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Functional microexons have not previously been described in filamentous fungi. Here, we describe a novel mechanism of transcriptional regulation in Trichoderma requiring the inclusion of a microexon from the Xlr2 gene. In low-glucose environments, a long mRNA including the microexon encodes a protein with a GAL4-like DNA-binding domain (Xlr2-α), whereas in high-glucose environments, a short mRNA that is produced encodes a protein lacking this DNA-binding domain (Xlr2-ß). Interestingly, the protein isoforms differ in their impact on cellulase and xylanase activity. Deleting the Xlr2 gene reduced both xylanase and cellulase activity and growth on different carbon sources, such as carboxymethylcellulose, xylan, glucose, and arabinose. The overexpression of either Xlr2-α or Xlr2-ß in T. virens showed that the short isoform (Xlr2-ß) caused higher xylanase activity than the wild types or the long isoform (Xlr2-α). Conversely, cellulase activity did not increase when overexpressing Xlr2-ß but was increased with the overexpression of Xlr2-α. This is the first report of a novel transcriptional regulation mechanism of plant-cell-wall-degrading enzyme activity in T. virens. This involves the differential expression of a microexon from a gene encoding a transcriptional regulator.

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PREMISE: Endophytic and mycorrhizal fungi are crucial in facilitating plant nutrition acquisition and stress tolerance. In epiphytic habitats, plants face nutrition and water stress, but their roots are mostly nonmycorrhizal and especially lacking in arbuscular mycorrhizal associations. Ophioderma pendulum is an epiphytic fern with a partially mycoheterotrophic lifestyle, likely heavily reliant on symbiotic fungi. To characterize fungal associations in the sporophyte of O. pendulum, we focused on leaves and roots of O. pendulum, seeking to reveal the fungal communities in these organs. METHODS: Roots and leaves from O. pendulum in a subtropical forest were examined microscopically to observe the morphology of fungal structures and determine the percentage of various fungal structures in host tissues. Fungal composition was profiled using metabarcoding techniques that targeted ITS2 of the nuclear ribosomal DNA. RESULTS: Roots were consistently colonized by arbuscular mycorrhizal fungi (Glomeromycota), especially Acaulospora. Unlike previous findings on epiphytic ferns, dark septate endophytes were rare in O. pendulum roots. Leaves were predominantly colonized by Ascomycota fungi, specifically the classes Dothideomycetes (46.88%), Eurotiomycetes (11.51%), Sordariomycetes (6.23%), and Leotiomycetes (6.14%). Across sampling sites, fungal community compositions were similar in the roots but differed significantly in the leaves. CONCLUSIONS: Ophioderma pendulum maintains stable, single-taxon-dominant communities in the roots, primarily featuring arbuscular mycorrhizal fungi, whereas the leaves may harbor opportunistic fungal colonizers. Our study underlines the significance of mycorrhizal fungi in the adaptation of epiphytic ferns.

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Plants establish symbiotic associations with arbuscular mycorrhizal fungi (AMF) to facilitate nutrient uptake, particularly in nutrient-limited conditions. This partnership is rooted in the plant's ability to recognize fungal signaling molecules, such as chitooligosaccharides (chitin) and lipo-chitooligosaccharides. In the legume Medicago truncatula, chitooligosaccharides trigger both symbiotic and immune responses via the same lysin-motif-receptor-like kinases (LysM-RLKs), notably CERK1 and LYR4. The nature of plant-fungal engagement is opposite according to the outcomes of immunity or symbiosis signaling, and as such, discrimination is necessary, which is challenged by the dual roles of CERK1/LYR4 in both processes. Here, we describe a LysM-RLK, LYK8, that is functionally redundant with CERK1 for mycorrhizal colonization but is not involved in chitooligosaccharides-induced immunity. Genetic mutation of both LYK8 and CERK1 blocks chitooligosaccharides-triggered symbiosis signaling, as well as mycorrhizal colonization, but shows no further impact on immunity signaling triggered by chitooligosaccharides, compared with the mutation of CERK1 alone. LYK8 interacts with CERK1 and forms a receptor complex that appears essential for chitooligosaccharides activation of symbiosis signaling, with the lyk8/cerk1 double mutant recapitulating the impact of mutations in the symbiosis signaling pathway. We conclude that this novel receptor complex allows chitooligosaccharides activation specifically of symbiosis signaling and helps the plant to differentiate between activation of these opposing signaling processes.

