- Email: [email protected]
LYK binding
Accepted Manuscript Genome-wide identification of Lysin-Motif Receptor-Like Kinase (LysM-RLK) gene family in Brachypodium distachyon and docking analysis of chitin/LYK binding Guzin Tombuloglu, Huseyin Tombuloglu, Emre Cevik, Hussain Sabit PII:
S0885-5765(19)30006-2
DOI:
https://doi.org/10.1016/j.pmpp.2019.03.002
Reference:
YPMPP 1405
To appear in:
Physiological and Molecular Plant Pathology
Received Date: 7 January 2019 Revised Date:
4 March 2019
Accepted Date: 5 March 2019
Please cite this article as: Tombuloglu G, Tombuloglu H, Cevik E, Sabit H, Genome-wide identification of Lysin-Motif Receptor-Like Kinase (LysM-RLK) gene family in Brachypodium distachyon and docking analysis of chitin/LYK binding, Physiological and Molecular Plant Pathology (2019), doi: https:// doi.org/10.1016/j.pmpp.2019.03.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Genome-wide identification of Lysin-Motif Receptor-Like Kinase (LysM-
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RLK) gene family in Brachypodium distachyon and docking analysis of
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chitin/LYK binding
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Guzin Tombuloglu1, Huseyin Tombuloglu2 *, Emre Cevik2, Hussain Sabit2
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Adnan Kahveci Mah, Mimar Sinan Cad., Mavisu evl, 7/28 Beylikduzu-Istanbul, Turkey
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Department of Genetics Research, Institute for Research and Medical Consultations (IRMC),
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P.O. Box 1982, 34221, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
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* e-mail: [email protected] Phone: +966 530725817 ORCID: 0000-0001-8546-2658
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Abstract
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Lysin-Motif Receptor-Like Kinase (LysM-RLK) family proteins have critical function in
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plant-microbe interaction. During infection, common peptide domain of these proteins,
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namely LysM, interacts with bacterial gylcans or fungal chitins which triggers; (1) plant
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immune responses, (2) plant defense responses, or (3) plant-microorganism symbiotic
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interactions. Since pathogenic diseases cause severe annual loss in cereals, a better
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understanding of the molecular basis of host-pathogen interactions is required to facilitate the
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development of new resistance strategies. Hence, this study focuses on LysM-RLK gene
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family in Brachypodium, a model plant to study cereal-pathogen interactions. Potential roles
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of the identified proteins during pathogen infection was assessed by digital gene expression
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analyses and protein modelling. We found that Brachypodium genome comprises of 11 LysM-
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RLK genes, which were further divided into four major groups, namely LYK (4), LysMe (2),
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LysMn (1), and LYP (4). They were mostly localized in plasma membrane and outer
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ACCEPTED MANUSCRIPT membrane (extracellular). BdLysMe genes were found to be tandem duplicated. Expression
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analyses showed that BdLYK2, BdLYK3, and BdLYK4 were up-regulated after Fusarium
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graminearum (Fg) infection. Modelling and docking analyses of BdLYK proteins with fungal
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chitin revealed two the most susceptible LYKs (BdLYK3 and BdLYK4) possibly having role
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in chitin recognition and induction of plant immune/defense responses. Results of this study
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can contribute to the understanding of plant-microbe interactions by assessing the structure
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and putative function of LysM-RLK proteins in Brachypodium, a model plant to study cereal-
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pathogen interactions.
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Keywords: LysM-RLK; Brachypodium distachyon; protein modelling; docking; chitin
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1. Introduction
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The molecular understanding of plant-microbe interaction is the hotspot of science to control
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the diseases in order to improve the pathogen resistance of plants (Poole 2017). Plant
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genomes evolved by millions of years to successfully gain control of unfavorable conditions
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like pathogenic infections (Imam et al. 2016; McDonald and Stukenbrock 2016). Infection of
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plants evokes some physiological and molecular responses in the cell, such as immune
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response. Upon infection, molecular markers secreted from the pathogen provokes innate
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immune responses known as pathogen-associated molecular patterns (PAMP)-triggered
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immunity (PTI). In addition, pathogens secrete effector molecules into the plant cell to
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suppress the PTI. Therefore, pathogens become a virulent for the host organism. This
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mechanism is called effector-triggered immunity (ETI) (Qiu et al. 2015).
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Many pathogen-derived glycans such as bacterial peptidoglycans, fungal chitin or
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rhizobacterial Nod factors (NFs) can activate the immune response or symbiosis (Gust et al.
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2012). For instance, as a characteristic component of fungal cell wall, chitin releases into the
ACCEPTED MANUSCRIPT plant cell after the pathogen infection and serve as a PAMP that activates immune response.
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Cell membrane localized lysin motif-receptor-like kinase (LysM-RLK) proteins detect these
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molecules and activates the mitogen-activated protein kinase (MAPK) cascade. Thus, the
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plant cell is warned against the pathogen and activates multiple defense responses, including
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PTI and generation of reactive oxygen species, defense gene activation, biosynthesis/signaling
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of plant stress/defense hormones, phytoalexin biosynthesis, and cell wall strengthening (Meng
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and Zhang 2013; Qui et al. 2015).
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In addition, LysM-RLK proteins is the key molecule mediating the recognition of
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glycans to initiate the PTI response during bacterial infection. LysM domain of those proteins
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has critical function for pathogen recognition. N-acetylglucosamine (GlcNAc) including
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peptidoglycan, an essential component of the bacterial cell wall, is the primary target for
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LysM domain for pathogen recognition. It was found even in all organisms except the
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Archaeal proteins (Buist et al. 2008). The size of the LysM domain is variable, which changes
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from 35 to 50 aa. LysM have a conserved three-dimensional βααβ structure with two α-
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helices surrounded by a two-stranded antiparallel β-sheet (Bateman and Bycroft, 2000; Liu et
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al. 2011; Gust et al. 2012). LsyM-RLK proteins harbor variable number of tandem LysM
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domains (one to three) (Zhang et al. 2009).
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So far, limited number of plant LysM-RLK proteins have been functionally identified.
