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Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: Medtr4g107930: CP3; Medtr4g079770: CP4; Medtr5g022560: CP2; Medtr4g079470: CP5; TC106667: Actine; Medtr1g099310.1: PR8; Medtr4g120970.1/Medtr6g033450.1: PR10; Medtr5g010640.1: PR5.3; Medtr8g096910.1: PR5.6; Medtr5g088770.1: PHYTOCYSTATIN32; Medtr2g026040.1: PHYTOCYSTATIN5; Medtr4g085800: DNF2; Medtr3g079850: SymCRK; MtrunA17_Chr7g0239441: RSD. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: Medtr4g107930: CP3; Medtr4g079770: CP4; Medtr5g022560: CP2; Medtr4g079470: CP5; TC106667: Actine; Medtr1g099310.1: PR8; Medtr4g120970.1/Medtr6g033450.1: PR10; Medtr5g010640.1: PR5.3; Medtr8g096910.1: PR5.6; Medtr5g088770.1: PHYTOCYSTATIN32; Medtr2g026040.1: PHYTOCYSTATIN5; Medtr4g085800: DNF2; Medtr3g079850: SymCRK; MtrunA17_Chr7g0239441: RSD.; International audience; Medicago (Medicago truncatula) establishes a symbiosis with the rhizobia Sinorhizobium sp, resulting in the formation of nodules where the bacteria fix atmospheric nitrogen. The loss of immunity repression or early senescence activation compromises symbiont survival and leads to the formation of nonfunctional nodules (fix−). Despite many studies exploring an overlap between immunity and senescence responses outside the nodule context, the relationship between these processes in the nodule remains poorly understood. To investigate this phenomenon, we selected and characterized three Medicago mutants developing fix− nodules and showing senescence responses. Analysis of specific defense (PATHOGENESIS-RELATED PROTEIN) or senescence (CYSTEINE PROTEASE) marker expression demonstrated that senescence and immunity seem to be antagonistic in fix− nodules. The growth of senescence mutants on non-sterile (sand/perlite) substrate instead of sterile in vitro conditions decreased nodule senescence and enhanced defense, indicating that environment can affect the immunity/senescence balance. The application of wounding stress on wild-type (WT) fix+ nodules led to the death of intracellular rhizobia and associated with co-stimulation of defense and senescence markers, indicating that in fix+ nodules the relationship between the two processes switches from opposite to synergistic to control symbiont survival during response to the stress. Our data show that the immune response in stressed WT nodules is linked to the repression of DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (SymCRK), and REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD), key genes involved in symbiotic immunity suppression. This study provides insight to understand the links between senescence and immunity in Medicago nodules. Analyses of Medicago mutants with non-functional nodules highlight the relationship and mechanisms controlling the establishment of the immune and senescence programs during nodule organogenesis.

As a major adverse environmental factor in most parts of the world, drought causes substantial crop yield losses. Rice (Oryza sativa) is one of the staple foods for more than one-half of the world’s population. Rice plants are sensitive to even mild drought stress and need almost twice the amount of water compared to wheat (Triticum aestivum) or maize (Zea mays). Arabidopsis (Arabidopsis thaliana) small GTPase Nucleolar GTP-binding protein 1 (AtNOG1) plays a role in biotic stress tolerance. Here, we created transgenic rice lines constitutively overexpressing AtNOG1-1 or AtNOG1-2. We also developed rice RNA interference (RNAi) lines that show downregulation of OsNOG1. AtNOG1-1 and AtNOG1-2 overexpressors showed enhanced drought tolerance without compromising grain yield, whereas OsNOG1-RNAi was more susceptible to drought when compared to wild-type plants. Analysis of physiological parameters showed increased cell sap osmolality, relative water content, and abscisic acid (ABA) level, but decreased leaf water loss in AtNOG1-1 or AtNOG1-2 overexpressor lines compared to the control. We found upregulation of several genes involved in ABA and jasmonic acid (JA) signaling, stomata regulation, osmotic potential maintenance, stress protection, and disease resistance in AtNOG1-1 and AtNOG1-2 overexpressor lines compared to the control. We elucidated the role of NOG1-2 and NOG1-1 in regulation of silica body formation around stomata to prevent transpirational water loss. These results provide an avenue to confer drought tolerance in rice.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula., Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.

Many plant species open their leaves during the daytime and close them at night as if sleeping. This leaf movement is known as nyctinasty, a unique and intriguing phenomenon that been of great interest to scientists for centuries. Nyctinastic leaf movement occurs widely in leguminous plants, and is generated by a specialized motor organ, the pulvinus. Although a key determinant of pulvinus development, PETIOLULE-LIKE PULVINUS (PLP), has been identified, the molecular genetic basis for pulvinus function is largely unknown. Here, through an analysis of knockout mutants in barrelclover (Medicago truncatula), we showed that neither altering brassinosteroid (BR) content nor blocking BR signal perception affected pulvinus determination. However, BR homeostasis did influence nyctinastic leaf movement. BR activity in the pulvinus is regulated by a BR-inactivating gene PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1), which is directly activated by PLP. A comparative analysis between M. truncatula and the non-pulvinus forming species Arabidopsis and tomato (Solanum lycopersicum) revealed that PLP may act as a factor that associates with unknown regulators in pulvinus determination in M. truncatula. Apart from exposing the involvement of BR in the functionality of the pulvinus, these results have provided insights into whether gene functions among species are general or specialized.

