Evolutionary analysis of plant jacalin-related lectins (JRLs) family and expression of rice JRLs in response to Magnaporthe oryzae

Journal of Integrative Agriculture 2018, 17(6): 1252–1266 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Evolutionary analysis of plant jacalin-related lectins (JRLs) family and expression of rice JRLs in response to Magnaporthe oryzae HAN Yi-juan, ZHONG Zhen-hui, SONG Lin-lin, Olsson Stefan, WANG Zong-hua, LU Guo-dong State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops/Key Laboratory of Biopesticide and Chemistry Biology, Ministry of Education/College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R.China

Abstract Jacalin-related lectins (JRLs) are widely distributed carbohydrate-binding proteins in the plant kingdom, which play key roles in development and pathogen defense. In this study, we profiled evolutionary trajectory of JRLs family in 30 plant species and identified domain diversification and recombination leading to different responsive patterns of JRLs in rice during defense against rice blast. All of 30 plant species analyzed in our study have two types of JRLs by containing either a single jacalin or repeated jacalin domains, while chimeric jacalins exist in more than half of the species, especially in the Poaceae family. Moreover, Poaceae species have evolved two types of unique chimeric JRLs by fusing the jacalin domain(s) with dirigent or NB_ARC domain, some of which positively regulate plant immunity. Seven Poaceae-specific JRLs are found in the rice genome. We further found expression of rice JRLs, including four Poaceae-specific JRLs, are induced by Magnaporthe oryzae infections at either early or late infection stages. Overall, the results present the evolutionary trajectory of JRLs in plant and highlight essential roles of Poaceae specific JRLs against pathogen attacks in rice. Keywords: jacalin-related lectins, phylogeny, Oryza sativa, Magnaporthe oryzae, infection responses

sub-families (Peumans et al. 2001). Among them, jacalin

1. Introduction Lectins, a group of proteins that have at least one noncatalytic carbohydrate binding site, bind reversibly to specific mono- or oligo-saccharides (Peumans and van Damme 1995; Vandenborre et al. 2011). Lectins have diverse functional roles in plants and animals (Peumans and van Damme 1995). Plant lectins can be classified into 12

proteins were first isolated from seeds of jack fruit (Moreira and Ainouz 1981). So far, at least 25 jacalin-related lectins (JRLs) have been identified in plants (Table 1). Among these, nine are chimeric JRLs that contained the exogenous domain dirigent which has important roles in plant secondary metabolism (Hosmani et al. 2013). The remaining 16 are merolectins or hololectins according to the classification scheme for lectins (Peumans and van Damme 1995). During the evolution, plants have to overcome various environmental challenges at different phases of their life cycles (Yamaji et al. 2012; Sandalio et al. 2015; Nejat et al.

Received 6 July, 2017 Accepted 28 September, 2017 Correspondence LU Guo-dong, E-mail: [email protected]; WANG Zong-hua, E-mail: [email protected] © 2018 CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: 10.1016/S2095-3119(17)61809-4

2017; Sahay et al. 2017). Duplication and rearrangement of adaptive genome modules play an important role for this process. Such events have increased the number of NBS-LRR (nucleotide- binding site plus leucine-rich repeat) genes involved in resistance (Richly et al. 2002). The plant

HAN Yi-juan et al. Journal of Integrative Agriculture 2018, 17(6): 1252–1266

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Table 1 Summary on already known plant jacalin-related lectins (JRLs) JRLs BGAF1

Domain Dirigent-jacalin

GenBank no. DQ975381

OsJAC1

Dirigent-jacalin

ABB51090

SbBGAF1 JPR TaHfr-1 TaJA1

Dirigent-jacalin Dirigent-jacalin Dirigent-jacalin Dirigent-jacalin

ABI24164 AF021256 AF483596.1 AY372111

TaVER2

Dirigent-jacalin

BAA32786

TaWC1 SalT

Dirigent-jacalin Jacalin

TAU32427 CAA81059

AtJAC1 JAL31

Jacalin Jacalin

NP_566547 NM_112514.3

JAL23

Jacalin

NM_001336766

PBP1/JAL30

Jacalin

AK319056

JAL22

Jacalin

NM_001161092

Banlec-1

Jacalin

AF001527

Calsepa

Jacalin

U56820

Heltuba

Jacalin

AF064029

Horcolin

Jacalin

AY033628

Morniga G

Jacalin

AY048576

PALb TaJRLL1 Ipomoelin

Jacalin Jacalin Jacalin

AB099933 HQ317136 D89823

Lem2

Jacalin

AY092765

1)

Species Zea mays

Description1) beta-Glucosidase aggregation

Reference Blanchard et al. (2001); Esen et al. (2000); Kittur et al. (2007) Oryza sativa Pathogen resistance Jiang et al. (2006); Weidenbach et al. (2016) Sorghum bicolor beta-Glucosidase aggregation Kitturet al. (2007) Hordeum vulgare beta-Glucosidase aggregation Wang et al. (2005a) Triticum aestivum Hessian fly-response gene Williams et al. (2002) Triticum aestivum Agglutinating activity and Ma et al. (2010, 2013); pathogen resistance Wang et al. (2005a) Triticum aestivum Vernalization signaling and Yong et al. (2003); spike development Feng et al. (2009); Xing et al. (2009) Triticum aestivum Biotic stresses response Görlach et al. (1996) Oryza sativa Agglutinating activity and abiotic Claes et al. (1990); stresses response Garcia et al. (1998) Arabidopsis thaliana Flowering time control Xiao et al. (2015) Arabidopsis thaliana Regulation on the size of ERNagano et al. (2008) bodies Arabidopsis thaliana Regulation on the size of ERNagano et al. (2008) bodies Arabidopsis thaliana Regulation on the size of ERNagano et al. (2008) bodies Arabidopsis thaliana Regulation on the size of ERNagano et al. (2008) bodies Musa acuminata Prevents viral infection Peumans et al. (2000); Liu et al. (2014) Calystegia sepium Agglutinating activity Van Damme et al. (1996); Bourne et al. (2004) Helianthus tuberosus Agglutinating activity Van Damme et al. (1999); Bourne et al. (1999) Hordeum vulgare Agglutinating activity Van Damme et al. (2004); and jasmonicacid-inducible Grunwald et al. (2007) Morus nigra Anti-tumor Van Damme et al. (2002); Poiroux et al. (2011) Phlebodium aureum Agglutinating activity Tateno et al. (2003) Triticum aestivum Pathogen resistance Xiang et al. (2011) Ipomoea batatas JA-mediated wound-induction Imanishi et al. (1997); and antioxidation Chang et al. (2012); Lin et al. (2014) Hordeum vulgare Salicylate-inducible Abebe et al. (2006)

