







































Highlights in Bioscience;


 
Highlights in BioScience             

ISSN: 2682-4043 

DOI:10.36462/H.BioSci.20219                                                                                                          
 

 

Highlights in BioScience                                                                                                                                                                 September 2020| Volume 3 

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Research Article 

 

 

Open Access 

 
 

 

 

 

1Department of Nucleic Acid and Protein 

Structure, Agricultural Genetic Engineering 

Research Institute (AGERI), ARC, Giza, 

Egypt 
2Department of Genetics, Faculty of 

Agriculture, Cairo University, Giza, Egypt 
3Department of Microbiology, Faculty of 

Science, Ain Shams University, 11566, 

Abbassia, Cairo, Egypt 

 

Contacts of Authors  

 

* To whom correspondence should be 

addressed: Ghada Abu El-Heba 

 

Citation: Hassan A., Abdallah A., A-bouzeid 

M. and Abu El-Heba G. (2020). blkn, a novel 

Medicago truncatula mutant achieving black 

nodule phenotype. Highlights in BioScience 

Volume 3. Article ID 20219. dio:10.36462/ 

H.BioSci.20219 

Received:  July 14, 2020 

 

Accepted:  August 27, 2020 

 

Published: September 10, 2020 
 

Copyright: © 2020 Hassan  et al. This is an 

open access article distributed under the terms 

of the Creative Commons Attribution License, 

which permits unrestricted use, distribution, 

and reproduction in any medium, provided the 

original author and source are credited. 

 

 

Data Availability Statement: All relevant data 

are within the paper and supplementary 

materials 

 

 

Funding: The authors have no support or 

funding to report. 

 

Competing interests: The authors declare that 

they have no competing interests. 

blkn, a novel Medicago truncatula mutant achieving black 

nodule phenotype 

 
Asmaa Hassan

1
, Naglaa Abdallah

2
, Mohamed A-bouzeid

3
 and Ghada Abu 

El-Heba
1
*  

 

Abstract 

        blkn is a Medicago truncatula mutant that is achieving null 

function-black nodule phenotype. blkn is a Tnt1-retrotransposon mutant, 

Tnt1 is Nicotiana tabacum retro-transposon which is replicated via RNA 

copy and integrated in plant genome. Interestingly, blkn exhibited double 

contents of phenolic compounds comparing to R108 wild type. The 

mutated black nodule is displaying cells abnormality in both infection 

and nitrogen fixation zones. Transverse section of blkn nodule doesn’t 

display clearly characteristic shape like the control and the symbiotic 

cells don't totally filled with bacteroids along with high lignification at 

the cell wall periphery. Our goal was blkn mutant; phenotype, 

physiological, and molecular characterizations. AFLP-based PCR method 

was used to detect the mutated gene(s) in this mutant line. About 25 

Tnt1-tagged fragments ranging from ~100 to ~500 bp were isolated, 

sequenced and submitted to Genbank. The Tnt1 insertion was precisely 

located next to the base number 303 post ATG start codon of M. 

truncatula L-type lectin-domain receptor kinase VII.2 gene encodes 

Lectin_LegB Receptor Like Kinase (MtLectinRLK). MtLectinRLK 

contains Lectin_legB domain, two transmembrane helix (TMhilex) and 

an extracellular Receptor Protein kinase (Pkinase). MtLectinRLK is an 

ancestry related to probable L-type lectin-domain containing receptor 

kinase Cicer arietinum, Trifolium pretense, Phaseolus vulgaris, Vigna 

radiate and Glycine soja.  

 

Keywords: blkn mutant, Medicago truncatula, lectin-domain, receptor kinase, 

Tnt1  retrotransposon, FSTs, AFLP. 

 

Introduction  

Legumes are considered as one of the main plant families for thousands of 

years. Their seeds contain large amount of protein therefore they are a vital plant 

protein supplier in food pyramid for human nutrition, animal feed and as a raw 

matter for industry [1]. Additionally, they are essential in agriculture as they can 

grow in limited nitrogen environment with no addition of organic or inorganic 

fertilizers. Legumes family has the ability to convert atmospheric nitrogen gas to 

ammonia throughout symbiotic association with soil bacteria commonly called 

rhizobia. Soil nitrogen fixing bacteria such as Mesorhizobium, Sinorhizobium, 

Bradyrhizobium, Rhizobium, and Azorhizobium have many common features 

such as; the rode shape, gram negative and non sporulated bacteria. Farming such 

biological nitrogen fixation family reduces expansive usage of fertilizers and 

hence decreasing environmental related pollution. The relation between legumes 

and rhizobia is beneficial symbiotic relationship.

https://creativecommons.org/licenses/by/4.0/


 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

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The plant provides micro-aerobic condition and 

energy supplier to bacteria, and then in return it obtains 

organic nitrogen compounds [2,3]. When nitrogen becomes 

limiting for growth, legume plant roots produce flavonoid 

compounds in the rhizosphere which are considered as 

signals to the compatible bacteria. Rhizobia reply to these 

compounds by secreting a lipochitin oligosaccharide called 

nod factor that activates several early steps in root hair 

infection progression. Then plant percepts this specific 

Node factor inducing alterations in the gene expression. 

Additionally, perception results in physical changes as plant 

root hair curling to quester the bacteria. Bacteroid is the 

form of bacteria which is considered as the function form 

that responsible for biological nitrogen fixation [3-6]. 

Total reprogramming in the gene expression appears 

in the two organisms during the nodule development and 

nitrogen fixation. Unfortunately, most of cultivated crucial 

legumes are weak model system for genetic study because 

of their large genome, difficult transformation, being out 

crossing and having polyploidy. Thus Medicago truncatula 

is considered as legume model plant which has many 

features to study the symbiosis mechanism between plant 

and bacteria for nitrogen fixation as it has diploid genome, 

produce numerous seeds, short life cycle and self-fertile 

plant [6,7].  Wide ranges of mutant collections have been 

developed from this plant by using many genetic tools such 

as T-DNA insertions, transposon, ionizing radiation, fast 

neutron bombardment and chemical mutagens [3,8]. The 

most effective transposon used for insertion mutagenesis in 

Medicago truncatula is Tobacco retrotransposon Tnt1 [9]. 

