GmACP expression is decreased in GmNORK knockdown transgenic soybean roots

Lijun Wang*,Lingwei Dengb

aCollege of Life Science,Yangtze University,Jingzhou 434025,China

bHeilongjiang Academy of Agricultural Sciences,Harbin 150086,China

Short communication

GmACP expression is decreased in GmNORK knockdown transgenic soybean roots

Lijun Wanga,*,1,Lingwei Dengb,1

aCollege of Life Science,Yangtze University,Jingzhou 434025,China

bHeilongjiang Academy of Agricultural Sciences,Harbin 150086,China

ARTICLEINFO

Article history:

Received 14 January 2016

Received in revised form 6 May 2016

Accepted 6 June 2016

Available online 11 June 2016

NORK

ACP

Soybean

Nodule

Rhizobium

NORK and soybean acyl carrier protein(ACP)both play important roles in nodulation. However,the relationship between Nod factor signaling and fatty acid(FA)biosynthesis during symbiotic development is unknown.In this study,an RNAi plasmid of GmNORK was constructed and transformed into soybean roots by Agrobacterium rhizogene-mediated hairy-root transformation.The nodule number decreased substantially in GmNORK knockdown soybean transgenic roots.To investigate the relationship between GmACP and Nod factor signaling,we measured GmACP expression levels in GmNORK RNAi soybean transgenic roots and found that rhizobia inoculation led to substantially reduced GmACP expression. Thus,FA biosynthesis was affected by Nod factor signaling during nodule development in soybean,a finding that provides valuable information that improves our understanding of the functions of GmNORK and GmACP in symbiotic signaling and nodule development.

?2016 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Soybean(Glycine max Merr.)in an important source of food and animal feed,and is the most common legume crop worldwide.Soybean is capable of establishing endosymbiotic relationships with nitrogen-fixing bacteria called rhizobia [1].The establishment of the legume-rhizobium symbiosis is a multistage process involving signal perception,signal transduction,and downstream developmental events that eventually give rise to a new organ,the nodule,in which the intracellular bacterial symbionts thrive[2].In nodules,the bacteria are enclosed in a membrane produced by the plant,giving rise to vesicular-like compartments called symbiosomes[3].Within the symbiosomes,the rhizobia differentiate into a nitrogen-fixing form called bacteroids[4]. These proceed to convert atmospheric nitrogen(N2)into ammonia(NH3)for plant use,in exchange for photosynthates produced by the host plant[5].

The formation of symbiotic nodules requires two parallel signaling pathways,one promoting nodule organogenesis and the other allowing bacterial infection to occur[6].These processes are coordinated in both spatial and temporal manners to ensure successful symbiotic development[7]. Both processes require the plant to be capable of recognizingthe Nod factor(NF).Nod factors are signaling molecules secreted by rhizobia,are perceived by the host plant through two plasma membrane-located receptor-like kinases,(RLK) NFR1 and NFR5[8,9],and activate a leucine-rich repeat(LRR) serine/threonine kinase that triggers downstream signaling cascades.The LRR serine/threonine kinase is one of the genes vital to nodule initiation.These receptor-like kinases have been given different names depending on the plant species from which they originate:SYMbiosis receptor-like kinase (SymRK)in Lotus japonicus,nodulation receptor kinase(NORK) in Medicago sativa,SYM19 in Pisum sativum,DMI2(Does not Make Infections 2)in Medicago truncatula,and NORKa/b in G.max[10–13].Recently,SYMRK/NORK was shown to be a co-receptor interacting with NFR5 and fine-tuning the symbiotic signaling cascade[14].The nodulation receptor kinase (NORK)gene is essential for Nod factor perception and transduction in M.sativa,as it is involved in the common symbiosis signaling pathway[11].It has been suggested that NORK participates in a protein complex with the Nod factor receptor that transduces the signaling of Nod factor receptors to subsequent stages[15,16].However,there is little mechanistic information available about the mechanism by which NORK functions.Protein NORK is a 919-amino acid LRR receptor-like kinase containing a 325-amino acid serine/ threonine kinase domain[17],and is an active kinase[18].