A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula. Cuticular wax is composed of very long chain fatty acids and their derivatives. Defects in cuticular wax often result in organ fusion, but little is known about the role of cuticular wax in compound leaf and flower development in Medicago truncatula. In this study, through an extensive screen of a Tnt1 retrotransposon insertion population in M. truncatula, we identified four mutant lines, named wrinkled flower and leaf (wfl) for their phenotype. The phenotype of the wfl mutants is caused by a Tnt1 insertion in Medtr3g105550, encoding 3-ketoacyl-CoA synthase (KCS), which functions as a rate-limiting enzyme in very long chain fatty acid elongation. Reverse transcription-quantitative PCR showed that WFL was broadly expressed in aerial organs of the wild type, such as leaves, floral organs, and the shoot apical meristem, but was expressed at lower levels in roots. In situ hybridization showed a similar expression pattern, mainly detecting the WFL transcript in epidermal cells of the shoot apical meristem, leaf primordia, and floral organs. The wfl mutant leaves showed sparser epicuticular wax crystals on the surface and increased water permeability compared with wild type. Further analysis showed that in wfl leaves, the percentage of C20:0, C22:0, and C24:0 fatty acids was significantly increased, the amount of cuticular wax was markedly reduced, and wax constituents were altered compared to the wild type. The reduced formation of cuticular wax and wax composition changes on the leaf surface might lead to the developmental defects observed in the wfl mutants. These findings suggest that WFL plays a key role in cuticular wax formation and in the late stage of leaf and flower development in M. truncatula.

Iron–sulfur (Fe–S) clusters are inorganic cofactors that are present in all kingdoms of life as part of a large number of proteins involved in several cellular processes, including DNA replication and metabolism. In this work, we demonstrate an additional role for two Fe–S cluster genes in biotic stress responses in plants. Eleven Fe–S cluster genes, including the NITROGEN FIXATION S-LIKE1 (NFS1) and its interactor FRATAXIN (FH), when silenced in Nicotiana benthamiana, compromised nonhost resistance to Pseudomonas syringae pv. tomato T1. NbNFS1 expression was induced by pathogens and salicylic acid. Arabidopsis (Arabidopsis thaliana) atnfs and atfh mutants, with reduced AtNFS1 or AtFH gene expression, respectively, showed increased susceptibility to both host and nonhost pathogen infection. Arabidopsis AtNFS1 and AtFH overexpressor lines displayed decreased susceptibility to infection by host pathogen P. syringae pv. tomato DC3000. The AtNFS1 overexpression line exhibited constitutive upregulation of several defense-related genes and enrichment of gene ontology terms related to immunity and salicylic acid responses. Our results demonstrate that NFS1 and its interactor FH are involved not only in nonhost resistance but also in basal resistance, suggesting a new role of the Fe–S cluster pathway in plant immunity.

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana. We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit. The pssRNAit server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.

SUMMARY Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon‐based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed‐derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1–5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1‐based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near‐saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses., Significance Statement Retrotransposable elements transpose from one position in the DNA to another during embryogenesis, causing mutations at the genomic landing site. Here we show that Tnt1, a retrotransposable element from tobacco, can transpose in a monocot plant, Brachypodium distachyon. Based on the analyses of Tnt1 insertions in B. distachyon, we estimated that 29 613 lines would be needed to achieve a 90% probability of tagging every gene in the B. distachyon genome.

Plants being sessile integrate information from a variety of endogenous and external cues simultaneously to optimize growth and development. This necessitates the signaling networks in plants to be highly dynamic and flexible. One such network involves heterotrimeric G-proteins comprised of Gα, Gβ, and Gγ subunits, which influence many aspects of growth, development, and stress response pathways. In plants such as Arabidopsis, a relatively simple repertoire of G-proteins comprised of one canonical and three extra-large Gα, one Gβ and three Gγ subunits exists. Because the Gβ and Gγ proteins form obligate dimers, the phenotypes of plants lacking the sole Gβ or all Gγ genes are similar, as expected. However, Gα proteins can exist either as monomers or in a complex with Gβγ, and the details of combinatorial genetic and physiological interactions of different Gα proteins with the sole Gβ remain unexplored. To evaluate such flexible, signal-dependent interactions and their contribution toward eliciting a specific response, we have generated Arabidopsis mutants lacking specific combinations of Gα and Gβ genes, performed extensive phenotypic analysis, and evaluated the results in the context of subunit usage and interaction specificity. Our data show that multiple mechanistic modes, and in some cases complex epistatic relationships, exist depending on the signal-dependent interactions between the Gα and Gβ proteins. This suggests that, despite their limited numbers, the inherent flexibility of plant G-protein networks provides for the adaptability needed to survive under continuously changing environments.

