Association of mycoheterotrophic Gentianaceae with specific Glomus lineages.

Some plant species took an alternative evolutionary pathway in which they lost their photosynthetic capacity to depend exclusively on carbon supplied by arbuscular mycorrhizal fungi (AMF) in an association called mycoheterotrophy. Among them is Voyriella parviflora, a species of the family Gentianaceae, which is found in tropical regions such as the Amazon basin. Here, we assessed the identity of AMF symbionts associated with this species. DNA was isolated from eight Gentianaceae specimens and from litter and surrounding roots of photosynthetic plants. The atp1 gene was amplified by Sanger sequencing to determine the taxonomic affiliation of the mycoheterotrophic plants. A 280 bp region of the 18S rRNA gene of AMF was amplified with primers NS31/AML2 by high-throughput sequencing. The mycoheterotrophic specimens were assigned to V. parviflora with a bootstrap support of 72%. Glomus was the most abundant AMF genus, both in the mycoheterotrophic plants and in the litter and roots of photosynthetic plants. In addition, a few Glomus genotypes were abundantly enriched in the mycoheterotrophic plants, with only a few specimens colonized by Gigaspora, Acaulospora, and Scutellospora in a low proportion. These genotypes formed a cluster within a larger clade, suggesting that V. parviflora shows a preferential association with a narrow Glomus lineage which is not phylogenetically close to a previously identified V. parviflora's associated lineage. Furthermore, detecting fungi from other families suggests that V. parviflora is colonized by other genera, although with low frequency. These findings provide new insights into the association between AMF and mycoheterotrophic species and highlight the importance of considering trap culture-independent approaches in understanding this symbiosis.

Transcriptomics and metabolomics reveal the changes induced by arbuscular mycorrhizal fungi in Panax quinquefolius L.

BACKGROUND: Panax quinquefolius L. is one of the most important foods and herbs because of its high nutritional value and medicinal potential. In our previous study we found that the ginsenoside content in P. quinquefolius was improved by arbuscular mycorrhizal fungi (AMFs). However, little research has been conducted on the molecular mechanisms in P. quinquefolius roots induced by AMFs colonization. To identify the metabolomic and transcriptomic mechanisms of P. quinquefolius induced by AMFs, non-mycorrhized (control) and mycorrhized (AMF) P. quinquefolius were used as experimental materials for comparative analysis of the transcriptome and metabolome. RESULTS: Compared with the control, 182 metabolites and 545 genes were significantly changed at the metabolic and transcriptional levels in AMFs treatment. The metabolic pattern of AMFs was changed, and the contents of ginsenosides (Rb1, Rg2), threonine, and glutaric acid were significantly increased. There were significant differences in the expression of genes involved in plant hormone signal transduction, glutathione metabolism, and the plant-pathogen interaction pathway. In addition, several transcription factors from the NAC, WRKY, and basic helix-loop-helix families were identified in AMFs versus the control. Furthermore, the combined analysis of 'transcriptomics-metabolomics' analysis showed that 'Plant hormone signal transduction', 'Amino sugar and nucleotide sugar metabolism' and 'Glutathione metabolism' pathways were the important enriched pathways in response to AMFs colonization. CONCLUSION: Overall, these results provide new insights into P. quinquefolius response to AMFs, which improve our understanding of the molecular mechanisms of P. quinquefolius induced by AMFs. © 2023 Society of Chemical Industry.

Mycorrhiza-induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity.

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.

Clonal spore populations in sporocarps of arbuscular mycorrhizal fungi.