Tnt1 is retro-transposon defined as group of moveable DNA 

elements which can transfer through RNA intermediate 

using copy-and- paste technique [5], Tnt1 is one of  long-

repeated terminal LTR retro-transposon, both ends of this 

subclass has internal direct repeats. Additionally its length 

is 5.3 Kb, it creates 5bp duplication in different sides during 

its inclusion, and it produces stable changes which 

effectively can move onto descendants [9].  

Samuel Roberts Noble Foundation in USA is the 

major source for the Medicago Tnt1-insertion mutant 

collections. Numerous mutant phenotypes have been 

separated, such as mutant has no ability to induce nodule 

(Nod−), mutants have less effective or inefficient nitrogen 

fixation nodule (Nod+Fix+/-), mutants with very high 

nodule number (Nod++) [3]. This study aimed to 

characterize blkn mutant, the Medicago truncatule Tnt1 

insertion mutagenesis mutant line. The mutant 

characterization includes; phenotype, physiological and 

molecular characterizations. 

Materials and Methods 

Plant Material: Medicago truncatula NF0478 is Tnt1 

mutant line was provided from Noble Foundation [9-11]. 

This line was produced in vitro through Agrobacterium 

tumefactions transformation [3]. In this study Medicago 

truncatula R108 was used as a wild type control plant.  

Bacterial strain: Sinorhizobium meliloti strain (Sm1021) 

[12] was used to inoculate seedlings of blkn as well as R108 

wild type.  

Seed germination and rhizobia inoculation: Both blkn 

mutant and R108 seeds were scraped with sand paper and 

the surface was sterilized with ten% (v/v) sodium 

hypochlorite for ten minutes. After 5 times washing using 

sterilize water, seeds were cultivated on basal nitrogen 

medium (BNM). Seven day old seedlings were inoculated 

with Sinorhizobium meliloti culture with an optical density 

0.1 at wave length 600 nm to examine the nodule phenotype 

according to Galibert 2001 [13].  

Genetic cross: Plants homozygous for blkn allele were 

crossed with R108 wild type plant. Mutant alleles were 

allowed to segregate through F1 self-fertilization. Genomic 

DNA extraction was carried out for F2 individuals and 

subjected to PCR amplification for analyzing the 

segregation pattern of blkn alleles.    

Nodule examination with light microscopy: Two weeks 

old nodules were directly fixed in 50ml solution containing 

25ml ethanol 95%, 5ml formaldehyde, 2.5ml glacial acetic 

acid and 17.5ml distilled water. The fixed nodules were 

embedded in paraffin wax. Blocks were divided with rotary 

microtome (Euromex USA).  Slides were stained with 

safranin red followed by light green dye. Finally, slides 

were scanned under light microscope (Axio vert.A1) [14]. 

Estimation of nitrogenase activity: Root nodules were 

washed with sterilized water and placed in bottles sealed 

with rubber. 10 ml of air was withdrawn from bottle and 

replaced by 10 ml of acetylene using plastic syringes. The 

bottles were incubated for one hour at 30
◦
 C thereafter, 2 ml 

gas samples were withdrawn and assayed for measuring 

ethylene concentration using liquid chromatography [15]. 

Determination of plant pigments: One hundred mg of 

fresh green leaves from each line with 10 biological replicas 

were frozen and grounded with liquid nitrogen rapidly. 

Chlorophyll pigment was obtained by 3 mL of 80% acetone 

including 1 mM potassium hydroxide then centrifuged at 

10,000 rpm for 10 min. Followed by supernatant 

quantification with a spectrophotometer device(bio 

spectrophotometer, ependorf) [16]. Chlorophyll and total 

carotenoid amounts were calculated based on [17]. 

Phenolic compounds Quantification: Total phenol conte-

nts were estimated by Folin Ciocalteu’s technique. One 

gram of each mutant and control leaves were extracted in 3 

ml of ethanol 95% and kept at 4°C/24 hrs in the dark, and 

then the samples were centrifuged at 13000rpm/5min. One 

ml of Supernatant was transferred to 5 ml of distilled water 

with 0.5ml of Folin Ciocalteu reagent (F-C) were mixed 

well, after that 1.5 ml of 20% of sodium carbonate 



 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

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(Na2CO3) was added and the total volume was adjusted to 

10 ml and was incubated for 2 hrs at room temp. Finally, 

spectrophotometer (bio spectrophotometer, ependorf) was 

used to read the samples at 765nm [18,19].Total contents of 

phenolic compounds were calculated from standard curve 

obtained with different concentration of salicylic acid (10 to 

100 mg/ml) 

AFLP type PCR for Tnt1 border characterization in 

blkn mutant: Genomic DNA of blkn seedlings in addition 

to control plants were isolated separately using i-genomic 

plant DNA extraction mini kit (iNtRON Biotechnology). 

DNA pool of  blkn isolated samples and DNA of control 

sample were separately double digested by EcoRI - MfeI 

and AseI - NdeI respectively according to enzyme producer 

protocol BioLabs®, ECO and ASE adaptors were ligated to 

the digested DNA using TaKaRa T4 DNA ligase separately. 

Amplification of Tnt1 borders was carried out by nested 

PCR to amplify all Tnt1 -Flanking Sequence Tags (FSTs) in 

blkn mutant line.  All PCR reactions were prepared using 

TaKaRa LA polymerase in Biorad . 

Cloning of amplified fragments: QIAquick PCR purificat-

ion Kit was used to purify the amplified tagged fragments 

before cloning into pGEM-Teasy vector (Promega®).  

After cloning, colony PCR method was done using Sp6 and 

T7 common oligonucleotide primers. Later fragments with 

different sizes were purified and sequenced. This method 

was used as confirmation test to success transformation.  

Data analysis: Tnt1 FSTs were analyzed against Medicago 

truncatule genome using National Center for Biotechnology 

Information data base NCBI (http://blast.ncbi.nlm.nih.gov 

and http://www.medicagohapmap.org/home/view). 

Statistical analysis: Data was exposed to analysis using 

spss20.0 (One-Way ANOVA) and the value of the least 

significant difference (LSD) was calculated at significance 

level of 5%.  