GAs play key roles in controlling cell proliferation through the GIBBERELLIN INSENSITIVE DWARF1/DELLA-mediated pathway. However, how DELLA proteins affect downstream pathways is not well understood. Therefore, discovering the signaling events downstream of DELLAs is key to better understanding the roles of GAs in plant development. Here, we discovered that miR396 is regulated by SLENDER RICE1 (SLR1) in controlling cell proliferation. The positive response of rice (Oryza sativa) GROWTH-REGULATING FACTORs (OsGRFs) to GAs was found to be caused by a negative response of miR396 to GAs. miR396 acts downstream of SLR1 and upstream of GA-induced cell-cycle genes. Rice INDETERMINATE DOMAIN2 (OsIDD2) directly binds the promoter of OsmiR396a and can interact with SLR1 in vivo and in vitro. Rice lines overexpressing miR396a (miR396OE) or OsIDD2 (OsIDD2OE) displayed dwarfism resulting from higher abundance of miR396 RNA. However, the stem elongation of OsIDD2OE plants could be significantly stimulated by applying exogenous GA(3), while that of miR396OE plants could not. Rice with OsIDD2 knocked down by RNA interference showed a slr1-like phenotype, in which the expression of miR396 was inhibited while its targets were enhanced. The protein levels of OsIDD2 were unaffected by GA in wild-type and OsIDD2OE plants, implying that OsIDD2 promotes the expression of miR396 and likely requires the coactivator of SLR1. Taken together, these results provided a close link between SLR1/OsIDD2 and GRFs via a negative regulator, miR396, and thus highlighted a molecular mechanism of GA-mediated cell proliferation in rice.

Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in four Medicago truncatula Tnt1-insertion mutants affected in arbuscular mycorrhizal colonisation. We located their Tnt1 insertions and identified alleles for two genes known to be involved in mycorrhization, RAM1 and KIN3. We compared the effects of the kin3-2 and ram1-4 mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting that RAM1 acts upstream of KIN3. Additionally, KIN3 appears to be involved in the suppression of plant defences in response to the fungal symbiont. KIN3 is located on the endoplasmic reticulum of arbuscule-containing cortical cells, and kin3-2 mutants plants hosted significantly fewer arbuscules than the wild type. KIN3 plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild-type plants, the kin3-2 mutant did not exhibit increased root colonisation under high nitrogen.

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388 888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene, AGAMOUS-LIKE FLOWER (AGLF), involved in flower development in the model legume Medicago truncatula. Loss-of-function of AGLF results in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C gene AGAMOUS (MtAG) in M. truncatula, the defects in floral organ identity are similar between aglf and mtag, but the floral indeterminacy is enhanced in the aglf mutant. Knockout of AGLF in the mutants of the class A gene MtAP1 or the class B gene MtPI leads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship of AGLF with the canonical A and B genes. Furthermore, we demonstrate that AGLF activates MtAG in transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.

Medicago truncatula is a model system for legume plants, which has substantially expanded the genome relative to the prototypical model dicot plant, Arabidopsis thaliana. An essential transcriptional regulator, FCP1 (transcription factor IIF-interacting RNA polymerase II carboxyl-terminal phosphatase 1) ortholog, is encoded by a single essential gene CPL4 (CTD-phosphatase-like 4), whereas M. truncatula genome contains four genes homologous to FCP1/AtCPL4, and splicing variants of MtCPL4 are observed. Functional diversification of MtCPL4 family proteins was analyzed using recombinant proteins (MtCPL4a1, MtCPL4a2, and MtCPL4b) produced in Arabidopsis cell culture system developed for plant protein overexpression. In vitro CTD phosphatase assay using highly purified MtCPL4 preparations revealed a potent CTD phosphatase activity in MtCPL4b, but not two splicing variants of MtCPL4a. On the other hand, in planta binding assay to RNA polymerase II (pol II) revealed a greater pol II-binding activity of both MtCPL4a variants. Our results indicate functional diversification of MtCPL4 isoforms and suggest the presence of a large number of functionally specialized CTD-phosphatase-like proteins in plants.

Plant leaves have evolved into diverse shapes and LATE MERISTEM IDENTITY1 (LMI1) and its putative paralogous genes encode homeodomain leucine zipper transcription factors that are proposed evolutionary hotspots for the regulation of leaf development in plants. However, the LMI1-mediated regulatory mechanism underlying leaf shape formation is largely unknown. MtLMI1a and MtLMI1b are putative orthologs of LMI1 in the model legume barrelclover (Medicago truncatula). Here, we investigated the role of MtLMI1a and MtLMI1b in leaf margin morphogenesis by characterizing loss-of-function mutants. MtLMI1a and MtLMI1b are expressed along leaf margin in a near-complementary pattern, and they redundantly promote development of leaf margin serrations, as revealed by the relatively smooth leaf margin in their double mutants. Moreover, MtLMI1s directly activate expression of SMOOTH LEAF MARGIN1 (SLM1), which encodes an auxin efflux carrier, thereby regulating auxin distribution along the leaf margin. Further analysis indicates that MtLMI1s genetically interact with NO APICAL MERISTEM (MtNAM) and the ARGONAUTE7 (MtAGO7)-mediated trans-acting short interfering RNA3 (TAS3 ta-siRNA) pathway to develop the final leaf margin shape. The participation of MtLMI1s in auxin-dependent leaf margin formation is interesting in the context of functional conservation. Furthermore, the diverse expression patterns of LMI1s and their putative paralogs within key domains are important drivers for functional specialization, despite their functional equivalency among species.

Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10‐silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA‐sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two‐hybrid assay. Silencing or mutants of genes encoding these RPL10‐interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation‐associated genes during nonhost pathogen infection., Ribosomal protein QM/RPL10 and its interacting proteins RRM, RPL30, RPL23, and RPS30 have extraribosomal functions and play a role in nonhost disease resistance.