ER, endoplasmic reticulum; JA, jasmonic acid.

lectin superfamily shows species-specific gene expansion

1996). Mannose-binding jacalin TaHfr-1 is transcriptionally

caused by tandem/segmental duplications. In Arabidopsis,

induced in wheat by Hessian fly larval infection (Williams

soybean, and rice, the tandem and segmental duplications

et al. 2002). Another mannose-specific jacalin-related

lead to lectin gene expression divergences under biotic

lectin-like gene (TaJRLL1) is induced by pathogen infection,

and abiotic stresses (Jiang et al. 2010). In wheat, JRLs

as well as salicylic acid (SA) and jasmonic acid (JA)

experienced a substantial diversification after the divergence

inducing treatments. Expression of TaJRLL1 in Arabidopsis

of wheat from other cereal species (Song et al. 2014).

enhances resistance against fungal diseases (Xiang et al.

JRLs are associated with plant responses to environmental

2011). Transient expression of the wheat JRL gene TaJA1

stresses and pathogen attacks. At the gene level, quite a

in barley also confers resistance against the powdery mildew

number of wheat JRLs genes are stress-inducible and

fungus (Weidenbach et al. 2016). In rice, SalT protein was

tissue-specific (Song et al. 2014). The wheat JRL protein

first isolated from salt-treated rice tissue of the indica variety

TaWC1 is an inducible gene that is up-regulated as part

Taicheng Native 1 (Claes et al. 1990). The SalT gene shows

of the wheat acquired resistance response (Görlach et al.

organ-specific expression and is induced by drought and

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hormone treatments. Another jacalin-related lectin protein OsJAC1 accumulates at fungal penetration sites and confers resistance against various rice pathogens (Jiang et al. 2006; Weidenbach et al. 2016). Jacalin proteins not only recognize carbohydrates for defense in plant, but also have roles during developmental processes. For example, the Arabidopsis jacalin-like gene AtJAC1 is a positive regulator in controlling timing of plant flowering (Xiao et al. 2015). Two pairs of heterodimer-stype jacalins (JAL31-JAL23 and JAL30-JAL22) have opposite effects on regulating the size of the PYK10 complex which serves as a major part of endoplasmic reticulum (ER) bodies and may be involved in Arabidopsis defense systems (Nagano et al. 2008). Although 28 JRLs have been predicted in the rice genome and some of the genes are responsive to various stresses (Jiang et al. 2010), details on gene composition, architecture, regulation, and function of rice JRLs are still unclear. In this study, we profiled and compared evolution and diversification of JRLs in 30 plant species. We found that rice contains seven Poaceae-specific JRLs by fusion with a dirigent domain or a NB_ARC domain. It is possible that the chimeric jacalins play specific roles in rice defense response against pathogens. The subsequent gene expression assays show that four of the Poaceae-specific OsJRLs genes as well as 19 of the mero-/ holo-jacalins have positive responses by being induced after Magnaporthe oryzae inoculation, indicating that these JRLs can indeed be an important part of the rice immune system directed against M. oryzae.

2. Materials and methods 2.1. Identification of jacalin domain proteins in plant genomes Genome wide protein sequences of Ananas comosus, Aquilegia coerulea, Brachypodium distachyon, Brassica oleracea, Brassica rapa, Carica papaya, Medicago truncatula, Mimulus guttatus, Physcomitrella patens, Populus trichocarpe, and Selaginella moellendorffii were downloaded from JGI Phytozome (https://phytozome.jgi. doe.gov/pz/portal.html). For Brassica napus, Glycine max, Sorghum bicolor, Triticum aestivum, Vitis vinifera, and Zea mays, the genome wide protein sequences were downloaded from Ensembl database (http:// plants.ensembl.org/index.htm). Genome wide amino acid sequences of Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii were downloaded from Cottongen (https://www.cottongen.org/). For Nicotiana benthamiana, Solanum tuberosum, and Solanum lycopersicum, genome wide protein sequences were downloaded from Sol Genomics Network Search (http:// solgenomics.net/). Arabidopsis lyrata genome was

downloaded from JGI Genome Portal (https://genome.jgi. doe.gov/Araly1/Araly1.home.html). Genome wide amino acid sequences of Arabidopsis thaliana were downloaded from TAIR (http://www.arabidopsis.org/). Genome wide amino acid sequences of Elaeis guineensis were downloaded from Eukaryotic Genome Annotation at NCBI (http://www.ncbi.nlm.nih.gov/genome/annotation_euk). Genome wide amino acid sequences of Hordeum vulgare was downloaded from MIPS Barley Genome Database (http://pgsb.helmholtzmuenchen.de/plant/ plantsdb. jspnfa). Genome of Lotus japonicus was downloaded from PlantGDB (https://plantgdb.org/LjGDB). Genome wide amino acid sequences of Musa nana was downloaded from Banana Genome Hub (http://banana-genomehub. southgreen.fr/). All annotated amino acid sequences of Oryza sativa were downloaded from RGAP MSU rice genome project database (http://rice.plantbiology.msu. edu/index.shtml). For those protein sequences, jacalin domain (PF01419.14) was then identified using the Hmmerscan Program in HMMER v3.1b2 with default settings (http://hmmer.org/). The obtained sequences were further manually compared with BlastP (E value 1e-10) citation (Altschul et al. 1997; Mistry et al. 2013).

2.2. Phylogenetic tree construction Multiple alignments of the full length sequences of JRLs were made using fast multiple sequence alignment software MAFTT (Multiple Alignment using Fast Fourier Transform) (Katoh and Standley 2013) with default parameters. For the large-scale alignment, the fast maximum-likelihood phylogenetic tree (Figs. 1–4) were generated using FastTree by JTT+CAT model (Price et al. 2009; Liu et al. 2011). In the phylogenetic tree, supporting values above 50 were kept and regarded reliable. Tree in Fig. 1-A was draw by Figtree and other trees in this paper were drawn by online itol tree (http://itol.embl.de/#).

2.3. Orthologous JRLs protein group Amino acids sequences from 30 plant species were used for an Orthofinder search (Emms and Kelly 2015) using default settings. The orthologous genes from each protein family were listed by group(s) (Appendices A–C).