Results 

Phenotype characterization 

blkn seedling Phenotype: For phenotype characterization, 

blkn mutant was tested against R108 for roots and shoots 

lengths, 10 biological replicas were used for each line. The 

lengths were measured seven days post germination on ½ 

MS medium, measurements were recorded every seven 

days’ time interval. blkn average root lengths were 

significally longer than R108 at (p=0.01). While the 

average shoot lengths in blkn was similar to that of R108 

(p>0.05) (Figure 1A and 1B). On the other hand, mature 

pods of blkn had different shape; it displayed compressed 

pod and shorter spines than R108 but the two lines showed 

anti-clock wise spines direction (Figure 1C and 1D) and 

the same number of seeds which is about from 5 to 8. The 

average number of produced pods has no significant 

difference between blkn and R108 at (p=0.2) (Figure 1E).

Figure 1. Phenotypic characters of M.truncatula wild type and blkn. (1A) comparison between average root lengths of blkn 

and R108 is indicating a significance difference between the two types of plants at (P = 0.01). (1B) the mean value of blkn 

shoot length was close to mean value of R108 shoot length at (p>0.05).  (1C)  mature pods of R108 is showing long spine with 

anti-clock wise spines direction. (1D) mature compacted  pods of blkn with short spines and anti-clock wise direction. (1E) the 

average number of the produced pods in blkn and R108 with no significant difference at (p=0.2).

http://blast.ncbi.nlm.nih.gov/
http://www.medicagohapmap.org/home/view


 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

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blkn nodule Phenotype: blkn nodules were examined three 

weeks post inoculation with Sinorhizobium meliloti strain 

(Sm1021) on BNM. blkn nodules showed null function-

black color phenotype with longer size than R108 nodule 

(Figure 2A). According to statically analysis, nodules 

number of blkn mutant is significally more than R108 at 

(p=0.03) as shown in (Figure 2B).  

 

Figure 2. (2A) one month-old blkn seedling showing black 

color nodule in a cluster arrangement. (2B) the average 

nodules number of blkn mutant was significally more than 

R108 wild type at p=0.03. 

 

Transfer sections of blkn nodule were compared to 

those of R108 through light microscopy examination. 

Nodules of R108 prompted by rhizobia displayed the 

characteristic layers and the cells in each layer have their 

definite structure and function (1, meristem zone; 2, 

bacterial infection zone; 3, nitrogen-fixing zone; 4, 

senescent zone) (Figure 3A).  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3. Nodule examination by light microscope. (3A) 

transfer section of wild type nodule, (3B) transfer section of 

blkn nodule. (1) bacterial infection zone; (2) nitrogen-fixing 

zone; (3) senescent zone. blkn transfer section is completely 

different from this of R108 in both number and size of cells 

in bacterial infection zone and nitrogen fixation zone.  

While the phenotype of blkn nodule that was induced 

with the same symbiotic bacteria strain showed an unusual 

nodule progressive process. The zones in blkn nodule didn’t 

have clearly characteristic shape like the control. Cells of 

infection zone were longer in size than those in the same 

layer of control nodule. Additionally, there was abnormal 

number of cells in nitrogen fixation zone although almost of 

these cells were free from bacteroid and had accumulated 

phenolic compounds. 

 

Physiological characterization 

Determination of blkn pigmentation: Plant pigment is any 

kind of colored material produced by the plant. Plant 

Pigments can absorb visible radiation between violet (380 

nm) and ruby-red (760 nm). Plant pigments responsible for 

the color to the leaves, the flowers, and the fruits and are 

essential in regulating photosynthesis and development. 

Plant has many different pigments found in several organic 

compounds. Chlorophyll, the green pigment that can absorb 

light energy as much as possible and Carotenoids, the 

pigments that can absorb blue and indigo light are the two 

most important pigmentation in the plant. The two pigments 

were estimated in blkn and R108 as well. Both type of 

chlorophyll (chlorophyll A and chlorophyll B) don’t show 

any significant difference between their content in blkn and 

R108 (p=0.7) for chlorophyll A and p=0.06 for chlorophyll 

B). While blkn produces more than double amounts of 

carotenoids than R108 with a significant differenc (p=0.02) 

as shown in (Figure 4A). 

Figure 4. (A) measurement of pigment contents in R108 

and blkn showing no significant difference in both types of 

chlorophylls (A and B) between R108 and blkn at (p=0.7) 

for chlorophyll A and  at (p=0.06) for chlorophyll . While 

blkn produces more than douple amounts of carotenoids 

than R108 with a significanc differenc at (p=0.02). (B) blkn 

roots contain more than double content of phenolic 

compounds than R108 (p=0.00).   



 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

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Phenolic compounds quantification in blkn: Plant phen-

olics are secondary native metabolites which perform a very 

wide range of physiological characters in plants. Higher 

plants produce several thousand of diverse phenolic 

compounds. Plant phenolics are fundamental defense 

compounds during different biotic and abiotic 

environmental stresses like; high and low temperatures, 

high light, ultraviolet radiation, different oxidants, nutrient 

deficiency, herbivores and pathogen infection. Due to the 

black nodule colour and the dark colour of blkn root, total 

phenols were estimated in root of blkn and R108 one day 

pre Rhizobium inoculation and 72 hrs post Rhizobium 

inoculation. The total phenolic compound estimation was 

performed colorimetrically using spectrophotometer. There 

was no significant differences between blkn and R108 pre 

rhizobia inoculation while blkn seedlings have the ability to 

accumulate large amount of phenolic compounds than R108  

and the difference was significantly at p=0.00 as shown in 

(Figure 4B). 

Molecular Characterization 

Tnt1-tagged loci identification and characterization in 

blkn mutant: To characterize Tnt1-flanking sequence tags 

in blkn mutant, oligonucleotide primers (LTR3, LTR4, 

LTR5, LTR6) for Tnt1 transposon  against AseI, AseII and  

EcoI, EcoII for ASE and ECO adaptor respectively 

[20]were used. All Tnt1 tagged fragments were separated 

and visualized on 1.5% agarose running at 80 V, data 

presented in (Figure 5A, B, C, and D). Tnt1-tagged 

fragments with different sizes were exposed to cloning in 

pGEM-Teasy and sequencing process. About 25 FSTs from 

blkn mutant line were isolated and subjected to data 

analysis against Genbank http://www.ncbi.nlm.nih.gov/ 

database.  Some of them were blasted with coding regions 

of medicago genome, others were blasted with non-coding 

regions and the rest were unknown sequences. FSTs 

corresponding to blkn mutant were submitted to Genbank 

http://www.ncbi.nlm.nih.gov/. Genbank accession numbers, 

E-value and reference genes are shown in (Table 1).