During the formation of symbiotic nodules,newly formed membranes(i.e.membranes needed for the growth of the infection thread and the membrane surrounding the intracellular rhizobial symbiont)generated by the host legume plant have been estimated to be 35-fold over that membranes formed in non-symbiotic plants[19].Fatty acids(FAs)and their derivatives are essential for plant development,acting as the basic components of membranes,energy storage components,signaling molecules,and structural components of surface layers.Biosynthesis of FA influences the development of root nodule symbiosis.In root tissue,FAs accumulate in nodules(e.g.palmitic and stearic acid in L.japonicus nodules),and levels of these compounds are strongly affected by rhizobial inoculation[20,21].They are also found on soybean root hairs[22].A key protein in plant FA biosynthesis is acyl carrier protein(ACP)[23].ACP,an essential cofactor protein,is one of the key components of fatty acid synthesis that takes place either in plastids or in mitochondria.A recent study showed that GmACP encodes a soybean acyl carrier protein that is involved in soybean nodulation[24].GmACP expression was detected in all soybean tissues,but at higher levels in nodules.GmACP knockdowns created via RNA interference show marked reductions in the production of nodules on soybean transgenic roots[24].NORK/SYMRK,the immediate downstream component of these Nod factor receptors,is central to the Nod factor signaling cascade [11,13].In addition to being part of the signaling pathway that occurs in root hairs,NORK participates in the later stages of nodule development by controlling the endocytic uptake of rhizobia from infection threads by host cells[10,25].However, it is unknown whether Nod factor signaling is associated with membrane biogenesis or FA biosynthesis during symbiotic development.

Here we find that rhizobial inoculation decreases the expression level of GmACP in GmNORK RNAi soybean transgenic roots.Our results provide important information furthering our understanding of the functions of GmNORK and GmACP in symbiotic signaling and nodule development.

2.Materials and methods

2.1.Alignment and phylogenetic analysis

NCBI BLAST searches using GmNORK1a and GmNORK1b detected several highly similar peptide sequences.Alignments of the SymRK(NORK)genes are presented as NCBI BLAST pairwise using the Newick format[26].

2.2.Plasmid construction and transformation

A 188-bp DNA fragment extending from 42 to 229 bp downstream of the NORK1b stop codon was amplified from soybean cultivar William 82 using NORK1b-RNAi F/R primer pairs(F:5′-GGGGACAAGTTTGTACAAA AAAGCAGGCTactgattt agtcatgatacatttcaaat-3′;R:5′-GGGGACCACTTTGTACAAGAAA GCTGGGTAatcaatttt gtttacgcaaattttacca-3′),and cloned into the binary vector pDONR222 to generate a gateway entry plasmid using a gateway BP reaction.The 188-bp DNA fragment was cloned into pCAM-GWi(GWY RNAi)using a gateway LR reaction to generate an NORK-RNAi plasmid.

An empty vector and CGT5200(GUS RNAi)were used as the RNAi control vectors,as described previously[27,28].CGT5200 (GUS RNAi)vector contains an RNAi construct that is designed with reference to GUS.

2.3.Agrobacterium rhizogenes-mediated hairy root transformation

The constructs were electroporated into A.rhizogenes strain K599 using a GenePulser apparatus with a pulse controller (Bio-Rad Laboratories,Hercules,CA,USA)and the following settings:25 μF,200 Ω,and 1.8 kV.After electroporation,100 μL LB medium was added to the competent cells,which were then allowed to recover at 30°C with shaking at 180 r min?1for at least 2 h,followed by plating onto LB agar supplemented with appropriate antibiotics.Plates were incubated at 28°C for 2–3 days.A.rhizogenes-mediated hairy root transformation was performed as described in the following protocol.

Seeds of the soybean cultivar Williams 82 were surfacesterilized using 10%bleach and rinsed several times with autoclaved distilled water(diH2O),once with 0.8%HCl for 10 min,and then several times with diH2O.Sterilized seeds were then sown on round 1%agar plates(20 cm diameter)and incubated in a growth chamber at 27°C and 80%humidity for 3 days in darkness and 3 days at 22°C with a light regime of 16 h light/8 h dark.

