The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula Qiong Li, Mei Li, Danping Zhang, Liangliang Yu, Junhui Yan*, Li Luo** Shanghai Key Laboratory of Bio-energy Crops, Center of Plant Science, School of Life Sciences, Shanghai University, Shanghai, 200444, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 December 2019 Accepted 4 December 2019 Available online xxx

Leguminous root nodules specifically induced by rhizobium species fix nitrogen gas to gain nitrogen sources, which is important in sustainable agriculture and ecological balance. Several peptide signals are reported to be involved in regulation of legume nodule number and development. There are fifteen genes coding Root Meristem Growth Factor (RGF) peptide in Medicago truncatula, herein we find the expression of MtRGF3 is significantly induced by Sinorhizobium meliloti with production of Nod factors. The gene promoter is active in nodule primordia, young nodules and the meristem region of mature nodules. Knock-down (RNAi) roots of the gene (MtRGF3-RNAi) formed more root nodules than the empty vector control, and the nodule number decreased in MtRGF3-overexpressing (MtRGF3-OX) roots. Exogenous addition of the synthesized peptide significantly promoted primary root growth and inhibited lateral root emergence, in addition, the peptide application reduced the number of infection threads, nodule primordia and root nodules of M. truncatula. We also found that tyrosine sulfation determines the biological activity of MtRGF3 functioning in nodulation process, and MtRGF3 peptide negatively regulates nodulation in a dosage manner. These results demonstrate that the MtRGF3 peptide is a novel regulator during nodulation of Medicago trucatula. © 2019 Elsevier Inc. All rights reserved.

Keywords: MtRGF3 Medicago truncatula Nodule development Root growth Lateral root emergence

1. Introduction The symbiosis between rhizobia and legumes benefits both of them, which results in the production of nodule where nitrogen is converted into ammonia. The balance between legume and rhizobia needs a systemic feedback regulatory system. A growing number of small secreted peptides (SSPs) are identified which function as critical regulators during nodulation [1]. The CLV3/ESRrelated (CLE) peptide generally consists of 12e13 amino acids, which are mainly located in the C-terminal domain of the precursor protein [2]. In Medicago truncatula, the expression of MtCLE12 and MtCLE13 was induced by inoculation of Sinorhizobium meliloti, and overexpression of each MtCLE12 or MtCLE13 gene abolished nodule formation [3]. The possible mechanism is that the CLE peptide or its precursor is transported in the xylem to the shoot, and is recognized by the leucine-rich receptor kinase Super Number Nodules

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Q. Li), [email protected] (M. Li), [email protected] (D. Zhang), [email protected] (L. Yu), junhuiyan@ shu.edu.cn (J. Yan), [email protected] (L. Luo).

(MtSUNN) in the leaf to promote production of a new suppression signal of nodulation [4]. For Lotus, nodulation leads to the transport of root-derived CLE peptides to shoots and which are perceived by receptor-like kinase HYPER-NODULATION ABERRANT ROOT FORMATION1 (HAR1) [5]. Shoot-synthesized microRNA miR2111 regulates the symbiosis suppressor TOO MUCH LOVE in roots to balance infection of nodulation events [6]. C-Terminally Encoded Peptides (CEPs) are another class of small peptides that regulate root and nodule development [7]. MtCEP1 expression is induced under low nitrogen conditions. Overexpression of MtCEP1 and exogenous addition of the CEP1 peptide of M. truncatula suppressed lateral root formation and increased nodules emergence [8]. MtCEP1 may be recognized by the receptor kinase Compact Root Architecture (CRA2), the mutant of the gene CRA2 increased the number of lateral roots and suppressed nodulation [9]. In addition, two small peptides, Devil 1 (MtDvL1) and Rapid Alkalinisation Factor 1 (MtRALF1), suppress formation of infection threads and development of nitrogen-fixing nodules [10]. Indeterminate root nodules of M. truncatula contain many Nodule specific Cysteine Rich (NCR) peptides, which play an important regulatory role in the terminal differentiation of bacteroids and nodule development at the later stages [11e13].

https://doi.org/10.1016/j.bbrc.2019.12.017 0006-291X/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Q. Li et al., The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.017