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5. blkn-Tnt1 border amplification. (5A) MfeI and EcoRI -double digestion the PCRII result using LTR4and EcoII 

oligonucleotide primers. (5B) EcoRI and  MfeI-double digestion PCRII result using LTR6 and EcOII oligonucleotide primers. 

(5C) NdeI and  Ase I -double digestion test PCRII result using LTR4 and AseII oligonucleotide primers. (5D) NdeI and  AseI -

double digestion PCRII test using LTR6 and AseII oligonucleotide primers. Lanes from 1-16 are, AG ,AT, AA, AC, CG, CT, 

CA, CC, GT, GG, GC, GA, TT, TG, TC, and TA  separately at the end of oligonucleotide primers. AseII and EcoII. 

M  1   2  3   4   5   6   7   8   9   10   11   12   13   14   15  

16  M 

M   1   2  3   4   5   6   7   8   9   10   11   12   13   14   

15  16  M 

A B 

M  1   2  3   4   5   6   7   8   9   10   11   12   13   14   15  16 M  M  1   2  3   4   5   6   7   8   9   10   11   12   13   14   15  16  M 

M  1   2  3   4   5   6   7   8   9   10   11   12   13   14   15  16 M  M  1   2  3   4   5   6   7   8   9   10   11   12   13   14   15  16 M  

A B 

C D 



 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

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Table1: blkn Tnt1-tagged sequences. 

Sequence   name      Length Accession number  Organism Reference E-value 

blkn2 560 MN529997 Medicago truncatula  MWMB01000023.1  0 

blkn3 291 MN529998 Medicago truncatula MWMB01000001.1  8.00E-152 

blkn4 289 MN529999 Medicago truncatula MWMB01000001.1  1.00E-125 

blkn5 288 MN530000 Medicago truncatula MWMB01000001.1  4.00E-150 

blkn6 291 MN530001 Medicago truncatula MWMB01000001.1  8.00E-152 

blkn8 284 MN530002 Medicago truncatula MWMB01000001.1  6.00E-148 

blkn9 171 MN530003 

Medicago truncatula 

XM_024776685.1  3.00E-79 probable L-type lectin-

domain containing receptor 

kinase VII.2 

blkn10 250 MN530004 Medicago truncatula MWMB01000001.1  4.00E-129 

blkn11 71 MN530005 Medicago truncatula MWMB01000031.1  1.00E-23 

blkn13 250 MN530006 Medicago truncatula CU179894.1  3.00E-106 

blkn15 251 MN530007 Medicago truncatula MWMB01000011.1  1.00E-129 

blkn16 292 MN530008 Medicago truncatula AC130200.30  2.00E-152 

blkn17 252 MN530009 Medicago truncatula MWMB01000011.1  3.00E-130 

blkn18 294 MN530010 Medicago truncatula AC130200.30  6.00E-103 

Blkn19 156 MN530011 Medicago truncatula MWMB01000006.1  5.00E-77 

Blkn20 255 MN530012 Medicago truncatula MWMB01000011.1  7.00E-132 

 

Medicago truncatula L-type lectin-domain receptor 

kinase locus is interrupted by Tnt1 insertion in all F2 

black nodule individuals  

From our data analysis, three Medicago truncatula 

Tnt1-insertion loci in blkn were subjected to additional 

investigation; blkn_2 (Tnt1 insertion located in F-box/LRR-

repeat protein), blkn_4 (Tnt1 insertion located in unknown 

protein) and blkn_9 (Tnt1 insertion located in L-type lectin-

domain receptor kinase). Two oligonucleotide primers 

blkn2–R (5-GCTTGGAAGAGTCTAAGGTTAA-3) and 

blkn4–R (5-CCAAGTTGACTTGTTTCATC-3) were used 

separately with LTR4 to test blkn_2 and blkn_4 insertion 

loci respectively. PCR was performed on genomic DNA 

extracted from F2 population entities resulting from blkn 

mutant and R108 wild type back cross. Only entities 

displaying black nodule phenotype with the other mutation 

phenotype characters were tested (about 1/4 population of 

F2 progeny).  

PCR result indicated that Tnt1 insertion wasn’t 

confirmed in all of the entities means that the insertion on 

those loci aren't charged for this mutation phenotype.  

Oligonucleotide primer pair Blkn9-F (5-CCCTCAAAAAAT 

ACCAACAAAACC-3) and blkn9-R (5-CAACTTTTCCAA 

TTCATCACCA-3) were designed to be used in compatible 

with the two LTRs oligonucleotides of Tnt1 to test the 

incidence of Tnt1 within the predicted mutated locus, L-type 

lectin-domain containing receptor kinase (BLKN_9), in all 

blkn mutant individuals. PCR result confirmed the incidence 

of the two Tnt1 side borders in all F2 entities displaying the 

mutation phenotype at the proper locus while showed a 

negative result in R108 entities. When using the two specific 

oligonucleotides for BLKN_9 locus amplification, only 

R108 gave the proper band at 358bp (data not shown) while 

the amplification at this position failed in all blkn mutant 

individuals (1/4 population of F2 progeny).  

The rest of segregation individuals which are showing 

wild type phenotype (3/4 population of F2 progeny) gave a 

ratio of 60/30 positive to negative PCR result for Tnt1 

border amplification. This result is obeying Mendel law, 

indicating that (BLKN_9) insertion could be accountable for 

null function black-nodule, phenol accumulation and other 

mutation phenotype. The mutation at this locus was 

confirmed also at RNA level, RT-PCR performed on all 

blkn mutant individuals using the two specific primers of the 

proper locus was giving negative result in all entities and 

positive amplification in R108 (data not shown). Tnt1 

insertion was precisely located next to the base number 303 

post ATG start codon of Medicago truncatula L-type lectin-

domain receptor kinase gene which is 2079 bp in length with 

one exon encode Lectin_LegB Receptor Like Kinase 692 

https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000023
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000001
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000001
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000001
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000001
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000001
https://www.ncbi.nlm.nih.gov/nucleotide/XM_024776685.1?report=genbank&log$=nuclalign&blast_rank=1&RID=SVNDUW9D015
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000001
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000031
https://www.ncbi.nlm.nih.gov/nucleotide/CU179894.1?report=genbank&log$=nuclalign&blast_rank=2&RID=S83XBG0Y014
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000011
https://www.ncbi.nlm.nih.gov/nucleotide/AC130200.30?report=genbank&log$=nuclalign&blast_rank=1&RID=REX0CU3V014
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000011
https://www.ncbi.nlm.nih.gov/nucleotide/AC130200.30?report=genbank&log$=nuclalign&blast_rank=1&RID=REYEG4PB01R
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000006
https://www.ncbi.nlm.nih.gov/nuccore/MWMB01000011


