# Identification of Immune Related LRR-Containing Genes in Maize (Zea mays L.) by Genome-Wide Sequence Analysis

Authors: Wei Song; Baoqiang Wang; Xinghua Li; Jianfen Wei; Ling Chen; Dongmin Zhang; Wenying Zhang; Ronggai Li
Journal: International Journal of Genomics
 (2015)

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## Abstract

A large number of immune receptors consist of nucleotide binding site-leucine rich repeat (NBS-LRR) proteins and leucine rich repeat-receptor-like kinases (LRR-RLK) that play a crucial role in plant disease resistance. Although many NBS-LRR genes have been previously identified inZea mays, there are no reports on identifying NBS-LRR genes encoded in the N-terminal Toll/interleukin-1 receptor (TIR) motif and identifying genome-wide LRR-RLK genes. In the present study, 151 NBS-LRR genes and 226 LRR-RLK genes were identified after performing bioinformatics analysis of the entire maize genome. Of these identified genes, 64 NBS-LRR genes and four TIR-NBS-LRR genes were identified for the first time. The NBS-LRR genes are unevenly distributed on each chromosome with gene clusters located at the distal end of each chromosome, while LRR-RLK genes have a random chromosomal distribution with more paired genes. Additionally, six LRR-RLK/RLPs including FLS2, PSY1R, PSKR1, BIR1, SERK3, and Cf5 were characterized in Zea mays for the first time. Their predicted amino acid sequences have similar protein structures with their respective homologues in other plants, indicating that these maize LRR-RLK/RLPs have the same functions as their homologues act as immune receptors. The identified gene sequences would assist in the study of their functions in maize.


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## Body

## 1. Introduction

Pathogens, including bacteria, fungi, oomycetes, and viruses, are a major threat to the global human food supply. They attack plants in an attempt to gain nutrients from them. During the course of evolution, both plants and pathogens have evolved methods to combat each other. Plants, like animals, are equipped with immune receptors for recognizing invading pathogens and activating innate immune responses [1, 2]. It is widely accepted that plant immune responses consist of two branches of resistance. The first branch involves plasma-membrane localized pattern recognition receptors (PRRs) that recognize the conserved microbial molecules referred to as pathogen-associated molecular patterns (PAMPs) [2, 3]. The second requires intracellular receptors that are proteins encoded by classical plant disease resistance (R) genes to detect the presence of pathogen proteins inside the host cell [4]. The largest group of plant immune receptors that were identified 20 years ago are cytoplasmic nucleotide binding site (NBS) leucine rich repeat (LRR) proteins encoded by R genes [5]. The NBS-LRR genes have been further subdivided into two main groups, based on their N-terminal structures [6–9]. The first group possesses a domain with homology to the intracellular signalling domains of theDrosophila Toll and mammalian interleukin-1 receptors and is referred to as TIR-NBS-LRRs or TNLs. The second, non-TNL, group is collectively known as CC-NBS-LRRs or CNLs, based on the presence of a predicted N-terminal coiled-coil domain in some, but not all, members of this group [10]. Studies on the structure of cloned R genes revealed that not all R genes encode NBS-LRR proteins. Several R genes encode transmembrane receptor-like kinase/proteins (RLK/RLP) that are one of the most important groups of cell surface receptors [4, 11]. A typical RLK contains an extracellular receptor domain (LRR) to perceive a specific signal, a transmembrane domain (TM) to anchor the protein within the membrane, and an intracellular cytoplasmic kinase domain to transduce the signal through autophosphorylation followed by further phosphorylation of downstream components to regulate gene expression [12–14].Since the discovery of the first plant RLK in 1990, a small number of RLKs have been functionally characterized, such as flagellin sensing 2 (FLS2), tyrosine-sulfated peptide receptors PSKR1 and PSY1R [15–17]. The extracellular LRR domains in these proteins that act as cell surface immune receptors or components of receptor complexes contribute to plant defense/pathogen-recognition. Further studies on the molecular structure and function of flagellin reveal that numerous signal perception and transduction systems are needed in plants to recognize all potential invaders [18]. Indeed, more than 400 genes encoding RLK sequences with various receptor configurations are present in the genomic sequence of theArabidopsis thaliana, of which 216 members contain an LRR in the extracellular domain [19]. In addition to RLKs, 149 R genes encoding NBS-LRR are also present in theArabidopsis thaliana genome [20]. Therefore, the LRR-containing receptors play a crucial role in intercellular communication and disease resistance in plant immunity.Since 1992 the first plant R gene,Hm1, which confers specific resistance against a leaf blight and ear mold disease of corn, was cloned. More than 100 R genes have been cloned from different plant species, of which approximately 80% encode R proteins with NBS-LRR domains [21]. To date, along with the development of bioinformatics technology and the availability of whole-genome sequences of several plants, such asArabidopsis, rice,Brachypodium distachyon, maize, and sorghum, numerous NBS-LRR genes have been revealed [10, 20, 22–24]. However, reports on how many RLK/RLP genes are present in different plant species are rare. To further understand the importance of R genes, this study aims at identifying the sequences of R genes containing NBS-LRR and RLKs from the sequenced maize genome. In doing so, the R genes may be made available for breeding purposes. The bioinformatics analysis of the R gene homologues provides a definitive resource for the ongoing functional and evolutionary studies of this large plant gene family. Meanwhile, maize (Zea maysL.) is a food staple in many regions of the world and is used for animal feed and ethanol fuel. It is the world’s most extensively grown crop and has the highest world-wide production in all cereal crops [http://faostat.fao.org/]. In addition to its economic value, maize is also an important model plant for studies in plant genetics, physiology, and development. Thus, our work will shed more light on the maize immune system and the findings will provide a strong groundwork for the isolation of candidate R genes in maize.


## 2. Methods

### 2.1. The Maize Genome

The complete genome sequence (RefGen_v3) ofZea mays (maize inbred line B73) collected in Ensemble (http://plants.ensembl.org/Zea_mays/Info/Index) was used in the genomic analysis of encoding LRR-receptor-like genes.


### 2.2. Identification of NBS-LRR and LRR-RLK Encoding Genes

Plant Gene Family Database (PGF-DB), a database for analysis of gene families fromOryza sativa,Sorghum bicolor,Zea mays, andArabidopsis thaliana, contains more than ten thousands gene families constructed by the Markov clustering (MCL) method and the complete linkage method of BLASTP. By using keyword “NB-LRR” to search for gene family in PGF-DB (http://rapdb.dna.affrc.go.jp/) we identified one relevant gene family, “NB-ARC.” This family includes a number of maize genes that are homologues of known rice NBS-LRR genes. The obtained maize genes were used as a set of candidate NBS-LRR genes to search the maize Ensemble database. First, the complete set of NBS-encoding genes was identified in the genome ofZea mays by reiterative process of using the NBS domain from the Pfam database (PF00931; http://pfam.sanger.ac.uk/search). The threshold expectation value (E-value) was set to 10−4 which corresponds to that described in rice andArabidopsis [20, 22]. In the second step, a set of LRR-encoding genes was identified using the conserved LRR-domain from the Pfam database (PF00560, PF08263, and PF12799) and then the encoding NBS-LRR genes were identified from the above two sets of NBS- and LRR-encoding genes. Sequences found multiple times were identified by multiple sequence alignments using Clustal W2 [25] and the redundant sequences were manually removed. All of the corresponding NBS-LRR candidate proteins were surveyed to determine whether they encoded TIR or CC motif. This survey was based on the Pfam database (http://pfam.xfam.org/search) and used SMART (Simple Modular Architecture Research Tool: http://smart.embl-heidelberg.de/) protein motif analysis and COILS, a program for detecting coiled coil (CC) domains (http://www.ch.embnet.org/software/COILS_form.html). The NBS-LRR candidate protein was filtered out in sequences lacking the TIR or CC motif.Using a similar method to retrieving NBS-encoding genes as described previously, a complete set of RLK-encoding genes were retrieved from the maize Ensemble database by using the protein kinase catalytic domain (PF00069) from the Pfam database. The LRR-RLK genes were then identified using the conserved LRR-domain (PF00560, PF08263, and PF12799) from the identified RLK-encoding genes.