Some arbuscular mycorrhizal (AM) fungal species known to form sporocarps (i.e., aggregations of spores) are polyphyletic in two orders, Glomerales and Diversisporales. Spore clusters (sporocarp-like structures) often formed in pot cultures or in vitro conditions are supposed to be clonal populations, while sporocarps in natural habitats with a fungal peridium are morphologically similar to those of epigeous sexual (zygosporic) sporocarps of Endogone species. Thus, in this study, we explored the genetics of sporocarpic spores of two AM fungi with a view to possibilities of clonal or sexual reproduction during sporocarps formation. To examine these possibilities, we investigated single-nucleotide polymorphisms (SNPs) in reduced genomic libraries of spores isolated from sporocarps molecularly identified as Rhizophagus irregularis and Diversispora epigaea. In addition, partial sequences of the MAT locus HD2 gene of R. irregularis were phylogenetically analyzed to determine the nuclear status of the spores. We found that most SNPs were shared among the spores isolated from each sporocarp in both species. Furthermore, all HD2 sequences from spores isolated from three R. irregularis sporocarps were identical. These results indicate that those sporocarps comprise clonal spores. Therefore, sporocarps with clonal spores may have different functions than sexual reproduction, such as massive spore production or spore dispersal via mycophagy.

Intracellular sequestration of cadmium and zinc in ectomycorrhizal fungus Amanita muscaria (Agaricales, Amanitaceae) and characterization of its metallothionein gene.

Amanita muscaria is an ectomycorrhizal mushroom that commonly grows at metal-polluted sites. Sporocarps from the lead smelter-polluted area near Príbram (Central Bohemia, Czech Republic) showed elevated concentrations of Cd and Zn. Size exclusion chromatography of the cell extracts of the sporocarps from both polluted and unpolluted sites indicated that substantial part of intracellular Cd and Zn was sequestered in 6-kDa complexes, presumably with metallothionein(s) (MT). When the cultured mycelial isolates were compared, those from Príbram were more Cd-tolerant and accumulated slightly less Cd and Zn than those from the unpolluted site. The analysis of the available A.muscaria sequence data returned a 67-amino acid (AA) MT encoded by the AmMT1 gene. Weak Cd and Zn responsiveness of AmMT1 in the mycelia suggested its metal homeostasis function in A.muscaria, rather than a major role in detoxification. The AmMT1 belongs to a ubiquitous peptide group in the Agaricomycetes consisting of 60-70-AA MTs containing seven cysteinyl domains and a conserved histidyl, features observed also in a newly predicted, atypical 45-AA RaMT1 of the Zn-accumulator Russula bresadolae in which the C-terminal cysteinyl domains VI and VII are missing. Heterologous expression in metal-sensitive yeast mutants indicated that AmMT1 and RaMT1 encode functional peptides that can protect cells against Cd, Zn, and Cu toxicity. The metal protection phenotype observed in yeasts with mutant variants of AmMT1 and RaMT1 further indicated that the conserved histidyl seems to play a structural, not metal binding role, and the cysteinyls of the C-terminal domains VI and VII are important for Cu binding. The data provide an important insight into the metal handling of site-associated ectomycorrhizal species disturbed by excess metals and the properties of MTs common in Agaricomycetes.

Tuber eburneum and Tuber mujicii: New pine-associated Tuber species from eastern North America.

The ectomycorrhizal truffle genus Tuber is widespread and diverse. Recent sampling of ascomata, ectomycorrhizal root tips, and environmental sequences has resulted in the identification of many Tuber species that cannot be assigned to described species and require formal description. Using morphological and molecular phylogenetic analysis, we describe two North American Tuber species associated with pines (Pinus spp.). Tuber eburneum, sp. nov., is an early-diverging taxon in the Melanosporum clade that differs substantially from all other taxa in that clade due to its light-colored peridium and gleba, lack of peridial warts, and peridial hairs that are ornamented with small, irregular protrusions. Tuber mujicii, sp. nov., is a whitish truffle species in the Puberulum clade. Although T. mujicii is morphologically similar to many related taxa, it can be distinguished by a combination of characters, including peridium color, spore size, number of ascospores per ascus, and number of reticulations across the spore surface.

New species of Tulasnella associated with Australian terrestrial orchids in the subtribes Megastylidinae and Thelymitrinae.