 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

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amino acids in length. MtLectinRLK consists of three 

domains; two transmembrane helix (TMhilex) the first is 

from 4bp to 26bp and the other from 288 to 310 predicted 

by TMHM (http://www.cbs.dtu.dk/services/TMHMM/). 

The second domain is Lectin_legB domain at the position 

from 22bp to 285bp, and third one is the extracellular 

Receptor Protein kinase (Pkinase) at position from 357bp to 

641bp as shown in (Figure 6). 

Figure 6. Mt-LectinRLK is 692 aa in length contains two 

transmembrane helixes (TMhelix) from 4 to 26 and from 

288 to 310, Lectin_legB domain at position from 22 to 

position 285, and extracellular Receptor Protein kinase 

(Pkinase) at position from 357 to position 641. 

 

Phylogenetic tree was constructed using EMBL-EBI 

(www.uniprot.org/align) as shown in (Figure 7). 

MtLectinRLK is an ancestry related to probable L-type 

lectin-domain containing receptor kinase VII.2 Cicer 

arietinum, Putative L-type lectin-domain containing receptor 

kinase-like protein Trifolium pretense, Protein kinase 

domain-containing protein Lupinus angustifolius, Protein 

kinase domain-containing protein Phaseolus vulgaris, 

probable L-type lectin-domain containing receptor kinase 

VII.2 isoform X1 Vigna radiate, Putative L-type lectin-

domain containing receptor kinase VII.2 Glycine soja, 

Putative L-type lectin-domain containing receptor kinase 

VII.2 Mucuna pruriens, Lectin-domain containing receptor 

kinase A4.2 Cajanus cajan. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 7. The predicted Medicago truncatula MtLectinRLK 

is ancestry related to probable L-type lectin-domain 

containing receptor kinase VII.2. Phylogenetic tree was 

constructed using EMBL-EBI (www.uniprot.org/align). 

Discussion 

Unique Medicago truncatula mutant line NF0478 is a 

Tnt1-retrotransposon mutant that is verified as black nodule 

phenotype mutant (blkn). We utilized reverse genetic tool to 

isolate and characterize the Medicago truncatula 

“knockout” mutations in a gene that is supposed to be 

involving in nodulation process and other physiological 

processes. Previously, about 2801 Tnt1 flanking sequence 

tags were isolated from 156 Medicago truncatula symbiotic 

mutant lines. The insertions were in nodulating genes like; 

NODULE INCEPTION (NIN), NODULATION 

SIGNALING PATHWAY (NSP1, NSP2), DOESN'T 

MAKE INFECTIONS (DMI1, DMI2 and DMI3) and a 

number of super-nodulation genes SUNN and SKL [3]. 

blkn the black nodule mutant is showing defective in 

infection zone and nitrogen fixation zone alongside cell wall 

periphery lignification in symbiotic organ. Characterization 

of blkn mutation depends mainly on phenotype monitoring 

and various biotechnology tools for investigation. Linking 

between mutation phenotype with the changes occurred in 

the native physiological process due to this mutation along 

with the molecular characterization gave us clear evidence 

about the defected protein responsible for the emergence of 

this mutation. More than ~25 Tnt1 insertion sites was 

isolated, sequenced and analyzed using Genbank (http:// 

www.ncbi.nlm.nih.gov/) database during our study. Some 

insertions were located within non-coding regions while the 

others were located in non-matching sequences.  

Our results enable us to confirm that the Medicago 

truncatula L-type lectin-domain receptor kinase locus is 

interrupted by the Tnt1 insertion in all blkn mutant line 

individuals. Tnt1 insertion locus was tested with PCR at 

both DNA and RNA levels and confirmed genetically 

through the segregation ratio of Tnt1 borders in F2 

population. Our results indicating that the Tnt1 insertion is 

precisely located next to the base number 303 post ATG 

start codon of Medicago truncatula L-type lectin-domain 

receptor kinase gene in blkn mutant line. This Medicago 

truncatula gene encodes Lec_legB-RLK which is 692 bp 

amino acids in length was scanned by http://pfam.xfam.org 

for functional domains. Our data showed that Medicago 

truncatula Lec_legB-RLK has two transmembrane helix 

domains (TMhilex) the first is from 4bp to 26bp and the 

other from 288 to 310 predicted by TMHM (http://www. 

cbs.dtu.dk/services/TMHMM/).  

The second is Lectin_legB domain (start from 22 to 

285). Each leguminous lectin subunit contains sole 

carbohydrate-binding site with beta pleated sheets consist of 

two conserved amino acids. The lectins are binding 

 galactose, mannose or glucose and this interaction requires 

a firmly bound manganese and calcium ions. The third 

conserved domain is the Protein kinase PK domain (start 

from 357 to 641) and it acts as switch on/off for majority of 

cellular activity in plant cell by phosphorylation and any 

defect in this domain can cause dysfunction abnormality. 

Native biotic environment of plants root contains both 

symbionts and pathogens. The plants are responding to 

http://www.cbs.dtu.dk/services/TMHMM/
http://www.uniprot.org/align
http://pfam.xfam.org/


 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

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Page 8 of 10 

various microbes either by establishing a symbiotic 

interaction with friendly symbionts bacteria and mycorrhiza 

[21], or by inducing plant innate immunity to defend against 

pathogens attack [22]. Medicago truncatula is an ultimate 

opportunity to study such crosstalk between symbiosis and 

defense in the environment. 