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For instance, rice chitin elicitor binding protein (CEBiP) is the first characterized membrane
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protein that binds to chitin and evoke chitin-inducible immune responses (Kaku et al. 2006;
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Kouzai et al. 2014). Knockdown of CEBiP gene resulted in increased susceptibility to blast
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fungus Magnaporthe oryzae in rice (Kaku et al. 2006; Kishimoto et al. 2010). Similarly,
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AtCERK1 was identified as an essential protein for chitin elicitor signalling in Arabidopsis
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(Miya et al. 2007). Of its rice ortholog, OsCERK1 was found to be involved in the regulation
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of chitin elicitor signalling (Shimizu et al. 2010). Other than chitin and its derivatives, PTI is
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ACCEPTED MANUSCRIPT induced by bacterial PGNs (plastoglandines). In Arabidopsis two LYP group members,
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namely LYM1 and LYM3 (or represented as LYP1 and LYP3), were identified as
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peptidoglycan receptors (Willmann et al. 2011). The third homolog protein LYM2 (AtCEBiP)
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is the major chitin-binding protein in the Arabidopsis membrane and involved in chitin
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perception (Shinya et al. 2012; Faulkner et al. 2013). In addition, Wan et al. (2012) suggested
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that LYK4 can be involved in Arabidopsis chitin signalling and plant innate immunity.
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Because of this perception and being mediator role of LysM RLKs, they are proposed to be
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the second major class of plant PRRs (pattern recognition receptor) after LRR-RP/LRR-RK
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family that mainly comprises sensors for proteinaceous pathogenic patterns (Gust et al. 2012).
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LysM proteins are not only serving as a receptor to trigger immune response, but also
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they have key role in plant-microorganism symbiotic interactions, such as leguminous plants
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with rhizobacterial species, or mycorrhizal fungi with the root system of a wide variety of
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plants (Fliegmann 2013; Sun et al. 2015). Microbial effector proteins secreted from pathogens
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have a LysM domain that binds LysM-proteins of host plant, which lead to suppression of the
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immune system. So, a symbiotic interaction can be maintained through this depletion. For
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instance, a LysM-containing effector protein `extracellular protein 6` (Ecp6) secreted from
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fungal pathogen Cladosporium fulvum, binds to chitin with a high specificity (de Jonge et al.
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2009, 2010). Together with, heterolog expression of two LysM-RLK genes (NFP and LYK3)
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from Medicago truncatula induced defense-like responses in Nicotiana benthamiana
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(Pietraszewska-Bogiel et al. 2013).
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For the cereals, pathogen-derived injury or loss is a challenge for producers. As a grass
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in the Pooideae subfamily of the Poaceae, Brachypodium distachyon exhibits fundamental
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genomics resource for C3 temperate cereals such as wheat, barley, and rye. In addition, it was
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suggested as a model pathosytem to understand the cereal-microbe interactions (Schneebei et
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al. 2014; Fitzgerald et al. 2015). Extensive genome and transcriptome-wide analysis of multi-
ACCEPTED MANUSCRIPT gene families has become possible thanks to the recent sequencing technologies (Bostancioglu
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et al. 2018; Tombuloglu et al. 2013; 2015; 2016; Tombuloglu 2018). In recent years, NBS
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(nucleotide-binding site) disease resistance genes and MLO (powdery mildew locus O) gene
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families were identified from the Brachypodium genome (Tan and Wu, 2012; Ablazov and
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Tombuloglu 2016). The current study focuses on LysM-RLK family members in
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Brachypodium for a better understating of plant-microbe interaction. Genome-wide
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identification exhibited the BdLysM-RLK members. Comparative phylogenetic analysis,
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digital expression profiling, homology modeling and docking analysis give insights about
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their potential roles during pathogen infection.
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Fig 1. Subcellular localization of BdLysM-RLK proteins. Among them, eight proteins are
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membrane localized; one extracellular, and two intracellular resided. Those proteins
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possess at least one and at most two LysM domains. According their domain
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architecture, they divided into four groups: LYKs (4), LYPs (4), LysMes (2), LysMn
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(1). LYPs, glycosylphosphatidylinositol (GPI)-anchored LysM proteins; LYKs, LysM
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receptor kinases; LysMe, extracellular LysM proteins; LysMn, intracellular non-
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secretory LysM proteins.
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2. Material and Methods
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2.1. Identification of LysM-RLK family members and LysM domain detection
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LysM-RLK family proteins from Arabidopsis was used to search for identification of LysM-
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RLK members in Brachypodium genome. The protein and gene sequences of AtLysM-RLK
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were
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(http://www.arabidopsis.org/). In total, 14 LysM-RLK members from Arabidopsis were used
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as query against the Brachypodium genome (v3.1) at the Phytozome database
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(https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Bdistachyon). A local blastP
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program (NCBI-Blast 2.7.1) was used to find the homology, with a score lower than e-10
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criterion. The candidate BdLysM-RLK members were searched for the required LysM
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domain (Pfam ID: PF01476), which is a characteristic module for the LysM-RLK family.
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HMM batch search in Pfam (http://www.pfam.sanger.ac.uk/) (Punta et al. 2011) and SMART
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(http://smart.embl-heidelberg.de) (Letunic et al. 2004) databases were used in order to verify
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the presence of a complete and conserved LysM domain (e-value < 10-5). The proteins with at
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least one LysM domain were selected as the family member. Also, according to the domain
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organisation (additional domain or GPI anchor), they were designated as LysMn, LYK, YLP,
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and LysMe. F-box-like (PF12937), protein tyrosine kinase (PF07714), and protein kinase
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domain (PF00069) were determined (e <10-5). Then, LysM domains were extracted from the
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protein sequences and conserved residues were shown by using the Weblogo analysis
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(http://weblogo.berkeley.edu/logo.cgi) (Crooks et al. 2004).
from
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Arabidopsis
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2.2. Phylogenetic analyses
ACCEPTED MANUSCRIPT Protein sequences of Arabidopsis and Brachypodium LysM-RLKs were aligned by MUSCLE
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and phylogenetic tree was constructed using UPGMA method (Sneath and Sokal 1973) with
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default parameters in Mega7 (Kumar et al. 2016). In addition, proteins belong to different
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LysM-RLK groups were aligned with ClustalW with BLOSUM as the protein weight matrix.