A.rhizogenes strain K599 carrying the respective constructs was inoculated into liquid LB medium and incubated overnight at 30°C with shaking at 200 r min?1.Approximately 500 μL of the overnight bacterial culture was plated onto LB plates with antibiotics and the bacteria were then grown overnight in a 30°C chamber.The 6-day-old seedlings were cut at the base of the hypocotyl and the shoots were dipped in the confluent bacterial lawn that had developed overnighton the plates.The seedlings were placed onto square Petri dishes with F?hraeus medium[29].Plantlets were incubated in the growth chamber at 22°C for 2 days in darkness. Plantlets were then transferred into plastic growth pouches containing 50 mL solid F?hraeus medium supplemented with CaCl2and KNO3and grown under a 16-h photoperiod for 5–7 days at 22°C.After 5–7 days,the composite plants were transferred into growth pouches with liquid F?hraeus medium supplemented with CaCl2and KNO3.Approximately 3 weeks after transformation,the formation of transgenic roots was checked using GFP fluorescence and nontransgenic roots were removed.The seedlings were then planted in a vermiculite:perlite mixture(3:1),grown under greenhouse conditions,and inoculated with the soybean rhizobial symbiont Bradyrhizobium japonicum.

Table 1–Sequences of primers for quantitative real-time RT-PCR.

2.4.Nodulation assay

To inoculate soybean plants with rhizobia,B.japonicum wild-type strain USDA110[30],or a derivative strain constitutively expressing β-glucuronidase(GUS)[31]were grown in liquid HM medium[32]supplemented with 50 μg mL?1tetracycline and 100 μg mL?1spectinomycin at 30°C for 3 days prior to inoculation.Cells were grown to an OD600between 0.5 and 1.0,then pelleted by centrifugation at 4000 rpm at 20°C for 10 min and resuspended in sterilized MilliQ water to a final OD600of 0.05.Composite plants growing in the vermiculite and perlite mixture were inoculated 2 days later with 2 mL of the respective B.japonicum strain.Plants were grown in the laboratory for 2 days for acclimatization and transferred into a greenhouse.

The nodulation phenotypes of the soybean roots inoculated with B.japonicum were analyzed 4 weeks post-inoculation (wpi)using a Leica M205 FA stereo microscope with a high-resolution Leica DFC295 color camera and Leica Application Suite Software.Only nodules formed on transgenic roots,as determined by expression of the GFP marker,were analyzed.

2.5.GUS assay

For histochemical GUS staining,the transgenic roots of plants were cut and placed into 15 mL Falcon tubes containing 10 mL GUS staining solution,as previously described[33].A vacuum was applied three times for 3 min,after which the roots were incubated at 37°C in the dark for 2 days.Infected roots and nodules were checked for blue staining using a stereomicroscope(Olympus SZX12;Olympus,Tokyo,Japan).The evaluation and documentation of nodules and primordia were performed using a stereomicroscope fitted with an Olympus DP10 camera.

2.6.Quantitative real-time RT-PCR analysis

Transgenic roots were collected 28 days after inoculation (dai).All samples were immediately frozen in liquid nitrogen and stored at?80°C until subsequent use.Total RNA was isolated from transgenic root tissues using TRIzol(Ambion, Austin,TX,USA),followed by a DNase treatment(Turbo DNase,Ambion),and 1 μg of root total RNA was used for complementary DNA synthesis using M-MLV Reverse Transcriptase(Promega,Madison,WI,USA),according to the manufacturer's instructions.

qPCR experiments were conducted on the 7500 System (Applied Biosystems),using primers for quantitative real-time RT-PCR(Table 1)and a reaction system with SYBRGreen mix (Bio-Rad),according to the manufacturer's instructions.The thermal profile of the qRT-PCR reactions was 50°C for 2 min, 95°C for 10 min,40 cycles of 95°C for 15 s,and 60°C for 1 min.The geometric means of cons4 and cons6,encoding an ATP-binding cassette transporter and an F-box protein[34], respectively,were used as reference genes to normalize the expression levels.The relative expression levels of genes compared to reference genes between the two samples was calculated using the 2?ΔΔCtmethod.The mean expression level of three different replicates was calculated.

2.7.Statistical analysis

Nodule number data were compared using a one-way analysis of variance(ANOVA).The expression levels of genes were analyzed using Student's t-test.