### 2.3. Gene Locations and Definitions of Gene Cluster and Gene Duplication

The positions of all the NBS-LRR and LRR-RLK genes on maize chromosomes were defined by Ensemble Genomes Search (http://plants.ensembl.org/Zea_mays/Info/Index) using the gene names. If two or more NBS-LRR/LRR-RLK genes resided within 200 kb, a gene cluster was defined based on Houb’s definition of a gene cluster [26]. If two of the paired genes were located in a cluster, this pair was designated as paired genes; if three or more genes were located within 200 kb, these genes were designated as multigenes. The gene-duplication events of NBS-LRR and LRR-RLK genes were also investigated in accordance with the below criteria when the encoded amino acid sequence was used as a query in BLASTP searches for possible homologues in theZea mays genome: (1) the sequence alignment covered >70% of the longer gene; (2) the aligned region had an 70% identity.


### 2.4. Alignment and Analysis of Sequence

Proteins encoded by LRR-RLK genes are crucial cell surface immune receptors for plant disease resistance. The LRR-RLK homologues in maize were found by searching the available maize nonredundant protein sequences database (taxid:4577) with BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The scoring parameters for BLASTP search were set as default: BLOSUM62 was used in the protein weight matrix, gap costs were set as 11 for existence and 1 for extension, and compositional adjustment was set as conditional compositional score matrix adjustment. The sequence with the highest score (the lowestE value and the highest identity) was categorized with LRR-RLK from other plants that had the same homology and phylogenetic grouping. Homology analysis of the maize LRR-RLK protein sequences with their homologue sequences was performed using MatGat software v2.02 [27]. The protein signature was also analyzed using the simple modular architecture research tool (SMART) [28]. Multiple sequence alignments were generated using Clustal W2 [25]. Based on a ClustalW multiple alignment phylogenetic tree, a similar one was constructed using the neighbor-joining method within the MEGA5 program [29] and bootstrapped 1,000 times.


## 2.1. The Maize Genome

The complete genome sequence (RefGen_v3) ofZea mays (maize inbred line B73) collected in Ensemble (http://plants.ensembl.org/Zea_mays/Info/Index) was used in the genomic analysis of encoding LRR-receptor-like genes.


## 2.2. Identification of NBS-LRR and LRR-RLK Encoding Genes

Plant Gene Family Database (PGF-DB), a database for analysis of gene families fromOryza sativa,Sorghum bicolor,Zea mays, andArabidopsis thaliana, contains more than ten thousands gene families constructed by the Markov clustering (MCL) method and the complete linkage method of BLASTP. By using keyword “NB-LRR” to search for gene family in PGF-DB (http://rapdb.dna.affrc.go.jp/) we identified one relevant gene family, “NB-ARC.” This family includes a number of maize genes that are homologues of known rice NBS-LRR genes. The obtained maize genes were used as a set of candidate NBS-LRR genes to search the maize Ensemble database. First, the complete set of NBS-encoding genes was identified in the genome ofZea mays by reiterative process of using the NBS domain from the Pfam database (PF00931; http://pfam.sanger.ac.uk/search). The threshold expectation value (E-value) was set to 10−4 which corresponds to that described in rice andArabidopsis [20, 22]. In the second step, a set of LRR-encoding genes was identified using the conserved LRR-domain from the Pfam database (PF00560, PF08263, and PF12799) and then the encoding NBS-LRR genes were identified from the above two sets of NBS- and LRR-encoding genes. Sequences found multiple times were identified by multiple sequence alignments using Clustal W2 [25] and the redundant sequences were manually removed. All of the corresponding NBS-LRR candidate proteins were surveyed to determine whether they encoded TIR or CC motif. This survey was based on the Pfam database (http://pfam.xfam.org/search) and used SMART (Simple Modular Architecture Research Tool: http://smart.embl-heidelberg.de/) protein motif analysis and COILS, a program for detecting coiled coil (CC) domains (http://www.ch.embnet.org/software/COILS_form.html). The NBS-LRR candidate protein was filtered out in sequences lacking the TIR or CC motif.Using a similar method to retrieving NBS-encoding genes as described previously, a complete set of RLK-encoding genes were retrieved from the maize Ensemble database by using the protein kinase catalytic domain (PF00069) from the Pfam database. The LRR-RLK genes were then identified using the conserved LRR-domain (PF00560, PF08263, and PF12799) from the identified RLK-encoding genes.


## 2.3. Gene Locations and Definitions of Gene Cluster and Gene Duplication

The positions of all the NBS-LRR and LRR-RLK genes on maize chromosomes were defined by Ensemble Genomes Search (http://plants.ensembl.org/Zea_mays/Info/Index) using the gene names. If two or more NBS-LRR/LRR-RLK genes resided within 200 kb, a gene cluster was defined based on Houb’s definition of a gene cluster [26]. If two of the paired genes were located in a cluster, this pair was designated as paired genes; if three or more genes were located within 200 kb, these genes were designated as multigenes. The gene-duplication events of NBS-LRR and LRR-RLK genes were also investigated in accordance with the below criteria when the encoded amino acid sequence was used as a query in BLASTP searches for possible homologues in theZea mays genome: (1) the sequence alignment covered >70% of the longer gene; (2) the aligned region had an 70% identity.


## 2.4. Alignment and Analysis of Sequence

Proteins encoded by LRR-RLK genes are crucial cell surface immune receptors for plant disease resistance. The LRR-RLK homologues in maize were found by searching the available maize nonredundant protein sequences database (taxid:4577) with BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The scoring parameters for BLASTP search were set as default: BLOSUM62 was used in the protein weight matrix, gap costs were set as 11 for existence and 1 for extension, and compositional adjustment was set as conditional compositional score matrix adjustment. The sequence with the highest score (the lowestE value and the highest identity) was categorized with LRR-RLK from other plants that had the same homology and phylogenetic grouping. Homology analysis of the maize LRR-RLK protein sequences with their homologue sequences was performed using MatGat software v2.02 [27]. The protein signature was also analyzed using the simple modular architecture research tool (SMART) [28]. Multiple sequence alignments were generated using Clustal W2 [25]. Based on a ClustalW multiple alignment phylogenetic tree, a similar one was constructed using the neighbor-joining method within the MEGA5 program [29] and bootstrapped 1,000 times.


## 3. Results

### 3.1. Identification and Classification of NBS-LRR and LRR-RLK Encoding Genes

Availability of the completeZea mays genome sequence (maize inbred line B73) has made it possible for the first time to identify all the LRR-containing receptor-like genes in this plant species [24]. From the first step of NBS-filter, a total of 217 NBS-encoding genes were identified in the genomic sequence of maize inbred line B73 and collected in Ensemble. In the second step of LRR-filter, 62 of the NBS-encoding genes were subsequently found not to be LRR-encoding genes. Of the remaining genes, 151 were identified to have an NBS-LRR structure and were surveyed using Pfam, SMART, and COILS to determine whether they encoded TIR, CC motifs. 147 genes were identified to contain a CC motif, but only four genes belong to the TNLs group (see Supplementary Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/231358). The fact that most of these genes belong to CNLs in maize genome further suggests that the monocots are likely to lack the TIR genes [10, 22, 23, 30]. 64 out of 151 NBS-LRR genes were identified for the first time in this study [31] (marked in asterisk in Supplementary Table S1). The majority of these newly found genes are located on chromosomes 2, 4, and 7 (11, 18, 7, resp.). Upon initial searching of the LRR-RLK genes, 1521 protein-kinase-encoding genes were identified in the maize genome. Of these genes, 226 genes were found to be LRR-RLK protein encoding genes in a following reiterative process (Supplementary Table S2).In maize whole-genome sequences, at least 39,469 protein-coding genes have been identified. NBS-LRR genes accounted for approximately 0.38% of all the protein-coding genes in this species. The relative proportion of these genes was over three times lower than that in the rice genome (1.23%) but slightly higher than that in the sorghum genome (0.18%) [22–24, 32, 33]. Additionally, the LRR-RLK encoding genes accounted for approximately 0.57% of maize protein-coding genes, 1.5 times higher than NBS-LRR genes. Furthermore, comparing with LRR-RLK gene numbers identified in maize, sorghum (Sorghum bicolor) had similar results with 208 [34] and 211 [35] in different studies. However the numbers in rice (Oryza sativa) varied widely from different studies which are 177 [34], 309 [36], and 353 [35], respectively.