International audience; The formation of nitrogen-fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen-fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin-motif receptor-like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild-type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild-type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan-defective strains was achieved by in trans rescue with a Nod factor-deficient S. meliloti mutant. While the Nod factor-deficient strain was always more abundant inside nodules, the succinoglycan-deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.

[EN] Legumes have unique features, such as compound inflorescences and a complex floral ontogeny. Thus, the study of regulatory genes in these species during inflorescence and floral development is essential to understand their role in the evolutionary origin of developmental novelties. The SUPERMAN (SUP) gene encodes a C2H2 zinc-finger transcriptional repressor that regulates the floral organ number in the third and fourth floral whorls of Arabidopsis thaliana. In this work, we present the functional characterization of the Medicago truncatula SUPERMAN (MtSUP) gene based on gene expression analysis, complementation and overexpression assays, and reverse genetic approaches. Our findings provide evidence that MtSUP is the orthologous gene of SUP in M. truncatula. We have unveiled novel functions for a SUP-like gene in eudicots. MtSUP controls not only the number of floral organs in the inner two whorls, but also in the second whorl of the flower. Furthermore, MtSUP regulates the activity of the secondary inflorescence meristem, thus controlling the number of flowers produced. Our work provides insight into the regulatory network behind the compound inflorescence and flower development in this angiosperm family., This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; BIO2016-75485-R). A.L.R. acknowledges a Santiago Grisolia fellowship (GRISOLIA 2017/168) from the Generalitat Valenciana. We thank Dr Beatriz Sabater (IBMCP, Valencia) for valuable suggestions and comments regarding evolutionary biology and Dr Javier Forment, head of the Bioinformatics Core Service at the IBMCP, for providing data from the OrthoVenn web platform. We thank Dr Lynne Yenush (IBMCP, Valencia) for English correction, and Dr Miguel A. Blazquez (IBMCP, Valencia) and Dr Javier Paz-Ares (CNB, Madrid) for critical reading of the manuscript. We would also like to acknowledge the technical assistance of Maria Victoria Palau in the glasshouse and Marisol Gascon in microscopy. Document

Puccinia novopanici is an important biotrophic fungal pathogen that causes rust disease in switchgrass. Lack of genomic resources for P. novopanici has hampered the progress toward developing effective disease resistance against this pathogen. Therefore, we have sequenced the whole genome of P. novopanici and generated a framework to understand pathogenicity mechanisms and identify effectors, repeat element invasion, genome evolution, and comparative genomics among Puccinia spp. in the future. Long- and short-read sequences were generated from P. novopanici genomic DNA by PacBio and Illumina technologies, respectively, and assembled a 99.9-Mb genome. Transcripts of P. novopanici were predicted from assembled genome using MAKER and were further validated by RNAseq data. The genome sequence information of P. novopanici will be a valuable resource for researchers working on monocot rusts and plant disease resistance in general.

Medicago truncatula was chosen by the legume community, along with Lotus japonicus, as a model plant to study legume biology. Since then, numerous resources and tools have been developed for M. truncatula. These include, for example, its genome sequence, core ecotype collections, transformation/regeneration methods, extensive mutant collections, and a gene expression atlas. This review aims to describe the different genetic and genomic tools and resources currently available for M. truncatula. We also describe how these resources were generated and provide all the information necessary to access these resources and use them from a practical point of view. © 2017 by John Wiley & Sons, Inc. Keywords: genomics; genome sequence; genetic diversity; insertion mutagenesis; medicago truncatula; mutagenesis

Summary The multidrug and toxin extrusion (MATE) transporter family comprises 70 members in the Medicago truncatula genome, and they play seemingly important, yet mostly uncharacterized, physiological functions. Here, we employed bioinformatics and molecular genetics to identify and characterize MATE transporters involved in citric acid export, Al3+ tolerance and Fe translocation. MtMATE69 is a citric acid transporter induced by Fe-deficiency. Overexpression of MtMATE69 in hairy roots altered Fe homeostasis and hormone levels under Fe-deficient or Fe-oversupplied conditions. MtMATE66 is a plasma membrane citric acid transporter primarily expressed in root epidermal cells. The mtmate66 mutant had less root growth than the wild type under Al3+ stress, and seedlings were chlorotic under Fe-deficient conditions. Overexpression of MtMATE66 rendered hairy roots more tolerant to Al3+ toxicity. MtMATE55 is involved in seedling development and iron homeostasis, as well as hormone signaling. The mtmate55 mutant had delayed development and chlorotic leaves in mature plants. Both knock-out and overexpression mutants of MtMATE55 showed altered Fe accumulation and abnormal hormone levels compared with the wild type. We demonstrate that the zinc-finger transcription factor MtSTOP is essentially required for MtMATE66 expression and plant resistance to H+ and Al3+ toxicity. The proper expression of two previously characterized MATE flavonoid transporters MtMATE1 and MtMATE2 also depends on several transcription factors. This study reveals not only functional diversity of MATE transporters and regulatory mechanisms in legumes against H+ and Al3+ stresses, but also casts light on their role in metal nutrition and hormone signaling under various stresses.