3.Results

3.1.GmNORK structure

Both GmNORK1a and GmNORK1b have signal peptides(SP) at the N-terminal,LRR,transmembrane(TM),and serine–threonine/tyrosine kinase(PK)domains(Fig.1A and B).Both genes encode 919 amino acids with 94.9%identity(Fig.1C).

3.2.Molecular phylogenetic analysis of GmNORK1a and GmNORK1b

To investigate the relationship between GmNORK1a and GmNORK1b and homologous proteins in soybean and other plantspecies,a phylogenetic tree was generated based on amino acid sequences(Fig.2).GmNORK1a and GmNORK1b are the closest homologs,likely derived from a common ancestor,and have close homologs in Phaseolus vulgaris(ADQ74920.1)and Sesbania rostrata(AAV88623.1).Both of these are legumes thatsupport rhizobial symbiosis.This result shows that SymRK (NORK)orthologs are divided among three groups:rhizobium (Fabaceae,legume),actinorhizal(non-legume)nodule-forming plants,and non-nodule-forming plants.Glycine max NORK and P.vulgaris SymRK are highly similar.Within the legume orthologs,Arachis hypogaea is the most distant ancestor,as the SymRK of A.hypogaea exhibits the lowest similarity to GmNORK (Fig.2).

Fig.1–Gene structure and protein sequences of GmNORK1a and GmNORK1b.(A)Genomic structure of GmNORKa with the indicated predicted protein domains.(B)Genomic structure of GmNORK1a and GmNORK1b with the indicated predicted protein domains.Exons are indicated as boxes and introns as black lines.SP,predicted signal peptide;EC,extracellular domain;LRR, leucine-rich repeat motif;TM,transmembrane domain;PK,protein kinase domain.(C)GmNORK1a and GmNORK1b show 94.9%identity to each other.Unconserved amino acids are indicated with box.

Fig.2–Molecular phylogenetic analysis.Monocot and dicot NORKs group into distinct subclades.In each dicot subclade, legume NORKs also assort into specific clusters.

3.3.The expression of GmNORK1a,GmNORK1b,and GmACP in soybean roots was reduced in GmNORK RNAi lines

Owing to the lack of homozygous knockout mutants for GmNORK1a and GmNORK1b in soybeans,RNA silencing was applied to investigate their functions in nodulation.Soybean roots were transformed with GmNORK RNAi,an empty vector,and a vector expressing an RNAi construct specifically targeted to GUS(GUS RNAi).An empty vector and GUS RNAi were used as control vectors.GmNORK1a and GmNORK1b expression levels in GmNORK RNAi were approximately 37% and 24%of those in control transgenic roots,respectively (Fig.3A and B).To determine whether GmACP is involved in GmNORK-associated nodulation,we measured GmACP expression levels in GmNORK RNAi lines.They were approximately 38%of that in the control transgenic roots(Fig.3C).

3.4.RNA silencing of GmNORK resulted in reduced nodule numbers

The roots of transgenic lines could be identified by the green fluorescent protein(GFP)marker expressed by the binary vector(Fig.4A).Nodule morphology was examined using GUS staining.The nodules of the RNAi roots showed no obvious structural changes relative to control nodules(Fig.4B and C). The number of nodules formed on GmNORK RNAi roots was 6.4%lower than that on the controls(P＜0.01)(Fig.4D).These results show that GmNORK1a and GmNORK1b are involved in nodule formation in soybean plants inoculated with B. japonicum.

4.Discussion

During the formation of symbiotic nodules,the combined surface area of the newly formed membranes generated by the host legume plant was approximately 21,500 m2per infected root nodule cell,an area estimated as 35-fold that is seen in plants with a basic,nonsymbiotic lifestyle [19].