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The Root meristem Growth Factor (RGF) found in Arabidopsis is a key regulator of root development [14e17]. Such peptides are encoded by a multi-gene family. They are formed by a series of post-translational processing (tyrosine sulfation, protease cleavage, etc.) [18]. Their precursors consist of an N-terminal signal peptide (SP), a variable region and a C-terminal domain. The active RGF peptide, 13-amino acid length, is located at the C-terminal of the precursor, wherein aspartic acid, tyrosine and proline are conserved, and tyrosine can be modified by sulfation. Overexpression of AtRGF genes promotes root elongation, inhibits lateral root growth, and induces wavy and agravitropic roots [15e17]. Three research groups demonstrated that RGF peptides can be recognized by multiple LRR receptor kinases, initiating downstream signaling processes [19e21]. We therefore investigated whether RGF peptides may regulate formation of nitrogen-fixing nodules and root development in legumes. To test this possibility, we systematically studied the MtRGF gene family using bioinformatic, physiological, genetic and biochemical techniques, and found that MtRGF3 and its encoded peptide, suppressed nitrogenfixing nodule development. While our study of this gene family was in progress, de Bang et al. [22] reported genome-wide identification of peptide involved in nodulation, which showed that the transcription levels of GLV1, GLV9 and GLV10 were induced by rhizobia, and the expression of GLV3, GLV5, GLV7, GLV8, GLV13, GLV14 and GLV15 were down-regulated after rhizobia inoculation. Sequence analysis revealed that GLV9 peptide shared the same sequences with MtRGF3 peptide that we have studied. However, our findings differed: we observed that MtRGF3 is active in root tip, nodule primodium and the apical zone of mature nodule, the exogenous application of synthetic MtRGF3 peptide suppressed nodulation and lateral root emergency, the nodule number decreased when MtRGF3 was overexpressed in Medicago roots, and the number increased in MtRGF3-RNAi transgenic roots. Taken together, the results demonstrate that MtRGF3 negatively regulate nodulation in M. truncatula.

2. Materials and methods 2.1. Preparation of plant materials M. truncatula (A17) seedlings grown in sterilized artificial soil (vermiculite: perlite ¼ 3:1) pots were inoculated with S. meliloti 1021 or the nodC mutant suspension or FM liquid medium (as a control), the plants were grown in greenhouse at 23  C with 16 h of light per day at 100 mmol s1 m2. To check the expression of MtRGF3 during the early nodulation processing, the roots were harvested at various days after inoculation (1, 3, 5 and 7 days). Additionally, root, leaf, and stem, from M. truncatula (A17) seedling grown for 3 weeks in green house; nodule, after inoculation with rhizobia for 3 weeks; pod/seed, young fruit harvested at 16 d after pollination (DAP); flower, fully open flowers.

2.2. RNA extraction and qRT-PCR Total RNA was extracted from plant tissues using RNAprep pure Plant kit (Tiangen biotech CO., LTD, DP437). Synthesis of first-strand cDNA and realtime PCR were performed using PrimeScript™ RT reagent Kit with gDNA Eraser and SYBR Green qPCR Master Mix SYBR Advantage (Takara, China, RR037Q). For gene expression analysis, qRT-PCR was performed according to the kit instructions (Takara, China), the housekeeping gene is MtACTIN, and the primers are shown in Table S1.

2.3. Plasmid construction For promoter activity assay, the promoter region (2044bp region upstream of the MtRGF3 start codon) was amplified by PCR and cloned into pENTR, and an LR reaction was then performed to clone the promoter region into the pKGWFS2 vector, which carries a GUSGFP fusion downstream of the cloning recombination region, named as MtRGF3pro:GUS-GFP. For MtRGF3 overexpression constructs, MtRGF3 CDS was amplified by PCR from M. truncatula cDNA with gene-specific primers and the amplified products were inserted into pENTR, then the constructs were used for recombination into the Gateway destination vector pK7GW2D (it contains a 35S cauliflower mosaic virus promoter and GFP reporter used as transformation marker) by LR reaction to create MtRGF3-OX. The MtRGF3m fragment was amplified using specific primers and MtRGF3m-OX vectors was obtained via the similar method. For RNAi construct, the MtRGF3 gene fragment (about 250 bp) was amplified by PCR from M. truncatula cDNA with gene-specific primers and then inserted into pENTR, and subsequently the construct was used for recombination into the Gateway destination vector pK7GWIWG2D(II) by LR reaction to create MtRGF3-RNAi, and pK7GWIWG2D(II) contains a 35S cauliflower mosaic virus promoter and GFP reporter used as transformation marker. All the primers used in vector construction are listed in Supplemental Material Table S1.