Tulasnella (Tulasnellaceae) is a genus of fungus that can form mycorrhizal associations with orchids (Orchidaceae). Here we used molecular phylogenetic analyses and morphological characteristics of pure cultures across four different media to support the description of five new Tulasnella species associated with commonly occurring and endangered Australian orchids. Tulasnella nerrigaensis associates with Calochilus; T. subasymmetrica and T. kiataensis with Thelymitra; and T. korungensis and T. multinucleata with Pyrorchis and Rimacola respectively. The newly described species were primarily delimited by analyses of five loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), C14436 (adenosine triphosphate [ATP] synthase), C4102 (glutamate synthase), C3304 (ATP helicase), and mt large subunit 16S rDNA (mtLSU). Tulasnella subasymmetrica is introduced for some isolates previously identified as T. asymmetrica, and this latter species is characterized from multilocus sequencing of a new isolate that matches ITS sequences from the ex-type culture. Morphological differences between the new species are slight. Tulasnella multinucleata has 6-12 nuclei per hyphal compartment which is the first instance of multinucleate rather than binucleate or trinucleate hyphal compartments in Tulasnella. The formal description of these species of Tulasnella will aid in future evolutionary and ecological studies of orchid-fungal interactions.

Two new Russula species (fungi) from dry dipterocarp forest in Thailand suggest niche specialization to this habitat type.

Dry dipterocarp forests are among the most common habitat types in Thailand. Russulaceae are known as common ectomycorrhizal symbionts of Dipterocarpaceae trees in this type of habitat. The present study aims to identify collections of Russula subsection Amoeninae Buyck from dry dipterocarp forests in Thailand. A multi-locus phylogenetic analysis placed Thai Amoeninae collections in two novel lineages, and they are described here as R. bellissima sp. nov. and R. luteonana sp. nov. The closest identified relatives of both species were sequestrate species suggesting that they may belong to drought-adapted lineages. An analysis of publicly available ITS sequences in R. subsect. Amoeninae did not confirm evidence of any of the new species occurring in other Asian regions, indicating that dry dipterocarp forests might harbor a novel community of ectomycorrhizal fungi. Macromorphological characters are variable and are not totally reliable for distinguishing the new species from other previously described Asian Amoeninae species. Both new species are defined by a combination of differentiated micromorphological characteristics in spore ornamentation, hymenial cystidia and hyphal terminations in the pileipellis. The new Amoeninae species may correspond to some Russula species collected for consumption in Thailand, and the detailed description of the new species can be used for better identification of edible species and food safety in the region.

Metabolic and antioxidant effects of inoculation with arbuscular mycorrhizal fungi in crops of flesh-coloured Solanum tuberosum treated with fungicides.

BACKGROUND: Solanum tuberosum tubers have higher content of phenolic compounds such as hydroxycinnamic acid derivatives (HCAD) and anthocyanins in coloured genotypes. The use of fungicides for crops is common, but there are few studies regarding the interaction of fungicides and arbuscular mycorrhizal fungi (AMF). Here, the AMF-plant interactions and the metabolic responses of three potato genotypes with different tuber colorations (VR808, CB2011-509 and CB2011-104) inoculated with Claroideoglomus claroideum (CC), Claroideoglomus lamellosum (HMC26) or Funneliformis mosseae (HMC7) were studied together with the use of the fungicides MONCUT (M) and ReflectXtra (R). Mycorrhizal traits, phenolic compound profiles and antioxidant activity (AA) were evaluated. RESULTS: Despite only two HCADs being identified, with 5-caffeolquinic acid the most abundant, four anthocyanins were detected only in purple potato genotypes. The anthocyanin and HCAD profiles, as well as AA, showed that the CB2011-104 genotype had better characteristics than the other genotypes, while VR808 and CB509 showed similar responses. The responses were dependent on the specific combinations of genotype, fungicide and the AMF strain, and generally showed better responses when colonized by AMFs. CONCLUSION: The three potato genotypes had differential responses depending on the inoculated AMFs and the fungicide applied before sowing, where the optimal combinations for antioxidant response, mycorrhization degree and performance were HMC26/R for VR808, HMC7/M for CB2011-509 and HMC26/M for CB2011-104. Our results suggest the existence of functional compatibility that can be registered as beneficial effects even at the genotypic level of the host regarding a specific AMF strain. © 2021 Society of Chemical Industry.

Identification and characterization of eight metallothionein genes involved in heavy metal tolerance from the ectomycorrhizal fungus Laccaria bicolor.

Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification. The increasing numbers of available genomic sequences of ectomycorrhizal (ECM) fungi enable deeper insights into the characteristics of MT genes in these fungi that form the most important symbiosis with the host trees in forest ecosystems. The aim of this study was to establish a comprehensive, genome-wide inventory of MT genes from the ECM fungus Laccaria bicolor. Eight MT genes in L. bicolor were cloned, and the expression patterns of their transcripts at various developmental stages based on expressed sequence tag (EST) counts were analyzed. The expression levels of four MTs were significantly increased during symbiosis stages. Quantitative real-time PCR (qRT-PCR) analysis revealed that transcripts of LbMT1 were dominant in free-living mycelia and strongly induced by excessive copper (Cu), cadmium (Cd), and hydrogen peroxide (H2O2). To determine whether these eight MTs functioned as metal chelators, we expressed them in the Cu- and Cd-sensitive yeast mutants, cup1∆ and yap1∆, respectively. All LbMT proteins provided similar levels of Cu(II) or Cd(II) tolerance, but did not affect by H2O2. Our findings provide novel data on the evolution and diversification of fungal MT gene duplicates, a valuable resource for understanding the vast array of biological processes in which these proteins are involved.

Transcriptomics of different tissues of blueberry and diversity analysis of rhizosphere fungi under cadmium stress.

Blueberry (Vaccinium ssp.) is a perennial shrub belonging to the family Ericaceae, which is highly tolerant of acid soils and heavy metal pollution. In the present study, blueberry was subjected to cadmium (Cd) stress in simulated pot culture. The transcriptomics and rhizosphere fungal diversity of blueberry were analyzed, and the iron (Fe), manganese (Mn), copper (Cu), zinc (Zn) and cadmium (Cd) content of blueberry tissues, soil and DGT was determined. A correlation analysis was also performed. A total of 84 374 annotated genes were identified in the root, stem, leaf and fruit tissue of blueberry, of which 3370 were DEGs, and in stem tissue, of which 2521 were DEGs. The annotation data showed that these DEGs were mainly concentrated in a series of metabolic pathways related to signal transduction, defense and the plant-pathogen response. Blueberry transferred excess Cd from the root to the stem for storage, and the highest levels of Cd were found in stem tissue, consistent with the results of transcriptome analysis, while the lowest Cd concentration occurred in the fruit, Cd also inhibited the absorption of other metal elements by blueberry. A series of genes related to Cd regulation were screened by analyzing the correlation between heavy metal content and transcriptome results. The roots of blueberry rely on mycorrhiza to absorb nutrients from the soil. The presence of Cd has a significant effect on the microbial community composition of the blueberry rhizosphere. The fungal family Coniochaetaceae, which is extremely extremelytolerant, has gradually become the dominant population. The results of this study increase our understanding of the plant regulation mechanism for heavy metals, and suggest potential methods of soil remediation using blueberry.

MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation.

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile.

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (13C and 15 N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in 13C but not in 15 N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 ± 8.2% of their C from fungal sources, using the 13C enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

Application of Virus-Induced Gene Silencing to Arbuscular Mycorrhizal Fungi.

Downregulation of AM fungal genes using a plant viral vector is feasible. A partial sequence of a target fungal gene is cloned into the multicloning site of CMV2-A1 vector developed from RNA2 of Cucumber mosaic virus Y strain, and the RNA2, together with RNA1 and RNA3 of the virus, are in vitro-transcribed. Inoculation of Nicotiana benthamiana with these viral RNAs results in reconstitution of the virus in the plant, which triggers silencing of the fungal gene. Here, we describe not only the methods but also several tips for successful application of virus-induced gene silencing to AM fungi.

Arsenic toxicity and its mitigation in ectomycorrhizal fungus Hebeloma cylindrosporum through glutathione biosynthesis.

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.

Species composition of arbuscular mycorrhizal communities changes with elevation in the Andes of South Ecuador.