Innate immunity in plants is triggered through 

recognition of microbe-associated molecular patterns 

(MAMPs) by plants pattern recognition receptors (PRRs) 

[23,24]. This recognition process elicits defense program 

cascades enable the plant to respond to the pathogen attack 

in a precisely and effective manner. Such recognition is 

switching on all the downstream protection including: 

production of reactive oxygen species (ROS), accumulation 

of salicylic acid signal,  inducing Nonexpressor of PR gene 

(NPR1) protein that convert into its monomeric form then 

translocate to the nucleus to activate PR–related genes, 

increasing Ca2+ influx, and finally activation of mitogen-

activated protein kinase (MAPK) cascade [22]. This fast 

response is leading to the resistance against wide range of 

pathogens attack [25]. Plant receptor-like kinases (RLKs) 

include a superfamily of trans-spanning proteins involved in 

pathogen detection and defense signaling transduction [26] 

which is closely related to ROS production [27]. 

Lectin receptor kinases (Lec-RKs) is one class of the 

plants RLKs that plays essential role through plant 

development and during the various stress responses. It 

contains an extracellular lectin domain and divided into 

three subclasses according to the diversity in this motif; G-

type (S-locus), C-type (calcium dependent) and L-type 

(legume) [28].  G-type lect-RKs are found in flowering 

plants and known as S-domain RLKs They are involving in 

self-incompatibility [29] .C-type Lec-RKs are calcium-

dependent lectin that are found regularly in the mammalian 

proteins and involving in pathogen recognition and trigger 

the various immune responses [30]. Arabidopsis has a single 

C- type Lec-RK motif but its function is not been 

established so far [28], while Arabidopsis contains about 45 

L-type Lec-RKs [28,31]. Lec-RKs were proposed to play 

essential role in both abiotic stress tolerance [32-34] and 

biotic stress signals transduction [28]. 

This study is reporting the knock out mutation of 

Medicago truncatula Lec_legB-RK gene (blkn), this 

mutation is driving null function black color root nodule 

phenotype. The mutated nodule is displaying abnormality in 

both infection zone and nitrogen fixation zone. Transverse 

section of the mutated nodule was displaying an empty 

symbiotic zone with higher lignification at the cell wall 

periphery compared with the wild type nodule phenotype. 

The released bacteriods in blkn infection zone were less than 

those in R108.   In addition, the total phenolic compound 

accumulation was dramatically and significantly excessive in 

blkn root 72 hours post inoculation with sinorhizobium 

comparing to R108.  

This ultimate modification wasn’t observed pre 

sinorhizobium inoculation. The first published mutation in 

lectin-RK was reported [35] in Arabidopsis LecRK-I.9. This 

mutation is exhibited an increasing susceptibility to 

Pseudomonas syringae, Phytophthora brassicae, and 

Phytophthora capsici, while the over expression of this gene 

enhanced the resistance of the plant against these three 

pathogens [35]. Although most of plant lectin receptor 

kinases function is not yet so clear, the role of these kinases 

in innate immunity is developing. Some other LRK were 

reported to be involving in plant protection against various 

pathogen attacks. Pi-d2 is G-LRK isolated from rice can 

provides defense against rice blast derived by 

Magnaporthegrisea, the parasitic fungal pathogen [37]. 

NbLRK1 is LecRK in Nicotiana benthamiana was suggested 

to recognize the INF1 elicitor of Phytophthora infestans and 

mediates plant defense [38]. While the expression of G-LRK 

is up-regulated in tobacco by lipopolysaccharides signal 

[39].  

According to our data, Lec_legB-RK switching off due 

to the Tnt1 insertion in blkn mutant line is resulting in 

defective nodule, so what is the proposed function of 

Medicago truncatula Lec_legB-RK? Our results in addition 

to the previous data enabled us to suggest that MtLec_legB-

RK is like a switch key. MtLec_legB-RK can recognize the 

symbionts microorganisms and directed the plant cells 

toward being involved in symbiotic interaction, in addition 

to recognize the invading microorganisms and trigger the 

signal defense pathway. Thus, the mutation of Medicago 

truncatula Lec_legB-RK altered the plant cells unable to 

distinguish between the symbionts and invaders. We are 

suggesting that in case of blkn mutant, although the 

Rhizobia-derived N-acetylglucosamine signal were normally 

perceived via LysM proteins [40] otherwise the mutant plant 

cells treated them as attackers instead of treating them as 

symbionts. Consequently, the mutant cells produced greater 

amount of phenolic compound in roots and nodules 

compared to those of wild type R108. 

Normally, such phenolic compounds are produced in 

plant cells due to several biotic and abiotic stress conditions 

such as: salt stress, drought, extreme temperature, nutrient 

deficiency, UV radiation, heavy metals and herbicides and 

other pathogen attacks. All these kind of plant stresses can 

induce unbalance between production of reactive oxygen 

species (ROS) and its scavengers. Plants (ROS) are 

extremely reactive, toxic, and can cause severe damage to 

various cell components; DNA, carbohydrates, proteins, and 

lipids leading to oxidative stress. Plants have their own 

enzymatic and non-enzymatic mechanisms for ROS 

detoxification. Peroxidase (POX) is one of main enzymatic 

system, it oxidize phenolic compound by using them as an 

electron donor [41,42]. So, what is the fate of high contents 

of phenolic compound in blkn mutant plant? According to 

[43], phenolics are oxidized by POX at H2O2 expense 



 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

Highlights in BioScience                                                                                                                                                                 September 2020| Volume 3 

http://bioscience.highlightsin.org/ 

Page 9 of 10 

throughout lignin formation. Thus, the strong lignin deposits 

around the cells wall periphery of both infection zone and 

nitrogen fixation zone confirmed our suggestion that the root 

cells in blkn mutant failed to distinguish between symbionts 

and attackers through producing a high amount of phenolic 

compound which is consider as ROS scavenging compound. 

In turn these phenolic compounds were oxidized into lignin 

via POX within the nitrogen fixation organ. This suggestion 

may also explain the low frequent bacteroids in infection 

zone of blkn mutant than R108 infection zone. This result is 

obeying with the previous published data by Roopashree 

[44]. They proposed that in native condition the legume 

lectin gene has two reverse functions depending on the 

perceived microorganisms and also the gene may be 

involved in the attachment of nitrogen-fixing bacteria to the 

legumes and in reverse function as a protector 

against different pathogen attacks. 

