In symbiotic plant-microbe interactions, the host invests considerable amounts of resources in the microbial partner. If the microbe does not reciprocate with a comparable symbiotic benefit, it is regarded as a cheater. The host responds to cheaters with negative feedback mechanisms (sanctions) to prevent fitness deficits resulting from being exploited. We study sanctioning in the symbiosis between Medicago truncatula and the nitrogen-fixing rhizobium Sinorhizobium meliloti. We manipulated the exchange of resources between the partners in three ways: by using mutant rhizobia defective in nitrogenase; replacing nitrogen in the atmosphere with argon gas; and supplying rich nitrogen fertilizer to the host. We follow the consequences of simulated cheating by examining the metabolome and proteome of both partners. We find that sanctioning occurs at multiple levels. In particular, we observe repression of essential symbiotic functions and changes in central metabolism that are likely to be relevant for microbial fitness and that could therefore contribute to sanctioning. In addition, sanctioning triggers a broad panel of defense markers. A thorough understanding of the multilevel phenomenon of sanctioning will be essential for its genetic dissection and for the breeding of elite legume crops with efficient symbiosis.

In plant-microbe interactions, the host plant invests considerable amounts of resources in the microbial partner until the symbiotic machinery is established. If the microbial partner does not reciprocate with a comparable symbiotic benefit, the interaction represents a parasitic relationship. This is thought to elicit a plant's response to prevent the selective disadvantage of being parasitized by such microbial cheaters. Indeed, negative feedback against bad mutualists, known as sanctioning, has been observed in interactions such as the arbuscular mycorrhizal and legume-rhizobium symbioses. Here, to study sanctioning by the plant host, we manipulate the exchange of resources between the model legume Medicago truncatula and its bacterial partner Sinorhizobium meliloti by three ways: mutating the bacterial nitrogenase enzyme, replacing nitrogen in the atmosphere with argon gas, and supplying high nitrate to the host. Then, we follow the consequences for the interaction by examining the metabolome, proteome, and phosphoproteome of nodules. We find that such cheating conditions result in sanctioning of the bacterial partner, and observe characteristic shifts including induced defense markers, repressed symbiotic markers, and changes in central metabolism that may be relevant for microbial fitness and that could therefore contribute to sanctioning. ### Competing Interest Statement The authors have declared no competing interest.

Type III protein secretion systems of nitrogen-fixing rhizobia deliver effector proteins into leguminous host cells to promote or inhibit the nodule symbiosis. However, mechanisms underlying effector-triggered inhibition of nodulation remain largely unknown. Nodulation outer protein T (NopT) of Sinorhizobium sp. NGR234 is an effector protease related to the Pseudomonas effector Avirulence protein Pseudomonas phaseolicola B (AvrPphB). Here, we constructed NGR234 mutants producing different NopT variants and found that protease activity of NopT negatively affects nodulation of smooth crotalaria (Crotalaria pallida). NopT variants lacking residues required for autocleavage and subsequent lipidation showed reduced symbiotic effects and were not targeted to the plasma membrane. We further noticed that Sinorhizobium fredii strains possess a mutated nopT gene. Sinorhizobium fredii USDA257 expressing nopT of NGR234 induced considerably fewer nodules in soybean (Glycine max) cv. Nenfeng 15 but not in other cultivars. Effector perception was further examined in NopT-expressing leaves of Arabidopsis (Arabidopsis thaliana) and found to be dependent on the protein kinase Arabidopsis AvrPphB Susceptible 1 (AtPBS1) and the associated resistance protein Arabidopsis Resistance to Pseudomonas syringae 5 (AtRPS5). Experiments with Nicotiana benthamiana plants indicated that the soybean homolog GmPBS1-1 associated with AtRPS5 can perceive NopT. Further analysis showed that NopT cleaves AtPBS1 and GmPBS1-1 and thus can activate these target proteins. Insertion of a DKM motif at the cleavage site of GmPBS1-1 resulted in increased proteolysis. Nodulation tests with soybeans expressing an autoactive GmPBS1-1 variant indicated that activation of a GmPBS1-1-mediated resistance pathway impairs nodule formation in cv. Nenfeng 15. Our findings suggest that legumes face an evolutionary dilemma of either developing effector-triggered immunity against pathogenic bacteria or establishing symbiosis with suboptimally adapted rhizobia producing pathogen-like effectors.

The intimate association of host and fungus in arbuscular mycorrhizal (AM) symbiosis can potentially trigger induction of host defence mechanisms against the fungus, implying that successful symbiosis requires suppression of defence. We addressed this phenomenon by using AM-defective vapyrin (vpy) mutants in Petunia hybrida, including a new allele (vpy-3) with a transposon insertion close to the ATG start codon. We explore whether abortion of fungal infection in vpy mutants is associated with the induction of defence markers, such as cell wall alterations, accumulation of reactive oxygen species (ROS), defence hormones and induction of pathogenesis-related (PR) genes. We show that vpy mutants exhibit a strong resistance against intracellular colonization, which is associated with the generation of cell wall appositions (papillae) with lignin impregnation at fungal entry sites, while no accumulation of defence hormones, ROS or callose was observed. Systematic analysis of PR gene expression revealed that several PR genes are induced in mycorrhizal roots of the wild-type, and even more in vpy plants. Some PR genes are induced exclusively in vpy mutants. Our results suggest that VPY is involved in avoiding or suppressing the induction of a cellular defence syndrome that involves localized lignin deposition and PR gene induction.