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Chitin oligomers (COs) are among the most common and active fungal elicitors of plant responses. Short-chain COs from symbiotic arbuscular mycorrhizal fungi activate accommodation responses in the host root, while long-chain COs from pathogenic fungi are acknowledged to trigger defence responses. The modulation of intracellular calcium concentration - a common second messenger in a wide variety of plant signal transduction processes - plays a central role in both signalling pathways with distinct signature features. Nevertheless, mounting evidence suggests that plant immunity and symbiosis signalling partially overlap at multiple levels. Here, we elaborate on recent findings on this topic, highlighting the nonbinary nature of chitin-based fungal signals, their perception and their interpretation through Ca2+ -mediated intracellular signals. Based on this, we propose that plant perception of symbiotic and pathogenic fungi is less clear-cut than previously described and involves a more complex scenario in which partially overlapping and blurred signalling mechanisms act upstream of the unambiguous regulation of gene expression driving accommodation or defence responses.

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Iron is an essential element for most organisms. Both plants and microorganisms have developed different mechanisms for iron uptake, transport and storage. In the symbiosis systems, such as rhizobia-legume symbiosis and arbuscular mycorrhizal (AM) symbiosis, maintaining iron homeostasis to meet the requirements for the interaction between the host plants and the symbiotic microbes is a new challenge. This intriguing topic has drawn the attention of many botanists and microbiologists, and many discoveries have been achieved so far. In this review, we discuss the current progress on iron uptake and transport in the nodules and iron homeostasis in rhizobia-legume symbiosis. The discoveries with regard to iron uptake in AM fungi, iron uptake regulation in AM plants and interactions between iron and other nutrient elements during AM symbiosis are also summarized. At the end of this review, we propose prospects for future studies in this fascinating research area.

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Lysin motif receptor-like kinases (LysM-RLKs) are involved in the perception of chitooligosaccharides (COs) and related lipochitooligosaccharides (LCOs) in plants. Expansion and divergence of the gene family during evolution have led to various roles in symbiosis and defense. By studying proteins of the LYR-IA subclass of LysM-RLKs of the Poaceae, we show here that they are high-affinity LCO-binding proteins with a lower affinity for COs, consistent with a role in LCO perception to establish arbuscular mycorrhiza (AM). In Papilionoid legumes, whole-genome duplication has resulted in two LYR-IA paralogs, MtLYR1 and MtNFP in Medicago truncatula, with MtNFP playing an essential role in root nodule symbiosis with nitrogen-fixing rhizobia. We show that MtLYR1 has retained the ancestral LCO-binding characteristic and is dispensable for AM. Domain swapping between the three LysMs of MtNFP and MtLYR1 and mutagenesis in MtLYR1 suggest that the MtLYR1 LCO-binding site is on the second LysM and that divergence in MtNFP led to better nodulation, but surprisingly with decreased LCO binding. These results suggest that divergence of the LCO-binding site has been important for the evolution of a role of MtNFP in nodulation with rhizobia.