### 3.2. Chromosomal Distribution of Maize NBS-LRR and NBS-RLK Encoding Genes

NBS-LRR type R genes were identified on each of ten maize chromosomes. The genes are either located separately on each individual chromosome or in gene clusters on a single chromosome (see Section2 for detailed definition). Their distribution on chromosomes is nonrandom and uneven. For example, chromosome 9 contains only two NBS-LRR genes, while chromosomes 10, 4, and 2 contain 32, 28, and 18 NBS-LRR genes, respectively, with about 21.19% of those genes locating on chromosome 10 (Figure 1(a) and Table 1). Notably, two larger clusters have been found on chromosome 10, with one containing 14 genes and the other containing 5 genes (Figure 1(a) and Table 2). Similar to the distribution of sorghum NBS-LRR gene clusters [23], most of the maize NBS-LRR gene clusters were located at the distal end of each chromosome (Figure 1).Table 1
The distribution of LRR-receptor genes on maize chromosome.
Chromosome
NBS-LRR gene
LRR-RLK gene
1
11
31
2
22
22
3
12
24
4
28
32
5
10
21
6
10
22
7
18
23
8
6
18
9
2
20
10
32
13
Total
151
226Table 2
Comparison of duplications of genes of NB-LRR and LRR-RLK in maize genome.
Gene type
NBS-LRR
LRR-RLK
Total number of genes
151
226
Single-gene families
94
117
Single-genes
94
117
Singletons
56
100
Multigene families
16
46
Multigenes
56
110
Maximal family members
14
5
Average members per family
3.5
2.4
Paired genes
14
36
Number of gene clusters
9
0
Clustered genes
42
0
Percentage of multigenes
37.09
48.67Figure 1
Physical locations of theZea mays R genes encoding LRR: (a) physical locations of NBS-LRR genes; (b) physical locations of LRR-RLK genes. The boxes above the chromosomes (Chr.; gray bars) indicate the approximate locations of each gene. Red boxes indicate the CC-NBS-LRR genes, while green boxes indicate TIR-NBS-LRR genes. Chromosome lengths are shown in megabase pairs on the scale at top.
(a)
(b)LRR-RLK genes are also located on each individual maize chromosome. However, their distribution on chromosomes is relatively even, unlike the clustered NBS-LRR genes (Figure1(b) and Table 2). The number of LRR-RLK genes on each chromosome is between 13 (on Chr. 10) and 32 (on Chr. 4).


### 3.3. Duplications of NBS-LRR and LRR-RLK Genes

During evolution, both segmental duplication and tandem duplication have contributed to the large number of gene families in plants [37]. The gene duplications have greatly expanded the NBS gene family in both monocot and dicot lineages [20, 22, 23]. In this study, the duplication of NBS-LRR and LRR-RLK genes was confirmed by a BLASTP comparison of all the predicted maize proteins against each other. A total of 56 out of the 151 NBS-LRR genes were duplicated and they were subsequently divided into 16 multigene families (Tables 2 and 3). The maximum number of family members was 14, which cluster at the distal end of chromosome 10 (Figure 1), and the average number of family members was 3.5.Table 3
The duplicated NBS-LRR genes in maize genome.
Number
Duplicated gene
Chr. location
1
GRMZM5G896901
1: 65769722–65771244
GRMZM2G454718
5: 55977976–55979514
2
GRMZM2G443525
1: 164103203–164108542
GRMZM2G044724
3: 85672960–85676903
3
GRMZM2G076474
2: 113990307–113993559
GRMZM2G142680
10: 94979016–94982685
4
GRMZM2G065692
2: 134668301–134679003
LOC100383650
2: 134763564–134769552
GRMZM2G003755
2: 134763419–134769548
5
GRMZM2G074496
2: 225896403–225899247
AC195587.4_FGP004
6: 78774141–78783661
6
GRMZM2G070503
2: 236854869–236856413
GRMZM2G026083
7: 2515278–2517156
GRMZM2G026189
7: 2525679–2527394
LOC103631898
7: 2539874–2544540
GRMZM2G403407
7: 2563208–2564699
LOC103631902
7: 2569872–2585021
GRMZM2G060714
7: 28599482–28602195
GRMZM5G827455
7: 2582504–2583985
7
GRMZM2G094664
2: 237541842–237545769
LOC103648313
2: 237541778–237547469
8
GRMZM2G064015
3: 132410799–132412650
GRMZM2G302279
3: 132637536–132639351
9
GRMZM2G005347
4: 203407679–203411878
GRMZM2G005452
4: 203456994–203463296
GRMZM2G167049
4: 203818715–203824772
10
GRMZM2G162098
5: 55459467–55463724
GRMZM2G091672
5: 55578326–55582323
GRMZM2G091696
5: 55590775–55599910
GRMZM2G105428
5: 55809843–55813411
11
GRMZM2G306727
6: 7232528–7235241
GRMZM2G334584
6: 8100151–8105044
12
LOC103630086
6: 128972997–128982259
GRMZM2G397785
10: 97377587–97383756
13
GRMZM2G169584
8: 155079613–155081884
GRMZM2G169571
8: 155119523–155121206
14
GRMZM2G180244
10: 2052395–2058076
GRMZM2G180254
10: 2127268–2133476
GRMZM2G004412
10: 2987324–2996389
GRMZM5G819919
10: 2842742–2846127
15
AC152495.1_FGP002
10: 3283523–3287419
AC152495.1_FGP003
10: 3299975–3307226
AC152495.1_FGP010
10: 3372154–3376038
AC152495.1_FGP015
10: 3404105–3405367
AC152495.1_FGP017
10: 3441445–3444666
GRMZM5G879178
10: 3445065–3449748
GRMZM2G069382
10: 3571990–3590706
GRMZM2G083246
10: 3645995–3648135
GRMZM2G143769
10: 3687121–3688770
GRMZM2G443939
10: 3700999–3705985
GRMZM2G349565
10: 3767328–3769922
GRMZM2G003625
10: 3849433–3854134
GRMZM2G061742
10: 3907357–3911655
GRMZM2G005134
10: 3983698–3989918
16
GRMZM2G319375
2: 206507648–206508569
GRMZM2G394261