Salmonellosis outbreaks associated with sprouted legumes have been a food safety concern for over two decades. Despite evidence that Salmonella enterica triggers biotic plant defense pathways, it has remained unclear how plant defenses impact Salmonella growth on sprouted legumes. We used Medicago truncatula mutants in which the gene for the flagellin receptor FLS2 was disrupted to demonstrate that plant defenses triggered by FLS2 elicitation do not impact the growth of Salmonella enterica serovar Typhimurium ATCC 14028S. As a control, we tested the growth of Salmonella enterica serovar Typhimurium LT2, which has a defect in rpoS that increases its sensitivity to reactive oxygen species. LT2 displayed enhanced growth on M. truncatula FLS2 mutants in comparison to wild-type M. truncatula. We hypothesize that these growth differences are primarily due to differences in 14028S and LT2 reactive oxygen species sensitivity. Results from this study show that FLS2-mediated plant defenses are ineffective in inhibiting growth of Salmonella entrica 14028S., This study demonstrates that FLS2-mediated plant defenses are ineffective in preventing growth of Salmonella enterica strain 14028S.

Cytokinin oxidase/dehydrogenases (CKXs) play a key role in the irreversible degradation of phytohormone cytokinin that is necessary for various plant growth and development processes. However, thus far, detailed investigations of the CKX gene family in the model legume Medicago truncatula are limited. In this study, we identified 9 putative CKX homologues with conserved FAD- and cytokinin-binding domains in the M. truncatula genome. We analyzed their phylogenetic relationship, gene structure, conserved domain, expression pattern, protein subcellular locations and other properties. The tissue-specific expression profiles of the MtCKX genes are different among different members and these MtCKXs also displayed different patterns in response to synthetic cytokinin 6-benzylaminopurine (6-BA) and indole-3-acetic acid (IAA), suggesting their diverse roles in M. truncatula development. To further understand the biological function of MtCKXs, we identified and characterized mutants of each MtCKX by taking advantage of the Tnt1 mutant population in M. truncatula. Results indicated that M. truncatula plants harboring Tnt1 insertions in each single MtCKX genes showed no morphological changes in aerial parts, suggesting functional redundancy of MtCKXs in M. truncatula shoot development. However, disruption of Medtr4g126160, which is predominantly expressed in roots, leads to an obvious reduced primary root length and increased lateral root number, indicating the specific roles of cytokinin in regulating root architecture. We systematically analyzed the MtCKX gene family at the genome-wide level and revealed their possible roles in M. truncatula shoot and root development, which shed lights on understanding the biological function of CKX family genes in related legume plants.

Optimising the timing of flowering contributes to successful sexual reproduction and yield in agricultural plants. FLOWERING LOCUS T (FT) genes, first identified in Arabidopsis thaliana (Arabidopsis), promote flowering universally, but the upstream flowering regulatory pathways can differ markedly among plants. Flowering in the model legume, Medicago truncatula (Medicago) is accelerated by winter cold (vernalisation) followed by long day (LD) photoperiods leading to elevated expression of the floral activator, FT-like gene FTa1. However, Medicago, like some other plants, lacks the activator CONSTANS (CO) and the repressor FLOWERING LOCUS C (FLC) genes which directly regulate FT and are key to LD and vernalisation responses in Arabidopsis. Conversely, Medicago has a VERNALISATION2-LIKE VEFS-box gene (MtVRN2). In Arabidopsis AtVRN2 is a key member of a Polycomb complex involved in stable repression of Arabidopsis FLC after vernalisation. VRN2-like genes have been identified in other eudicot plants, but their function has never been reported. We show that Mtvrn2 mutants bypass the need for vernalisation for early flowering in LD conditions in Medicago. Investigation of the underlying mechanism by transcriptome analysis reveals that Mtvrn2 mutants precociously express FTa1 and other suites of genes including floral homeotic genes. Double-mutant analysis indicates that early flowering is dependent on functional FTa1. The broad significance of our study is that we have demonstrated a function for a VRN2-like VEFS gene beyond the Brassicaceae. In particular, MtVRN2 represses the transition to flowering in Medicago by regulating the onset of expression of the potent floral activator, FTa1.

The C function gene AGAMOUS (AG) encodes for a MADS-box transcription factor required for floral organ identity and floral meristem (FM) determinacy in angiosperms. Unlike Arabidopsis, most legume plants possess two AG homologs arose by an ancient genome duplication event. Recently, two euAGAMOUS genes, MtAGa and MtAGb, were characterized and shown to fulfill the C function activity in the model legume Medicago truncatula. Here, we reported the isolation and characterization of a new mtaga allele by screening the Medicago Tnt1 insertion mutant collection. We found that MtAGa was not only required for controlling the stamen and carpel identity but also affected pod and seed development. Genetic analysis indicated that MtAGa and MtAGb redundantly control Medicago floral organ identity, but have minimal distinct functions in regulating stamen and carpel development in a dose-dependent manner. Interestingly, the stamens and carpels are mostly converted to numerous vexillum-like petals in the double mutant of mtaga mtagb, which is distinguished from Arabidopsis ag. Further qRT-PCR analysis in different mtag mutants revealed that MtAGa and MtAGb can repress the expression of putative A and B function genes as well as MtWUS, but promote putative D function genes expression in M. truncatula. In addition, we found that the abnormal dorsal petal phenotype observed in the mtaga mtagb double mutant is associated with the upregulation of CYCLOIDEA (CYC)-like TCP genes. Taken together, our data suggest that the redundant MtAGa and MtAGb genes of M. truncatula employ a conserved mechanism of action similar to Arabidopsis in determining floral organ identity and FM determinacy but may have evolved distinct function in regulating floral symmetry by coordinating with specific floral dorsoventral identity factors.