2.4. Plant growth and inoculation assay Rice plants were grown in a growth chamber at 28°C with 16 h light and 8 h darkness. M. oryzae conidia were produced by growing cultures on rice bran medium (4% rice bran, 2% agar, pH 6.0) for 5 days in the dark followed

HAN Yi-juan et al. Journal of Integrative Agriculture 2018, 17(6): 1252–1266

by exposure to 12 h light/dark cycles for further 2 days at 25°C (Chen et al. 2008). For the plant inoculation assays, conidia were harvested from the rice bran plate cultures with M. oryzae isolates and suspended to a concentration of 1×105 spores mL–1 in 0.2% Tween 20 in distilled water. Plants at the four-leaf-stage were then spray-inoculated with conidia suspension (Chen et al. 2008). Rice leaf samples were harvested at 0, 24, and 48 hours post inoculation (hpi).

2.5. Expression profile and quantitative RT-PCR analysis For RNA-seq, publicly available RNA-seq data for M. oryzae

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and rice interaction were obtained from NCBI GEO database (www.ncbi.nlm.nih.gov/geo/). Sequencing reads were mapped to rice reference sequence with Tophat, Cufflinks, and Cuffmerge to extract all possible exons (Trapnell et al. 2012). Expression abundance were calculated by RSEM with default parameters (Li et al. 2011). The RPKM values were log transformed, and genes were clustered according to their expression patterns using the R package Pheatmap. For real-time PCR, the total RNAs were extracted using RNAiso Plus (TaKaRa, China) and quantified by Nanodrop (Thermo, USA). The first chains of cDNA were synthesized using ReverTraAce qPCR RT Master Mix with gDNA Remover Kit (TOYOBO, Japan) according to manufacturer’s

A

Clade 1

Clade 2

Clade 3

Clade 4

B Family

Species (gene number)

Family

Arabidopsis lyrata (46) Brassicaceae

Arabidopsis thaliana (51)

Brassica rapa (53)

Leguminosae

Solanaceae

Malvaceae

Lotus japonicus (7) Medicago truncatula (3)

Selaginellaceae

Mimulus guttatus (8)

Solanum lycopersicum (8)

Funariaceae

Physcomitrella patens (8)

Solanum tuberosum (12)

Ranunculaceae

Gossypium arboreum (3)

Vitaceae Caricaceae

Carica papaya (2)

Gossypium hirsutum (6) Gossypium raimondii (3)

Species (gene number) Brachypodium distachyon (21) Hordeum vulgare (22)

Poaceae

Oryza sativa (30) Sorghum bicolor (22)

Selaginella moellendorffii (10)

Scrophulariaceae

Nicotiana benthamiana (19)

Family

Glycine max (6)

Brassica napus (123) Brassica oleracea (52)

Species (gene number)

Triticum aestivum (40) Zea mays (20) Bromeliaceae

Ananas comosus (15)

Aquilegia coerulea (5)

Arecaceae

Elaeis guineensis (16)

Vitis vinifera (3)

Musaceae

Musa nana (23)

Salicaceae

Populus trichocarpa (10)

Fig. 1 Phylogenetic relationship of jacalin-related lectins (JRLs) of rice and 29 other plant species. A, the phylogenetic tree was divided into four clades, each represented by different colors of the surrounding dots. Blue, yellow, purple, and green represent clades 1, 2, 3, and 4, respectively. B, the 15 leaf colors are used to distinguish the 15 families that shown in the columns below the tree.

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instructions. Quantitative RT-PCR amplifications were performed in 10 μL reactions containing 5 μL of 2×SYBR Premix ExTaq (TliRNaseH plus, TaKaRa), 10 ng of cDNA template and 0.75 μL of each primer (10 μmol L–1). The reactions were then subjected to Eppendorf Mastercycler Realplex Real-Time PCR System (Eppendorf, Germany). The reaction settings were 95°C for 5 min; 40 cycles of 95°C for 20 s, 56°C for 15 s. The cycle threshold value of each gene was normalized to the internal reference gene OsActin. The relative expression of each gene was calculated by the 2–ΔΔCt method (Livak and Schmittgen 2001; Schmittgen and Livak 2008).

3. Results 3.1. Identification and phylogeny of JRLs in 30 selected plant species To analyze the evolutionary relationships of JRLs, combined BlastP and Pfam Search Program which were used to identify JRLs in 30 different plants across 15 monocots-/ dicots-families. A total of 651 JRLs have been identified in 30 plant species (Fig. 1-B). It’s noticeable the total number of

JLRs varied in different plant species and this variation is not related with genome size. The largest two families include Brassicaceae with 324 JRLs and Poaceae with 157 JRLs. The number of JRLs per species in Brassicaceae ranges from 46 to 123, while in Poaceae from 20 to 41. The lowest number is found in C. papaya that only contains two JRLs. Classic JRLs have only one jacalin domain, while JRLs proteins identified in this study contain three sub-types with the following domain-combinations: merojacalin, holojacalin, and chimeric jacalin, according to the classification of lectin (Peumans and Damme 1995). Merojacalin contains only one jacalin domain. Holojacalin contains at least two identical jacalin domains without fusion with other domains. Chimeric jacalin have one or more jacalin domains that combine(s) with other types of domains such as dirigent, Pkinase, NB_ARC or Kelch_1/2/3/6 found in this study (Appendix D). The lower plant P. patens (a moss) has only seven JRLs, while five out of the seven JRLs are chimeric jacalin with Pkinase_Tyr domain which is otherwise mainly found in Poaceae species. In the fern plant S. moellendorffi, seven out of eight JRLs are merojacalins while the last one is a holojacalin. In higher plants, Solanaceae species have merojacalin as the major

Group B (Clade 1)

0.5

Group A (Clade 4)

Fig. 2 Branch-length view of phylogenic tree of jacalin-related lectins (JRLs) from rice and 29 other plant species. Chimeric jacalins, holojacalins, and merojacalins are shown with red, blue, and black branches, respectively.

