SSH (subtractive suppressive hybridization) library, representing plant genes up-regulated during mycorrhiza-induced control of X. index, and of described grapevine defence genes showed activation of chitinase 1b, pathogenesis-related 10, glutathione S-transferase, stilbene synthase 1, 5-enolpyruvyl shikimate-3-phosphate synthase, and a heat shock proein 70-interacting protein in association with the observed local and/or systemic induced bioprotection against the nematode. Overall, the data suggest priming of grapevine defence responses by the AM fungus and transmission of a plant-mediated signal to non-mycorrhizal tissues. Grapevine gene responses during AM-induced local and systemic bioprotection against X. index point to biological processes that are related either to direct effects on the nematode or to protection against nematode-imposed stress to maintain root tissue integrity.

Arbuscular mycorrhizal symbiosis elicits shoot proteome changes that are modified during cadmium stress alleviation in Medicago truncatula.

BACKGROUND: Arbuscular mycorrhizal (AM) fungi, which engage a mutualistic symbiosis with the roots of most plant species, have received much attention for their ability to alleviate heavy metal stress in plants, including cadmium (Cd). While the molecular bases of Cd tolerance displayed by mycorrhizal plants have been extensively analysed in roots, very little is known regarding the mechanisms by which legume aboveground organs can escape metal toxicity upon AM symbiosis. As a model system to address this question, we used Glomus irregulare-colonised Medicago truncatula plants, which were previously shown to accumulate and tolerate heavy metal in their shoots when grown in a substrate spiked with 2 mg Cd kg(-1). RESULTS: The measurement of three indicators for metal phytoextraction showed that shoots of mycorrhizal M. truncatula plants have a capacity for extracting Cd that is not related to an increase in root-to-shoot translocation rate, but to a high level of allocation plasticity. When analysing the photosynthetic performance in metal-treated mycorrhizal plants relative to those only Cd-supplied, it turned out that the presence of G. irregulare partially alleviated the negative effects of Cd on photosynthesis. To test the mechanisms by which shoots of Cd-treated mycorrhizal plants avoid metal toxicity, we performed a 2-DE/MALDI/TOF-based comparative proteomic analysis of the M. truncatula shoot responses upon mycorrhization and Cd exposure. Whereas the metal-responsive shoot proteins currently identified in non-mycorrhizal M. truncatula indicated that Cd impaired CO2 assimilation, the mycorrhiza-responsive shoot proteome was characterised by an increase in photosynthesis-related proteins coupled to a reduction in glugoneogenesis/glycolysis and antioxidant processes. By contrast, Cd was found to trigger the opposite response coupled the up-accumulation of molecular chaperones in shoot of mycorrhizal plants relative to those metal-free. CONCLUSION: Besides drawing a first picture of shoot proteome modifications upon AM symbiosis and/or heavy metal stress in legume plants, the current work argues for allocation plasticity as the main driving force for Cd extraction in aboveground tissues of M. truncatula upon mycorrhization. Additionally, according to the retrieved proteomic data, we propose that shoots of mycorrhizal legume plants escape Cd toxicity through a metabolic shift implying the glycolysis-mediated mobilization of defence mechanisms at the expense of the photosynthesis-dependent symbiotic sucrose sink.

Identification of in planta-expressed arbuscular mycorrhizal fungal proteins upon comparison of the root proteomes of Medicago truncatula colonised with two Glomus species.

In the absence of sequenced genomes for arbuscular mycorrhizal (AM) fungi, their obligatory biotrophy makes their intra-radical biology especially recalcitrant to functional analyses. Because tandem mass spectrometry-based proteomics enables fungal gene product identifications in phyla lacking genomic information, we have compared as a way to enlarge the coverage of in planta expressed-mycorrhiza-related proteins, the root proteome responses of Medicago truncatula upon colonisation with two AM fungi, Glomus mosseae and G. intraradices, using two-dimensional electrophoresis. In contrast to phosphate fertilization, mycorrhization led to specific changes in the abundance of 99 spots, including 42 overlapping modifications between G. mosseae- and G. intraradices-colonised roots. The 32 confident identifications that could be retrieved following tandem mass spectrometry encompassed 21 fungal proteins whose homology-inferred functions were found to complement the working models so far proposed for the intra-radical functioning of AM fungi with regard to carbon utilization, energy generation, redox homeostasis and protein turnover-related processes.

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices.

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.

On the mechanisms of cadmium stress alleviation in Medicago truncatula by arbuscular mycorrhizal symbiosis: a root proteomic study.