7: 158297463–158301857In contrast to NBS-LRR genes, 46 multigene families that contain 110 out of 226 LRR-RLK genes were identified in the maize genome (Tables2 and 4). Among these multigene families, the percentage in LRR-RLK genes (48.67%) was higher than that in NBS-LRR genes (37.09%), demonstrating that almost half of the LRR-RLK genes were duplicated. Furthermore, the maximal number of family members in LRR-RLK genes was lower than that in NBS-LRR genes (5 and 14, resp.; Table 2). The average number of LRR-RLK members per multigene family was 2.4 and also lower than that of NBS-LRR members per multigene family. This result revealed that LRR-RLK genes are highly diverse NBS-LRR genes within the maize genome. More interestingly, no pairs of NBS-LRR genes were found on duplicated chromosomal segments, while 36 pairs of LRR-RLK genes were found. This indicates that the expansion of LRR-RLK genes may have been created via duplication and, subsequently, diversifying selection.Table 4
The duplicated LRR-RLK genes in maize genome.
Number
Duplicated gene
Chr. location
1
GRMZM2G371137
1: 5803454–5808250
GRMZM2G172014
9: 153777111–153781389
2
GRMZM2G009818
1: 9800492–9804655
GRMZM2G438840
9: 152523091–152526601
GRMZM2G168603
5: 68680529–68684140
3
GRMZM2G153393
1: 17640006–17655596
GRMZM2G330907
9: 150263840–150275563
GRMZM2G004572
1: 141080538–141086039
4
GRMZM2G038165
1: 26120398–26128604
GRMZM2G069201
3: 185481180–185489362
GRMZM2G089461
6: 154565943–154575228
GRMZM2G174585
6: 154839078–154847341
5
GRMZM2G043584
1: 30258404–30262357
GRMZM2G141517
7: 4082077–4086056
GRMZM2G072569
1: 282595023–282599365
6
GRMZM5G886952
1: 38791249–38799875
GRMZM2G479243
9: 140610164–140617939
7
GRMZM2G084587
1: 76878185–76889886
GRMZM2G122717
4: 27843728–27845162
8
GRMZM2G093809
1: 180006011–180009342
GRMZM2G080503
3: 92276172–92279783
9
GRMZM2G461278
1: 180366146–180370208
GRMZM2G147857
3: 93326318–93329971
10
GRMZM2G428554
1: 210641732–210645615
GRMZM2G011806
4: 61742558–61747904
11
GRMZM2G001812
1: 233516803–233520836
GRMZM2G112309
5: 30237719–30241622
12
GRMZM2G137788
1: 268802186–268806287
GRMZM2G158359
5: 10425114–10429156
13
GRMZM2G114276
2: 16005190–16009431
GRMZM2G016477
10: 136139046–136143214
14
GRMZM2G021619
2: 36708164–36710640
GRMZM2G167280
10: 126603529–126608907
15
GRMZM2G073928
2: 161907176–161910550
GRMZM2G432642
7: 18436556–18440330
16
GRMZM2G163724
2: 164310419–164313687
GRMZM2G072868
7: 50383762–50386524
17
GRMZM2G002569
2: 182995589–182999190
GRMZM2G084248
7: 117574984–117578965
18
AC233861.1_FG001
2: 189738061–189741584
GRMZM2G149051
7: 130969891–130973647
GRMZM2G039431
4: 197994710–197998218
GRMZM2G131609
6: 129696721–129700755
GRMZM2G463493
8: 17060563–17064940
19
GRMZM2G172429
2: 201545881–201550118
GRMZM2G313643
7: 150075579–150080212
20
GRMZM2G162781
2: 213192905–213197565
GRMZM2G081857
7: 167142415–167147144
21
GRMZM2G169681
2: 219686483–219692413
GRMZM2G068398
4: 10223589–10229102
22
GRMZM2G349875
2: 221419815–221427366
GRMZM2G104384
4: 17311139–17323011
23
GRMZM2G012861
3: 2763089–2767869
GRMZM2G127687
8: 26863212–26867537
24
GRMZM2G010693
3: 12429859–12434725
GRMZM2G067675
8: 17304016–17309182
25
GRMZM2G463574
3: 16573593–16577181
GRMZM2G421669
8: 14848451–14851761
26
GRMZM2G138338
3: 147442361–147446195
GRMZM2G059117
8: 157002878–157006630
27
GRMZM2G447447
3: 161622655–161626810
GRMZM2G306771
5: 123289050–123293128
28
GRMZM2G145753
3: 184727722–184731545
GRMZM2G128315
6: 154358844–154362599
29
GRMZM2G465771
3: 185367968–185371929
AC218972.3_FG004
8: 170198252–170201068
AC214817.3_FG004
6: 154514150–154517066
30
GRMZM2G078926
3: 209624891–209634088
GRMZM2G059497
8: 149851364–149863364
31
GRMZM2G339540
3: 224241290–224255475
GRMZM2G050548
8: 141765271–141771284
32
GRMZM2G438007
4: 65769675–65773352
GRMZM2G092604
7: 79119386–79123848
33
GRMZM2G150024
4: 124660672–124667062
GRMZM2G115420
5: 176261637–176267226
GRMZM2G384439
10: 71477986–71484046
GRMZM5G870959
10: 121772419–121777860
34
GRMZM2G071396
4: 138248437–138252721
GRMZM2G702599
4: 139022453–139031123
GRMZM2G039665
4: 139156285–139159606
35
GRMZM2G150930
4: 165794972–165797318
GRMZM2G171114
6: 103870389–103873917
36
GRMZM2G100234
4: 226544149–226547671
GRMZM2G176206
5: 151922862–151926238
37
GRMZM2G451007
4: 237861330–237864849
GRMZM2G177570
5: 51462007–51465569
GRMZM2G474777
6: 88899974–88903683
GRMZM2G104425
5: 81113414–81119465
38
GRMZM2G322348
4: 240249146–240253001
GRMZM2G123314
8: 14843475–14846908
39
GRMZM2G126161
5: 42533252–42537728
GRMZM2G100858
6: 91599104–91604620
40
GRMZM2G463904
5: 211800909–211813877
GRMZM5G809695
6: 108369697–108377055
GRMZM2G082855
9: 24074354–24081173
41
GRMZM2G125081
6: 5162305–5165886
GRMZM2G045981
9: 146683625–146687407
42
GRMZM2G162531
6: 97773823–97791863
GRMZM2G071573
9: 98256956–98262572
43
GRMZM2G449817
6: 104839663–104842894
GRMZM2G350918
9: 74480747–74484663
44
GRMZM2G349665
6: 116081434–116084931
GRMZM2G145720
9: 35179613–35185362
GRMZM2G019317
5: 204644182–204648555
45
GRMZM2G149201
6: 130782221–130784726
GRMZM2G151738
8: 89697764–89700024
46
GRMZM2G046729
9: 146340642–146344213
AC233893.1_FG006
9: 146407378–146411588
47
GRMZM2G009770
9: 152025490–152029185
GRMZM5G839644
10: 6940302–6943942