2.4. RGF peptide activity assays The peptides were synthesized by the Scilight-Peptide Company (Beijing, China), and dissolved in sterile water, then the specific peptide solution was added to Fahraeus medium (FM medium) before pouring the medium. The following peptide sequences were assayed: RGF3, DY(SO3H)SPARKKS (Hyp)IHN; Random, SHPKY(SO3H)(Hyp)KNSDIRA, the amino acid sequences of MtRGF3 were randomized; RGF3 unsulfated, DYSPARKKS(Hyp)IHN; Y(SO3H), sulfated tyrosin; Hyp, hydroxyproline. Sterilization and germination of M. truncatula (A17) seeds were performed as described by Liu et al. [23], then germinated seeds of M. truncatula were arranged neatly on a solid FM plate that contains appropriate peptide (one row per plate, 10 per row). S. mliloti 1021 was suspended in FM solution (OD600 ¼ 0.05) and flood-inoculated onto roots. To minimize the exposure roots to light, the plate was three quarter wrapped in black paper pockets and placed vertically in a 23  C incubator (with 16 h of light per day at 100 mmol s1 m2) for one or three weeks for nodulation assays. For infection threads and nodule primordia assay, the seedlings grown on FM solid medium supplemented with appropriate peptides were inoculated with S. meliloli 1021 carrying pXLGD4 (lacZ) for 7 days. For nodulation assay, the FM plates were added aminoethoxyvinylglycine (AVG, Sigma) up to 0.5 mM final concentration. Alternatively, these seedlings are not inoculated with rhizobia, and cultured on the FM plates containing peptides and nitrogen sources for 1e2 weeks to observe root growth and development.

2.5. Hairy root transformation The corresponding constructs were transformed into the Agrobacterium rhizogenes strain ARqua1. The hairy roots transformation was performed as previously described by Lei et al. [24] and the transgenic roots were detected by the presence of green fluorescent protein.

Please cite this article as: Q. Li et al., The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.017

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2.6. Histochemical staining X-gal staining for observations of infection threads and nodule primordia was performed as described by Chen et al. [25]. X-gluc staining for checking the activity of MtRGF3pro:GUS-GFP in transgenic roots was done as described by Lei et al. [24]. 3. Results 3.1. The expression of MtRGF3 is induced by rhizobia There are fifteen members of RGF family in M. truncatula, termed as MtRGF1-MtRGF15 (Fig. S1). The pre-propeptides of MtRGFs contain N-terminal signal peptide and C-terminal RGF domain. The RGF motifs are 13e16 amino acids long and contain conserved aspartic acid (D), hydroxylated proline (Hyp) and sulfated tyrosine (YeSO3H) residues. We then searched the expression data of these genes from the MtGEA database (Fig. S2) and evaluated the gene expression levels of MtRGFs in roots inoculated with rhizobia by qRT-PCR (Fig. S3), the results showed that expression of MtRGF3 was strongly induced after inoculation of rhizobia; the induction even was up to 8 folds compared with the blank control (Fig. S2 and Fig. 1A). To further determine whether transcriptional induction of MtRGF3 is associated with Nod factors, we inoculated the S. meliloti nodC null mutant with M. truncatula roots and analyzed the transcript levels. qRT-PCR results showed that the expression level of MtRGF3 was not induced by the nodC mutant at 1dpi, 3dpi, 5dpi and 7 dpi (Fig. 1A), suggesting Nod factors synthesized by rhizobia involved in the rhizobia-induced expression pattern of MtRGF3. To complement expression pattern of MtRGF3 from qRT-PCR and database, the promoter activity was determined in MtRGF3pro:GUS-GFP transgenic roots transformed

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via Agrobacterium rhizogenes. The results showed that the MtRGF3 promoter presented the highest activity in the root meristem region, nodule primordia, young root nodules and the apical zones of mature root nodules (Fig. 1DeG). We further prepared the paraffin sections for mature root nodules strained by X-Gluc. Microscopic observations indicated that the promoter was high-level activated in meristem and infected zones (Fig. 1H). Additionally, the transcript level of MtRGF3 in mature root nodules was evaluated by tissue dissection and qRT-PCR, and the results showed that this gene predominantly expressed in apical meristem and infection zones (Fig. 1B), which is consistent with the data from laser capture micro-dissection and RNA-seq (Fig. 1C) [26]. 3.2. Exogenous application of synthetic MtRGF3 peptide suppresses nodulation in M. truncatula Exogenous addition of RGF peptides results in root phenotype of Arabidopsis [15,16]. To check the biological activity of MtRGF3 peptide, we synthesized 13-aa peptides derived from the RGF domain of MtRGF3 precursors, the synthetic peptide sequences: RGF3, DY(SO3H)SPARKKS(Hyp)IHN; Random, SHPKY(SO3H)(Hyp) KNSDIRA, the amino acid sequences of MtRGF3 were randomized; RGF3 unsulfated, DYSPARKKS(Hyp)IHN. Then the peptides were added into FM medium (working concentration is 1 mM) for M. truncatula roots phenotype assay (Fig. S4). We found that the root length treated by synthetic MtRGF3 peptide is 20% longer than that of the random peptide treatment. The number of lateral roots on MtRGF3 peptide treated plate was fewer than both the random and unsulfated MtRGF3 treatment, which revealed that MtRGF3 peptide inhibited the occurrence of lateral roots and the tyrosine sulfation modification was indispensable. Since the MtRGF3 peptide promoted the primary root growth and inhibited the lateral