FAs are actively involved in the development of root nodule symbiosis.As the key building blocks of lipids,FAs(e.g.palmitic and stearic acid in L.japonicus nodules) accumulated at higher levels in nodules than in root tissue, and FA levels were strongly affected by rhizobial inoculation [20,21].A key protein involved in plant FA biosynthesis is ACP[23].Previous studies have shown that ACP is involved in the feedback regulation of FA biosynthesis.In Escherichia coli,long-chain(C16–C18)acylACP inhibited ACCase and the β-ketoacyl acyl carrier protein(ACP)synthase subunit[35,36]. The interaction of Acyl-CoA or long-chain acyl-ACP with transcription factors resulted in the repression of bacterial FA biosynthetic genes,including ACCase[37].GmACP displayed a higher level of transcript accumulation in soybean nodules 4 weeks post-inoculation(wpi)[24].RNAi-dependent gene silencing of GmACP expression led to decreased nodule numbers on inoculated transgenic soybean roots.Total FA contents,including palmitic and stearic acids,were reduced in GmACP-RNAi transgenic tissue[24],showing that GmACP is essential for nodulation.

Fig.3–The expression of GmNORK1a,GmNORK1b,and GmACP in soybean roots was reduced in GmNORK RNAi lines.(A)Relative expression levels of GmNORK1a in roots of soybean transformed with negative control vector,GmNORK RNAi plasmids. (B)GmNORK1b relative expression levels in roots of soybean transformed with negative control vector,GmNORK RNAi plasmids. (C)GmACP relative expression levels in roots of soybean transformed with negative control vector,GmNORK RNAi plasmids. **Significant at P＜0.01.Bars indicate the standard error of the mean of 3 plants.Control indicates the geometric mean of GUS RNAi and Empty vector.

NORK/SYMRK,a component acting immediately downstream of these Nod factor receptors,is central to the Nod factor signaling cascade[11,13].Several interacting NFRs and SYMRK/NORK proteins have been identified,including SINA4 (Seven In Absentia 4),HMGR1(3-hydroxy-3-methylglutaryl-CoA reductase 1),PUB1(Plant U-box protein 1)and SYMREM1 (Symbiotic Remorin 1).How the signal is transferred from the plasma membrane to the nuclear envelope remains unclear. Three nucleoporin(NUP85,NUP133,and NENA)and two cation channel proteins(CASTOR and POLLUX)play a role in generating symbiotic calcium oscillations.The nucleuslocalized Ca2+/calmodulin-independent protein kinase andCYCLOPS are used in decoding these calcium oscillations, and activate a transcription factor(TF)for symbiosis-associated gene expression[14].However,the mechanism by which Nod factor signaling affects FA biosynthesis remains unknown.

Fig.4–RNA silencin g of GmNORK resulted in reduced nodule numbers.(A)Representative transgenic root and nodules expressing GUS RNAi,EVRNAi,and GmNORK RNAi constructs.Bar length,1 mm.(B)Stained micrographs of nodule derived from RNAi transgenic roots at 21 days after inoculation with Bradyrhizobium japonicum.Bar length,1 mm.(C)Stained micrographs of nodule sections derived from RNAi transgenic roots at 21 days after inoculation with B.japonicum.The nodule morphology was similar among nodules formed on roots expressing GUS RNAi,Empty vector(EV),and GmNORK RNAi constructs.Bar length, 1 mm.EV RNAi indicates Empty vector RNAi.(D)Nodulation was measured as nodule number per transgenic root.The nodule numbers per root in GmNORKRNAi transgenic lines decreased significantly compared to those in the GUSRNAi and empty vector transgenic lines.**Denotes significant differences at P＜0.01.Bars indicate the standard error of the mean of 30 plants.

In this study,we found that GmACP expression levels were decreased in GmNORK RNAi transgenic soybean roots,suggesting that GmNORK affects FA biosynthesis by reducing GmACP expression levels.GmNORK and GmACP probably function in root hairs prior to the release of rhizobial infection threads, given that the nodule numbers on GmNORK and GmACP RNAi roots decreased significantly.GmNORK and GmACP may function in root hair infection,from the time when rhizobia are entrapped in a root hair curl to the point where infection thread growth is initiated,and finally through the induction of plasma membrane invagination.The details of the mechanism behind the entire process await further elucidation.

Acknowledgments

This study was supported by the PhD Early Development Program of Yangtze University(0121).We thank Yongqing Jiao (Oil Crops Research Institute,Chinese Academy of Agricultural Sciences,Wuhan,China)for reviewing the manuscript.

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E-mail address:ljwang516@126.com(L.Wang).

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science,CAAS.1These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.cj.2016.05.005

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