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Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number
Title: Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number
The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. The autoregulation of nodulation (AON) pathway negatively regulates the number of nodules formed in response to the carbon/nitrogen metabolic status of the shoot and root by long-distance signaling to and from the shoot and root. Central to AON signaling in the shoots ofMedicago truncatulais SUNN, a leucine-rich repeat receptor-like kinase with high sequence similarity with CLAVATA1 (CLV1), part of a class of receptors inArabidopsisinvolved in regulating stem cell populations in the root and shoot. This class of receptors inArabidopsisincludes the BARELY ANY MERISTEM family, which, like CLV1, binds to CLE peptides and interacts with CLV1 to regulate meristem development.M. truncatulacontains five members of theBAMfamily, but onlyMtBAM1andMtBAM2are highly expressed in the nodules 48 hours after inoculation. Plants carry mutations in individualMtBAMs, and several doubleBAMmutant combinations all displayed wild-type nodule number phenotypes. However,Mtbam2suppressed thesunn-5hypernodulation phenotype and partially rescued the short root length phenotype ofsunn-5 when present in asunn-5background. Grafting determined thatbam2suppresses supernodulation from the roots, regardless of theSUNNstatus of the root. Overexpression ofMtBAM2in wild-type plants increases nodule numbers, while overexpression ofMtBAM2in somesunnmutants rescues the hypernodulation phenotype, but not the hypernodulation phenotypes of AON mutantrdn1-2orcrn. Relative expression measurements of the nodule transcription factor MtWOX5 downstream of the putativebam2 sunn-5complex revealed disruption of meristem signaling; while bothbam2andbam2 sunn-5influenceMtWOX5expression, the expression changes are in different directions. We propose a genetic model wherein the specific root interactions of BAM2/SUNN are critical for signaling in nodule meristem cell homeostasis inM. truncatula.
Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood.
Results
We report a new hypernodulating mutant,defective in autoregulation, with disruption of a gene,DAR(Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. Thedar-1mutant produces ten-fold more nodules than wild type, similar to AON mutants with disruptedSUNNgene function. As insunnmutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished indar. Furthermore,dar-1also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlikeSUNNwhich functions in the shoot to control nodulation,DARfunctions in the root.
Conclusions
DARencodes a membrane protein that is a member of a small protein family inM. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis.DARgene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.
Schnabel, Elise L.; Chavan, Suchitra A.; Gao, Yueyao; Poehlman, William L.; Feltus, Frank Alex; Frugoli, Julia A.(
, Current Issues in Molecular Biology)
Nodule number regulation in legumes is controlled by a feedback loop that integrates nutrient and rhizobia symbiont status signals to regulate nodule development. Signals from the roots are perceived by shoot receptors, including a CLV1-like receptor-like kinase known as SUNN in Medicago truncatula. In the absence of functional SUNN, the autoregulation feedback loop is disrupted, resulting in hypernodulation. To elucidate early autoregulation mechanisms disrupted in SUNN mutants, we searched for genes with altered expression in the loss-of-function sunn-4 mutant and included the rdn1-2 autoregulation mutant for comparison. We identified constitutively altered expression of small groups of genes in sunn-4 roots and in sunn-4 shoots. All genes with verified roles in nodulation that were induced in wild-type roots during the establishment of nodules were also induced in sunn-4, including autoregulation genes TML2 and TML1. Only an isoflavone-7-O-methyltransferase gene was induced in response to rhizobia in wild-type roots but not induced in sunn-4. In shoot tissues of wild-type, eight rhizobia-responsive genes were identified, including a MYB family transcription factor gene that remained at a baseline level in sunn-4; three genes were induced by rhizobia in shoots of sunn-4 but not wild-type. We cataloged the temporal induction profiles of many small secreted peptide (MtSSP) genes in nodulating root tissues, encompassing members of twenty-four peptide families, including the CLE and IRON MAN families. The discovery that expression of TML2 in roots, a key factor in inhibiting nodulation in response to autoregulation signals, is also triggered in sunn-4 in the section of roots analyzed, suggests that the mechanism of TML regulation of nodulation in M. truncatula may be more complex than published models.
Despite of important functions of strigolactones (SLs) and karrikins (KARs) in plant development, plant–parasite and plant–fungi interactions, their roles in soybean–rhizobia interaction remain elusive. SL/KAR signaling genesGmMAX2a, GmD14s,andGmKAIsare activated by rhizobia infection. GmMAX2a restoredatmax2root hair defects and soybean root hairs were changed inGmMAX2aoverexpression (GmMAX2a‐OE) or knockdown (GmMAX2a‐KD) mutants.GmMAX2a‐KDgave fewer, whereasGmMAX2a‐OEproduced more nodules than GUS hairy roots. Mutation ofGmMAX2ain itsKDorOEtransgenic hairy roots affected the rhizobia infection‐induced increases in early nodulation gene expression. Both mutant hairy roots also displayed the altered auxin, jasmonate and abscisic acid levels, as further verified by transcriptomic analyses of their synthetic genes. Overexpression of an auxin synthetic geneGmYUC2aalso affected SL and KAR signaling genes. GmMAX2a physically interacted with SL/KAR receptors GmD14s, GmKAIs, and GmD14Ls with different binding affinities, depending on variations in the critical amino acids, forming active D14/KAI‐SCFMAX2complexes. The knockdown mutant roots of the nodule‐specifically expressingGmKAIs andGmD14Ls gave fewer nodules, with altered expression of several early nodulation genes. The expression levels ofGmKAIs, andGmD14Ls were markedly changed inGmMAX2amutant roots, so did their target repressor genesGmD53s andGmSMAX1s. Thus, SL and KAR signaling were involved in soybean–rhizobia interaction and nodulation partly through interactions with hormones, and this may explain the different effects of MXA2 orthologs on legume determinate and indeterminate nodulation. The study provides fresh insights into the roles of GmMAX2‐mediated SL/KAR signaling in soybean root hair and nodule formation.
Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. InMedicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors ofSinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b ofM. truncatulaexpressed inEscherichia colican release lipodisaccharides from Nod factors. Analysis ofM. truncatulamutant plants indicated that MtCHIT5b, together with MtNFH1, degradesS. melilotiNod factors in the rhizosphere.MtCHIT5bexpression was induced by treatment of roots with purified Nod factors or inoculation with rhizobia. MtCHIT5b with a fluorescent tag was detected in the infection pocket of root hairs. Nodulation of aMtCHIT5bknockout mutant was not significantly altered whereas overexpression ofMtCHIT5bresulted in fewer nodules. Reduced nodulation was observed whenMtCHIT5bandMtNFH1were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation ofM. truncatulais regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.
Transport systems are crucial in many plant processes, including plant–microbe interactions. Nodule formation and function in legumes involve the expression and regulation of multiple transport proteins, and many are still uncharacterized, particularly for nitrogen transport. Amino acids originating from the nitrogen-fixing process are an essential form of nitrogen for legumes. This work evaluates the role of MtN21 (henceforth MtUMAMIT14), a putative transport system from the MtN21/EamA-like/UMAMIT family, in nodule formation and nitrogen fixation inMedicago truncatula. To dissect this transporter’s role, we assessed the expression ofMtUMAMIT14using GUS staining, localized the corresponding protein inM. truncatularoot and tobacco leaf cells, and investigated two independentMtUMAMIT14mutant lines. Our results indicate that MtUMAMIT14 is localized in endosomal structures and is expressed in both the infection zone and interzone of nodules. Comparison of mutant and wild-typeM. truncatulaindicates MtUMAMIT14, the expression of which is dependent on the presence ofNIN, DNF1,andDNF2, plays a role in nodule formation and nitrogen-fixation. While the function of the transporter is still unclear, our results connect root nodule nitrogen fixation in legumes with the UMAMIT family.
Thomas, Jacklyn, and Frugoli, Julia. Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number. Retrieved from https://par.nsf.gov/biblio/10535907. Frontiers in Plant Science 14. Web. doi:10.3389/fpls.2023.1334190.
Thomas, Jacklyn, & Frugoli, Julia. Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number. Frontiers in Plant Science, 14 (). Retrieved from https://par.nsf.gov/biblio/10535907. https://doi.org/10.3389/fpls.2023.1334190
Thomas, Jacklyn, and Frugoli, Julia.
"Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number". Frontiers in Plant Science 14 (). Country unknown/Code not available: Frontiers Media SA. https://doi.org/10.3389/fpls.2023.1334190.https://par.nsf.gov/biblio/10535907.
@article{osti_10535907,
place = {Country unknown/Code not available},
title = {Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number},
url = {https://par.nsf.gov/biblio/10535907},
DOI = {10.3389/fpls.2023.1334190},
abstractNote = {The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. The autoregulation of nodulation (AON) pathway negatively regulates the number of nodules formed in response to the carbon/nitrogen metabolic status of the shoot and root by long-distance signaling to and from the shoot and root. Central to AON signaling in the shoots ofMedicago truncatulais SUNN, a leucine-rich repeat receptor-like kinase with high sequence similarity with CLAVATA1 (CLV1), part of a class of receptors inArabidopsisinvolved in regulating stem cell populations in the root and shoot. This class of receptors inArabidopsisincludes the BARELY ANY MERISTEM family, which, like CLV1, binds to CLE peptides and interacts with CLV1 to regulate meristem development.M. truncatulacontains five members of theBAMfamily, but onlyMtBAM1andMtBAM2are highly expressed in the nodules 48 hours after inoculation. Plants carry mutations in individualMtBAMs, and several doubleBAMmutant combinations all displayed wild-type nodule number phenotypes. However,Mtbam2suppressed thesunn-5hypernodulation phenotype and partially rescued the short root length phenotype ofsunn-5 when present in asunn-5background. Grafting determined thatbam2suppresses supernodulation from the roots, regardless of theSUNNstatus of the root. Overexpression ofMtBAM2in wild-type plants increases nodule numbers, while overexpression ofMtBAM2in somesunnmutants rescues the hypernodulation phenotype, but not the hypernodulation phenotypes of AON mutantrdn1-2orcrn. Relative expression measurements of the nodule transcription factor MtWOX5 downstream of the putativebam2 sunn-5complex revealed disruption of meristem signaling; while bothbam2andbam2 sunn-5influenceMtWOX5expression, the expression changes are in different directions. We propose a genetic model wherein the specific root interactions of BAM2/SUNN are critical for signaling in nodule meristem cell homeostasis inM. truncatula.},
journal = {Frontiers in Plant Science},
volume = {14},
publisher = {Frontiers Media SA},
author = {Thomas, Jacklyn and Frugoli, Julia},
}
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