Fig. 1. Expression pattern and promoter activity of MtRGF3 in M. truncatula seedlings were induced by S. meliloti 1021. (A) The total RNA was extracted from M. truncatula roots after 1-, 3-, 5- and 7- day inoculation of S. meliloti 1021 or the nodC mutant (without Nod factors) and the transcript level was evaluated by qRT-PCR. Control, nitrogen-free buffer. Three biological replicates were performed; error bars, ±SE. (B) The mature nodules were dissected into three parts by hand. Zone I þ II, the apical meristem and infection zones; Zone III, the nitrogen-fixation zone; Zone IV, the senescence zone. The expression level was evaluated by qPCR. The experiments were repeated for three times, and fifteen nodules were collected each time. (C) The data was collected from published RNA-Seq data (https://iant.toulouse.inra.fr/symbimics/). (DeH) Expression of the MtRGF3pro:GUS-GFP fusion in transgenic roots. The GUS activity detected by X-Gluc staining in a primary root (D), a nodule primordia (E), a young nodule (F) and in a mature nodule (G and H). White bars, 1 cm; black bar, 100 mm.

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roots emergence in M. truncatula plants, and the expression of MtRGF3 was induced by rhizobia, so it indicates that the peptide promotes M. truncatula nodulation just like the LjPSK peptide [27]. We used MtRGF3, unsulfated MtRGF3 and the random peptide for the plant nodulation assay (Fig. 2A and B). After three-week inoculation, the counting result showed that 1 mM and 10 mM of the MtRGF3 peptide significantly suppressed root nodule formation compared with the same dosage of the random peptide, but 0.5 mM of the MtRGF3 peptide did not (Fig. 2C), which demonstrates the appropriate dosage of the MtRGF3 peptide suppresses nodule development. We also found that tyrosine sulfation modification is important for its function in nodulation, as shown in Fig. 2D, there are fewer nodules on MtRGF3 treated plates compared with the random and unsulfated MtRGF3 treatment. To determine whether MtRGF3 peptide affects the early stage of nodulation, we checked the infection threads and nodule primordia on MtRGF3 peptide plates, and the results showed that both the infection threads (Fig. 2E) and nodule primordia emergence (Fig. 2F) were suppressed by MtRGF3 peptide. 3.3. MtRGF3 is a suppressor for M. truncatula nodulation To determine the biological function of MtRGF3, we attempted to isolate the right Tnt1 insertion mutant. However, we haven’t got any. Therefore, we constructed MtRGF3-RNAi and MtRGF3-OX vectors, which were used for hairy root transformation of M. truncatula. After three-week inoculation of S. meliloti 1021 with the transgenic roots, the expression level of MtRGF3 was evaluated

by qRT-PCR and nodules were counted. The results showed that the transcripts of MtRGF3 in RNAi hairy roots were significantly reduced, and nodules induced by rhizobia had significant increase compared with the roots transformed by empty vectors (Fig. 3AeD). To exclude the possibility that expression of other MtRGF genes was also interfered in hairy roots of MtRGF3-RNAi, we evaluated the expression level of MtRGF5, MtRGF7 and MtRGF8 (abundantly expressing in roots) by qRT-PCR. The results showed that there was no significant alteration on transcription of these genes in RNAi roots (Fig. S5). Correspondingly, on the MtRGF3 overexpression roots, fewer nodules were induced by S. meliloti 1021, as the expression level of MtRGF3 significantly elevated (Fig. 3EeI). As tyrosine and proline are the conserved and bioactive amino acid in RGF domain, and the exogenous application peptide on nodulation showed that tyrosine sulfation determined MtRGF3 peptide bioactivity in nodulation. So we got the mutated MtRGF3 (MtRGF3m) sequence which coded an MtRGF3 peptide with amino acid substitutions as Y to N and P to A in its RGF domain. Interestingly, nodule formation was not altered on the MtRFG3m overexpression roots compared with vector control, though the expression level had a significant increase, which suggests that the active RGF domain encoded by the gene is the function unit. These results revealed that MtRGF3 is a suppressor for M. truncatula nodulation. 4. Discussion Tyrosine sulfation catalyzed by tyrosylprotein sulfotransferase