The arbuscular mycorrhizal (AM) symbiosis belongs to the strategies plants have developed to cope with adverse environmental conditions including contamination by heavy metals such as cadmium (Cd). In the present work, we report on the protective effect conferred by AM symbiosis to the model legume Medicago truncatula grown in presence of Cd, and on the 2-D-based proteomic approach further used to compare the proteomes of M. truncatula roots either colonised or not with the AM fungus Glomus intraradices in Cd-free and Cd-contaminated substrates. The results indicated that at the proteome level, 9 out of the 15 cadmium-induced changes in nonmycorrhizal roots were absent or inverse in those Cd-treated and colonized by G. intraradices, including the G. intraradices-dependent down-accumulation of Cd stress-responsive proteins. Out of the twenty-six mycorrhiza-related proteins that were identified, only six displayed changes in abundance upon Cd exposure, suggesting that part of the symbiotic program, which displays low sensitivity to Cd, may be recruited to counteract Cd toxicity through the mycorrhiza-dependent synthesis of proteins having functions putatively involved in alleviating oxidative damages, including a cyclophilin, a guanine nucleotide-binding protein, an ubiquitin carboxyl-terminal hydrolase, a thiazole biosynthetic enzyme, an annexin, a glutathione S-transferase (GST)-like protein, and a S-adenosylmethionine (SAM) synthase.

Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions.

To gain further insight into the role of the plant genome in arbuscular mycorrhiza (AM) establishment, we investigated whether symbiosis-related plant genes affect fungal gene expression in germinating spores and at the appressoria stage of root interactions. Glomus intraradices genes were identified in expressed sequence tag libraries of mycorrhizal Medicago truncatula roots by in silico expression analyses. Transcripts of a subset of genes, with predicted functions in transcription, protein synthesis, primary or secondary metabolism, or of unknown function, were monitored in spores and germinating spores and during interactions with roots of wild-type or mycorrhiza-defective (Myc-) mutants of M. truncatula. Not all the fungal genes were active in quiescent spores but all were expressed when G. intraradices spores germinated in wild-type M. truncatula root exudates or when appressoria or arbuscules were formed in association with wild-type M. truncatula roots. Most of the fungal genes were upregulated or induced at the stage of appressorium development. Inactivation of the M. truncatula genes DMI1, DMI2/MtSYM2, or DMI3/MtSYM13 was associated with altered fungal gene expression (nonactivation or inhibition), modified appressorium structure, and plant cell wall responses, providing first evidence that cell processes modified by symbiosis-related plant genes impact on root interactions by directly modulating AM fungal activity.

An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen.

Mechanisms of root penetration by arbuscular mycorrhizal (AM) fungi are unknown and investigations are hampered by the lack of transformation systems for these unculturable obligate biotrophs. Early steps of host infection by hemibiotrophic fungal phytopathogens, sharing common features with those of AM fungal colonization, depend on the transcription factor STE12. Using degenerated primers and rapid amplification of cDNA ends, we isolated the full-length cDNA of an STE12-like gene, GintSTE, from Glomus intraradices and profiled GintSTE expression by real-time and in situ RT-PCR. GintSTE activity and function were investigated by heterologous complementation of a yeast ste12Delta mutant and a Colletotrichum lindemuthianum clste12Delta mutant. * Sequence data indicate that GintSTE is similar to STE12 from hemibiotrophic plant pathogens, especially Colletotrichum spp. Introduction of GintSTE into a noninvasive mutant of C. lindemuthianum restored fungal infectivity of plant tissues. GintSTE expression was specifically localized in extraradicular fungal structures and was up-regulated when G. intraradices penetrated roots of wild-type Medicago truncatula as compared with an incompatible mutant. Results suggest a possible role for GintSTE in early steps of root penetration by AM fungi, and that pathogenic and symbiotic fungi may share common regulatory mechanisms for invasion of plant tissues.

A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation.

One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc(++), Nod (-/+) phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.

Development and activity of Glomus intraradices as affected by co-existence with Glomus claroideum in one root system.