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Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) are able to provide key ecosystem services, protecting plants against biotic and abiotic stresses. Here, we hypothesized that a combination of AMF (Rhizophagus clarus) and PGPR (Bacillus sp.) could enhance 33P uptake in maize plants under soil water stress. A microcosm experiment using mesh exclusion and a radiolabeled phosphorus tracer (33P) was installed using three types of inoculation: i) only AMF, ii) only PGPR, and iii) a consortium of AMF and PGPR, alongside a control treatment without inoculation. For all treatments, a gradient of three water-holding capacities (WHC) was considered i) 30% (severe drought), ii) 50% (moderate drought), and iii) 80% (optimal condition, no water stress). In severe drought conditions, AMF root colonization of dual-inoculated plants was significantly lower compared to individual inoculation of the AMF, whilst 33P uptake by dual-inoculated plants or plants inoculated with bacteria was 2.4-fold greater than the uninoculated treatment. Under moderate drought conditions the use of AMF promoted the highest 33P uptake by plants, increasing it by 2.1-fold, when compared to the uninoculated treatment. Without drought stress, AMF showed the lowest 33P uptake and, overall, plant P acquisition was lower for all inoculation types when compared to the severe and moderate drought treatments. The total shoot P content was modulated by the water-holding capacity and inoculation type, with the lowest values observed under severe drought and the highest values under moderate drought. The highest soil electrical conductivity (EC) values were found under severe drought in AMF-inoculated plants and the lowest EC for no drought in single or dual-inoculated plants. Furthermore, water-holding capacity influenced the total soil bacterial and mycorrhizal abundance over time, with the highest abundances being found under severe and moderate drought. This study demonstrates that the positive influence of microbial inoculation on 33P uptake by plants varied with soil water gradient. Furthermore, under severe stress conditions, AMF invested more in the production of hyphae, vesicles and spore production, indicating a significant carbon drain from the host plant as evidenced by the lack of translation of increased 33P uptake into biomass. Therefore, under severe drought the use of bacteria or dual-inoculation seems to be more effective than individual AMF inoculation in terms of 33P uptake by plants, while under moderate drought, the use of AMF stood out.

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Plants interacting with mutualistic fungi (MF) or antagonistic fungi (AF) can form associations with bacteria. We assessed whether the performance gain conferred by mutualistic bacteria to fungal-associated plants is affected by the interaction between symbiont traits, type of bacterial-protective traits against AF and abiotic/biotic stresses. Results showed that (A) performance gain conferred by bacteria to MF-associated plants was greater when symbionts promoted distinct rather than similar plant functions, (B) bacterial-based alleviation of the AF's negative effect on plants was independent of the type of protective trait, (C) bacteria promoted a greater performance of symbiotic plants in presence of biotic, but not abiotic, stress compared to stress-free situations. The plant performance gain was not affected by any fungal-bacterial trait combination but optimised when bacteria conferred resistance traits in biotic stress situations. The effects of bacteria on fungal-associated plants were controlled by the interaction between the symbionts' functional traits and the relationship between bacterial traits and abiotic/biotic stresses.

