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Significance Oscillations in intracellular calcium concentration play an essential role in the regulation of multiple cellular processes. In plants capable of root endosymbiosis with nitrogen-fixing bacteria and/or arbuscular mycorrhizal fungi, nuclear localized calcium oscillations are essential to transduce the microbial signal. Although the ion channels required to generate the nuclear localized calcium oscillations have been identified, their mechanisms of regulation are unknown. Here, we combined proteomics and engineering approaches to demonstrate that the calcium-bound form of the calmodulin 2 (CaM2) associates with CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15s), closing the channels and providing the negative feedback to sustain the oscillatory mechanism. We further unraveled that the engineered CaM2 accelerates early endosymbioses and enhanced root nodule symbiosis but not arbuscular mycorrhization.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2. These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

How cells control the proper delivery of vesicles and their associated cargo to specific plasma membrane (PM) domains upon internal or external cues is a major question in plant cell biology. A widely held hypothesis is that expansion of plant exocytotic machinery components, such as SNARE proteins, has led to a diversification of exocytotic membrane trafficking pathways to function in specific biological processes. A key biological process that involves the creation of a specialized PM domain is the formation of a host–microbe interface (the peri-arbuscular membrane) in the symbiosis with arbuscular mycorrhizal fungi. We have previously shown that the ability to intracellularly host AM fungi correlates with the evolutionary expansion of both v- (VAMP721d/e) and t-SNARE (SYP132α) proteins, that are essential for arbuscule formation in Medicago truncatula. Here we studied to what extent the symbiotic SNAREs are different from their non-symbiotic family members and whether symbiotic SNAREs define a distinct symbiotic membrane trafficking pathway. We show that all tested SYP1 family proteins, and most of the non-symbiotic VAMP72 members, are able to complement the defect in arbuscule formation upon knock-down/-out of their symbiotic counterparts when expressed at sufficient levels. This functional redundancy is in line with the ability of all tested v- and t-SNARE combinations to form SNARE complexes. Interestingly, the symbiotic t-SNARE SYP132α appeared to occur less in complex with v-SNAREs compared to the non-symbiotic syntaxins in arbuscule-containing cells. This correlated with a preferential localization of SYP132α to functional branches of partially collapsing arbuscules, while non-symbiotic syntaxins accumulate at the degrading parts. Overexpression of VAMP721e caused a shift in SYP132α localization toward the degrading parts, suggesting an influence on its endocytic turn-over. These data indicate that the symbiotic SNAREs do not selectively interact to define a symbiotic vesicle trafficking pathway, but that symbiotic SNARE complexes are more rapidly disassembled resulting in a preferential localization of SYP132α at functional arbuscule branches.

Arbuscular mycorrhizal (AM) fungi and rhizobium bacteria are accommodated in specialized membrane compartments that form a host-microbe interface. To better understand how these interfaces are made, we studied the regulation of exocytosis during interface formation. We used a phylogenetic approach to identify target soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) that are dedicated to symbiosis and used cell-specific expression analysis together with protein localization to identify t-SNAREs that are present on the host-microbe interface in Medicago truncatula. We investigated the role of these t-SNAREs during the formation of a host-microbe interface. We showed that multiple syntaxins are present on the peri-arbuscular membrane. From these, we identified SYNTAXIN OF PLANTS 13II (SYP13II) as a t-SNARE that is essential for the formation of a stable symbiotic interface in both AM and rhizobium symbiosis. In most dicot plants, the SYP13II transcript is alternatively spliced, resulting in two isoforms, SYP13IIα and SYP13IIβ. These splice-forms differentially mark functional and degrading arbuscule branches. Our results show that vesicle traffic to the symbiotic interface is specialized and required for its maintenance. Alternative splicing of SYP13II allows plants to replace a t-SNARE involved in traffic to the plasma membrane with a t-SNARE that is more stringent in its localization to functional arbuscules.