HAN Yi-juan et al. Journal of Integrative Agriculture 2018, 17(6): 1252–1266

sub-type (21 out of 39), and then holojacalin (12 out of 39). Besides merojacalin and holojacalin, N. benthamiana has three chimeric jacalins with NB_ARC domain which is absent in tomato or potato. Leguminosae, Malvaceae, A. coerulea, M. guttatus, V. vinifera, C. papaya, and P. trichocarpa contain less number of jacalins (from two to 10) and have different distributions of merojacalin and holojacalin. For example, in Leguminosae, soybean also has three sub-type jacalins, two for each type of jacalin proteins. M. truncatula contains only one merojacalin and one holojacalin. L. japonicus has six merojacalins, one holojacalin, and two chimeric jacalins with DIOX_N and OG-FeII_Oxy domains. The last appears conserved at the N-terminal region by containing 2-oxoglutarate/Fe(II)-dependent dioxygenase that catalyzes O-demethylation (Hagel and Facchini 2010). In five selected Brassicaceae species, more than half (about 55%) of JRLs are holojacalins. Merojacalins are the second most abundant with 28%. The remaining JRLs are chimeric jacalins that mainly contain F-box associated (FBA) domain and Kelch-repeat domain with 3 and 11%, respectively. For monocot species, the major subtype is merojacalin. A. comosus, M. nana, and E. guineensis have two subtype jacalins: merojacalin and holojacalin with proportions 11:4, 20:1, and 10:4, respectively. In Poaceae, JRLs are present as two main subtypes: merojacalins (50%) and chimeric jacalins (40%), while holojacalins only constitutes 10%. A phylogenetic maximum-likelihood tree was generated based on a multiple alignments of full-length amino acid sequences of JRLs by FastTree to investigate evolutionary relationships (Fig. 1-A). The phylogeny of 651 JRLs resolves the four expected groups. The majority of Brassicaceae JRLs are included in clade 1 (Fig. 1-A) along with a few Poaceae JRLs. We further made the alignments of JRLs in this clade and found that Poaceae JRLs shared high value of 20–83% identity with certain Brassicaceaeunique JRLs (Appendix E), suggesting that horizontal gene transfer and homoplasy may exist between Brassicaceae and Poaceae. In clade 2 (Fig. 1-A), Brassicaceae JRLs are nested with P. trichocarpa, M. guttatus, and A. coerulea. Clade 3 (Fig. 1-A) contains JRLs from all 30 examined species, including moss, fern, dicots, and monocots, and seems evolutionally conserved, indicating that JRLs in this clade could have been present before the divergence of monocots and dicots. JRLs in clade 4 are only present within as Poaceae species (Fig. 1-A). To further describe the distribution of different types of JRLs in 30 selected genomes, we made a more detailed view of the distribution of merojacalins, holojacalins, and chimeric jacalins (Fig. 2) based on the data presented in Fig. 1. Poaceae JRLs in clade 4 (Fig. 1-A) are highly prone to have chimeric jacalins (55%) compared to merojacalins (37%) and holojacalins (8%) (Fig. 2, Group A). The

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integrated domains of these jacalin proteins are listed in Table 2 and mainly found to be Pkinase, dirigent, and NB_ARC domains. In contrast, JRLs in clade 1 (Fig. 1-A) mainly contain holojacalins (60%), followed by merojacalins (20%) and chimeric jacalins (20%) (Fig. 2, Group B). From dicots to monocots, chimeric jacalins in clade 4 show more evolutionary differentiations than the other clades, which could be a special adaption in Poaceae species. These results suggest the number and domain combination of JRLs in different plant species are highly variable and JRLs in Poaceae species have experienced different diversification compared with Brassicaceae species.

3.2. Evolution and divergence of JRLs in Poaceae To further characterize JRLs in Poaceae, we investigated the phylogeny of jacalin-related lectins in grass species. A maximum likelihood phylogenic tree is constructed by using the full-length amino acid sequences. Six groups of JLRs are categorized by containing representative domains from six grass species, including O. sativa, Z. mays, H. vulgare, T. aestivum, S. bicolor, and B. distachyon (Fig. 3). Clade 6 (Fig. 3) contains 6 wheat and barley JRLs with four types of domain integrations: merojacalins (20%), holojacalins (50%) and chimeric jacalins (30%) among which the integrated domains are fused to single-jacalin domains (10%) and multiple-jacalin domains (20%) (Table 3). Clade 1 (Fig. 3) is the largest group by consisting of 60 JRL proteins across all six species: six from maize, eight from sorghum, 10 from wheat, 11 from B. distachyon, 11 from barley, and 14 from rice. Of the JRLs located in clade 1 (Fig. 3), 80% contain a single jacalin domain, compared with 13% containing repeated jacalin domains (Table 3). Only two JRLs (Os11g06570 and Os04g12390) contain other domains, such as Peptidase_C43, Peptidase_C48, DUF4216, DUF4218, or Transposase domains. Similarly, most of JRLs in clade 3 (Fig. 3) are found coding for only one jacalin domain (94%) except for wheat JRL coding for one jacalin containing Pkinase-jacalin-jacalin domains (Traes_4AL_015A5C163.3) that is found as a chimeric jacalin (Fig. 3, leaves labeled with dash lines). The other three groups (Fig. 3) (clades 2, 4, and 5) all contain chimeric jacalins. Most of these proteins encode for other types of domains, such as dirigent, Pkinase, and NB_ARC. Clade 5 (Fig. 3) contains 10 chimeric jacalins and one merojacalin that code for only one single jacalin domain across four cereal species but is not present in B. distachyon and barley: one from sorghum, two from wheat, three from maize, and five from rice. Chimeric jacalins in this clade are present as jacalin-NB_ARC and multiple jacalins-NB_ARC, which comprise 45 and 45% of all jacalins, respectively (Table 3). This clade forms two paired monophyletic

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represented) jacalin domains among all leaves in this clade. The other JRLs, Br_4g13990 and Traes_5BS_60FAB49FB.1, encode for three and one jacalin domains, respectively, without any exogenous domains. Two monophyletic pairs Traes_2AL_F5E021100.1–Traes_2DL_59C23B50A.2 and Os11g32210.1–Os11g32170.1 are formed in this clade. Traes_2AL_F5E021100.1–Traes_2DL_59C23B50A.2 share high amino acid sequence identity which may be a result of segmental duplication events which also occurred in the Os11g32210.1–Os11g32170.1 pair. Clade 2 (Fig. 3) is comprised of 48 JRLs proteins from six grass species: three from barley, five from rice, eight from sorghum, nine from maize, 11 from B. distachyon, and 12 from wheat. Maize JRLs cluster in two sub-clans

subgroups present as paralogs: Os11g39480–Os11g39530 and GRMZM2G355098_P01–GRMZM2G368663_P01. The maize pair with a high bootstrap (100%) assembles jacalin and NB_ARC in a similar way, indicating a duplication event occurred before the maize-rice divergence. The rice pair did not share much common in domain arrangements. Os11g39530 contains two jacalin domains and one NB_ARC domain while Os11g39480 has only one jacalin domain, which may due to an occasional loss during duplications. Clade 4 (Fig. 3) is composed of 10 JRLs proteins, one from B. distachyon, one from sorghum, two from barley, three from rice, and three from wheat. Eight of the chimeric jacalins harbor a kinase domain across all the grass species. A Pkinase domain is fused to single (50%) or multiple (30% Species: Zea mays Oryza sativa Sorghum bicolor Hordeum vulgare Triticum aestivum Brachypodium distachyon