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The phylogeny was evaluated using the Neighbor-Joining (NJ) method. 1000 replication as
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the bootstrap method, Poisson correction model and complete deletion parameters were
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applied to construct the trees. Also, the predicted BdLysM-RLK protein sequences were
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aligned with LysM-RLK members from Arabidopsis (14), Glycine max (47), Medicago
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truncatula (17), and Oryza sativa (20), and a comparative phylogenetic tree was constructed
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by using the same parameters. The protein alignments were visualized by using Jalview
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program (Waterhouse et al. 2009). Gene duplication analysis was performed by aligning the
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BdLysM-RLK
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(http://www.ebi.ac.uk/Tools/msa/clustalw2/) (Thompson et al. 1994). The genes were
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accepted as duplicated according to the following criteria: the similarity of the coding
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nucleotide sequences > 80 % and the identity between the sequences > 80 % (Gu et al. 2002;
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Yang et al. 2008).
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2.3. Digital expression analyses of LysM-RLK genes
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Tissue spesific expressions of each LysM-RLK genes were represented by calculating the
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relative expression values (log2) obtained from Brachypodium eFP expression browser
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(http://bar.utoronto.ca/efp_brachypodium/cgi-bin/efpWeb.cgi; Sibout et al. 2017: Winter et al.
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2007). RStudio (version 1.0.143) program was used to build up the heatmaps. Also, in order
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to show LysM-RLK expression changes upon fungal infection, we assessed a GEO dataset
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(GPL15863) including the Brachypodium seedlings infected by either Fusarium graminearum
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(Fusarium head blight (FHB) causing agent) (named as DON+) or a F. graminearum strain
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with reduced virulence on Brachypodium spikes (named as DON-). The expression data was
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normalized by GCRMA algoritm (Bolstad et al. 2003).
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2.4. Modelling of BdLYKs and docking analysis with chitin ligand
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Three dimensional structure of four BdLysM-LYK proteins was generated using the I-
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TASSER tool (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (Yang et al. 2015). Chitin
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structure (ZINC24425833) was downloaded from Zinc database (http://zinc.docking.org/;
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Irwin et al. 2012). 1-CLICK DOCKING program (https://mcule.com/apps/1-click-docking/)
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was used to target the ligand. Hydrogen atoms and Gasteiger charges were added in the
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molecules. LysM domain was targeted to show the binding affinity of LysM with chitin
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molecule. Protein secondary structure was submitted to Deep-view Swiss-pdb Viewer tool
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(http://spdbv.vital-it.ch/) (Guex and Peitsch 1997). PyMOL 2.2 program was used to visulize
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the molecular structures and surface interactions (DeLano 2002).
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3. Results and Discussion
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3.1. Identification and domain organization of LysM-RLK proteins in Brachypodium
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Genome-wide identification of Lysin-Motif Receptor-Like (LysM-RLK) family members
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revealed that Brachypodium genome harbors 11 LysM-RLK genes. The amino acid sequences
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were listed in Supplementary Table 2. Major differences in terms of conserved domain
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organization separated them into four major groups: LYK (4), LysMe (2), LysMn (1), and
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LYP (4) (Supplementary Fig. 1). Characteristic motif and domain structures of each group are
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shown in Fig. 1 and Table 1. Accordingly, LYKs are composed of one LysM domain and
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additional single protein tyrosine kinase or protein kinase domain. LYPs harbored two
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extracellular tandem LysM domains. Some of these proteins are located as transmembrane,
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and some are attached to the membrane with a GPI (glycosylphosphatidylinositol) anchor.
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The two LysMe proteins had a single LysM domain and they are localized in intracellular or
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extracellular matrix. LysMn protein harbored single LysM and F box-like domain with
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intracellular localization. Exon and intron structures were varied among the LysM-RLK members (Table 2).
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However, four genes (BdLYK3, BdLYK4, BdLysMe1, and BdLysMe2) are composed of single
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exon, seems to escape alternative splicing. Subcellular localization of LysM-RLK proteins
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were predicted with CELLO program and revealed membrane, chloroplast, cytoplasm,
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nucleus, and extracellular resident proteins. This observation agrees with the previous
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observation by Gust et al. (2012) who depicted that LysM-RLK members localize according
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their signal peptides. Also, transmembrane helices (TMH) of the LysM proteins were
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predicted using PRED-TMR algorithm which supports the cellular localizations of LysM
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proteins (Table 2). In general, functionally characterized LysMe proteins reside inside or
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outside of the cell. However, BdLysMe1 and BdLysMe2 were predicted to include one
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transmembrane helix and may potentially localize in the cell membrane.
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All BdLysM-RLK family proteins have at least one LysM domain with changing
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amino acid length (29 to 43) (Table 1). In addition to LysM domain, LYP members include
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additional LysM domain. Also, F-box-like, protein tyrosine kinase and protein kinase
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domains were the characteristic modules for LysMn, and LYK, respectively. LysMe members
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were comprised of single LysM domain (Table 1; Fig. 1). The number of LysM domains
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within individual protein range from one to twelve across kingdoms (Zhang et al 2009). This
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number can confer flexibility for binding and target selection. In addition, multiple LysM
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modules (1 to 6) increase the binding to peptidoglycan (Mesnage et al. 2014). In rice,
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OsCERK1 contains only one conserved LysM domain and does not bind to
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chitooligosaccharides. Instead, heterodimerization with OsCEBiP (containing two LysM
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domains) is required for chitin binding and innate immunity response (Shimizu et al. 2010).
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ACCEPTED MANUSCRIPT Moreover, the number of LysM in plants is variable for LYK proteins, which ranges from one
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to three (Zhang et al. 2009). Chitin binding site of AtLYK5 was formed by three LysM
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modules with a binding energy of −8.9 kcal mol−1 (Cao et al. 2014). On the other hand, it was
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reported that the second LysM of AtCERK1 is essential for chitin binding (Liu et al. 2012).
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Similarity, the second LysM of OsCEBiP is essential for chitin binding (Hayafune et al.