Arbuscular mycorrhizal fungi (AMF) are the most prominent mycobionts of plants in the tropics, yet little is known about their diversity, species compositions and factors driving AMF distribution patterns. To investigate whether elevation and associated vegetation type affect species composition, we sampled 646 mycorrhizal samples in locations between 1000 and 4000 m above sea level (masl) in the South of Ecuador. We estimated diversity, distribution and species compositions of AMF by cloning and Sanger sequencing the 18S rDNA (the section between AML1 and AML2) and subsequent derivation of fungal OTUs based on 99% sequence similarity. In addition, we analyzed the phylogenetic structure of the sites by computing the mean pairwise distance (MPD) and the mean nearest taxon difference (MNTD) for each elevation level. It revealed that AMF species compositions at 1000 and 2000 masl differ from 3000 and 4000 masl. Lower elevations (1000 and 2000 masl) were dominated by members of Glomeraceae, whereas Acaulosporaceae were more abundant in higher elevations (3000 and 4000 masl). Ordination of OTUs with respect to study sites revealed a correlation to elevation with a continuous turnover of species from lower to higher elevations. Most of the abundant OTUs are not endemic to South Ecuador. We also found a high proportion of rare OTUs at all elevations: 79-85% of OTUs occurred in less than 5% of the samples. Phylogenetic community analysis indicated clustering and evenness for most elevation levels indicating that both, stochastic processes and habitat filtering are driving factors of AMF community compositions.

Heterologous expression of Zn-binding peptide RaZBP1 from Russula bresadolae does not overcome Zn and Cd detoxification mechanisms in Hebeloma mesophaeum.

Homeostatic mechanisms preventing the toxicity of heavy metal ions in cells involve, among others, compartmentalization and binding with peptidaceous ligands, particularly the cysteinyl-rich metallothioneins (MTs). We have previously shown that in natural conditions Zn-overaccumulating ectomycorrhizal (EM) fungus Russula bresadolae stores nearly 40% of Zn bound with cysteinyl- and hystidyl-containing RaZBP peptides, which resemble MTs, while the detoxification of Zn and Cd in EM Hebeloma mesophaeum relies upon compartmentalization in small vesicles and vacuoles, respectively. Here, we examined the performance of RaZBP1 gene expressed in H. mesophaeum mycelium with respect to handling of Zn and Cd. Expression of RaZBP1 impaired growth of the mycelium on low-Zn medium by 60%, the growth was partly ameliorated upon the addition of Zn and remained considerable up to 2 mmol/L Zn, while the growth of the wild-type and control mycelia transformed with empty T-DNA was severely reduced in the presence of 0.5 mmol/L Zn; furthermore, RaZBP1 slightly added to Cd tolerance in the range of Cd concentrations of 0.625 to 8 µmol/L. Staining of Zn- or Cd-exposed hyphal cells with Zn- or Cd-specific fluorescent tracers did not indicate that the expression of RaZBP1 would redirect the flow of the metals away from their innate sinks. Size exclusion chromatography of extracted metal species revealed that the complexes corresponding to Zn/Cd-RaZBP1 are present only in minute levels. Considering that RaZBP1 inhibited growth at low Zn, and despite the benefit that it provided to H. mesophaeum in the presence of high Zn and moderate Cd, these data indicate that the binding of excess Zn and Cd with RaZBP1 is not a trait that would be outright transmitted to H. mesophaeum.

Effector proteins of Rhizophagus proliferus: conserved protein domains may play a role in host-specific interaction with different plant species.

Arbuscular mycorrhizal (AM) fungi show high promiscuity in terms of host. Effector proteins expressed by AM fungi are found important in establishing interaction with host. However, the mechanistic underlying host-specific interactions of the fungi remain unknown. The present study aimed (i) to identify effectors encoded by Rhizophagus proliferus and (ii) to understand molecular specificity encoded in effectors for interaction with specific plant species. The effectors predicted from the whole genome sequence were annotated by homology search in NCBI non-redundant protein, Interproscan, and pathogen-host interaction (PHI) databases. In total, 416 small secreted peptides (SSPs) were predicted, which were effector peptides with presence of nuclear localization signal, small cysteine-rich, and repeat-containing proteins domains. Similar to the functionally validated SP7 effectors in Rhizophagus irregularis, two proteins (RP8598 and RP23081) were identified in R. proliferus. To understand whether interaction between SP7 and the plant target protein, ERF19, is specific in nature, we examined protein-peptide interaction using in silico molecular docking. Pairwise interaction of RP8598 and RP23081 with the ethylene-responsive factors (ERF19) coded by five different plant species (Lotus japonicus, Solanum lycopersicum, Ocimum tenuiflorum, Medicago truncatula, Diospyros kaki) was investigated. Prediction of high-quality interaction of SP7 effector with ERF19 protein expressed only by specific plant species was observed in in silico molecular docking, which may reiterate the role of effectors in host specificity. The outcomes from our study indicated that sequence precision encoded in the effector peptides of AM fungi and immunomodulatory proteins of host may regulate host specificity in these fungi.