Abstract Most land plants entertain a mutualistic symbiosis known as arbuscular mycorrhiza with fungi (Glomeromycota) that provide them with essential mineral nutrients, in particular phosphate (Pi), and protect them from biotic and abiotic stress. Arbuscular mycorrhizal (AM) symbiosis increases plant productivity and biodiversity and is therefore relevant for both natural plant communities and crop production. However, AM fungal populations suffer from intense farming practices in agricultural soils, in particular Pi fertilization. The dilemma between natural fertilization from AM symbiosis and chemical fertilization has raised major concern and emphasizes the need to better understand the mechanisms by which Pi suppresses AM symbiosis. Here, we test the hypothesis that Pi may interfere with AM symbiosis via the phytohormone gibberellic acid (GA) in the Solanaceous model systems Petunia hybrida and Nicotiana tabacum. Indeed, we find that GA is inhibitory to AM symbiosis and that Pi may cause GA levels to increase in mycorrhizal roots. Consistent with a role of endogenous GA as an inhibitor of AM development, GA-defective N. tabacum lines expressing a GA-metabolizing enzyme (GA methyltransferase—GAMT) are colonized more quickly by the AM fungus Rhizoglomus irregulare, and exogenous Pi is less effective in inhibiting AM colonization in these lines. Systematic gene expression analysis of GA-related genes reveals a complex picture, in which GA degradation by GA2 oxidase plays a prominent role. These findings reveal potential targets for crop breeding that could reduce Pi suppression of AM symbiosis, thereby reconciling the advantages of Pi fertilization with the diverse benefits of AM symbiosis.

The VAPYRIN (VPY) gene in Medicago truncatula and Petunia hybrida is required for arbuscular mycorrhizal (AM) symbiosis. The moss Physcomitrella patens has a close homolog (VPY-like, VPYL), although it does not form AM. Here, we explore the phylogeny of VPY and VPYL in land plants, and study the expression and developmental function of VPYL in Ppatens We show that VPYL is expressed primarily in the protonema, the early filamentous stage of moss development, and later in rhizoids arising from the leafy gametophores and in adult phyllids. Knockout mutants have specific phenotypes in branching of the protonema and in cell division of the leaves (phyllids) in gametophores. The mutants are responsive to auxin and strigolactone, which are involved in regulation of protonemal branching, indicating that hormonal signaling in the mutants is not affected in hormonal signaling. Taken together, these results suggest that VPYL exerts negative regulation of protonemal branching and cell division in phyllids. We discuss VPY and VPYL phylogeny and function in land plants in the context of AM symbiosis in angiosperms and development in the moss.

Arbuscular mycorrhiza (AM) is a symbiosis between plants and AM fungi that requires the intracellular accommodation of the fungal partner in the host. For reciprocal nutrient exchange, AM fungi form intracellular arbuscules that are surrounded by the peri-arbuscular membrane. This membrane, together with the fungal plasma membrane, and the space in between, constitute the symbiotic interface, over which nutrients are exchanged. Intracellular establishment of AM fungi requires the VAPYRIN protein which is induced in colonized cells, and which localizes to numerous small mobile structures of unknown identity (Vapyrin-bodies). In order to characterize the identity and function of the Vapyrin-bodies we pursued a dual strategy. First, we co-expressed fluorescently tagged VAPYRIN with a range of subcellular marker proteins, and secondly, we employed biochemical tools to identify interacting partner proteins of VAPYRIN. As an important tool for the quantitative analysis of confocal microscopic data sets from co-expression of fluorescent proteins, we developed a semi-automated image analysis pipeline that allows for precise spatio-temporal quantification of protein co-localization and of the dynamics of organelle association from movies. Taken together, these experiments revealed that Vapyrin-bodies have an endosomal identity with trans-Golgi features, and that VAPYRIN interacts with a symbiotic R-SNARE of the VAMP721 family, that localizes to the same compartment.

Arbuscular mycorrhiza (AM) is the most common symbiotic association of plants with microbes. AM fungi occur in the majority of natural habitats and they provide a range of important ecological services, in particular by improving plant nutrition, stress resistance and tolerance, soil structure and fertility. AM fungi also interact with most crop plants including cereals, vegetables, and fruit trees, therefore, they receive increasing attention for their potential use in sustainable agriculture. Basic research of the past decade has revealed the existence of a dedicated recognition and signaling pathway that is required for AM. Furthermore, recent evidence provided new insight into the exchange of nutritional benefits between the symbiotic partners. The great potential for application of AM has given rise to a thriving industry for AM-related products for agriculture, horticulture, and landscaping. Here, we discuss new developments in these fields, and we highlight future potential and limits toward the use of AM fungi for plant production.

A knowledge gap still remains concerning the in situ influences of nanoparticles on plant systems, partly due to the absence of soil microorganisms. Arbuscular mycorrhizal fungi (AMF) can form a mutualistic symbiosis with the roots of over 90% of land plants. This investigation sought to reveal the responses of mycorrhizal clover (Trifolium repens) to silver nanoparticles (AgNPs) and iron oxide nanoparticles (FeONPs) along a concentration gradient of each. FeONPs at 3.2 mg/kg significantly reduced mycorrhizal clover biomass by 34% by significantly reducing the glomalin content and root nutrient acquisition of AMF. In contrast, no negative effects of AgNPs at concentrations over 0.1 mg/kg were observed; however, AgNPs at 0.01 mg/kg inhibited mycorrhizal clover growth. In response to the elevated AgNPs content, the ability of AMF to alleviate AgNPs stress (via increased growth and ecological behaviors) was enhanced, which decreased Ag content and the activities of antioxidant enzymes in plants. These results were further supported by X-ray microcomputed tomography. Our findings suggest that in soil ecosystem, the influence of nanometals on plant systems would be more complicated than expected, and more attention should be focused on plant responses in combination with those of soil microorganisms.