The co-existence of two arbuscular mycorrhizal fungal (AMF) species, Glomus intraradices and Glomus claroideum, in the root systems of plants was investigated in a greenhouse experiment aimed at reconstructing interactions during an early stage of primary succession on a coal-mine spoil bank in Central Europe. Two plant species, Tripleurospermum inodorum and Calamagrostis epigejos, were inoculated either with one or both AMF species. Fungal development, determined by trypan blue and alkaline phosphatase staining as well as by PCR amplification of rRNA genes with species-specific primers, and the expression of five genes with different metabolic functions in the intraradical structures of G. intraradices were followed after 6 and 9 weeks of cultivation. The two AMF closely co-existed in the root systems of both plants possibly through similar colonisation rates and competitivity. Inoculation with the two fungi, however, did not bring any additional benefit to the host plants in comparison with single inoculation; moreover, plant growth depression observed after inoculation with G. claroideum persisted also in mixed inoculation. The expression of all the assayed G. intraradices genes was affected either by host plant or by co-inoculation with G. claroideum. The effects of both factors depended on the time of sampling, which underlines the importance of addressing this topic in time-course studies.

Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR.

Gene expression profiling based on tissue extracts gives only limited information about genes associated with complex developmental processes such as those implicated in fungal interactions with plant roots during arbuscular mycorrhiza development and function. To overcome this drawback, a direct fluorescent in situ RT-PCR methodology was developed for spatial mapping of gene expression in different presymbiotic and symbiotic structures of an arbuscular mycorrhizal fungus. Transcript detection was optimized by targeting the LSU rRNA gene of Glomus intraradices and monitoring expression of a stearoyl-CoA-desaturase gene that is consistently expressed at high levels in spores, hyphae, arbuscules and vesicles. This method was further validated by localizing expression of fungal peptidylprolyl isomerase and superoxide dismutase genes, which are expressed to different extents in fungal structures. Direct fluorescent in situ RT-PCR offers new perspectives for the sensitive analysis of fungal developmental processes that occur during functional differentiation in symbiotic arbuscular mycorrhiza interactions.

Impact of a pesticide cocktail (fenhexamid, folpel, deltamethrin) on the abundance of Glomeromycota in two agricultural soils.

Pesticide contamination of the environment can result from agricultural practices. Persistence of pesticide residues is a threat to the soil biota including plant roots and beneficial microorganisms, which support an important number of soil ecosystem services. Arbuscular mycorrhizal fungi (AMF) are key symbiotic microorganisms contributing to plant nutrition. In the present study, we assessed whether AMF could indicate eventual side effects of pesticides when directly applied to field soils. We evaluated the ecotoxicological impact of a cocktail of three commonly used agricultural pesticides (fenhexamid, folpel, deltamethrin) on the abundance and composition of the AMF community in vineyard (Montagne de Saint-Emilion) and arable (Martincourt) soils subjected to different agricultural practices. The dissipation of applied pesticides was monitored by multiresidual analyses to determine the scenario of exposure of the AMF community. Diversity analysis before application of the pesticide cocktail showed that the AMF communities of vineyard soils, subjected to mechanical weeding or grass cover, and of the arable soil subjected to intensive agriculture, were dominated by Glomerales. Ribotypes specific to each soil and to each agricultural practice in the same soil were found, with the highest abundance and diversity of AMF being observed in the vineyard soil with a grass-cover. The abundance of the global AMF community (Glomeromycota) and of three taxa of AMF (Funneliformis mosseae, Claroideoglomus etunicatum/C. claroideum) was evaluated after pesticide application. The abundance of Glomeromycota decreased in both soils after pesticide application while the abundance of Claroideoglomus and F. mosseae decreased only in the arable soil. These results show that higher doses of pesticide exposure did not affect the global abundance, but altered the composition, of the AMF community. Resilience of the AMF community composition was observed only in the vineyard soil, where F. mosseae was the most tolerant taxon to pesticide exposure.

Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis.

We report on a large-scale expressed sequence tag (EST) sequencing and analysis program aimed at characterizing the sets of genes expressed in roots of the model legume Medicago truncatula during interactions with either of two microsymbionts, the nitrogen-fixing bacterium Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus intraradices. We have designed specific tools for in silico analysis of EST data, in relation to chimeric cDNA detection, EST clustering, encoded protein prediction, and detection of differential expression. Our 21 473 5'- and 3'-ESTs could be grouped into 6359 EST clusters, corresponding to distinct virtual genes, along with 52 498 other M.truncatula ESTs available in the dbEST (NCBI) database that were recruited in the process. These clusters were manually annotated, using a specifically developed annotation interface. Analysis of EST cluster distribution in various M.truncatula cDNA libraries, supported by a refined R test to evaluate statistical significance and by 'electronic northern' representation, enabled us to identify a large number of novel genes predicted to be up- or down-regulated during either symbiotic root interaction. These in silico analyses provide a first global view of the genetic programs for root symbioses in M.truncatula. A searchable database has been built and can be accessed through a public interface.

Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula.

Suppressive subtractive hybridization and expressed sequence tag sequencing identified 29 plant genes which are upregulated during the appressorium stage of mycorrhiza establishment between Medicago truncatula J5 (Myc+) and Glomus mosseae. Eleven genes coding plant proteins with predicted functions in signal transduction, transcription, and translation were investigated in more detail for their relation to early events of symbiotic interactions. Expression profiling showed that the genes are activated not only from the appressorium stage up to the fully established symbiosis in the Myc+ genotype of M. truncatula, but also when the symbionts are not in direct cell contact, suggesting that diffusible fungal molecules (Myc factors) play a, role in the induction of a signal-transduction pathway. Transcript accumulation in roots of a mycorrhiza-defective Myc- dmi3 mutant of M. truncatula is not modified by appressorium formation or diffusible fungal molecules, indicating that the signal transduction pathway is required for a successful G. mosseae-M. truncatula interaction leading to symbiosis development. The symbiotic nodulating bacterium Sinorhizobium meliloti does not activate the 11 genes, which supposes early discrimination by plant roots between the microbial symbionts.

Ultrastructure of cortical root cells of Alnus incana infected by Penicillium nodositatum.

Penicillium nodositatum Valla penetrates Alnus incana (L.) Moench roots through deformed root hairs. It then colonizes the cortical cells and induces the formation of myconodules which have structures similar to those of young actinorhizas. The ultrastructure of infected cortical cells has been compared with that of cells infected by pathogenic or symbiotic fungi as well as with that of cells infected by Frankia. Our observations show that an infected host cell shows few visible signs of resistance to the fungal invasion. As soon as P. nodositatum enters the cell, it is surrounded by a wall-like pecto-cellulosic matrix produced by the host cell. The host plasmalemma, invaginated around the endophyte, keeps its integrity as it does in symbiotic associations. At the beginning of the infection, the host cytoplasm and the fungus are intact but when the cell is completely invaded, the host cytoplasm becomes disorganized and subsequently the fungus also senesces, as in roots of Ericaceae infected by an endomycorrhizal fungus or as in older tissues of actinorhizas. P. nodositatum could survive the dead host cell by using its own reserves. Uninfected neighbouring host cells remain intact. P. nodositatum can be considered to behave as a neutral microsymbiont of A. incana, in a way similar to that of a compatible but ineffective Frankia strain.

Electrophoretic and immunological studies on acid phosphatase from a mycorrhizal fungus of Erica hispidula L.

Wall-bound acid phosphatases of an endomycorrhizal fungus of Erica hispidula L. were separated by PAGE, electrotransferred to nitrocellulose membranes and glycoprotein characteristics studied using Concanavalin A. An antiserum was raised against the major high-molecular-weight acid phosphatase and its specificity demonstrated by immunoblotting and ELISA. The antiserum reacted very little with protein extracts of other cellular fractions and was unable to bind to wall-bound protein antigens of a mycorrhizal isolate from K. mauritanica. Ultrastructural localization of acid phosphatase by cytoenzymology and indirect immunogold labelling showed it to be almost exclusively associated with the fungal wall and septa of living hyphae, whilst in senescent hyphae it was also present within the disorganized cytoplasm.

Targeted proteomics to identify cadmium-induced protein modifications in Glomus mosseae-inoculated pea roots.

• Arbuscular mycorrhiza (AM) can increase plant tolerance to heavy metals. A targeted proteomic approach was used to determine the putative identity of some of the proteins induced/modulated by cadmium (Cd) and to analyse the impact of the mycorrhizal process. • The effect of Cd (100 mg Cd kg-1  substrate) applied either at planting or 15 d later on two pea (Pisum sativum) genotypes, differing in sensitivity to Cd inoculated or not with the AM fungus Glomus mosseae, was studied at three levels: plant biomass production, development of G. mosseae and root differential protein display with one- and two-dimensional gel electrophoresis (1-DE and 2-DE) analyses. • Cd-induced growth inhibition was significantly alleviated by mycorrhiza in the Cd-sensitive genotype. The AM symbiosis modulated the expression of several proteins, identified by liquid chromatography-tandem mass spectrometry, newly induced and upregulated or downregulated by Cd. • The protective effect of AM symbiosis towards Cd stress was observed in the Cd-sensitive genotype. Our results demonstrate the usefulness of proteomics to better understand the possible role of AM symbiosis in detoxification/response mechanisms towards Cd in pea plants.