But if the Lec_legB-RK kinase is involved in Rhizobia 

attachment to the root of legumes, we should ask what type 

of component that is interacting with Lec_legB-RK kinase 

domain? Also which component is phosphorylated by these 

kinases?  In fact this gene needs more investigation to 

identify its function in a more precise manner in our 

perspective work.         

Acknowledgments 

The authors appreciate Dr. Pascal Ratet for NF0478 

mutant line multiplication, R108 wild type and 

Sinorhizobium meliloti  providing and for generous help. 

References 

1. Graham PH, Vance CP. Legumes: importance and 

constraints to greater use. Plant physiology. 2003 Mar 1; 

131(3):872-7. 

2. Kosuta S, Hazledine S, Sun J, Miwa H, Morris RJ, 

Downie JA, Oldroyd GE. Differential and chaotic calcium 

signatures in the symbiosis signaling pathway of legumes. 

Proceedings of the National Academy of Sciences. 2008 

Jul 15; 105 (28):9823-8. 

3. Pislariu CI, Murray JD, Wen J, Cosson V, Muni RR, 

Wang M, Benedito VA, Andriankaja A, Cheng X, Jerez 

IT, Mondy S. A Medicago truncatula tobacco retrotran-

sposon insertion mutant collection with defects in nodule 

development and symbiotic nitrogen fixation. Plant 

Physiology. 2012 Aug 1; 159(4):1686-99. 

4. Udvardi MK, Day DA. Metabolite transport across 

symbiotic membranes of legume nodules. Annual review 

of plant biology. 1997 Jun; 48 (1):493-523. 

5. Kumar A, Bennetzen JL. Plant retrotransposons. Annual 

review of genetics. 1999 Dec;33 (1):479-532. 

6. Cook DR. Medicago truncatula-a model in the making!. 

Current opinion in plant biology. 1999 Aug;2 (4):301-4. 

7. Udvardi MK, Tabata S, Parniske M, Stougaard J. Lotus 

japonicus: legume research in the fast lane. Trends in 

plant science. 2005 May 1; 10 (5):222-8. 

8. Scholte M, d'Erfurth I, Rippa S, Mondy S, Cosson V, 

Durand P, Breda C, Trinh H, Rodriguez-Llorente I, 

Kondorosi E, Schultze M. T-DNA tagging in the model 

legume Medicago truncatula allows efficient gene 

discovery. Molecular Breeding. 2002 Dec 1;10(4):203-15. 

9. d'Erfurth I, Cosson V, Eschstruth A, Lucas H, Kondorosi 

A, Ratet P. Efficient transposition of the Tnt1 tobacco 

retrotransposon in the model legume Medicago 

truncatula. The Plant Journal. 2003 Apr;34(1):95-106. 

10. Tadege M, Wen J, He J, Tu H, Kwak Y, Eschstruth A, 

Cayrel A, Endre G, Zhao PX, Chabaud M, Ratet P. 

Large‐scale insertional mutagenesis using the Tnt1 

retrotransposon in the model legume Medicago 

truncatula. The Plant Journal. 2008 Apr;54 (2):335-47. 

11. Sun L, Ge Y, Bancroft AC, Cheng X, Wen J. FNBtools: 

A software to identify homozygous lesions in deletion 

mutant populations. Frontiers in plant science. 2018 Jul 

10;9:976. 

12. Galibert F, Finan TM, Long SR, Pühler A, Abola P, 

Ampe F, Barloy-Hubler F, Barnett MJ, Becker A, 

Boistard P, Bothe G. The composite genome of the 

legume symbiont. Science. Sinorhizobium meliloti 2001 

Jul 27;293(5530):668-72. 

13. Ehrhardt DW, Atkinson EM. Depolarization of alfalfa 

root hair membrane potential by Rhizobium meliloti Nod 

factors. Science. 1992 May 15;256(5059):998-1000. 

14. Elkamali HH, Eltahir AS, Yousif IS, Khalid AM, Elneel 

EA. Comparative Anatomical Study of the Stems and 

Leaflets of Tribulus longipetalous, T. pentandrus and T. 

terrestris (Zygophyllaceae). Open Access Library 

Journal. 2016 Aug 30;3(8):1-5. 

15. Summerfield RJ, Dart PJ, Huxley PA, Eaglesham AR, 

Minchin FR, Day JM. Nitrogen nutrition of cowpea 

(Vigna unguiculata). I. Effects of applied nitrogen and 

symbiotic nitrogen fixation on growth and seed yield. 

Experimental Agriculture. 1977 Apr;13(2):129-42. 

16. Schelbert S, Aubry S, Burla B, Agne B, Kessler F, 

Krupinska K, Hörtensteiner S. Pheophytin pheophorbide 

hydrolase (pheophytinase) is involved in chlorophyll 

breakdown during leaf senescence in Arabidopsis. The 

Plant Cell. 2009 Mar 1;21(3):767-85. 

17. Lichtenthaler HK, Buschmann C. Chlorophylls and 

carotenoids: Measurement and characterization by 

UV‐VIS spectroscopy. Current protocols in food 

analytical chemistry. 2001 Aug;1(1):F4-3. 

18. Patel A, Patel A, Patel A, Patel NM. Determination of 

polyphenols and free radical scavenging activity of 

Tephrosia purpurea linn leaves (Leguminosae). 

Pharmacognosy Research. 2010 May;2(3):152. 

19. Ainsworth EA, Gillespie KM. Estimation of total 

phenolic content and other oxidation substrates in plant 

tissues using Folin–Ciocalteu reagent. Nature protocols. 

2007 Apr;2(4):875-7. 

20. Ratet P, Porcedu A, Tadege M, Mysore KS. Insertional 

mutagenesis in M. truncatula using Tnt1 retrotransposon. 

21. Oldroyd GE, Murray JD, Poole PS, Downie JA. The 

rules of engagement in the legume-rhizobial symbiosis. 

Annual review of genetics. 2011 Dec 15;45:119-44. 

22. Nicaise V, Roux M, Zipfel C. Recent advances in 

PAMP-triggered immunity against bacteria: pattern 

recognition receptors watch over and raise the alarm. 

Plant physiology. 2009 Aug 1;150(4):1638-47. 