### 3.4. Molecular Characterization and Phylogenetic Analysis of Maize LRR-RLKs

To identify the homologues of the LRR-RLKs in maize, we used known plant LRR-RLK protein sequences to search the maize protein database. Out of the 226 identified LRR-RLK genes, six homologues were found, including FLS2 (GRMZM2G080041), Cf5 (GRMZM2G107872), PSY1R (GRMZM2G177570), PSKR1 (GRMZM2G080537), BIR1 (GRMZM2G121565), and OsSERK1 (GRMZM2G150024). All the predicted amino acid sequences of these genes contain a signal peptide, extracellular LRR domains, a single-pass transmembrane domain (TM), and an intracellular kinase domain except for Cf5 (Figure2). Maize FLS2, OsSERK1, PSY1R, and PSKR1 are typically LRR-serine/threonine protein kinases, while BIR1 is dual-specificity serine/threonine/tyrosine kinase but Cf5 is a transmembrane LRR-receptor-like protein. At the amino acid level, the identified maize LRR-RLK/RLPs shared the highest identity (or similarity) within family members (Table 5). Meanwhile, maize LRR-RLK/RLP proteins share a higher identity (or similarity) to their monocots homologues than dicots homologues.Table 5
Amino acid identities and similarities of LRR-RLK homologues from different plantsa.
Gene
Organism
Identity (%)
Similarity (%)
FLS2
Zea mays
100.0
—
Sorghum bicolor
87.6
92.4
Oryza sativa Indica
68.8
80.9
Oryza sativa Japonica
68.5
80.7
Triticum aestivum
70.7
81.0
Triticum urartu
46.0
53.4
Brachypodium distachyon
56.1
67.3
Nicotiana sylvestris
47.3
67.1
Arabidopsis thaliana
44.4
66.0
Cf5
Zea mays
100.0
—
Sorghum bicolor
98.2
98.8
Oryza sativa Japonica
91.1
95.5
Setaria italica
94.5
97.3
Brachypodium distachyon
90.0
96.7
Arabidopsis thaliana
73.6
83.9
OsSERK1
Zea mays
100.0
Oryza sativa Indica
87.0
91.2
Vitis vinifera
83.9
88.9
Solanum tuberosum
83.5
89.0
PSKR1
Zea mays
100.0
Sorghum bicolor
89.0
94.4
Oryza sativa Japonica
77.5
87.5
Morus notabilis
53.1
71.2
Brassica rapa
47.8
65.4
Vitis vinifera
53.0
71.3
Arabidopsis thaliana
50.7
70.5
PSY1R
Zea mays
100.0
Sorghum bicolor
91.5
94.9
Setaria italica
81.9
90.0
Oryza sativa Indica
62.9
77.1
Brassica rapa
46.3
64.3
Glycine max
48.3
66.5
Nicotiana sylvestris
47.2
66.1
Arabidopsis thaliana
46.6
65.1
BIR1
Zea mays
100.0
Sorghum bicolor
65.5
82.3
Arabidopsis thaliana
57.8
74.7
aThe accession numbers of the sequences used for the homology analysis are listed in Figure 3.Figure 2
Schematic diagram of the protein features of the identified LRR-RLKs. This diagram was constructed using the predicted amino acid (aa) sequences from retrieved proteins and SMART online program. The size of each protein is presented with a grey line. The predicted domains are highlighted in each protein. The signal peptides are shown as red boxes, while the low complexity regions and transmembrane regions are shown as pink boxes and blue boxes, respectively. LRRNT_2: leucine rich repeats domains that consist of 2–45 motifs of 20–30 amino acids in length that generally fold into a horseshoe shape. LRR: leucine rich repeat domain; S_TKc: serine/threonine protein kinases, catalytic domain; STYKc: dual-specificity serine/threonine/tyrosine kinase domain.In order to elucidate the relationships among the maize LRR-RLK/RLP genes with their homologues in other plants, the encoded amino acid sequences of LRR-RLK/RLP genes (see Supplementary Material 2) were used to construct a neighbor-joining phylogenetic tree with the multiple sequence alignments (Figure3). The phylogenetic tree showed that all of the six maize LRR-RLK/RLP protein sequences were grouped with their respective homologues, confirming their phylogenetic relationships. The tree also indicated that there were two distinct clades: one clade includes PSY1R, PSKR1, BIR1, and OsSERK1 which are typical LRR-RLKs and the other includes FLS2 and Cf5, both known as PRRs in dicots as they contain a more similar LRR domain. This is supported by the fact that these maize LRR-RLK/RLPs are grouped with their homologues from other plant species. It can be concluded that the maize LRR-RLK/RLPs are more close to that from monocots than dicots members.Figure 3
Unrooted phylogenetic tree of the seven identified LRR-RLKs showing the relationship with their homologues in other plants. This tree was constructed from a ClustalW generated multiple sequence alignment of amino acid sequences using the neighbor-joining method in MEGA5. Node values indicate percentage bootstrap values derived from 10,000 replications. The maize LRR-RLKs reported in this study are shaded in grey. The accession numbers of the sequences used for the phylogenetic analysis are as follows:Zea mays PSY1R [XP 008644981], PSKR1 [XP 008645956], BIR1 [NP 001147794], OsSERK1 [XP 008678722], Cf5 [NP 001132758], and FLS2 [XP 008668880];Sorghum bicolor PSY1R [XP 002437497], PSKR1 [XP 002454207], BIR1 [XP 002450551], Cf5 [XP 002465654], and FLS2 [Sb06g028760];Setaria italica PSY1R [XP 004966176] and Cf5 [XP 004985227];Oryza sativa Indica Group PSY1R [AAU12600] and FLS2 [CAH68341];Oryza sativa Japonica Group PSKR1 [BAD23737], OsSERK1 [AAU88198], Cf5 [NP 001049352], and FSL2 [CAE02151];Brachypodium distachyon Cf5 [XP 003558509] and FLS2 [BRADI5G21960];Triticum aestivum FLS2 [6E8764762];Triticum urartu FLS2 [EMS63184];Arabidopsis thaliana PSY1R [NP 177374], PSKR1 [NP 178330], BIR1 [NP 568696], Cf5 [NP 200932], and FLS2 [NP 199445];Brassica rapa PSY1R [XP 009105890] and PSKR1 [XP 009129163];Glycine max PSY1R [XP 003520891];Nicotiana sylvestris PSY1R [XP 009759322] and FLS2 [XP 009801818];Morus notabilis PSKR1 [EXC37937];Vitis vinifera PSKR1 [XP 002273186] and OsSERK1 [XP 002270847];Solanum tuberosum OsSERK1 [NP 001275293].


## 3.1. Identification and Classification of NBS-LRR and LRR-RLK Encoding Genes

Availability of the completeZea mays genome sequence (maize inbred line B73) has made it possible for the first time to identify all the LRR-containing receptor-like genes in this plant species [24]. From the first step of NBS-filter, a total of 217 NBS-encoding genes were identified in the genomic sequence of maize inbred line B73 and collected in Ensemble. In the second step of LRR-filter, 62 of the NBS-encoding genes were subsequently found not to be LRR-encoding genes. Of the remaining genes, 151 were identified to have an NBS-LRR structure and were surveyed using Pfam, SMART, and COILS to determine whether they encoded TIR, CC motifs. 147 genes were identified to contain a CC motif, but only four genes belong to the TNLs group (see Supplementary Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/231358). The fact that most of these genes belong to CNLs in maize genome further suggests that the monocots are likely to lack the TIR genes [10, 22, 23, 30]. 64 out of 151 NBS-LRR genes were identified for the first time in this study [31] (marked in asterisk in Supplementary Table S1). The majority of these newly found genes are located on chromosomes 2, 4, and 7 (11, 18, 7, resp.). Upon initial searching of the LRR-RLK genes, 1521 protein-kinase-encoding genes were identified in the maize genome. Of these genes, 226 genes were found to be LRR-RLK protein encoding genes in a following reiterative process (Supplementary Table S2).In maize whole-genome sequences, at least 39,469 protein-coding genes have been identified. NBS-LRR genes accounted for approximately 0.38% of all the protein-coding genes in this species. The relative proportion of these genes was over three times lower than that in the rice genome (1.23%) but slightly higher than that in the sorghum genome (0.18%) [22–24, 32, 33]. Additionally, the LRR-RLK encoding genes accounted for approximately 0.57% of maize protein-coding genes, 1.5 times higher than NBS-LRR genes. Furthermore, comparing with LRR-RLK gene numbers identified in maize, sorghum (Sorghum bicolor) had similar results with 208 [34] and 211 [35] in different studies. However the numbers in rice (Oryza sativa) varied widely from different studies which are 177 [34], 309 [36], and 353 [35], respectively.


## 3.2. Chromosomal Distribution of Maize NBS-LRR and NBS-RLK Encoding Genes

NBS-LRR type R genes were identified on each of ten maize chromosomes. The genes are either located separately on each individual chromosome or in gene clusters on a single chromosome (see Section2 for detailed definition). Their distribution on chromosomes is nonrandom and uneven. For example, chromosome 9 contains only two NBS-LRR genes, while chromosomes 10, 4, and 2 contain 32, 28, and 18 NBS-LRR genes, respectively, with about 21.19% of those genes locating on chromosome 10 (Figure 1(a) and Table 1). Notably, two larger clusters have been found on chromosome 10, with one containing 14 genes and the other containing 5 genes (Figure 1(a) and Table 2). Similar to the distribution of sorghum NBS-LRR gene clusters [23], most of the maize NBS-LRR gene clusters were located at the distal end of each chromosome (Figure 1).Table 1
The distribution of LRR-receptor genes on maize chromosome.
Chromosome
NBS-LRR gene
LRR-RLK gene
1
11
31
2
22
22
3
12
24
4
28
32
5
10
21
6
10
22
7
18
23
8
6
18
9
2
20
10
32
13
Total
151
226Table 2
Comparison of duplications of genes of NB-LRR and LRR-RLK in maize genome.
Gene type
NBS-LRR
LRR-RLK
Total number of genes
151
226
Single-gene families
94
117
Single-genes
94
117
Singletons
56
100
Multigene families
16
46
Multigenes
56
110
Maximal family members
14
5
Average members per family
3.5
2.4
Paired genes
14
36
Number of gene clusters
9
0
Clustered genes
42
0
Percentage of multigenes
37.09
48.67Figure 1
Physical locations of theZea mays R genes encoding LRR: (a) physical locations of NBS-LRR genes; (b) physical locations of LRR-RLK genes. The boxes above the chromosomes (Chr.; gray bars) indicate the approximate locations of each gene. Red boxes indicate the CC-NBS-LRR genes, while green boxes indicate TIR-NBS-LRR genes. Chromosome lengths are shown in megabase pairs on the scale at top.
(a)
(b)LRR-RLK genes are also located on each individual maize chromosome. However, their distribution on chromosomes is relatively even, unlike the clustered NBS-LRR genes (Figure1(b) and Table 2). The number of LRR-RLK genes on each chromosome is between 13 (on Chr. 10) and 32 (on Chr. 4).