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2014). However, in Brachypodium, all BdLYK members composed of single LysM module.
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Further studies are required to understand LYK interaction model in Brachypodium during
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the activation of immune system.
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ACCEPTED MANUSCRIPT Fig 2. Chromosome distribution of LysM-RLK genes in Brachypodium genome. Tandem
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duplicated genes were labelled with blue rectangular. Position of the genes were stated
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with blue and red arrows representing (–) or (+) strand, respectively. The chromosome
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size was shown at the bottom of each representative chromosome as mega base (Mb)
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pair.
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3.2. Characteristics of LysM-RLKs in Brachypodium
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The information regarding protein characteristics (pI, GRAVY score, MW, and length) of
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each BdLysM-RLK members were retrieved from SwissProt and Expasy’s ProtParam
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(Gasteiger, 2005) databases and listed in Table 2. Analysis showed that they are varied and
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well-separated between each subgroup (LysMe, LYK, LysMn, and LYP). For instance, LYK
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proteins (LYK1 to LYK4) were relatively large proteins than the members from other
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subgroups in terms of amino acid length (average is 658 aa) and molecular weight (average is
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70 kDa). LysMe subgroup harbored small sized protein members with an average protein
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length of 96 aa. The isoelectric points (pI) are varied between 8.39 (BdLYP1) to 4.55
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(BdLYP4). According to pI value, most of the proteins (eight members, 72.7%) showed acidic
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character. Hydropathicity (GRAVY) index ranged between -0.4 (BdLysMn) and 0.4
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(BdLysMe1). Two of the BdLysMe members (BdLysMe1 and BdLysMe2) have the highest
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GRAVY score showing hydrophobic character. Additionally, most of the LysM-RLK proteins
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(eight members, 72.7%) exhibited hydrophobic nature.
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3.3. Chromosome distribution and duplication analysis of LysM-RLK genes in
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Brachypodium
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According to the chromosomal distribution, we found that LysM-RLK genes resided in all
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chromosomes except chromosome 5. The positions of genes and strand (- or +) was shown in
ACCEPTED MANUSCRIPT Fig. 2. One tandem duplication was estimated between LysMe group members (LysMe1 and
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LysMe2) (Fig. 2; Supplementary Table 1). This duplication event contributed to the
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expansion of LysMe subgroup that may lead to neofunctionalization of these genes during
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pathogen infection. Previous reports indicate that the total number of LysM-RLK members
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varies between plant species (Table 3). So far, LysM-RLK families have been identified in
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Arabidopsis (14), Glycine max (47), Medicago truncatula (17), and Oryza sativa (20) (Zhang
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et al. 2009) (Table 3). Accordingly, Brachypodium genome harbors the smallest number of
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LysM-RLK proteins (11) among the plants identified for LysM-RLK family. The great
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difference of the number of LysM-RLK members in those species is likely due to the different
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rounds of genome duplication events occurred during the species diversification (Shin-Han
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and Bleecker 2003).
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BdLysM-RLK proteins were depicted with red circles. Functionally verified
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Arabidopsis proteins recognizing either chitin or PGN were labelled with blue and red
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colored triangle, respectively.
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3.4. Phylogenetic analysis of BdLysM-RLK proteins
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Brachypodium orthologs of functionally characterized LysM-RLK proteins from different
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plants can give insights about their potential roles. For this purpose, we compared the critical
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residues and domain sequences of known LysM-RLK family members with currently
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identified Brachypodium ones. In Fig. 3, phylogenetic tree shows the evolutionary
ACCEPTED MANUSCRIPT relationship between Arabidopsis and Brachypodium LysM-RLK proteins. Subgroups were
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well separated from each other. Functionally validated Arabidopsis LysM-RLK members are
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shown in the tree, which can provide information about the possible functions of BdLysM-
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RLKs targeting PGN or chitin. Accordingly, two functionally characterized proteins (AtLYP1
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and AtLYP3) which recognize the PGNs were resided in the LYP subgroup. Three proteins
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recognize the chitin (AtCERK1, AtLYK4, and AtLYK5) which harbored in the LYK
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subgroup. It is likely that LYP and LYK subgroups are specialized for PGN and chitin
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targeting, respectively. This can help to identify their Brachypodium orthologs, which is
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functionalized for the pathogen recognition during infection. Additionally, a comprehensive
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phylogenetic analysis was conducted to show the relationship of BdLysM-RLK family
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proteins with the selected plant species (G. max, Arabidopsis, M. truncatula, O. sativa) (Fig.
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Fig 4. Comparative phylogenetic analyses of LysM-RLK proteins from five selected plants;
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G. max, Arabidopsis (blue) M. truncatula (yellow), O. sativa (pink) and Brachypodium
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(red). NJ (Neighbor-joining) trees were shown for each distinct group, separately: (a)
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LYK, (b) LysMe (c) LYP, and (d) LysMn.
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3.5. Expressions of BdLysM-RLK genes upon Fusarium infection and tissues
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Analysis of BdLysM-RLK gene expressions in 44 different developmental stages and tissues
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were represented in Fig. 5a. Tissue specific, spatial and temporal expression patterns were
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detected. In addition, to understand the response of LysM-RLK family members to fungal
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infection, we analyzed the GEO database revealing the gene repertoire after Fusarium
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graminearum (Fg), Fusarium head blight (FHB) causing plant fungal pathogen, infection.
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Digital expression analysis of Brachypodium spikes infected by two different Fusarium
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strains, either DON+ (Fg ph-1, produce toxin) or DON- (Fg delta tri5 mutant, unable to
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produce toxin) showed that BdLYK2, BdLYK3, and BdLYK4 were up-regulated after infection
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(Fig. 5b). Interestingly, the induction was higher in Fg DON- than the Fg DON+ samples.
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Also, BdLYK4 expression in Fusarium-infected plants was dramatically increased (4 times)
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compared to the ones from mock-inoculated plants (Fig. 5b). In the phylogenetic analyses,
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BdLYK2, BdLYK3, and BdLYK4 were resided in the same clade with AtLYK4 and AtLYK5
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(Fig. 3). Previous studies demonstrated that AtLYK4 is important for chitin recognition
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during fungal infection (Wan et al. 2012). Moreover, AtLYK5 has been proposed as a major
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chitin receptor (Cao et al. 2014). In addition to fungal responsive expressional changes of
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BdLYK4, its sequence similarity (40-45%) and phylogenetic proximity with AtLYK4 and
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AtLYK5 pointed out that BdLYK4 could be the most susceptible gene in Brachypodium,
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which may play role in chitin recognition.