23. Jones JD, Dangl JL. The plant immune system. nature. 

2006 Nov;444(7117):323-9. 



 

 

Hassan et al., 2020                                                                                               blkn, a novel Medicago truncatula mutant achieving black nodule phenotype 

 

 

Highlights in BioScience                                                                                                                                                                 September 2020| Volume 3 

http://bioscience.highlightsin.org/ 

Page 10 of 10 

24. Tsuda K, Katagiri F. Comparing signaling mechanisms 

engaged in pattern-triggered and effector-triggered 

immunity. Current opinion in plant biology. 2010 Aug 

1;13(4):459-65. 

25. Boller T, Felix G. A renaissance of elicitors: perception 

of microbe-associated molecular patterns and danger 

signals by pattern-recognition receptors. Annual review 

of plant biology. 2009 Jun 2;60:379-406. 

26. Tang D, Wang G, Zhou JM. Receptor kinases in plant-

pathogen interactions: more than pattern recognition. 

The Plant Cell. 2017 Apr 1;29(4):618-37. 

27. Kimura S, Waszczak C, Hunter K, Wrzaczek M. Bound 

by fate: the role of reactive oxygen species in receptor-

like kinase signaling. The Plant Cell. 2017 Apr 

1;29(4):638-54. 

28. Bouwmeester K, Govers F. Arabidopsis L-type lectin 

receptor kinases: phylogeny, classification, and 

expression profiles. Journal of experimental botany. 

2009 Nov 1;60(15):4383-96. 

29. Sherman-Broyles S, Boggs N, Farkas A, Liu P, Vrebalov 

J, Nasrallah ME, Nasrallah JB. S locus genes and the 

evolution of self-fertility in Arabidopsis thaliana. The 

Plant Cell. 2007 Jan 1;19(1):94-106. 

30. Cambi A, Koopman M, Figdor CG. How C‐type lectins 

detect pathogens. Cellular microbiology. 2005 

Apr;7(4):481-8. 

31. Herve C, Dabos P, Galaud JP, Rougé P, Lescure B. 

Characterization of an Arabidopsis thaliana Gene that 

Defines a New Class of Putative Plant Receptor Kinases 

with an Extracellular Lectin-like Domain. Journal of 

molecular biology. 1996 May 24;258(5):778-88. 

32. Garcia-Hernandez M, Berardini T, Chen G, Crist D, 

Doyle A, Huala E, Knee E, Lambrecht M, Miller N, 

Mueller LA, Mundodi S. TAIR: a resource for integrated 

Arabidopsis data. Functional and  integrative genomics. 

2002 Nov 1;2(6):239-53. 

33. He XJ, Zhang ZG, Yan DQ, Zhang JS, Chen SY. A salt-

responsive receptor-like kinase gene regulated by the 

ethylene signaling pathway encodes a plasma membrane 

serine/threonine kinase. Theoretical and Applied 

Genetics. 2004 Jul 1;109(2):377-83. 

34. Joshi A, Dang HQ, Vaid N, Tuteja N. Pea lectin 

receptor-like kinase promotes high salinity stress 

tolerance in bacteria and expresses in response to stress 

in planta. Glycoconjugate journal. 2010 Jan 1;27(1):133-

50. 

35. Bouwmeester K, De Sain M, Weide R, Gouget A, 

Klamer S, Canut H, Govers F. The lectin receptor kinase 

LecRK-I. 9 is a novel Phytophthora resistance 

component and a potential host target for a RXLR 

effector. PLoS Pathog. 2011 Mar 31;7(3):e1001327. 

36. Balagué C, Gouget A, Bouchez O, Souriac C, Haget N, 

Boutet‐Mercey S, Govers F, Roby D, Canut H. The 

Arabidopsis thaliana lectin receptor kinase LecRK‐I. 9 

is required for full resistance to Pseudomonas syringae 

and affects jasmonate signalling. Molecular plant 

pathology. 2017 Sep;18(7):937-48. 

37. Chen X, Shang J, Chen D, Lei C, Zou Y, Zhai W, Liu G, 

Xu J, Ling Z, Cao G, Ma B. AB‐lectin receptor kinase 

gene conferring rice blast resistance. The Plant Journal. 

2006 Jun;46(5):794-804. 

38. Kanzaki H, Saitoh H, Takahashi Y, Berberich T, Ito A, 

Kamoun S, Terauchi R. NbLRK1, a lectin-like receptor 

kinase protein of Nicotiana benthamiana, interacts with 

Phytophthora infestans INF1 elicitin and mediates INF1-

induced cell death. Planta. 2008 Nov 1;228(6):977-87. 

39. Sanabria NM, van Heerden H, Dubery IA. Molecular 

characterisation and regulation of a Nicotiana tabacum 

S-domain receptor-like kinase gene induced during an 

early rapid response to lipopolysaccharides. Gene. 2012 

Jun 10;501(1):39-48. 

40. Rey T, Nars A, Bonhomme M, Bottin A, Huguet S, 

Balzergue S, Jardinaud MF, Bono JJ, Cullimore J, 

Dumas B, Gough C. NFP, a L ys M protein controlling N 

od f actor perception, also intervenes in M edicago 

truncatula resistance to pathogens. New Phytologist. 

2013 May;198(3):875-86. 

41. De Gara L, de Pinto MC, Tommasi F. The antioxidant 

systems vis-à-vis reactive oxygen species during plant–

pathogen interaction. Plant Physiology and 

Biochemistry. 2003 Oct 1;41(10):863-70. 

42. Takahama U. Oxidation of vacuolar and apoplastic 

phenolic substrates by peroxidase: physiological 

significance of the oxidation reactions. Phytochemistry 

Reviews. 2004 Jan 1;3(1-2):207-19. 

43. Ralph J, Lundquist K, Brunow G, Lu F, Kim H, Schatz 

PF, Marita JM, Hatfield RD, Ralph SA, Christensen JH, 

Boerjan W. Lignins: natural polymers from oxidative 

coupling of 4-hydroxyphenyl-propanoids. 

Phytochemistry reviews. 2004 Jan 1;3(1-2):29-60. 

44. Roopashree S, Singh SA, Gowda LR, Rao AGA. Dual-

function protein in plant defence : seed lectin from 

Dolichos biflorus ( horse gram ) exhibits lipoxygenase 

activity. Biochem J. 2006;639:629–39. 