## 3.3. Duplications of NBS-LRR and LRR-RLK Genes

During evolution, both segmental duplication and tandem duplication have contributed to the large number of gene families in plants [37]. The gene duplications have greatly expanded the NBS gene family in both monocot and dicot lineages [20, 22, 23]. In this study, the duplication of NBS-LRR and LRR-RLK genes was confirmed by a BLASTP comparison of all the predicted maize proteins against each other. A total of 56 out of the 151 NBS-LRR genes were duplicated and they were subsequently divided into 16 multigene families (Tables 2 and 3). The maximum number of family members was 14, which cluster at the distal end of chromosome 10 (Figure 1), and the average number of family members was 3.5.Table 3
The duplicated NBS-LRR genes in maize genome.
Number
Duplicated gene
Chr. location
1
GRMZM5G896901
1: 65769722–65771244
GRMZM2G454718
5: 55977976–55979514
2
GRMZM2G443525
1: 164103203–164108542
GRMZM2G044724
3: 85672960–85676903
3
GRMZM2G076474
2: 113990307–113993559
GRMZM2G142680
10: 94979016–94982685
4
GRMZM2G065692
2: 134668301–134679003
LOC100383650
2: 134763564–134769552
GRMZM2G003755
2: 134763419–134769548
5
GRMZM2G074496
2: 225896403–225899247
AC195587.4_FGP004
6: 78774141–78783661
6
GRMZM2G070503
2: 236854869–236856413
GRMZM2G026083
7: 2515278–2517156
GRMZM2G026189
7: 2525679–2527394
LOC103631898
7: 2539874–2544540
GRMZM2G403407
7: 2563208–2564699
LOC103631902
7: 2569872–2585021
GRMZM2G060714
7: 28599482–28602195
GRMZM5G827455
7: 2582504–2583985
7
GRMZM2G094664
2: 237541842–237545769
LOC103648313
2: 237541778–237547469
8
GRMZM2G064015
3: 132410799–132412650
GRMZM2G302279
3: 132637536–132639351
9
GRMZM2G005347
4: 203407679–203411878
GRMZM2G005452
4: 203456994–203463296
GRMZM2G167049
4: 203818715–203824772
10
GRMZM2G162098
5: 55459467–55463724
GRMZM2G091672
5: 55578326–55582323
GRMZM2G091696
5: 55590775–55599910
GRMZM2G105428
5: 55809843–55813411
11
GRMZM2G306727
6: 7232528–7235241
GRMZM2G334584
6: 8100151–8105044
12
LOC103630086
6: 128972997–128982259
GRMZM2G397785
10: 97377587–97383756
13
GRMZM2G169584
8: 155079613–155081884
GRMZM2G169571
8: 155119523–155121206
14
GRMZM2G180244
10: 2052395–2058076
GRMZM2G180254
10: 2127268–2133476
GRMZM2G004412
10: 2987324–2996389
GRMZM5G819919
10: 2842742–2846127
15
AC152495.1_FGP002
10: 3283523–3287419
AC152495.1_FGP003
10: 3299975–3307226
AC152495.1_FGP010
10: 3372154–3376038
AC152495.1_FGP015
10: 3404105–3405367
AC152495.1_FGP017
10: 3441445–3444666
GRMZM5G879178
10: 3445065–3449748
GRMZM2G069382
10: 3571990–3590706
GRMZM2G083246
10: 3645995–3648135
GRMZM2G143769
10: 3687121–3688770
GRMZM2G443939
10: 3700999–3705985
GRMZM2G349565
10: 3767328–3769922
GRMZM2G003625
10: 3849433–3854134
GRMZM2G061742
10: 3907357–3911655
GRMZM2G005134
10: 3983698–3989918
16
GRMZM2G319375
2: 206507648–206508569
GRMZM2G394261
7: 158297463–158301857In contrast to NBS-LRR genes, 46 multigene families that contain 110 out of 226 LRR-RLK genes were identified in the maize genome (Tables2 and 4). Among these multigene families, the percentage in LRR-RLK genes (48.67%) was higher than that in NBS-LRR genes (37.09%), demonstrating that almost half of the LRR-RLK genes were duplicated. Furthermore, the maximal number of family members in LRR-RLK genes was lower than that in NBS-LRR genes (5 and 14, resp.; Table 2). The average number of LRR-RLK members per multigene family was 2.4 and also lower than that of NBS-LRR members per multigene family. This result revealed that LRR-RLK genes are highly diverse NBS-LRR genes within the maize genome. More interestingly, no pairs of NBS-LRR genes were found on duplicated chromosomal segments, while 36 pairs of LRR-RLK genes were found. This indicates that the expansion of LRR-RLK genes may have been created via duplication and, subsequently, diversifying selection.Table 4
The duplicated LRR-RLK genes in maize genome.
Number
Duplicated gene
Chr. location
1
GRMZM2G371137
1: 5803454–5808250
GRMZM2G172014
9: 153777111–153781389
2
GRMZM2G009818
1: 9800492–9804655
GRMZM2G438840
9: 152523091–152526601
GRMZM2G168603
5: 68680529–68684140
3
GRMZM2G153393
1: 17640006–17655596
GRMZM2G330907
9: 150263840–150275563
GRMZM2G004572
1: 141080538–141086039
4
GRMZM2G038165
1: 26120398–26128604
GRMZM2G069201
3: 185481180–185489362
GRMZM2G089461
6: 154565943–154575228
GRMZM2G174585
6: 154839078–154847341
5
GRMZM2G043584
1: 30258404–30262357
GRMZM2G141517
7: 4082077–4086056
GRMZM2G072569
1: 282595023–282599365
6
GRMZM5G886952
1: 38791249–38799875
GRMZM2G479243
9: 140610164–140617939
7
GRMZM2G084587
1: 76878185–76889886
GRMZM2G122717
4: 27843728–27845162
8
GRMZM2G093809
1: 180006011–180009342
GRMZM2G080503
3: 92276172–92279783
9
GRMZM2G461278
1: 180366146–180370208
GRMZM2G147857
3: 93326318–93329971
10
GRMZM2G428554
1: 210641732–210645615
GRMZM2G011806
4: 61742558–61747904
11
GRMZM2G001812
1: 233516803–233520836
GRMZM2G112309
5: 30237719–30241622
12
GRMZM2G137788
1: 268802186–268806287
GRMZM2G158359
5: 10425114–10429156
13
GRMZM2G114276
2: 16005190–16009431
GRMZM2G016477
10: 136139046–136143214
14
GRMZM2G021619
2: 36708164–36710640
GRMZM2G167280
10: 126603529–126608907
15
GRMZM2G073928
2: 161907176–161910550
GRMZM2G432642
7: 18436556–18440330
16
GRMZM2G163724
2: 164310419–164313687
GRMZM2G072868
7: 50383762–50386524
17
GRMZM2G002569
2: 182995589–182999190
GRMZM2G084248
7: 117574984–117578965
18
AC233861.1_FG001
2: 189738061–189741584
GRMZM2G149051
7: 130969891–130973647
GRMZM2G039431
4: 197994710–197998218
GRMZM2G131609
6: 129696721–129700755
GRMZM2G463493
8: 17060563–17064940
19
GRMZM2G172429
2: 201545881–201550118
GRMZM2G313643
7: 150075579–150080212
20
GRMZM2G162781
2: 213192905–213197565
GRMZM2G081857
7: 167142415–167147144
21
GRMZM2G169681
2: 219686483–219692413
GRMZM2G068398
4: 10223589–10229102
22
GRMZM2G349875
2: 221419815–221427366
GRMZM2G104384
4: 17311139–17323011
23
GRMZM2G012861
3: 2763089–2767869
GRMZM2G127687
8: 26863212–26867537
24
GRMZM2G010693
3: 12429859–12434725
GRMZM2G067675
8: 17304016–17309182
25
GRMZM2G463574
3: 16573593–16577181
GRMZM2G421669
8: 14848451–14851761
26
GRMZM2G138338
3: 147442361–147446195
GRMZM2G059117
8: 157002878–157006630
27
GRMZM2G447447
3: 161622655–161626810
GRMZM2G306771
5: 123289050–123293128
28
GRMZM2G145753
3: 184727722–184731545
GRMZM2G128315
6: 154358844–154362599
29
GRMZM2G465771
3: 185367968–185371929
AC218972.3_FG004
8: 170198252–170201068
AC214817.3_FG004
6: 154514150–154517066
30
GRMZM2G078926
3: 209624891–209634088
GRMZM2G059497
8: 149851364–149863364
31
GRMZM2G339540
3: 224241290–224255475
GRMZM2G050548
8: 141765271–141771284
32
GRMZM2G438007
4: 65769675–65773352
GRMZM2G092604
7: 79119386–79123848
33
GRMZM2G150024
4: 124660672–124667062
GRMZM2G115420
5: 176261637–176267226
GRMZM2G384439
10: 71477986–71484046
GRMZM5G870959
10: 121772419–121777860
34
GRMZM2G071396
4: 138248437–138252721
GRMZM2G702599
4: 139022453–139031123
GRMZM2G039665
4: 139156285–139159606
35
GRMZM2G150930
4: 165794972–165797318
GRMZM2G171114
6: 103870389–103873917
36
GRMZM2G100234
4: 226544149–226547671
GRMZM2G176206
5: 151922862–151926238
37
GRMZM2G451007
4: 237861330–237864849
GRMZM2G177570
5: 51462007–51465569
GRMZM2G474777
6: 88899974–88903683
GRMZM2G104425
5: 81113414–81119465
38
GRMZM2G322348
4: 240249146–240253001
GRMZM2G123314
8: 14843475–14846908
39
GRMZM2G126161
5: 42533252–42537728
GRMZM2G100858
6: 91599104–91604620
40
GRMZM2G463904
5: 211800909–211813877
GRMZM5G809695
6: 108369697–108377055
GRMZM2G082855
9: 24074354–24081173
41
GRMZM2G125081
6: 5162305–5165886
GRMZM2G045981
9: 146683625–146687407
42
GRMZM2G162531
6: 97773823–97791863
GRMZM2G071573
9: 98256956–98262572
43
GRMZM2G449817
6: 104839663–104842894
GRMZM2G350918
9: 74480747–74484663
44
GRMZM2G349665
6: 116081434–116084931
GRMZM2G145720
9: 35179613–35185362
GRMZM2G019317
5: 204644182–204648555
45
GRMZM2G149201
6: 130782221–130784726
GRMZM2G151738
8: 89697764–89700024
46
GRMZM2G046729
9: 146340642–146344213
AC233893.1_FG006
9: 146407378–146411588
47
GRMZM2G009770
9: 152025490–152029185
GRMZM5G839644
10: 6940302–6943942