Virus-induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built-in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword-based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org. Noble Research Institute and NSF IOS-1025642

Like Arabidopsis thaliana, the flowering of the legume Medicago truncatula is promoted by long day (LD) photoperiod and vernalization. However, there are differences in the molecular mechanisms involved, with orthologs of two key Arabidopsis thaliana regulators, FLOWERING LOCUS C (FLC) and CONSTANS (CO), being absent or not having a role in flowering time function in Medicago. In Arabidopsis, the MADS-box transcription factor gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (AtSOC1), plays a key role in integrating the photoperiodic and vernalization pathways. In this study, we set out to investigate whether the Medicago SOC1 genes play a role in regulating flowering time. Three Medicago SOC1 genes were identified and characterized (MtSOC1a–MtSOC1c). All three MtSOC1 genes, when heterologously expressed, were able to promote earlier flowering of the late-flowering Arabidopsis soc1-2 mutant. The three MtSOC1 genes have different patterns of expression. However, consistent with a potential role in flowering time regulation, all three MtSOC1 genes are expressed in the shoot apex and are up-regulated in the shoot apex of plants in response to LD photoperiods and vernalization. The up-regulation of MtSOC1 genes was reduced in Medicago fta1-1 mutants, indicating that they are downstream of MtFTa1. Insertion mutant alleles of Medicago soc1b do not flower late, suggestive of functional redundancy among Medicago SOC1 genes in promoting flowering.

Plants face several challenges by bacterial, fungal, oomycete, and viral pathogens during their life cycle. In order to defend against these biotic stresses, plants possess a dynamic, innate, natural immune system that efficiently detects potential pathogens and initiates a resistance response in the form of basal resistance and/or resistance (R)-gene-mediated defense, which is often associated with a hypersensitive response. Depending upon the nature of plant–pathogen interactions, plants generally have two main defense mechanisms, host resistance and nonhost resistance. Host resistance is generally controlled by single R genes and less durable compared with nonhost resistance. In contrast, nonhost resistance is believed to be a multi-gene trait and more durable. In this review, we describe the mechanisms of host and nonhost resistance against fungal and bacterial plant pathogens. In addition, we also attempt to compare host and nonhost resistance responses to identify similarities and differences, and their practical applications in crop improvement.

Non-homologous end joining (NHEJ) is the major model proposed for Agrobacterium T-DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR), play an important role in 'classical' (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a 'backup' (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant-specific DNA Ligase VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T-DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T-DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus-induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T-DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T-DNA integration. Thus, T-DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.

Most potential pathogens fail to establish virulence for a plethora of plants found in nature. This intrinsic property to resist pathogen virulence displayed by organisms without triggering canonical resistance (R) genes has been termed nonhost resistance (NHR). While host resistance involves recognition of pathogen elicitors such as avirulence factors by bona fide R proteins, mechanism of NHR seems less obvious, often involving more than one gene. We can generally describe NHR in two steps: 1) pre-invasive resistance, either passive or active, which can restrict the pathogen from entering the host, and 2) post-invasive resistance, an active defense response that often results in hypersensitive response like programmed cell death and reactive oxygen species accumulation. While PAMP-triggered-immunity (PTI) is generally effective against nonhost pathogens, effector-triggered-immunity (ETI) can be effective against both host and nonhost pathogens. Prolonged interactions between adapted pathogens and their resistant host plants results in co-evolution, which can lead to new pathogen strains that can be virulent and cause disease on supposedly resistant host. In this context, engineering durable resistance by manipulating genes involved in NHR is an attractive approach for sustainable agriculture. Several genes involved in NHR have been characterized for their role in plant defense. In this review, we report genes involved in NHR identified to date and highlight a few examples where genes involved in NHR have been used to confer resistance in crop plants against economically important diseases.

Brachypodium distachyon is a model grass species for economically important cereal crops. Efforts are in progress to develop useful functional genomic resources in Brachypodium. A tobacco retrotransposon, Tnt1, has been used successfully in recent past to generate insertional mutagenesis in several dicot plant species. Tnt1 retrotransposon replicates, transposes, and inserts at multiple random genomic locations in the plant genome. Transposition occurs only during somatic embryogenesis but not during seed transmission. We developed Brachypodium transgenic plants that can express the Tnt1 element. Here, we describe an efficient tissue culture-based approach to generate Tnt1 insertional mutant population using transgenic Brachypodium line expressing the Tnt1 retrotransposon.

Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis.