Structural Variations in LysM Domains of LysM-RLK PsK1 May Result in a Different Effect on Pea⁻Rhizobial Symbiosis Development.

Lysin-motif receptor-like kinase PsK1 is involved in symbiosis initiation and the maintenance of infection thread (IT) growth and bacterial release in pea. We verified PsK1 specificity in relation to the Nod factor structure using k1 and rhizobial mutants. Inoculation with nodO and nodE nodO mutants significantly reduced root hair deformations, curling, and the number of ITs in k1-1 and k1-2 mutants. These results indicated that PsK1 function may depend on Nod factor structures. PsK1 with replacement in kinase domain and PsSYM10 co-production in Nicotiana benthamiana leaves did not induce a hypersensitive response (HR) because of the impossibility of signal transduction into the cell. Replacement of P169S in LysM3 domain of PsK1 disturbed the extracellular domain (ECD) interaction with PsSYM10's ECD in Y2H system and reduced HR during the co-production of full-length PsK1 and PsSYM0 in N. benthamiana. Lastly, we explored the role of PsK1 in symbiosis with arbuscular mycorrhizal (AM) fungi; no significant differences between wild-type plants and k1 mutants were found, suggesting a specific role of PsK1 in legume⁻rhizobial symbiosis. However, increased sensitivity to a highly aggressive Fusarium culmorum strain was found in k1 mutants compared with the wild type, which requires the further study of the role of PsK1 in immune response regulation.

Emerging roles of tetraspanins in plant inter-cellular and inter-kingdom communication.

Inter-cellular and inter-kingdom signaling systems of various levels of complexity regulate pathogenic and mutualistic interactions between bacteria, parasites, and fungi and animal and plant hosts. Inter-kingdom interactions between mutualistic bacteria such as rhizobia and legumes during nodulation and between fungi and plants during mycorrhizal associations, are characterized by the extensive exchange of molecular signals, which allow nitrogen and phosphate assimilation, respectively. A novel aspect of this signaling exchange is the existence of specific structures, the exosomes, that carry important molecules that shape the plant-pathogen interactions. Exosomes contain a wide array of molecules, such as lipids, proteins, messenger RNA, and microRNAs, that play important roles in cell-to-cell communication in animal and plant cells by affecting gene expression and other physiological activity in distant cells within the same organism (e.g., during cancer metastases and neuron injuries). In plant cells, it has been recently reported that exosomes go beyond organism boundaries and inhibit a pathogenic interaction in plants. Plant produce and send exosomes loaded with specific small miRNA which inhibit the pathogen infection, but the pathogen can also produce exosomes carrying pro-pathogenic proteins and microRNAs. Therefore, exosomes are the important bridge regulating the signal exchange. Exosomes are small membrane-bound vesicles derived from multivesicular bodies (MVBs), which carries selected cargos from the cytoplasm (protein, lipids, and microRNAs) and under certain circumstances, they fuse with the plasma membrane, releasing the small vesicles as cargo-carrying exosomes into the extracellular space during intercellular and inter-kingdom communication. Animal and plant proteomic studies have demonstrated that tetraspanin proteins are an integral part of exosome membranes, positioning tetraspanins as essential components for endosome organization, with key roles in membrane fusion, cell trafficking, and membrane recognition. We discuss the similarities and differences between animal tetraspanins and plant tetraspanins formed during plant-microbe interactions and their potential role in mutualistic communication.