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e

1

{

★ ★ ★ ★ ★ ★ ★ ★ ★ ★

Cl

W1

★ ★

6 Clade

Clade

★ ★ ★

M2

Jacalin

★

{

Dirigent

4

★ ★

★

★

★

NB_ARC DUF4216

★ ★

★ ★ ★ ★ ★

★ ★★

Transposase_21

de

5

e3

{

Clade 2

d Cla

M1

★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

Cla

★

Pkinase DUF4218

★★ ★ ★★

B3

Peptidase_S10

Peptidase_C43

Peptidase_C48

Transpos_assoc

Motile_Sperm

Fig. 3 Phylogeny of jacalin-related lectins (JRLs) in grass genomes. Sequences of JRLs from six grass species were used to generate a phylogenic tree by FastTree. The branches were divided into six clades with colors (blue, prune, purple, olive, green, gold). The leaf colors (orange, green, red, purple, black, and blue) were used to distinguish the respective six species: Brachypodium distachyon, Hordeum vulgare, Oryza sativa, Sorghum bicolor, Triticum aestivum, and Zea mays. The grass specific genes are marked with stars (containing dirigent-jacalin, NB_ARC-jacalin, or Pkinase domain).

HAN Yi-juan et al. Journal of Integrative Agriculture 2018, 17(6): 1252–1266

1259

(M1 and M2). The M1 proteins are closely related to the

for dirigent-jacalin chimeric proteins. In all these JRLs,

sorghum JRL Sb09g001880 and the M2 proteins are

the dirigent domain is fused with only a single jacalin

related to Sb02g007740, Sb02g007735, Sb02g007750,

domain. Using protein sequence clustering analysis, we

and Sb02g007760 (Fig. 3). Those JRLs are more related

found a total 53 of JRLs are only found in the six grass

to each other than to JRLs from the same species from

species (Fig. 3). A total of 40 of the grass-specific JRLs

other clades, suggesting that maize and sorghum have a

that contain dirigent domain are found in clade 2 (Fig. 3).

common ancestor and the duplication already existed prior

The remaining eight JRLs are found in clade 4 and contain

the divergence of maize-sorghum. A sub-branch containing

a Pkinase domain (Fig. 3). In a group comparison, there

rice JRLs (including Os12g09720.1, Os12g09700.1,

is a particularly high number of grass-specific JRLs found

Os12g14440.1, and Os12g1270.1) is located closely to

in Group A (Fig. 2) that contains dirigent domain jacalins

maize-sorghum. Duplication of the rice JRLs in this sub-

(dash leaves with stars in clade 2 or 4 of Fig. 4). Rice

branch may have occurred before the ancestor of maize-

JRLs (Os02g19890.1, Os11g32170.1, Os11g32210.1,

sorghum separated from rice. In contrast, the wheat sub-

Os12g09720.1, Os12g09700.1, Os12g14440.1, and

branch W1 JRLs probably have duplicated more recently

Os12g12720.1) are only found in grass species. Together,

than the divergence of wheat from other grass species

our results indicate that a rapid domain recombination of

(Fig. 3).

JLRs has occurred in Poaceae, and a unique group has

Domain analysis showed that 39 out of 48 JRLs encodes

been identified in rice genome.

92 Os12g09700.1 97 Os12g09720.1 Os12g14440.1 57 Os12g12720.1 74 Os12g05020.1 HvLEM2 TaJRLL1 74 84 Os01g24710.2 91 Os01g252801 Os06g12180.1 68 99 Os06g07250.1 99 Os06g07300.1 Os03g28160.1 Os04g22900.2 80 Os05g05170.1 94 Os11g39490.1 Os11g39530.1 94 97 Os04g30040.2 Os11g39420.1 100 90 100 Os11g32170.1 82 Os11g32210.1 86 Os02g19890.2 70 92 Os11g39480.1 Os10g04270.1 Os01g25160.1 94 87 Os04g03320.1 Os04g03360.1 99 Os01g51050.1 Os05g43240.3 At01g58160 81 96 Os04g12390.1 Os04g26320.1 100 Os11g06570.1 CaMBL1 OslecRK 91

A

B

C

D

E

F G H

I

0.5

Fig. 4 Phylogeny of rice jacalin-related lectins (JRLs). The phylogenetic tree was constructed based on multiple alignments of full-length amino acid sequences using Multiple Alignment using Fast Fourier Transform (MAFFT). The resulting alignment was then used to generate a maximum-likelihood tree by FastTree. The scale bar shows 0.5 amino acid substitutions per site. CaMBL1 and OslecRK are set as out-group. Rice JRLs are divided into nine subgroups, from A to I.

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3.3. Jacalin-related lectins in rice Comparison of JRLs in Poaceae indicates the rice genome contain a unique group of dirigent domain jacalins. We next focus on rice and analyze phylogeny of rice JRLs. In this species, two more jacalins are found than those presented in the report by Jiang et al. (2010). All JRLs in rice (OsJRLs) contain full open reading frames and are distributed on nine of 12 chromosomes, with none found on chromosomes 7 to 9 (Table 4). The deduced protein products of the 30 OsJRLs range from 12.2 to 73.5 kDa with pI value from 4.8 to 9.4. All

OsJRLs contain typical jacalin domain structures and some jacalin domains are fused to other types of domains. Most of the rice JRLs (16 out of 30) have only one jacalin domain as merojacalins. Thirteen out of 30 OsJRLs are chimeric proteins that have recruited NB_ARC, Pkinase, dirigent or Peptidase domain to the jacalin domain(s) and cluster on chromosome 11 or 12 (Table 4). Only one OsJRLs is a holojacalin and it contains three repeated jacalin domains. In order to evaluate the evolutionary relationship among the 30 OsJRLs, we performed a phylogenetic analysis based on full amino acid sequences and compared with