At the heart of endosymbiosis microbes are hosted inside living cells in specialized membrane compartments that from a host-microbe interface, where nutrients and signal are efficiently exchanged. Such symbiotic interfaces include arbuscules produced by arbuscular mycorrhiza (AM) and organelle-like symbiosomes formed during the rhizobium-legume symbiosis. Also during pathogenic interactions, microbes such as biotrophic fungi and oomycetes are hosted in specialized membrane compartments called haustoria. The formation of such new membrane compartments requires a major reorganization of the host endomembrane system, with a special role for the targeting of secretory/exocytotic vesicles and their cargo to the newly forming interfaces. In this thesis, I studied how exocytotic membrane traffic is regulated to facilitate the formation and maintenance of a host-microbe interface. Therefore, I especially focussed on the role of SNARE (Soluble NSF Attachment Protein Receptor) proteins, as key components of the exocytotic machinery, in symbiotic interface formation. In Chapter 1, I introduce the different symbioses in which host-microbe interfaces are formed, and the role of the host-microbe interface in these symbioses. Further, I introduce the evolutionary relationship between the different symbioses: AM symbiosis is the most ancient endosymbiosis in plants, which provided the blueprint for different symbioses that evolved later; other symbiotic microbes including rhizobia co-opted the signalling program and adaptations to membrane trafficking required for arbuscule formation, to be hosted inside cells. Finally, I will introduce the symbiosis dedicated SNAREs as key regulators of exocytosis to form a host-microbe interface. In Chapter 2, we tested the long-standing hypothesis that pathogens make use of the AM symbiotic program to allow the formation of haustoria. To test this, we set up a pathosystem using the biotrophic oomycete Phytophthora palmivora that is able to form haustoria in Medicago truncatula root cells. Using M. truncatula mutants impaired in AM and rhizobium symbioses, we demonstrated that neither the common symbiotic signalling genes, nor symbiosis dedicated regulators of vesicle trafficking are required for haustorium formation. This showed that biotrophic pathogens like P. palmivora, do not hijack the symbiotic program to be accommodated inside plant cells. In Chapter 3, we identified the t-SNARE SYP132α as a key regulator of both arbuscule and symbiosome formation. During vesicle fusion, a vesicle SNARE (v-SNARE) on the vesicle forms a complex with a target membrane SNAREs (t-SNAREs) on the target membrane. Previous work in our lab identified specific exocytotic v-SNAREs required for arbuscule and symbiosome formation. We identified the t-SNARE counterpart SYP132, and demonstrated that in most dicot plants SYP132 is spliced into two spliceforms; SYP132α and SYP132β. Interestingly, alternative splicing of SYP132 leading to the dominant use of a SYP132α-specific last exon coincides with the accommodation of AM fungi in arbuscule forming root cortex cells and rhizobium bacteria in nodule cells. Using a spliceform-specific RNAi construct, we showed that SYP132α is specifically required for the formation of a stable host-microbe interface in both AM symbiosis and rhizobium symbiosis. Furthermore, we showed that during arbuscular collapse, the two spliceforms localize differently to healthy and degrading arbuscule branches. These results indicated that alternative splicing of SYP132 allows plants to replace a t-SNARE involved in traffic to the plasma membrane with a t-SNARE that is more stringent in its localization to functional arbuscules. The evolutionary expansion of SNAREs in plants has been hypothesized to have allowed the adaptation of exocytosis to different biological processes. In Chapter 4, we studied what makes the symbiotic SNAREs so special in comparison to their non-symbiotic family members, of which many are also expressed in arbuscule cells. We hypothesized that symbiotic SNAREs define a distinct secretory pathway, that ensures specificity of protein delivery to the host-microbe interface. We show that all tested SYP1 family proteins, and most of the non-symbiotic VAMP72 members, were able to complement the defect in arbuscule formation upon knock-down of their symbiotic counterparts when expressed at sufficient levels. This functional redundancy is in line with the ability of all tested v- and t-SNARE combinations to form SNARE complexes at the peri-arbuscular membrane. This showed that the symbiotic SNAREs do not selectively interact to define a distinct vesicle trafficking pathway, but that their essential role in arbuscule formation can be largely explained by their dominant expression level. Interestingly, the symbiotic t-SNARE SYP132α appeared to occur less in SNARE complexes with v-SNAREs compared to the non-symbiotic syntaxins in the arbuscule cells, suggesting a more strict regulation of symbiotic SNARE complexes at the interface. Since the alternative splicing of SYP132 does not affect the total transcript levels, we hypothesized that there must be a functional difference between SYP132α and –β, potentially leading to subtle phenotypes that may have gone undetected in the Agrobacterium rhizogenes mediated complementation approach applied in Chapter 4. In Chapter 5, we therefore generated and characterized a stable mutant line in which all SYP132 transcripts are constitutively spliced into the non-symbiotic SYP132β form. Although this mutant is normally colonized by AM fungi, with no effects on arbuscule morphology, it has a severely reduced biomass after mycorrhization. This hints to a yet unknown role for SYP132α to control arbuscule functionality, and offers an explanation for the evolutionary conservation of the SYP132 alternative splicing in dicot plants. Finally, using fluorescent timer fusions to both SYP132 isoforms, we showed that the difference in localization of the two proteins during arbuscular collapse is the result of a different (endocytic) turnover of the two spliceforms at the healthy/functional arbuscule branches, possibly due to a difference in interactions with VAMPs. Together, our data show that, although both SYP132 isoforms can mediate arbuscule formation, SYP132α is functionally different from SYP132β, which may reveal new aspects of the control of nutrient exchange. In Chapter 6, I discuss the data generated during my thesis research in relation to additional symbiosis dedicated regulators of exocytosis, as well as in relation to other biological processes that depend on specific secretory SNAREs. Following our conclusion that the symbiotic SNAREs do not mark a separate exocytosis pathway, but are functionally different from non-symbiotic SNAREs, I will speculate on the possible scenarios in which symbiosis dedicated SNAREs are specialized for host-microbe interface functionality.