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Fig 5. Heatmap analysis of the BdLysM-RLK genes shows tissue specific expression. Color
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legend represents the log2 calculated relative expressions (a). Response of BdLysM-
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RLK genes after Fusarium graminearum (Fg) infection (b). DON+ and DON-
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represent the Brachypodium plants infected by different Fg strains with high or reduced virulence on Brachypodium spikes, respectively. Expressions were normalized by GCRMA method.
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LysMe1 expression did not change significantly upon Fg infection or mock
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inoculation. Interestingly BdLYP1 and BdLYP4 were decreased after Fg DON+ and DON-
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infection. Recent studies demonstrated that AtLYP2 and AtLYP3 are involved in PGN
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2013). However, they are not responsive to chitin oligosaccarides (Willmann et al. 2011;
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Shinya et al. 2012). Similarly, our analysis showed that BdLYP1 and BdLYP4 expression did
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not increase after Fg infection, even their expression were found to be decreased. These
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results suggested that BdLYP proteins could be spesific to bacterial PGNs rather than fungal
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chitin.
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Fig 6. Interaction of the LysM domain of the BdLYK proteins and triacetylchitotriose
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(ZINC24425833). a. LYK1 b. LYK2 c. LYK3 d. LYK4. Yellow lines depict hydrogen
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bonds formed between ligand atoms and their corresponding residues atoms.
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3.6. Chitin and LYK-LysM interaction
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during infection. Previous reports proved that AtCERK1, AtLYK4, and AtLYK5 can bind to
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chitin (Petutschnig et al. 2010; Shinya et al. 2012; Wan et al. 2012; Cao et al. 2014).
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However, AtLYK2, AtLYK3 and AtLYK5 do not appear to be involved in chitin elicitor
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signalling (Wan et al. 2012). To assess this interaction between BdLYKs and chitin molecules
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(Supplementary Fig. 2), modeling and docking studies were conducted. Three-dimensional
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structure of the proteins was computationally predicted and LysM domain was targeted by
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triacetylchitotriose (ZINC24425833). 1-Click Docking program calculates the binding
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affinities of -6.0, -7.8, -6.8, and -5.2 kcal/mol for BdLYK1, BdLYK2, BdLYK3, and
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BdLYK4, respectively (Fig. 6). Among these, BdLYK2 has the highest binding affinity (-7.8
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kcal/mol). It was -8.6 and -9.2 for AtLYK4-chitotetraose and AtCERK1-chitotetraose
347
interaction, respectively (Tanaka et al. 2013). The predicted three-dimensional models show
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that LysM domain of BdLYK3 and BdLYK4 has very similar structures (Fig. 6). However,
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their predicted chitin interacting residues are variable: R178, N223, and I231 for BdLYK3;
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and H196, L198, A250, D251, and L253 for BdLYK4 (Fig. 7a, b). Also, LysM domain
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architecture (including the first and the second LysM domains) from all identified BdLysM-
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RLK proteins exhibits a variable sequence pattern (Fig. 7c), which enables the LysM domain
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unique for target selection and suggesting that the proteins have been evolved to recognize
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variable targets.
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Fig 7. Alignment of LysM modules from BdLYK3, BdLYK4, AtCERK1, and AtLYK4. The
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conserved amino acid residues are labelled with blue font according to the percent
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identity score. Chitin binding residues were labelled with red rectangular (a): Docking
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model between triacetylchitotriose and proteins, BdLYK3 and BdLYK4 (b): Weblogo analysis shows the LysM domains resided in all BdLysM-RLK family members, including the first and the second LysMs for BdLYPs (c): Analysis show some
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conserved residues among the BdLYKs, which are abundant in between BdLYK3 and
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BdLYK4. However, LysM domain exhibits sequence variance within the BdLysM-
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RLK members.
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Compliance with ethical standards This article does not contain any studies with human
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participants or animals performed by any of the authors.
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Conflict of Interest All authors declare that they have no conflict of interest.