Table 2 Characteristics of jacalin-related lectins (JRLs) from rice genome1) JRLs Os01g24 710 Os01g25 160 Os01g25 280 Os01g51 050 Os02g19 890 Os03g28 160 Os04g03 320 Os04g03 360 Os04g12 390 Os04g22 900 Os04g26 320 Os04g30 040 Os05g05 170 Os05g43 240 Os06g07 250 Os06g07 300 Os06g12 180 Os10g04 270 Os11g06 570 Os11g32 170 Os11g32 210 Os11g39 420 Os11g39 480 Os11g39 490 Os11g39 530 Os12g05 020 Os12g09 700 Os12g09 720 Os12g12 720 Os12g14 440 1)

Chr. no. 1 1 1 1 2 3 4 4 4 4 4 4 5 5 6 6 6 10 11 11 11 11 11 11 11 12 12 12 12 12

Amino acid (aa) 145 160 144 243 1 072 201 183 141 1 269 150 331 723 152 660 183 209 145 143 2 505 1 384 1 386 474 130 837 597 124 307 258 260 306

ORF length (bp) 438 483 435 732 3 219 606 552 426 3 810 453 996 2 172 459 1 983 552 630 438 432 7 518 4 155 4 161 1 425 393 2 514 1 794 375 924 777 783 921

MW (g mol–1) 15 172.1 17 409.8 14 872 26 378.9 121 833 21 076.7 19 095.6 15 401.1 144 755 16 342.7 39 095.9 78 434.4 15 308.3 72 551.9 19 306.3 21 498 15 144.2 15 640.5 285 602 155 067 154 461 53 852.4 14 191.8 91 828.8 65 590.9 13 393.2 33 352.8 28 600.8 27 918.8 33 012.4

CDS coordinates (5´→3´) 13 904 490–13 903 153 14 211 106–14 210 479 14 280 746–14 282 187 29 324 340–29 323 220 11 708 374–11 701 937 16 209 134–16 210 085 1 411 107–1 412 422 1 444 093–1 444 933 6 842 367–6 833 910 12 989 860–12 996 145 15 345 795–15 342 548 17 929 442–17 940 490 2 520 751–2 521 623 25 158 992–25 163 447 3 470 973–3 475 963 3 504 057–3 508 740 6 521 987–6 523 338 1 999 595–1 999 064 3 159 728–3 175 505 18 999 281–18 990 892 19 029 390–19 022 758 23 475 516–23 459 658 23 507 170–23 506 616 23 513 127–23 508 365 23 545 497–23 537 272 2 179 174–2 180 895 5 118 098–5 116 345  5 135 246–5 132 814 7 011 245–7 012 771 8 239 429–8 241 153

pI

Domain (no.)

4.8 Jacalin (1) 5.7 Jacalin (1) 7.5 Jacalin (1) 9.8 Jacalin (1) 7.9 NB_ARC (1), jacalin (1) 5.3 Jacalin (1) 8.7 Jacalin (1) 8.2 PeptidaseC47 (1), jacalin (1) 6.9 Jacalin (1) 8.4 Jacalin (1) 7.4 Jacalin (1) 5.8 Pkinase (1), jacalin (3) 7.8 Jacalin (1) 8.3 Jacalin (3) 8.5 Jacalin (1) 6.3 Jacalin (1) 6.3 Jacalin (1) 7.1 Jacalin (1) 6.4 PeptidaseC48 (1), jacalin (1) 6.9 NB_ARC (1), jacalin (3) 6.7 NB_ARC (1), jacalin (3) 7.4 Pkinase (1), jacalin (1) 4.8 Jacalin (1) 6.7 Pkinase (1), jacalin (3) 7.9 Pkinase (1), jacalin (2) 9.4 Jacalin (1) 8.6 Dirigent (1), jacalin (1) 9.0 Dirigent (1), jacalin (1) 8.7 Dirigent (1), jacalin (1) 4.8 Dirigent (1), jacalin (1)

ORF, open reading frame; MW, molecular weight; CDS, coding sequence; pI, isoelectric point.

Table 3 Distribution of grass chimeric jacalins in group A of Fig. 3 Species No. of JRLs in each genome1) No. of chimeric jacalins in group A Exogenous domains (no.) Zea mays 20 5 Pkinase (3), dirigent (2) Sorghum bicolor 22 8 Pkinase (1), dirigent (6), NB-ARC (1) Brachypodium distachyon 23 8 Dirigent (7), peptidase_S10 (1) Oryza sativa 30 11 Pkinase (4), dirigent (4), NB-ARC (3) Hordeum vulgare 21 7 Pkinase (1), dirigent (3), NB-ARC (2), B3 (1) Triticum aestivum 40 14 Pkinase (5), dirigent (7), NB-ARC (2) 1)

JRLs, jacalin-related lectins.

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Table 4 Summary of jacalin domains found in each clade (Fig. 4) of grass phylogeny Clade 6 5 4 3 2 1 1)

No. of total JRLs1) 10 11 10 17 49 60

No. of merojacalins 2 1 1 0 7 48

No. of holojacalins 5 0 1 1 1 10

No. of chimeric jacalin with a singlejacalin domain 1 5 5 16 41 2

No. of chimeric jacalin with repeated-jacalin domains 2 5 3 0 0 0

JRLs, jacalin-related lectins.

barley HvLEM2, wheat TaJRLL1, Arabidopsis At01g58160, and pepper CaMBL1 (Fig. 4). The last two are used as out-group. Nine clades (A–I) (Fig. 4) are identified as monophyletic subgroups with high bootstrap (≥80%), suggesting that OsJRLs in the same clade may share the same evolutionary origin. The additional two JRLs (Os12g05020 and Os05g05170) form single branches. Different types of jacalins distributes into subfamilies. In subfamilies B, C, F, G, and H, OsJRLs are present as merojacalins. Subfamilies A, D, and E are enriched in chimeric jacalins with dirigent, Pkinase or NB_ARC domain, respectively. In addition, seven sister pairs paralogous with rice JRLs are found with strong bootstrap (≥92%), Os12g09700.1–Os12g09720, Os06g07250.1– O s 0 6 g 7 3 0 0 . 1 , O s 11 g 3 9 4 9 0 . 1 – O s 11 g 3 9 5 3 0 . 1 , O s 4 g 3 0 0 4 0 . 2 – O s 11 g 3 9 4 2 0 . 1 , O s 11 g 3 2 1 7 0 . 1 – Os11g32210.1, Os04g03320.1–Os04g03360.1, and Os04g12390.1–Os04g26320.1, indicating that a clear paralogous pattern of JRLs divergence caused by duplication occurs in rice.