## 3.4. Molecular Characterization and Phylogenetic Analysis of Maize LRR-RLKs

To identify the homologues of the LRR-RLKs in maize, we used known plant LRR-RLK protein sequences to search the maize protein database. Out of the 226 identified LRR-RLK genes, six homologues were found, including FLS2 (GRMZM2G080041), Cf5 (GRMZM2G107872), PSY1R (GRMZM2G177570), PSKR1 (GRMZM2G080537), BIR1 (GRMZM2G121565), and OsSERK1 (GRMZM2G150024). All the predicted amino acid sequences of these genes contain a signal peptide, extracellular LRR domains, a single-pass transmembrane domain (TM), and an intracellular kinase domain except for Cf5 (Figure2). Maize FLS2, OsSERK1, PSY1R, and PSKR1 are typically LRR-serine/threonine protein kinases, while BIR1 is dual-specificity serine/threonine/tyrosine kinase but Cf5 is a transmembrane LRR-receptor-like protein. At the amino acid level, the identified maize LRR-RLK/RLPs shared the highest identity (or similarity) within family members (Table 5). Meanwhile, maize LRR-RLK/RLP proteins share a higher identity (or similarity) to their monocots homologues than dicots homologues.Table 5
Amino acid identities and similarities of LRR-RLK homologues from different plantsa.
Gene
Organism
Identity (%)
Similarity (%)
FLS2
Zea mays
100.0
—
Sorghum bicolor
87.6
92.4
Oryza sativa Indica
68.8
80.9
Oryza sativa Japonica
68.5
80.7
Triticum aestivum
70.7
81.0
Triticum urartu
46.0
53.4
Brachypodium distachyon
56.1
67.3
Nicotiana sylvestris
47.3
67.1
Arabidopsis thaliana
44.4
66.0
Cf5
Zea mays
100.0
—
Sorghum bicolor
98.2
98.8
Oryza sativa Japonica
91.1
95.5
Setaria italica
94.5
97.3
Brachypodium distachyon
90.0
96.7
Arabidopsis thaliana
73.6
83.9
OsSERK1
Zea mays
100.0
Oryza sativa Indica
87.0
91.2
Vitis vinifera
83.9
88.9
Solanum tuberosum
83.5
89.0
PSKR1
Zea mays
100.0
Sorghum bicolor
89.0
94.4
Oryza sativa Japonica
77.5
87.5
Morus notabilis
53.1
71.2
Brassica rapa
47.8
65.4
Vitis vinifera
53.0
71.3
Arabidopsis thaliana
50.7
70.5
PSY1R
Zea mays
100.0
Sorghum bicolor
91.5
94.9
Setaria italica
81.9
90.0
Oryza sativa Indica
62.9
77.1
Brassica rapa
46.3
64.3
Glycine max
48.3
66.5
Nicotiana sylvestris
47.2
66.1
Arabidopsis thaliana
46.6
65.1
BIR1
Zea mays
100.0
Sorghum bicolor
65.5
82.3
Arabidopsis thaliana
57.8
74.7
aThe accession numbers of the sequences used for the homology analysis are listed in Figure 3.Figure 2
Schematic diagram of the protein features of the identified LRR-RLKs. This diagram was constructed using the predicted amino acid (aa) sequences from retrieved proteins and SMART online program. The size of each protein is presented with a grey line. The predicted domains are highlighted in each protein. The signal peptides are shown as red boxes, while the low complexity regions and transmembrane regions are shown as pink boxes and blue boxes, respectively. LRRNT_2: leucine rich repeats domains that consist of 2–45 motifs of 20–30 amino acids in length that generally fold into a horseshoe shape. LRR: leucine rich repeat domain; S_TKc: serine/threonine protein kinases, catalytic domain; STYKc: dual-specificity serine/threonine/tyrosine kinase domain.In order to elucidate the relationships among the maize LRR-RLK/RLP genes with their homologues in other plants, the encoded amino acid sequences of LRR-RLK/RLP genes (see Supplementary Material 2) were used to construct a neighbor-joining phylogenetic tree with the multiple sequence alignments (Figure3). The phylogenetic tree showed that all of the six maize LRR-RLK/RLP protein sequences were grouped with their respective homologues, confirming their phylogenetic relationships. The tree also indicated that there were two distinct clades: one clade includes PSY1R, PSKR1, BIR1, and OsSERK1 which are typical LRR-RLKs and the other includes FLS2 and Cf5, both known as PRRs in dicots as they contain a more similar LRR domain. This is supported by the fact that these maize LRR-RLK/RLPs are grouped with their homologues from other plant species. It can be concluded that the maize LRR-RLK/RLPs are more close to that from monocots than dicots members.Figure 3
Unrooted phylogenetic tree of the seven identified LRR-RLKs showing the relationship with their homologues in other plants. This tree was constructed from a ClustalW generated multiple sequence alignment of amino acid sequences using the neighbor-joining method in MEGA5. Node values indicate percentage bootstrap values derived from 10,000 replications. The maize LRR-RLKs reported in this study are shaded in grey. The accession numbers of the sequences used for the phylogenetic analysis are as follows:Zea mays PSY1R [XP 008644981], PSKR1 [XP 008645956], BIR1 [NP 001147794], OsSERK1 [XP 008678722], Cf5 [NP 001132758], and FLS2 [XP 008668880];Sorghum bicolor PSY1R [XP 002437497], PSKR1 [XP 002454207], BIR1 [XP 002450551], Cf5 [XP 002465654], and FLS2 [Sb06g028760];Setaria italica PSY1R [XP 004966176] and Cf5 [XP 004985227];Oryza sativa Indica Group PSY1R [AAU12600] and FLS2 [CAH68341];Oryza sativa Japonica Group PSKR1 [BAD23737], OsSERK1 [AAU88198], Cf5 [NP 001049352], and FSL2 [CAE02151];Brachypodium distachyon Cf5 [XP 003558509] and FLS2 [BRADI5G21960];Triticum aestivum FLS2 [6E8764762];Triticum urartu FLS2 [EMS63184];Arabidopsis thaliana PSY1R [NP 177374], PSKR1 [NP 178330], BIR1 [NP 568696], Cf5 [NP 200932], and FLS2 [NP 199445];Brassica rapa PSY1R [XP 009105890] and PSKR1 [XP 009129163];Glycine max PSY1R [XP 003520891];Nicotiana sylvestris PSY1R [XP 009759322] and FLS2 [XP 009801818];Morus notabilis PSKR1 [EXC37937];Vitis vinifera PSKR1 [XP 002273186] and OsSERK1 [XP 002270847];Solanum tuberosum OsSERK1 [NP 001275293].


