Exploring the potential role of four Rhizophagus irregularis nuclear effectors: opportunities and technical limitations.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts that interact with the roots of most land plants. The genome of the AMF model species Rhizophagus irregularis contains hundreds of predicted small effector proteins that are secreted extracellularly but also into the plant cells to suppress plant immunity and modify plant physiology to establish a niche for growth. Here, we investigated the role of four nuclear-localized putative effectors, i.e., GLOIN707, GLOIN781, GLOIN261, and RiSP749, in mycorrhization and plant growth. We initially intended to execute the functional studies in Solanum lycopersicum, a host plant of economic interest not previously used for AMF effector biology, but extended our studies to the model host Medicago truncatula as well as the non-host Arabidopsis thaliana because of the technical advantages of working with these models. Furthermore, for three effectors, the implementation of reverse genetic tools, yeast two-hybrid screening and whole-genome transcriptome analysis revealed potential host plant nuclear targets and the downstream triggered transcriptional responses. We identified and validated a host protein interactors participating in mycorrhization in the host.S. lycopersicum and demonstrated by transcriptomics the effectors possible involvement in different molecular processes, i.e., the regulation of DNA replication, methylglyoxal detoxification, and RNA splicing. We conclude that R. irregularis nuclear-localized effector proteins may act on different pathways to modulate symbiosis and plant physiology and discuss the pros and cons of the tools used.

Strigolactones repress nodule development and senescence in pea.

Strigolactones are a class of phytohormones that are involved in many different plant developmental processes, including the rhizobium-legume nodule symbiosis. Although both positive and negative effects of strigolactones on the number of nodules have been reported, the influence of strigolactones on nodule development is still unknown. Here, by means of the ramosus (rms) mutants of Pisum sativum (pea) cv Terese, we investigated the impact of strigolactone biosynthesis (rms1 and rms5) and signaling (rms3 and rms4) mutants on nodule growth. The rms mutants had more red, that is, functional, and larger nodules than the wild-type plants. Additionally, the increased nitrogen fixation and senescence zones with consequently reduced meristematic and infection zones indicated that the rms nodules developed faster than the wild-type nodules. An enhanced expression of the nodule zone-specific molecular markers for meristem activity and senescence supported the enlarged, fast maturing nodules. Interestingly, the master nodulation regulator, NODULE INCEPTION, NIN, was strongly induced in nodules of all rms mutants but not prior to inoculation. Determination of sugar levels with both bulk and spatial metabolomics in roots and nodules, respectively, hints at slightly increased malic acid levels early during nodule primordia formation and reduced sugar levels at later stages, possibly the consequence of an increased carbon usage of the enlarged nodules, contributing to the enhanced senescence. Taken together, these results suggest that strigolactones regulate the development of nodules, which is probably mediated through NIN, and available plant sugars.

Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility.

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.

Histone Deacetylases Regulate MORE AXILLARY BRANCHED 2-Dependent Germination of Arabidopsis thaliana.

Under specific conditions, the germination of Arabidopsis thaliana is dependent on the activation of the KARRIKIN INSENSITIVE 2 (KAI2) signaling pathway by the KAI2-dependent perception of karrikin or the artificial strigolactone analogue, rac-GR24. To regulate the induction of germination, the KAI2 signaling pathway relies on MORE AXILLARY BRANCHED 2- (MAX2-)dependent ubiquitination and proteasomal degradation of the repressor protein SUPPRESSOR OF MAX2 1 (SMAX1). It is not yet known how the degradation of SMAX1 proteins eventually results in the regulation of seed germination, but it has been hypothesized that SMAX1-LIKE generally functions as transcriptional repressors through the recruitment of co-repressors TOPLESS (TPL) and TPL-related, which in turn interact with histone deacetylases. In this article, we show the involvement of histone deacetylases HDA6, HDA9, HDA19 and HDT1 in MAX2-dependent germination of Arabidopsis, and more specifically, that HDA6 is required for the induction of DWARF14-LIKE2 expression in response to rac-GR24 treatment.

MAX2-dependent competence for callus formation and shoot regeneration from Arabidopsis thaliana root explants.