Rapid transient elevation of cytoplasmic calcium (Ca(2+)) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca(2+) levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca(2+) sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca(2+) biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca(2+) levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca(2+) signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca(2+)signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca(2+) signaling pathways in plant cells.

Binding of specific microbial epitopes [MAMPs (microbe-associated molecular patterns)] to PRRs (pattern recognition receptors) and subsequent receptor kinase activation are key steps in plant innate immunity. One of the earliest detectable events after MAMP perception is a rapid and transient rise in cytosolic Ca2+ levels. In plants, knowledge about the signalling events leading to Ca2+ influx and on the molecular identity of the channels involved is scarce. We used a transgenic Arabidopsis thaliana line stably expressing the luminescent aequorin Ca2+ biosensor to monitor pharmacological interference with Ca2+ signatures following treatment with the bacterial peptide MAMPs flg22 and elf18, and the fungal carbohydrate MAMP chitin. Using a comprehensive set of compounds known to impede Ca2+-transport processes in plants and animals we found strong evidence for a prominent role of amino acid-controlled Ca2+ fluxes, probably through iGluR (ionotropic glutamate receptor)-like channels. Interference with amino acid-mediated Ca2+ fluxes modulates MAMP-triggered MAPK (mitogen-activated protein kinase) activity and affects MAMP-induced accumulation of defence gene transcripts. We conclude that the initiation of innate immune responses upon flg22, elf18 and chitin recognition involves apoplastic Ca2+ influx via iGluR-like channels.

In biotrophic plant-microbe interactions, microbes infect living plant cells, in which they are hosted in a novel membrane compartment, the host-microbe interface. To create a host-microbe interface, arbuscular mycorrhizal (AM) fungi and rhizobia make use of the same endosymbiotic program. It is a long-standing hypothesis that pathogens make use of plant proteins that are dedicated to mutualistic symbiosis to infect plants and form haustoria. In this report, we developed a Phytophthora palmivora pathosystem to study haustorium formation in Medicago truncatula roots. We show that P. palmivora does not require host genes that are essential for symbiotic infection and host-microbe interface formation to infect Medicago roots and form haustoria. Based on these findings, we conclude that P. palmivora does not hijack the ancient intracellular accommodation program used by symbiotic microbes to form a biotrophic host-microbe interface.

Interaction of key regulators of exocytosis with potassium channels enhances both secretion and K+ uptake, making these processes intertwined and jointly coordinated.

Plants host many different microbes within their cells. These endosymbiotic relationships are characterized by the formation of new specialized membrane compartments inside the plant cells in which the microbes live and where nutrients and signals are efficiently exchanged. Such symbiotic interfaces include arbuscules produced by arbuscular mycorrhiza (AM), organelle-like symbiosomes formed during the rhizobium-legume symbiosis, and haustoria produced by biotrophic fungi and oomycetes. The formation and maintenance of such new membrane compartments require a major reorganization of the host endomembrane system. In the last decade, much progress has been made in understanding how arbuscules, symbiosomes, and haustoria are formed. In this chapter, we will summarize the recent developments in each field, with a major focus on the AM and rhizobial endosymbiosis. It has become clear that rhizobia have co-opted a signalling pathway as well as a cellular mechanism to make the interface membrane compartment from the ancient and most successful AM symbiosis. Both AM symbiosis and rhizobium symbiosis depend on the secretion of lipo-chito-oligosaccharides that trigger a symbiotic signalling cascade, which is required for both arbuscule and symbiosome formation. In both interactions a shared specific exocytosis pathway is recruited to facilitate the formation of the symbiotic interface resulting in a membrane compartment with distinct protein composition. Given the structural similarity of haustoria to arbuscules, similar mechanisms are envisioned to be involved in the formation of a haustorium.