## 4. Discussions

A genomic analysis of the disease-resistance genes encoding NBS has been extensively investigated in many plant species. However no TIR-NBS-LRR genes were reported in maize and the LRR-RLK genes were rarely characterized, although the first plant RLK was identified in maize twenty years ago [38]. In the present study, we discovered for the first time four TIR-NBS-LRR genes out of 151 NBS-LRR genes and 226 LRR-RLK genes from the maize whole-genomic sequence database and further characterized six proteins encoded by LRR-RLK/RLP genes. The putative LRR domain in these proteins is likely to act as a ligand-binding domain to recognize pathogens as part of the maize immune system.The maize genome sequence data employed in the present study is more accurate than in previous studies as gaps were also sequenced, which enabled us to estimate the actual number of NBS-LRR encoding genes. Thus, additional 64 NBS-LRR genes in maize were identified compared to 109 [31] or 95 [39] NBS-LRR genes in previous investigations. It is interesting that the newly found genes on chromosome 7 are in clusters or pairs, indicating that the local tandem duplications are possibly responsible for gene expansion. 226 LRR-RLK genes were discovered in maize in this study, which is in line with the number ofSorghum bicolor (211) andArabidopsis thaliana (213), but much less than that ofOryza sativa (353) [35].Previous studies have shown that rice diverged from the progenitors of maize and sorghum about 60 million years ago, whereas maize and sorghum evolved from a common ancestor about 25 million years ago [40]. The NBS-LRR genes are much less frequent in maize than in rice or sorghum, in which 480 and 211 NBS-LRR genes were identified, respectively [22, 23]. This suggests that loss of NBS-LRR genes in maize and sorghum was rapid after the split with rice especially in maize. The distribution of the number of NBS-LRR genes on each chromosome confirmed previous findings, although the total number found was different [31]. For example, chromosome 10 contains the greatest number of NBS-LRR genes, whereas chromosome 9 has the least. Additionally, NBS-LRR genes are most abundant on maize chromosome 10, and their homologues are located in chromosome 11 in rice and chromosome 8 in sorghum [33, 41]. Clustering of NBS-LRR genes in the genomic regions in various species suggests that there are chromosomal hot spots in which the NBS-LRR genes are duplicated. Additionally, 14 NBS-LRR genes are located in maize chromosome 10, suggesting that these genes originated by tandem duplications and subsequently evolved under selective pressure in this region. Maize NBS-LRR genes mostly encode the CC-type of N-terminal domains. Only four maize TIR-encoding genes (GRMZM2G319375, GRMZM2G402165, GRMZM2G132403, and GRMZM2G302279) were identified. Similar findings were also reported in other monocots, such as rice (only one TIR-encoding gene identified), sorghum (two TIR-encoding genes), andBrachypodium distachyon (none identified) [10, 22, 33]. In contrast, the TIR encoding genes have extensively expanded in dicots, with 98 found in Arabidopsis and 78 in poplar [20, 30]. The CC-type NBS-LRR genes from dicots and monocots tend to cluster together on chromosomes suggesting that the CC-type NBS-LRR genes originated before the divergence of the monocots and dicots [20, 22, 23]. Additionally, TIR-type genes are likely to have been lost from the grass species rather than having arisen from plant evolution after the monocot/dicot separation as there are less TIR-type genes in monocots than dicots, although the reason remains unclear [42, 43].In maize, 27.8% of the NBS-LRR genes are located in gene clusters, a much lower proportion than in sorghum at 97% [23]. It has also been reported that the highest proportion of clustered NBS-LRR genes has been found in Arabidopsis and rice [20, 22]. Moreover, there is potentially less duplication and fewer multigene families in maize than sorghum and rice. This peculiar distribution in maize might be caused by more dispersed-recombination than duplication as the maize genome is approximately 3 times bigger than the sorghum genome [33]. In contrast, the LRR-RLK genes are not distributed in clusters but are scattered throughout the maize genome. Nearly 50% of LRR-RLK genes have been duplicated, indicating that a whole-genome wide duplication event could result in the expansion of LRR-RLK genes in maize. The difference genome location and copy number between NBS-LRR genes and LRR-RLK genes suggest that they may have evolved from two different ancestors and have different roles in acting as receptors in plant immune systems. Additionally, the genome-wide distribution of maize NBS-LRR and LRR-RLK genes indicates that a large number of different loci are related to the immune system and that the maize resistance system is very complex.The number of LRR-RLK genes found in Arabidopsis was similar to that in maize. More than 400 genes were identified with RLK configurations that can be classified into at least 21 structural classes based on their extracellular domains, in which the LRR-RLKs represented the largest group consisting of 216 members [19]. The presence of LRR-RLK genes in both dicot and monocot suggests that the size of this gene family may have been similar to the present-day level before the diversification of the land plant lineages.The plant RLKs have been functionally characterised in some studies and are implicated to be involved in a diverse range of signalling processes, including brassinosteroid signalling via brassinosteroid insensitive 1 (BRI1) [44], recognition of flagellin by FLS2 [15], and bacterial resistance mediated by Xa21 [11]. So far, none of 226 LRR-RLKs in maize have been functionally studied. The LRR-RLK genes identified in this paper will be invaluable for gene function analysis. In addition, six maize LRR-RLK/RLPs were found to contain an extracellular ligand-binding domain (LRR domain), a single membrane-spanning domain, and a C-terminal intracellular protein kinase domain (tyrosine or serine/threonine rich region) and thus it is likely that they have the same function as their homologues in other plant species. For example, FLS2 functions as an immune receptor sensing the bacterial flagellin in maize as it does in Arabidopsis. The extracellular LRR domain to recognize the peptide fig22 [45], maize BIR1, as a BAK1- (BRI1-associated receptor kinase 1-) interacting receptor-like kinase, works together with BAK1 to negatively regulate cell death and defense responses [12, 46, 47]. Other than OsSERK1 (GRMZM2G150024 on Chr. 4), two more SERK (Somatic Embryogenesis Receptor-Like Kinase) family members, named ZmSERK1 (GRMZM5G870959 on Chr. 10) and ZmSERK2 (GRMZM2G115420 on Chr. 5), had been characterised in maize previously [48]. Besides the role in somatic embryogenesis, the SERK family members have been associated with R-gene resistance, such as Mi-1 against potato aphids in tomato plants [49] and Xa21 againstXanthomonas oryzaepv.oryzae (Xoo) in rice [50]. Although three members of the SERK family have been found in maize, whether they have the same functions in disease resistance as seen in tomato and rice remains to be determined.In conclusion, this study has identified a number of the maize LRR-receptor-like genes and characterised a number of LRR-RLK/RLP genes based on their structural domains, physical chromosomal locations, and phylogenetic relationships. These sequences will aid in the study of their functions in maize.



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*Source: 231358-2015-10-22.xml*