3.4. Expression patterns of rice JRLs genes in response to rice blast disease Gene expression patterns offer important clues of gene functions. To investigate possible involvement of JRLs in the response to M. oryzae, we examined the expression of OsJRLs genes in rice during M. oryzae infection (at time 0, 24 and 48 hpi) using RNA sequencing. Two M. oryzae isolates (Guy11 and FJ81278) are used to inoculate the susceptible rice cultivar Nipponbare in this assay. Most of the OsJRLs show detectable expression in the assay except seven genes (Os01g25160, Os04g03320, Os04g03360, Os11g06570, Os11g32170, Os11g39480, and Os12g12720) (Fig. 5). The inducible genes include 12 merojacalins, one holojacalin, and 10 chimeric jacalins. Of the 10 chimeric jacalins, three are Poaceae specific dirigent-jacalins. Of the remaining, four chimeric jacalins contain NB_ARC and three contain Pkinase domains. The expression patterns of the three types of jacalins in rice are different. The 19 OsJRLs are divided into six groups depending on their

expression patterns. Compared to the control treatment, Guy11 and FJ81278 strains up-regulate most of the OsJRLs in group 1 (Os5g43240 and Os12g09700) and group 2 (Os10g04270, Os11g39490, and Os01g25280) at 48 hpi. Group 3 (Os04g12390 and Os03g28160) shows marked down-regulation at 24 hpi for both Guy11 and FJ81278 inoculations but a slower return to control levels at 48 hpi for FJ81278 than that for Guy11. Group 5 (Os01g51050, Os12g05020, Os04g26320, and Os04g22900) are downregulated by M. oryzae infections but with slower response in the FJ81278 treatment. OsJRLs in Group 4 (Os06g07300 and Os06g12180) are strongly up-regulated by FJ81278 but not by Guy11. Group 6 JRLs are up-regulated at 24 hpi and then returns down to control levels at 48 hpi with a stronger up-regulation for Guy11 inoculated rice. To verify these results, eight OsJRLs genes were then examined by real-time PCR for their expression in Nipponbare rice after inoculation with M. oryzae Guy11. These eight gene were responsive to M. oryzae challenge mainly at 24 or 48 hpi. The transcriptional profiles of Os01g24710, Os06g07250, and Os11g32210 are all elevated after 12 hpi and reached the maximum at 24 hpi. Os12g14440 (also known as OsJAC1) has a much earlier response and reach the peak at 12 hpi, which is consistent with the finding by Jiang (2006) and Weidenbach (2016). Os01g25280, Os05g43240, Os10g04270, and Os12g09720 have similar expression pattern by being up-regulated at 48 hpi.

4. Discussion 4.1. Phylogeny of plant JRLs Jacalin-related lectin proteins are found in many plants, some of which have been characterized and take roles in enhancing tolerance against environmental stresses and pathogens attacks (Xiao et al. 2005; Jiang et al. 2006; Xiang et al. 2011; Ma et al. 2013; Weidenbach et al. 2016). In this study, we increase the number of studied plants since more genomes are now available and analyzed the distribution, phylogenic relationship, and domain composition of JRLs

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Os11g39490

Group 2

Os04g12390

10 0

Group 3

Os03g28160

Group 5

Os04g26320 Os04g22900

Os11g32210 Os06g07250 Os04g30040 Os11g39530 8N48

8N24

GN48

0N

GN24

Os05g05170

Os12g14440

0

4 Relative level

Os12g14440

Group 6

12 24 48 Time (hpi) Os10g04270

72

1 0

12

24 48 Time (hpi)

12

72

24 48 Time (hpi)

Os01g25280

2 1 0

2

0

12

24 48 Time (hpi) Os12g09720

24 48 Time (hpi)

72

6 4 2 0

72

12

Os05g43240

8

3

0

4

0

72

4

8

2

, merojacalin , holojacalin , chimeric jacaliq

0

5

3

0

5 0

72

5 0

Os11g39420 Os01g24710

12 24 48 Time (hpi)

10

Os01g51050 Os12g05020

0

15

Group 4

Os06g12180

Relative level

Os06g07300

10

20

Os01g25280

Relative level

Os10g04270

Group 1

Os11g32210

6

Relative level

Os12g09700

Relative level

Os05g43240

30

M.oryzae

Os06g07250

15

Relative level

Os12g09720

Water Os01g24710

40

Relative level

B

Os02g19890

1.5 1.0 0.5 0 –0.5 –1.0 –1.5

Relative level

A

0

12

24 48 Time (hpi)

72

6 4 2 0

0

12

24

48

72

Time (hpi)

Fig. 5 Expression profiles of rice jacalin-related lectins (OsJRLs) following rice blast inoculation. A, heat map of 23 OsJRLs genes regulated by Magnaporthe oryzae inoculations. Color represents relative expression level for RNA sequencing. Scale ranges from blue (low) to red (high). Up-regulation (dark red) and down-regulation (dark blue) are described by log2fold changes relative to the mean expression of the selected genes in three stages: uninoculated rice (0N), inoculated with Guy 11 at 24 hours post inoculation (hpi) (GN24), inoculated with Guy11 at 48 hpi (GN48), inoculated with FJ81278 at 24 hpi (8N24), and inoculated with FJ81278 at 48 hpi (8N48). B, qRT-PCR expression of eight selected OsJRLs genes in susceptible rice in exposure to Guy 11. Error bars represent the standard deviation from three replications.

in plants across 15 monocots-/dicots-families. JRLs are ubiquitous in all examined genomes but with various gene number expansions. For example, Brassicacea species contained many more JRLs (from 46 to 123) than the other 25 plants (from 2 to 41), indicating that the expansion of JRLs in Brassicacea species has been faster than those in the other plants, such as moss, fern, and other angiosperms. In Poaceae, rice has a small size genome (380 Mb) but contains 30 JRL genes (latest version of MSU rice genome project) (Table 2), suggesting that rice JRLs genes have experienced a faster gene expansion rate compared to B. distachyon, H. vulgare, S. bicolor, and Z. mays. For rice and maize, both of which have polyploid origin, whole-genome duplication occurred in the common rice and maize ancestor (at 70–80 mya (million years ago)) followed by genome diploidization (including some loss of duplicated genes) before the divergence of rice-maize (at 50 mya) from the polyploid ancestor (Gaut et al. 1997; Paterson et al. 2004; Wang et al. 2005; Llaca et al. 2011). The genome contraction most probably has continued further in the maize line and removed more duplicated genes, during at least two more polyploidization-contraction events (Paterson et al. 2004; Llaca et al. 2011), while in rice more of the originally duplicated genes were retained instead of diversifying to gain new functions. Ancient polyploidization and subsequent diploidization (loss) of

many duplicated gene copies have shaped the genomes of all Poaceae cereal, forage, and biomass crops.