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Legumes are able to meet their nitrogen need by establishing nitrogen-fixing symbiosis with rhizobia. Nitrogen fixation is performed by rhizobia, which has been converted to bacteroids, in newly formed organs, the root nodules. In the model legume Medicago truncatula, nodule cells are invaded by rhizobia through transcellular tubular structures called infection threads (ITs) that are initiated at the root hairs. Here, we describe a novel M. truncatula early symbiotic mutant identified as infection-related epidermal factor (ief), in which the formation of ITs is blocked in the root hair cells and only nodule primordia are formed. We show that the function of MtIEF is crucial for the bacterial infection in the root epidermis but not required for the nodule organogenesis. The IEF gene that appears to have been recruited for a symbiotic function after the duplication of a flower-specific gene is activated by the ERN1-branch of the Nod factor signal transduction pathway and independent of the NIN activity. The expression of MtIEF is induced transiently in the root epidermal cells by the rhizobium partner or Nod factors. Although its expression was not detectable at later stages of symbiosis, complementation experiments indicate that MtIEF is also required for the proper invasion of the nodule cells by rhizobia. The gene encodes an intracellular protein of unknown function possessing a coiled-coil motif and a plant-specific DUF761 domain. The IEF protein interacts with RPG, another symbiotic protein essential for normal IT development, suggesting that combined action of these proteins plays a role in nodule infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation of plant vegetative tissues by pathogenic Agrobacterium spp., previous reports have indicated that T-DNA sequences originating from an ancestral Agrobacterium sp. are present in the genomes of all cultivated sweet potato (Ipomoea batatas) varieties analyzed. Expression of an Agrobacterium-derived agrocinopine synthase (ACS) gene was detected in leaf and root tissues of sweet potato, suggesting that the plant can produce agrocinopine, a sugar-phosphodiester opine considered to be utilized by some strains of Agrobacterium spp. in crown gall. To validate the product synthesized by Ipomoea batatas ACS (IbACS), we introduced IbACS into tobacco under a constitutive promoter. High-voltage paper electrophoresis followed by alkaline silver nitrate staining detected the production of an agrocinopine-like substance in IbACS1-expressing tobacco, and further mass spectrometry and nuclear magnetic resonance analyses of the product confirmed that IbACS can produce agrocinopine A from natural plant substrates. The partially purified compound was biologically active in an agrocinopine A bioassay. A 16S ribosomal RNA amplicon sequencing and meta-transcriptome analysis revealed that the rhizosphere microbial community of tobacco was affected by the expression of IbACS. A new species of Leifsonia (actinobacteria) was isolated as an enriched bacterium in the rhizosphere of IbACS1-expressing tobacco. This Leifsonia sp. can catabolize agrocinopine A produced in tobacco, indicating that the production of agrocinopine A attracts rhizosphere bacteria that can utilize this sugar-phosphodiester. These results suggest a potential role of IbACS conserved among sweet potato cultivars in manipulating their microbial community.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic 'Candidatus Liberibacter' spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of 'Ca. L. asiaticus' bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that 'Ca. L. asiaticus' BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. 'Ca. L. asiaticus' BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of 'Ca. L. asiaticus' acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Beneficial rhizobacteria can stimulate changes in plant root development. Although root system growth is mediated by multiple factors, the regulated distribution of the phytohormone auxin within root tissues plays a principal role. Auxin transport facilitators help to generate the auxin gradients and maxima that determine root structure. Here, we show that the plant-growth-promoting rhizobacterial strain Bradyrhizobium japonicum IRAT FA3 influences specific auxin efflux transporters to alter Arabidopsis thaliana root morphology. Gene expression profiling of host transcripts in control and B. japonicum-inoculated roots of the wild-type A. thaliana accession Col-0 confirmed upregulation of PIN2, PIN3, PIN7, and ABCB19 with B. japonicum and identified genes potentially contributing to a diverse array of auxin-related responses. Cocultivation of the bacterium with loss-of-function auxin efflux transport mutants revealed that B. japonicum requires PIN3, PIN7, and ABCB19 to increase lateral root development and utilizes PIN2 to reduce primary root length. Accelerated lateral root primordia production due to B. japonicum was not observed in single pin3, pin7, or abcb19 mutants, suggesting independent roles for PIN3, PIN7, and ABCB19 during the plant-microbe interaction. Our work demonstrates B. japonicum's influence over host transcriptional reprogramming during plant interaction with this beneficial microbe and the subsequent alterations to root system architecture.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Fusaricidins produced by Paenibacillus polymyxa are important lipopeptide antibiotics against fungi. The fusGFEDCBA (fusaricidin biosynthesis) operon is responsible for synthesis of fusaricidins. However, the regulation mechanisms of fusaricidin biosynthesis remain to be fully clarified. In this study, we revealed that fusaricidin production is controlled by a complex regulatory network including KinB-Spo0A-AbrB. Evidence suggested that the regulator AbrB represses the transcription of the fus gene cluster by direct binding to the fus promoter, in which the sequences (5'-AATTTTAAAATAAATTTTGTGATTT-3') located from -136 to -112 bp relative to the transcription start site is required for this repression. Spo0A binds to the abrB promoter that contains the Spo0A-binding sequences (5'-TGTCGAA-3', 0A box) and in turn prevents the further transcription of abrB. The decreasing concentration of AbrB allows for the derepression of the fus promoter repressed by AbrB. The genome of P. polymyxa WLY78 contains two orthologs (named Kin1508 and Kin4833) of Bacillus subtilis KinB, but only Kin4833 activates sporulation and fusaricidin production, indicating that this kinase may be involved in phosphorylating Spo0A to initiate sporulation and regulate the abrB transcription. Our results reveal that Kin4833 (KinB), Spo0A, and AbrB are involved in regulation of fusaricidin production and a signaling mechanism that links fusaricidin production and sporulation.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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