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2. Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development., Facultad de Ciencias Exactas

The genome sequence of Medicago truncatula was published and released in 2011. A Tnt1 insertional mutant population with 21,700 independently regenerated lines was completed in 2012 at The Samuel Roberts Noble Foundation. With an estimated 25 insertions per line, the Tnt1 mutant population harbors more than 500,000 insertions in the M. truncatula genome. Based on the genome size, average gene length, and random insertion of Tnt1into the genome, the mutant population affects about 90% of genes in the M. truncatula genome. Therefore, the mutant population enables functional characterization of most genes in the M. truncatula genome. From 2006 to 2011, we sequenced about 33,000 flanking sequence tags (FSTs) from 2600 Tnt1 lines using TAIL-PCR followed by TA cloning, plasmid isolation, and traditional Sanger sequencing. To accelerate FST sequencing, we developed a two-dimensional DNA pooling strategy coupled with next-generation sequencing and produced about 380,000 FSTs from all 21,700 lines in a relatively short time. All FSTs are BLAST searchable in a web-based database. One can quickly search the database to find M. truncatula mutant lines with Tnt1 insertions in most genes of interest.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

The LOS2 gene in Arabidopsis encodes an enolase with 72% amino acid sequence identity with human ENO1. In mammalian cells, the α-enolase (ENO1) gene encodes both a 48 kDa glycolytic enzyme and a 37 kDa transcriptional suppressor protein that are targeted to different cellular compartments. The tumor suppressor c-myc binding protein (MBP-1), which is alternatively translated from the second start codon of ENO1 transcripts, is preferentially localized in nuclei while α-enolase is found in the cytoplasm. We report here that an Arabidopsis MBP-1-like protein (AtMBP-1) is alternatively translated from full-length LOS2 transcripts using a second start codon. Like mammalian MBP-1, this truncated form of LOS2 has little, if any, enolase activity, indicating that an intact N-terminal region of LOS2 is critical for catalysis. AtMBP-1 has a short half-life in vivo and is stabilized by the proteasome inhibitor MG132, indicating that it is degraded via the ubiquitin-dependent proteasome pathway. Arabidopsis plants that over-express AtMBP-1 are hypersensitive to abscisic acid (ABA) during seed germination and show defects in vegetative growth and lateral stem development. AtMBP-1 interacts directly with the E3 ubiquitin ligase AtSAP5 and co-expression of these proteins resulted in destabilization of AtMBP-1 in vivo and abolished the developmental defects associated with AtMBP-1 over-expression. Thus, AtMBP-1 is as a bona fide alternative translation product of LOS2. Accumulation of this factor is limited by ubiquitin-dependent destabilization, apparently mediated by AtSAP5.

Nodulation in legumes involves the coordination of epidermal infection by rhizobia with cell divisions in the underlying cortex. During nodulation, rhizobia are entrapped within curled root hairs to form an infection pocket. Transcellular tubes called infection threads then develop from the pocket and become colonized by rhizobia. The infection thread grows toward the developing nodule primordia and rhizobia are taken up into the nodule cells, where they eventually fix nitrogen. The epidermal and cortical developmental programs are synchronized by a yet-to-be-identified signal that is transmitted from the outer to the inner cell layers of the root. Using a new allele of the Medicago truncatula mutant Lumpy Infections, lin-4, which forms normal infection pockets but cannot initiate infection threads, we show that infection thread initiation is required for normal nodule development. lin-4 forms nodules with centrally located vascular bundles similar to that found in lateral roots rather than the peripheral vasculature characteristic of legume nodules. The same phenomenon was observed in M. truncatula plants inoculated with the Sinorhizobium meliloti exoY mutant, and the M. truncatula vapyrin-2 mutant, all cases where infections arrest. Nodules on lin-4 have reduced expression of the nodule meristem marker MtCRE1 and do not express root-tip markers. In addition, these mutant nodules have altered patterns of gene expression for the cytokinin and auxin markers CRE1 and DR5. Our work highlights the coordinating role that bacterial infection exerts on the developing nodule and allows us to draw comparisons with primitive actinorhizal nodules and rhizobia-induced nodules on the nonlegume Parasponia andersonii.

Cold acclimation is an important process by which plants respond to low temperature and enhance their winter hardiness. C-REPEAT BINDING FACTOR1 (CBF1), CBF2, and CBF3 genes were shown previously to participate in cold acclimation in Medicago truncatula In addition, MtCBF4 is transcriptionally induced by salt, drought, and cold stresses. We show here that MtCBF4, shown previously to enhance drought and salt tolerance, also positively regulates cold acclimation and freezing tolerance. To identify molecular factors acting upstream and downstream of the MtCBF4 transcription factor (TF) in cold responses, we first identified genes that are differentially regulated upon MtCBF4 overexpression using RNAseq Digital Gene Expression Profiling. Among these, we showed that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC15 (MtCAS15) gene. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified based on a yeast one-hybrid screen as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression. In addition, another MYB TF, MtMYB61, identified as an interactor of MtMYB3, can relieve the inhibitory effect of MtMYB3 on MtCBF4 transcription. This study, therefore, supports a model describing how MtCBF4 is regulated by antagonistic MtMYB3/MtMYB61 TFs, leading to the up-regulation of downstream targets such as MtCAS15 acting in cold acclimation in M. truncatula.

Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised in nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens.