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Table 1. Domain structure of BdLysM-RLK proteins. Domain1
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113 197 200 217 127 114 107 119 111 54 48
156 240 243 244 171 160 156 165 164 97 87
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PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476
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LysM LysM LysM LysM LysM LysM LysM LysM LysM LysM LysM
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Bradi1g11687.2 Bradi4g16350.1 Bradi3g06770.1 Bradi3g51790.1 Bradi2g40627.1 Bradi4g37090.1 Bradi1g46200.1 Bradi1g76177.2 Bradi3g57756.2 Bradi2g51930.1 Bradi2g51851.1
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BdLysMn BdLYK3 BdLYK4 BdLYK2 BdLYK1 BdYLP1 BdYLP4 BdYLP2 BdYLP3 BdLysMe1 BdLysMe2
Domain Pfam ID Start End
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Prot ID
Domain2 HMM HMM from Domain length /to 1 44 44 F-box-like 10 44 44 Protein tyrosine kinase 1 44 44 Protein kinase domain 1 29 44 Protein kinase domain 1 44 44 Protein kinase domain 1 44 44 LysM 5 44 44 LysM 1 44 44 LysM 4 44 44 LysM 3 42 44 5 39 44
Pfam ID Start End
PF12937 PF07714 PF00069 PF00069 PF00069 PF01476 PF01476 PF01476 PF01476
39 386 403 398 273 178 176 183 184
78 644 671 655 548 221 218 226 226
HMM HMM from length /to 5 39 48 47 259 260 44 258 264 13 258 264 2 259 264 1 44 44 1 44 44 1 44 44 1 44 44
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Table 2. Characteristics of LysM-RLK in Brachypodium
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Subs Gravy Gene ID Exon Localization ** TMH* pI MW (kD) Score Chr Strand Chr Location BdLYK1 Bradi2g40627.1 10 PM Chlr Cyt 2 5,74 63.60 -0.045 2 + 40700069..40703030 52555458..52559509 BdLYK2 Bradi3g51790.1 4 PM Chlr 1 6,34 73.14 0.003 3 + 17136796..17139097 BdLYK3 Bradi4g16350.1 1 PM Chlr 2 8,65 70.29 0.003 4 + BdLYK4 Bradi3g06770.1 1 PM 3 6,87 73.63 -0.001 3 + 5002793..5004886 51247464..51248121 BdLysMe1 Bradi2g51930.1 1 Extracellular 1 5,45 10.43 0.462 2 + BdLysMe2 Bradi2g51851.1 1 Extracellular 1 5,04 9.56 0.303 2 51206759..51207037 BdLysMn Bradi1g11687.2 2 Nucl 0 7,77 28.09 -0.402 1 8694155..8697817 42224657..42227694 BdLYP1 Bradi4g37090.1 2 Extracellular 1 8,39 38.44 0.354 4 + BdLYP2 Bradi1g76177.2 4 Extracellular 1 6,05 37.56 0.225 1 73079251..73081951 57189190..57192732 BdLYP3 Bradi3g57756.2 5 Extracellular 1 5,11 43.48 0.258 3 + 44732649..44736017 BdLYP4 Bradi1g46200.1 6 Extracellular 0 4,55 39.81 0.305 1 * TMH transmembrane helices were predicted using PRED-TMR algorithm (Pasquier et al. 1999) http://athina.biol.uoa.gr/PRED-TMR/
Nucl (bp) 2962 4062 2302 2094 658 279 3663 3038 2701 3643 3369
CDS (bp) Prot (aa) 1734 577 2049 682 2031 676 2094 697 308 101 279 92 768 255 1122 373 1089 362 1302 433 1194 397
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** Predicted using CELLO v.2.5: subCELlular LOcalization predictor http://cello.life.nctu.edu.tw/ (Yu et al. 2006). PM, plasma membrane; Chlr, chloroplast; Nucl, nucleus; Cyt, cytoplasm.
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Mm Type Bd At Gm Mt Os 9 LYK 4 5 21 8 6 2 LYP 4 3 4 2 6 NA LysMe 2 3 16 5 4 NA LysMn 1 3 6 2 4 11 Total 11 14 47 17 20 Lv et al. (2018) Reference This study Zhang et al. (2009) Bd, B. distacyon; At, A. thaliana; Gm, G. max; Mt, M. truncatula; Os, O. sativa; Mm, Morus multicaulis; NA, not applied.
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Table 3. Number of LysM-RLK family members from various plants.
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Highlights Brachypodium genome comprises of 11 LysM-RLK genes
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BdLysMe genes were found to be tandem duplicated
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Fungal chitin and LYK interaction was modelled by docking analyses
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BdLYK3 and BdLYK4 are the most susceptible LYKs for chitin perception
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S0885-5765(19)30006-2
DOI:
https://doi.org/10.1016/j.pmpp.2019.03.002
Reference:
YPMPP 1405
To appear in:
Physiological and Molecular Plant Pathology
Received Date: 7 January 2019 Revised Date:
4 March 2019
Accepted Date: 5 March 2019
Please cite this article as: Tombuloglu G, Tombuloglu H, Cevik E, Sabit H, Genome-wide identification of Lysin-Motif Receptor-Like Kinase (LysM-RLK) gene family in Brachypodium distachyon and docking analysis of chitin/LYK binding, Physiological and Molecular Plant Pathology (2019), doi: https:// doi.org/10.1016/j.pmpp.2019.03.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Genome-wide identification of Lysin-Motif Receptor-Like Kinase (LysM-
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RLK) gene family in Brachypodium distachyon and docking analysis of
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chitin/LYK binding
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Guzin Tombuloglu1, Huseyin Tombuloglu2 *, Emre Cevik2, Hussain Sabit2
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Adnan Kahveci Mah, Mimar Sinan Cad., Mavisu evl, 7/28 Beylikduzu-Istanbul, Turkey
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Department of Genetics Research, Institute for Research and Medical Consultations (IRMC),
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P.O. Box 1982, 34221, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
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* e-mail: [email protected] Phone: +966 530725817 ORCID: 0000-0001-8546-2658
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Abstract
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Lysin-Motif Receptor-Like Kinase (LysM-RLK) family proteins have critical function in
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plant-microbe interaction. During infection, common peptide domain of these proteins,
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namely LysM, interacts with bacterial gylcans or fungal chitins which triggers; (1) plant
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immune responses, (2) plant defense responses, or (3) plant-microorganism symbiotic
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interactions. Since pathogenic diseases cause severe annual loss in cereals, a better
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understanding of the molecular basis of host-pathogen interactions is required to facilitate the
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development of new resistance strategies. Hence, this study focuses on LysM-RLK gene
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family in Brachypodium, a model plant to study cereal-pathogen interactions. Potential roles
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of the identified proteins during pathogen infection was assessed by digital gene expression
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analyses and protein modelling. We found that Brachypodium genome comprises of 11 LysM-
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RLK genes, which were further divided into four major groups, namely LYK (4), LysMe (2),
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LysMn (1), and LYP (4). They were mostly localized in plasma membrane and outer
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ACCEPTED MANUSCRIPT membrane (extracellular). BdLysMe genes were found to be tandem duplicated. Expression
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analyses showed that BdLYK2, BdLYK3, and BdLYK4 were up-regulated after Fusarium
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graminearum (Fg) infection. Modelling and docking analyses of BdLYK proteins with fungal
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chitin revealed two the most susceptible LYKs (BdLYK3 and BdLYK4) possibly having role
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in chitin recognition and induction of plant immune/defense responses. Results of this study
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can contribute to the understanding of plant-microbe interactions by assessing the structure
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and putative function of LysM-RLK proteins in Brachypodium, a model plant to study cereal-
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pathogen interactions.