4.2. Domain combination of Poaceae JRLs JRLs from 30 selective genomes are investigated with different combinations of jacalin and other domains. In dicots, the predominating types are different for species except in the Brassicaceae family. Five Brassicaceae species encode for more holojacalins than the remaining two. Most of the holojacalins contain more than two identical jacalin domains. In other dicots species, the distributions of the major jacalin types are not associated with evolutionary relationships (Fig. 1), which may be due to the limited number of genomes we investigated. JRLs of dicots are different from that of monocots, but both of them have similarity with mosses. Monocot species have merojacalins as their major type, chimeric jacalins as the second most abundant, holojacalins as the third. Six holojacalins from five grass species contain more than two repeated jacalin domains. This result is different from what have been found by other authors (Song et al. 2014) and it may be due to updated versions of the selected grass genomes. Poaceae species contain a higher proportion of chimeric jacalins (40%) than dicots in this study. The jacalin domains in grass chimeric jacalins are covalently

HAN Yi-juan et al. Journal of Integrative Agriculture 2018, 17(6): 1252–1266

linked to dirigent, Pkinase, NB_ARC or B3 domain and so on. Two types of chimeric jacalins are specific to grass species (Fig. 3). One type is dirigent-jacalin domains found in all six grass species which further confirmed the results reported by Song (2014). Another type is NB_ARC-jacalin from wheat, sorghum, barley, and rice. Proteins with dirigent or disease-response domain exists in plants and have been implicated in plant defense (Ralph et al. 2007; Seneviratne et al. 2015; Weidenbach et al. 2016). Jacalins proteins with N-terminal dirigent domains have only been found in Poaceae species (Song et al. 2014) but we have now confirmed this by a more detailed study. Rice OsJAC1 (Os12g14440) gene is encoded with a Poaceae-specific jacalin-related lectin and is required for resistance against rice pathogens by containing dirigent and jacalin domains (Jiang et al. 2006; Weidenbach et al. 2016). In wheat, dirigent-JRL TaHfr-1 (Hessian fly-response gene1) shows a positive transcription response to virulent Hessian fly larvae infection (Williams et al. 2002). Two dirigent-lead jacalins in wheat TaJRLL1 and TaJA1 (Traes_2BS_A1F541056.1) both confer enhanced resistance against pathogenic fungi in wheat immunity (Wang and Ma 2005; Xiang et al. 2011; Ma et al. 2013). Many plant resistance genes contain NBARC (nucleotide-binding proteins involved in ubiquitination) domains that regulate defense responses (De Schutter and Van Damme 2015). In this study, eight of the NB_ARCjacalins are only found in grass species. It is possible that NB_ARC domain in JRLs protect grass species from pathogen attacks. From monocots to dicots, the proportion of chimeric jacalins increase while holojacalins decrease, it is possible that JRLs with multiple copies of jacalin domains will not fuse to other domains and be more prone to be lost during evolution to acquire new functions as JRLs with single domains. In rice, JRLs genes are distributed over nine out of 12 chromosomes. Six JRLs clusters with 27 genes in total were found in the rice genome. Ten rice chimeric jacalins encoded with NB_ARC, Pkinase or dirigent domain are found in a cluster on chromosomes 11 and 12. Three pairs of these 10 JRLs were present as monophyletic groups. Since rice diverged from wheat-barley much later than that from maize lineage 50 mya (Kellogg 2001), newly evolved rice monophyletic pairs may have occurred after the divergence of rice from wheat-barley or maize-sorghum.

4.3. Poaceae JRLs genes in response to pathogens Some already known JRLs genes that encode with merojacalins or chimeric jacalins are regulated by biotic or abiotic stresses. For example, rice merojacalin SalT and chimeric jacalin OsJAC1 both show positive responses to several stress treatments, including salt, drought, cold,

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salicylic acid (SA), methyl jasmonate (Me-JA), and abscisic acid (ABA) (Claes et al. 1990; Garcia et al. 1998; De Souza Filhoa et al. 2003; Jiang et al. 2006; Weidenbach et al. 2016). In wheat, JRLs show different response patterns toward abiotic agents suggesting that TaJRLs may have complicated roles in plant defense response (Song et al. 2014). In the present study, we examined the transcripts of rice JRLs gene in response to rice blast disease. Most of the rice JRLs (23) show transcriptional responses to M. oryzae inoculations while the remaining (7) was not responsive. Some of these nonresponsive genes were previously also not found to respond to M. oryzae (Bagnaresi et al. 2012). These genes can be expressed in other tissues or under other environmental conditions. The expression patterns of OsJRLs are basically the same in response to both M. oryzae Guy11 and FJ81278 strains, although it appears like that rice reacts slower to FJ81278 than to Guy11. Most of the responsive genes are up-regulated at specific stages of infections, such as OsJRLs genes in groups 1, 2, 4, and 6 (Fig. 5). Some OsJRLs genes (groups 3 and 5) are suppressed after M. oryzae inoculation, especially at the early infection stage (Group 3 at 24 hpi). Rice JRLs are largely characterized by structure diversity and pathogen inducible-expression, which also applies to other plants, such as wheat (Song et al. 2014). Poaceae have evolved unique JRLs genes by recruiting exogenous domains for certain adaptations. All detected Poaceaespecific OsJRL genes responded to M. oryzae inoculation in this study (Fig. 5). The detailed functions of these unique JRLs genes should be investigated in future studies.

5. Conclusion We identified 651 jacalin-related lectins and analyzed the evolutionary trajectory and domain diversification in 30 plants species. We found the number and domain combination of JRLs proteins are highly variable in those plants. Poaceae species have evolved some Poaceae-specific JRLs. Rice JRLs genes displayed variable transcriptional changes upon M. oryzae infections.

Acknowledgements We are graceful to MSc Lin Lianyu of Fujian Agriculture and Forestry University for assistance in sequencing result analysis. This project is funded by the National Key Research and Development Program of China (2016YFD0100600) and the National Natural Science Foundation of China (U1405212). Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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