[EN] PISTILLATA (PI) is a member of the B-function MADS-box gene family, which controls the identity of both petals and stamens in Arabidopsis thaliana. In Medicago truncatula (Mt), there are two PI-like paralogs, known as MtPI and MtNGL9. These genes differ in their expression patterns, but it is not known whether their functions have also diverged. Describing the evolution of certain duplicated genes, such as transcription factors, remains a challenge owing to the complex expression patterns and functional divergence between the gene copies. Here, we report a number of functional studies, including analyses of gene expression, protein-protein interactions, and reverse genetic approaches designed to demonstrate the respective contributions of each M. truncatula PI-like paralog to the B-function in this species. Also, we have integrated molecular evolution approaches to determine the mode of evolution of Mt PI-like genes after duplication. Our results demonstrate that MtPI functions as a master regulator of B-function in M. truncatula, maintaining the overall ancestral function, while MtNGL9 does not seem to have a role in this regard, suggesting that the pseudogenization could be the functional evolutionary fate for this gene. However, we provide evidence that purifying selection is the primary evolutionary force acting on this paralog, pinpointing the conservation of its biochemical function and, alternatively, the acquisition of a new role for this gene., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; BIO2009-08134 and BIO2012-39849-CO2-01). MAF was supported by a grant from the MINECO (BFU2012-36346). We wish to thank Drs Santiago F. Elena Fito (IBMCP, CSIC-UPV), Jose V. Gimeno Alcaniz (IATA, CSIC), Javier Paz-Ares (CNB, CSIC), and Carlos Alonso-Blanco (CNB, CSIC) for valuable suggestions and comments in the initial stages of this work. The technical assistance of Rafael Martinez-Pardo in the greenhouse is gratefully acknowledged.

International audience; Legumes improve their mineral nutrition through nitrogen-fixing root nodule symbioses with soil rhizobia. Rhizobial infection of legumes is regulated by a number of transcription factors, including ERF Required for Nodulation1 (ERN1). Medicago truncatula plants defective in ERN1 are unable to nodulate, but still exhibit early symbiotic responses including rhizobial infection. ERN1 has a close homolog, ERN2, which shows partially overlapping expression patterns. Here we show that ern2 mutants exhibit a later nodulation phenotype than ern1, being able to form nodules but with signs of premature senescence. Molecular characterization of the ern2-1 mutation reveals a key role for a conserved threonine for both DNA binding and transcriptional activity. In contrast to either single mutant, the double ern1-1 ern2-1 line is completely unable to initiate infection or nodule development. The strong ern1-1 ern2-1 phenotype demonstrates functional redundancy between these two transcriptional regulators and reveals the essential role of ERN1/ERN2 to coordinately induce rhizobial infection and nodule organogenesis. While ERN1/ERN2 act in concert in the root epidermis, only ERN1 can efficiently allow the development of mature nodules in the cortex, probably through an independent pathway. Together, these findings reveal the key roles that ERN1/ERN2 play at the very earliest stages of root nodule development.

Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)-mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-ray cross complementation group4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod−), 51 mutants with totally ineffective nodules (Nod+ Fix−), 17 mutants with partially ineffective nodules (Nod+ Fix+/−), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/− Fix−), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/− Fix+), and 11 supernodulating mutants (Nod++Fix+/−). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN’T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod− lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

• Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex. • We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. • We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. • We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome.

Bacterial pathogens colonize a host plant by growing between the cells by utilizing the nutrients present in apoplastic space. While successful pathogens manipulate the plant cell membrane to retrieve more nutrients from the cell, the counteracting plant defense mechanism against nonhost pathogens to restrict the nutrient efflux into the apoplast is not clear. To identify the genes involved in nonhost resistance against bacterial pathogens, we developed a virus-induced gene-silencing-based fast-forward genetics screen in Nicotiana benthamiana. Silencing of N. benthamiana SQUALENE SYNTHASE, a key gene in phytosterol biosynthesis, not only compromised nonhost resistance to few pathovars of Pseudomonas syringae and Xanthomonas campestris, but also enhanced the growth of the host pathogen P. syringae pv tabaci by increasing nutrient efflux into the apoplast. An Arabidopsis (Arabidopsis thaliana) sterol methyltransferase mutant (sterol methyltransferase2) involved in sterol biosynthesis also compromised plant innate immunity against bacterial pathogens. The Arabidopsis cytochrome P450 CYP710A1, which encodes C22-sterol desaturase that converts β-sitosterol to stigmasterol, was dramatically induced upon inoculation with nonhost pathogens. An Arabidopsis Atcyp710A1 null mutant compromised both nonhost and basal resistance while overexpressors of AtCYP710A1 enhanced resistance to host pathogens. Our data implicate the involvement of sterols in plant innate immunity against bacterial infections by regulating nutrient efflux into the apoplast.

Tnt1 (transposable element if Nicotiana tabaccum cell type 1) is one of the very few active LTR retrotransposons used for gene tagging in plants. In the model legume Medicago truncatula, Tnt1 has been effectively used as a gene knock-out tool to generate several very useful mutants. stenofolia (stf) is such a mutant identified by Tnt1 insertion in a WUSCHEL-like homeobox transcription factor. STF is required for blade outgrowth, leaf vascular patterning and female reproductive organ development in barrel medic and woodland tobacco. Using transcript profiling and metabolite analysis, we uncovered that mutant leaves are compromised in steady-state levels of multiple phytohormones, sugar metabolites and derivatives including flavonoids and polyamines. In the lam1 mutant (caused by deletion of the STF ortholog in Nicotiana sylvestris), while glucose, fructose, mannose, galactose, myo-inositol and aromatic aminoacids are dramatically reduced, sucrose is comparable to wild-type levels, and glutamine, proline, putrescine, nicotine and sorbitol are highly increased. We demonstrated that both stf and lam1 mutants accumulate reduced levels of free auxin and ABA in their leaves, and ectopic expression of STF in tobacco leads to auxin and cytokinin overproduction phenotypes including formation of tumors on the roots and crown. These data suggest that STF mediated integration of metabolic and hormonal signals are required for lateral organ morphogenesis and elaboration.