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Keywords: LysM-RLK; Brachypodium distachyon; protein modelling; docking; chitin
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1. Introduction
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The molecular understanding of plant-microbe interaction is the hotspot of science to control
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the diseases in order to improve the pathogen resistance of plants (Poole 2017). Plant
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genomes evolved by millions of years to successfully gain control of unfavorable conditions
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like pathogenic infections (Imam et al. 2016; McDonald and Stukenbrock 2016). Infection of
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plants evokes some physiological and molecular responses in the cell, such as immune
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response. Upon infection, molecular markers secreted from the pathogen provokes innate
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immune responses known as pathogen-associated molecular patterns (PAMP)-triggered
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immunity (PTI). In addition, pathogens secrete effector molecules into the plant cell to
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suppress the PTI. Therefore, pathogens become a virulent for the host organism. This
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mechanism is called effector-triggered immunity (ETI) (Qiu et al. 2015).
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Many pathogen-derived glycans such as bacterial peptidoglycans, fungal chitin or
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rhizobacterial Nod factors (NFs) can activate the immune response or symbiosis (Gust et al.
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2012). For instance, as a characteristic component of fungal cell wall, chitin releases into the
ACCEPTED MANUSCRIPT plant cell after the pathogen infection and serve as a PAMP that activates immune response.
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Cell membrane localized lysin motif-receptor-like kinase (LysM-RLK) proteins detect these
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molecules and activates the mitogen-activated protein kinase (MAPK) cascade. Thus, the
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plant cell is warned against the pathogen and activates multiple defense responses, including
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PTI and generation of reactive oxygen species, defense gene activation, biosynthesis/signaling
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of plant stress/defense hormones, phytoalexin biosynthesis, and cell wall strengthening (Meng
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and Zhang 2013; Qui et al. 2015).
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In addition, LysM-RLK proteins is the key molecule mediating the recognition of
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glycans to initiate the PTI response during bacterial infection. LysM domain of those proteins
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has critical function for pathogen recognition. N-acetylglucosamine (GlcNAc) including
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peptidoglycan, an essential component of the bacterial cell wall, is the primary target for
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LysM domain for pathogen recognition. It was found even in all organisms except the
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Archaeal proteins (Buist et al. 2008). The size of the LysM domain is variable, which changes
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from 35 to 50 aa. LysM have a conserved three-dimensional βααβ structure with two α-
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helices surrounded by a two-stranded antiparallel β-sheet (Bateman and Bycroft, 2000; Liu et
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al. 2011; Gust et al. 2012). LsyM-RLK proteins harbor variable number of tandem LysM
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domains (one to three) (Zhang et al. 2009).
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So far, limited number of plant LysM-RLK proteins have been functionally identified.
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For instance, rice chitin elicitor binding protein (CEBiP) is the first characterized membrane
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protein that binds to chitin and evoke chitin-inducible immune responses (Kaku et al. 2006;
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Kouzai et al. 2014). Knockdown of CEBiP gene resulted in increased susceptibility to blast
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fungus Magnaporthe oryzae in rice (Kaku et al. 2006; Kishimoto et al. 2010). Similarly,
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AtCERK1 was identified as an essential protein for chitin elicitor signalling in Arabidopsis
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(Miya et al. 2007). Of its rice ortholog, OsCERK1 was found to be involved in the regulation
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of chitin elicitor signalling (Shimizu et al. 2010). Other than chitin and its derivatives, PTI is
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ACCEPTED MANUSCRIPT induced by bacterial PGNs (plastoglandines). In Arabidopsis two LYP group members,
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namely LYM1 and LYM3 (or represented as LYP1 and LYP3), were identified as
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peptidoglycan receptors (Willmann et al. 2011). The third homolog protein LYM2 (AtCEBiP)
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is the major chitin-binding protein in the Arabidopsis membrane and involved in chitin
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perception (Shinya et al. 2012; Faulkner et al. 2013). In addition, Wan et al. (2012) suggested
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that LYK4 can be involved in Arabidopsis chitin signalling and plant innate immunity.
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Because of this perception and being mediator role of LysM RLKs, they are proposed to be
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the second major class of plant PRRs (pattern recognition receptor) after LRR-RP/LRR-RK
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family that mainly comprises sensors for proteinaceous pathogenic patterns (Gust et al. 2012).
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LysM proteins are not only serving as a receptor to trigger immune response, but also
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they have key role in plant-microorganism symbiotic interactions, such as leguminous plants
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with rhizobacterial species, or mycorrhizal fungi with the root system of a wide variety of
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plants (Fliegmann 2013; Sun et al. 2015). Microbial effector proteins secreted from pathogens
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have a LysM domain that binds LysM-proteins of host plant, which lead to suppression of the
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immune system. So, a symbiotic interaction can be maintained through this depletion. For
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instance, a LysM-containing effector protein `extracellular protein 6` (Ecp6) secreted from
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fungal pathogen Cladosporium fulvum, binds to chitin with a high specificity (de Jonge et al.
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2009, 2010). Together with, heterolog expression of two LysM-RLK genes (NFP and LYK3)
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from Medicago truncatula induced defense-like responses in Nicotiana benthamiana
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(Pietraszewska-Bogiel et al. 2013).
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For the cereals, pathogen-derived injury or loss is a challenge for producers. As a grass
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in the Pooideae subfamily of the Poaceae, Brachypodium distachyon exhibits fundamental
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genomics resource for C3 temperate cereals such as wheat, barley, and rye. In addition, it was
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suggested as a model pathosytem to understand the cereal-microbe interactions (Schneebei et
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al. 2014; Fitzgerald et al. 2015). Extensive genome and transcriptome-wide analysis of multi-
ACCEPTED MANUSCRIPT gene families has become possible thanks to the recent sequencing technologies (Bostancioglu
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et al. 2018; Tombuloglu et al. 2013; 2015; 2016; Tombuloglu 2018). In recent years, NBS
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(nucleotide-binding site) disease resistance genes and MLO (powdery mildew locus O) gene
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families were identified from the Brachypodium genome (Tan and Wu, 2012; Ablazov and
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Tombuloglu 2016). The current study focuses on LysM-RLK family members in
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Brachypodium for a better understating of plant-microbe interaction. Genome-wide
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identification exhibited the BdLysM-RLK members. Comparative phylogenetic analysis,
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digital expression profiling, homology modeling and docking analysis give insights about
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their potential roles during pathogen infection.