Although the division of the pericycle cells initiates both lateral root development and root-derived callus formation, these developmental processes are affected differently in the strigolactone and karrikin/KARRIKIN INSENSITIVE 2 (KAI2) ligand signalling mutant more axillary growth 2 (max2). Whereas max2 produces more lateral roots than the wild type, it is defective in the regeneration of shoots from root explants. We suggest that the decreased shoot regeneration of max2 originates from delayed formation of callus primordium, yielding less callus material to regenerate shoots. Indeed, when incubated on callus-inducing medium, the pericycle cell division was reduced in max2 and the early gene expression varied when compared with the wild type, as determined by a transcriptomics analysis. Furthermore, the expression of the LATERAL ORGAN BOUNDARIES DOMAIN genes and of callus-induction genes was modified in correlation with the max2 phenotype, suggesting a role for MAX2 in the regulation of the interplay between cytokinin, auxin, and light signalling in callus initiation. Additionally, we found that the in vitro shoot regeneration phenotype of max2 might be caused by a defect in KAI2, rather than in DWARF14, signalling. Nevertheless, the shoot regeneration assays revealed that the strigolactone biosynthesis mutants max3 and max4 also play a minor role.

Flemish soils contain rhizobia partners for Northwestern Europe-adapted soybean cultivars.

In Europe, soybean (Glycine max) used for food and feed has to be imported, causing negative socioeconomic and environmental impacts. To increase the local production, breeding generated varieties that grow in colder climates, but the yield using the commercial inoculants is not satisfactory in Belgium because of variable nodulation efficiencies. To look for indigenous nodulating strains possibly adapted to the local environment, we initiated a nodulation trap by growing early-maturing cultivars under natural and greenhouse conditions in 107 garden soils in Flanders. Nodules occurred in 18 and 21 soils in the garden and greenhouse experiments respectively. By combining 16S rRNA PCR on single isolates with HiSeq 16S metabarcoding on nodules, we found a large bacterial richness and diversity from different soils. Furthermore, using Oxford Nanopore Technologies sequencing of DNA from one nodule, we retrieved the entire genome of a Bradyrhizobium species, not previously isolated, but profusely present in that nodule. These data highlight the need of combining diverse identification techniques to capture the true nodule rhizobial community. Eight selected rhizobial isolates were subdivided by whole-genome analysis in three genera containing six genetically distinct species that, except for two, aligned with known type strains and were all able to nodulate soybean in the laboratory.

Interactions between soil compositions and the wheat root microbiome under drought stress: From an in silico to in planta perspective.

As wheat (Triticum aestivum) is an important staple food across the world, preservation of stable yields and increased productivity are major objectives in breeding programs. Drought is a global concern because its adverse impact is expected to be amplified in the future due to the current climate change. Here, we analyzed the effects of edaphic, environmental, and host factors on the wheat root microbiomes collected in soils from six regions in Belgium. Amplicon sequencing analysis of unplanted soil and wheat root endosphere samples indicated that the microbial community variations can be significantly explained by soil pH, microbial biomass, wheat genotype, and soil sodium and iron levels. Under drought stress, the biodiversity in the soil decreased significantly, but increased in the root endosphere community, where specific soil parameters seemingly determine the enrichment of bacterial groups. Indeed, we identified a cluster of drought-enriched bacteria that significantly correlated with soil compositions. Interestingly, integration of a functional analysis further revealed a strong correlation between the same cluster of bacteria and ß-glucosidase and osmoprotectant proteins, two functions known to be involved in coping with drought stress. By means of this in silico analysis, we identified amplicon sequence variants (ASVs) that could potentially protect the plant from drought stress and validated them in planta. Yet, ASVs based on 16S rRNA sequencing data did not completely distinguish individual isolates because of their intrinsic short sequences. Our findings support the efforts to maintain stable crop yields under drought conditions through implementation of root microbiome analyses.

Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor.

The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke-derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/ß-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. In addition, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14, and KAI2 protein network by tandem affinity purification in Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were copurified, among which were general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than in D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination under suboptimal conditions and seedling development. In addition, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic SL analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14, and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL-related phenotypes and, hence, provide another link with the light pathway.

Unraveling new molecular players involved in the autoregulation of nodulation in Medicago truncatula.

The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.

Quantitative Tandem Affinity Purification, an Effective Tool to Investigate Protein Complex Composition in Plant Hormone Signaling: Strigolactones in the Spotlight.

Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein-protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones.

MtNRLK1, a CLAVATA1-like leucine-rich repeat receptor-like kinase upregulated during nodulation in Medicago truncatula.

Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.

Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures.

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species.

Strigolactones spatially influence lateral root development through the cytokinin signaling network.

Strigolactones are important rhizosphere signals that act as phytohormones and have multiple functions, including modulation of lateral root (LR) development. Here, we show that treatment with the strigolactone analog GR24 did not affect LR initiation, but negatively influenced LR priming and emergence, the latter especially near the root-shoot junction. The cytokinin module ARABIDOPSIS HISTIDINE KINASE3 (AHK3)/ARABIDOPSIS RESPONSE REGULATOR1 (ARR1)/ARR12 was found to interact with the GR24-dependent reduction in LR development, because mutants in this pathway rendered LR development insensitive to GR24. Additionally, pharmacological analyses, mutant analyses, and gene expression analyses indicated that the affected polar auxin transport stream in mutants of the AHK3/ARR1/ARR12 module could be the underlying cause. Altogether, the data reveal that the GR24 effect on LR development depends on the hormonal landscape that results from the intimate connection with auxins and cytokinins, two main players in LR development.

Strigolactones as an auxiliary hormonal defence mechanism against leafy gall syndrome in Arabidopsis thaliana.

Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures-including the strigolactone response-in reaction to bacterial cytokinins to constrain the activity of R. fascians.

From lateral root density to nodule number, the strigolactone analogue GR24 shapes the root architecture of Medicago truncatula.

In the rhizosphere, strigolactones not only act as crucial signalling molecules in the communication of plants with parasitic weeds and arbuscular mycorrhiza, but they also play a key role in regulating different aspects of the root system. Here we investigated how strigolactones influence the root architecture of Medicago truncatula. We provide evidence that addition of the synthetic strigolactone analogue GR24 has an inhibitory effect on the lateral root density. Moreover, treatment with GR24 of Sinorhizobium meliloti-inoculated M. truncatula plants affects the nodule number both positively and negatively, depending on the concentration. Plants treated with 0.1 µM GR24 had a slightly increased number of nodules, whereas concentrations of 2 and 5 µM strongly reduced it. This effect was independent of the autoregulation of nodulation mechanism that is controlled by SUPER NUMERIC NODULE. Furthermore, we demonstrate that GR24 controls the nodule number through crosstalk with SICKLE-dependent ethylene signalling. Additionally, because the expression of the nodulation marker EARLY NODULATION11 was strongly reduced in GR24-treated plants, we concluded that strigolactones influence nodulation at a very early stage of the symbiotic interaction.

Role of LONELY GUY genes in indeterminate nodulation on Medicago truncatula.

LONELY GUY (LOG) genes encode cytokinin riboside 5'-monophosphate phosphoribohydrolases and are directly involved in the activation of cytokinins. To assess whether LOG proteins affect the influence of cytokinin on nodulation, we studied two LOG genes of Medicago truncatula. Expression analysis showed that MtLOG1 and MtLOG2 were upregulated during nodulation in a CRE1-dependent manner. Expression was mainly localized in the dividing cells of the nodule primordium. In addition, RNA interference revealed that MtLOG1 is involved in nodule development and that the gene plays a negative role in lateral root development. Ectopic expression of MtLOG1 resulted in a change in cytokinin homeostasis, triggered cytokinin-inducible genes and produced roots with enlarged vascular tissues and shortened primary roots. In addition, those 35S:LOG1 roots also displayed fewer nodules than the wild-type. This inhibition in nodule formation was local, independent of the SUPER NUMERIC NODULES gene, but coincided with an upregulation of the MtCLE13 gene, encoding a CLAVATA3/EMBRYO SURROUNDING REGION peptide. In conclusion, we demonstrate that in M. truncatula LOG proteins might be implicated in nodule primordium development and lateral root formation.

pFiD188, the linear virulence plasmid of Rhodococcus fascians D188.

Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.

Transcriptional and post-transcriptional regulation of a NAC1 transcription factor in Medicago truncatula roots.

• Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. • The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. • MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. • This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.

Rhodococcus fascians impacts plant development through the dynamic fas-mediated production of a cytokinin mix.

The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.