Fig. 2. MtRGF3 peptide suppressed root nodule formation in M. truncatula. (A, B and D) M. truncatula (A17) seedlings inoculated with S. meliloti 1021 for 3-weeks were grown on FM agar plate supplemented with synthetic peptides (1 mM). (C) The effect of various concentration MtRGF3 peptides on nodule formation after inoculation with S. meliloti 1021 for 3 weeks. (E and F) The infection thread and nodule primordia were suppressed by 1 mM MtRGF3, the infection threads and nodule primordia were scored 7 dpi (days post inoculation) with S. meliloli 1021 carring pXLGD4 (lacZ) after LacZ staining. Scale bars ¼ 1 cm *, the significant difference in t-test (P < 0.05), the number of nodules, infection threads and primordia were counted from three experiments (n > 40 independent M. truncatula roots were used per peptide treatment in each experiment); Error bars, ±SE.

Please cite this article as: Q. Li et al., The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.017

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Fig. 3. M. truncatula nodulation phenotypes of MtRGF3 transgenic roots. (A, B, E, F and G) The nodulation phenotypes of MtRGF3-RNAi, MtRGF3-OX and MtRGF3m-OX roots after inoculation of S. meliloti 1021 compared with the vector controls, the left panel, bright-filed images; the right panel, fluorescence microscopy images; Scale bars ¼ 1 cm. (C and H) Expression level of MtRGF3 and MtRGF3m in M. truncatula transgenic roots detected by qRT-PCR. (D and I) Statistic analysis of nodules number on MtRGF3-RNAi, overexpression of MtRGF3 roots after inoculation of S. meliloti 1021. *, the significant difference in t-test (P < 0.05), the qPCR detection and nodule assay were performed in three independent experiments (n > 30 independent transgenic roots were used per construct in each experiment); Error bars, ±SE. MtRGF3m, the N terminal RGF domain of MtRGF3 with substitutions of Y as N, P as A.

(TPST) is important modification for the maturation and biological function of peptide hormones, PSK, PSY and RGF in plants [18]. In Arabidopsis, sulfated RGF8/GLV6 peptide induced the slanted roots while its unmodified counterpart did not show this biological function. However, post-translational tyrosine sulfation is not always the essential modification for the biological activity of all RGF peptides; for example, the gravity phenotype of Arabidopsis roots grown on plate containing RGF1/GLV11p is similar to its sulfation modified counterpart [28]. In our study, the application of exogenous synthetic MtRGF3-sulfated peptide induces less lateral roots and longer primary root growth, but the MtRGF3-unsulfated only affect lateral roots emergence. We also found that the tyrosine sulfation modified MtRGF3 peptide suppressed nodulation, and its unmodified counterpart did not alter nodulation. In Arabidopsis, both GLV5-overexpressing and exogenous GLV5 peptide addition lead to gravitropic defects [28], but it is not all the RGF peptides totally mimic the corresponding gene overexpression phenotypes, such as the addition of GLV6 cannot resulted the strong agravitropic phenotype appeared in GLV6overexpression roots [15]. In our study, the expression of MtRGF3 was reduced significantly in MtRGF3-RNAi transgenic hairy roots and resulted in the increase of nodule numbers, which is consistent to the nodulation assay treated by exogenous sulfated MtRGF3 peptide. The nodules number was significantly altered in MtRGF3OX transgenic hairy roots with the high transcript level of the corresponding gene. In M. truncatula, symbiotic nodulation is negatively regulated by CLE, RALF and DVL peptides, and positively regulated by CEP

peptides [3,7,8,10]. In Lotus japonicus, PSK positively regulates symbiotic nodulation [27]. In our study, MtRGF3 peptide promoted root length and inhibited lateral roots number, what is more, which attenuated nodulation as a new SSPs regulator in M. truncatula. It is known that RGF peptide influence root meristem development through binding the LRR-RKs RGFR and regulating the AP transcription factor PLTs [19e21,29]. Does the MtRGF3 peptide suppress nodulation through this RGFR-PLT signaling cascade? Future research directed at the identification of stable mutants for the MtRGF3 and RGF receptors will shed light on the mechanism of this peptide family in nodulation. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgments This research was supported by the Natural Science Foundation of China (grants 31370277, 31570241 and 31900214). Drs. Jeremy Murray and Chengwu Liu critical reviewed the manuscript. Dr. Ertao Wang gifted the strain of ARqua1. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.017.

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Please cite this article as: Q. Li et al., The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.017