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Fig 1. Subcellular localization of BdLysM-RLK proteins. Among them, eight proteins are
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membrane localized; one extracellular, and two intracellular resided. Those proteins
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possess at least one and at most two LysM domains. According their domain
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architecture, they divided into four groups: LYKs (4), LYPs (4), LysMes (2), LysMn
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(1). LYPs, glycosylphosphatidylinositol (GPI)-anchored LysM proteins; LYKs, LysM
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receptor kinases; LysMe, extracellular LysM proteins; LysMn, intracellular non-
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secretory LysM proteins.
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2. Material and Methods
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2.1. Identification of LysM-RLK family members and LysM domain detection
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LysM-RLK family proteins from Arabidopsis was used to search for identification of LysM-
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RLK members in Brachypodium genome. The protein and gene sequences of AtLysM-RLK
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were
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(http://www.arabidopsis.org/). In total, 14 LysM-RLK members from Arabidopsis were used
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as query against the Brachypodium genome (v3.1) at the Phytozome database
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(https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Bdistachyon). A local blastP
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program (NCBI-Blast 2.7.1) was used to find the homology, with a score lower than e-10
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criterion. The candidate BdLysM-RLK members were searched for the required LysM
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domain (Pfam ID: PF01476), which is a characteristic module for the LysM-RLK family.
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HMM batch search in Pfam (http://www.pfam.sanger.ac.uk/) (Punta et al. 2011) and SMART
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(http://smart.embl-heidelberg.de) (Letunic et al. 2004) databases were used in order to verify
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the presence of a complete and conserved LysM domain (e-value < 10-5). The proteins with at
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least one LysM domain were selected as the family member. Also, according to the domain
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organisation (additional domain or GPI anchor), they were designated as LysMn, LYK, YLP,
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and LysMe. F-box-like (PF12937), protein tyrosine kinase (PF07714), and protein kinase
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domain (PF00069) were determined (e <10-5). Then, LysM domains were extracted from the
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protein sequences and conserved residues were shown by using the Weblogo analysis
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(http://weblogo.berkeley.edu/logo.cgi) (Crooks et al. 2004).
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Arabidopsis
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2.2. Phylogenetic analyses
ACCEPTED MANUSCRIPT Protein sequences of Arabidopsis and Brachypodium LysM-RLKs were aligned by MUSCLE
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and phylogenetic tree was constructed using UPGMA method (Sneath and Sokal 1973) with
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default parameters in Mega7 (Kumar et al. 2016). In addition, proteins belong to different
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LysM-RLK groups were aligned with ClustalW with BLOSUM as the protein weight matrix.
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The phylogeny was evaluated using the Neighbor-Joining (NJ) method. 1000 replication as
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the bootstrap method, Poisson correction model and complete deletion parameters were
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applied to construct the trees. Also, the predicted BdLysM-RLK protein sequences were
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aligned with LysM-RLK members from Arabidopsis (14), Glycine max (47), Medicago
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truncatula (17), and Oryza sativa (20), and a comparative phylogenetic tree was constructed
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by using the same parameters. The protein alignments were visualized by using Jalview
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program (Waterhouse et al. 2009). Gene duplication analysis was performed by aligning the
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BdLysM-RLK
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(http://www.ebi.ac.uk/Tools/msa/clustalw2/) (Thompson et al. 1994). The genes were
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accepted as duplicated according to the following criteria: the similarity of the coding
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nucleotide sequences > 80 % and the identity between the sequences > 80 % (Gu et al. 2002;
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Yang et al. 2008).
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2.3. Digital expression analyses of LysM-RLK genes
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Tissue spesific expressions of each LysM-RLK genes were represented by calculating the
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relative expression values (log2) obtained from Brachypodium eFP expression browser
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(http://bar.utoronto.ca/efp_brachypodium/cgi-bin/efpWeb.cgi; Sibout et al. 2017: Winter et al.
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2007). RStudio (version 1.0.143) program was used to build up the heatmaps. Also, in order
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to show LysM-RLK expression changes upon fungal infection, we assessed a GEO dataset
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(GPL15863) including the Brachypodium seedlings infected by either Fusarium graminearum
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(Fusarium head blight (FHB) causing agent) (named as DON+) or a F. graminearum strain
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with reduced virulence on Brachypodium spikes (named as DON-). The expression data was
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normalized by GCRMA algoritm (Bolstad et al. 2003).
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2.4. Modelling of BdLYKs and docking analysis with chitin ligand
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Three dimensional structure of four BdLysM-LYK proteins was generated using the I-
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TASSER tool (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (Yang et al. 2015). Chitin
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structure (ZINC24425833) was downloaded from Zinc database (http://zinc.docking.org/;
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Irwin et al. 2012). 1-CLICK DOCKING program (https://mcule.com/apps/1-click-docking/)
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was used to target the ligand. Hydrogen atoms and Gasteiger charges were added in the
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molecules. LysM domain was targeted to show the binding affinity of LysM with chitin
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molecule. Protein secondary structure was submitted to Deep-view Swiss-pdb Viewer tool
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(http://spdbv.vital-it.ch/) (Guex and Peitsch 1997). PyMOL 2.2 program was used to visulize
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the molecular structures and surface interactions (DeLano 2002).
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3. Results and Discussion
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3.1. Identification and domain organization of LysM-RLK proteins in Brachypodium
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Genome-wide identification of Lysin-Motif Receptor-Like (LysM-RLK) family members
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revealed that Brachypodium genome harbors 11 LysM-RLK genes. The amino acid sequences
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were listed in Supplementary Table 2. Major differences in terms of conserved domain
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organization separated them into four major groups: LYK (4), LysMe (2), LysMn (1), and
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LYP (4) (Supplementary Fig. 1). Characteristic motif and domain structures of each group are
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shown in Fig. 1 and Table 1. Accordingly, LYKs are composed of one LysM domain and
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additional single protein tyrosine kinase or protein kinase domain. LYPs harbored two
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extracellular tandem LysM domains. Some of these proteins are located as transmembrane,
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and some are attached to the membrane with a GPI (glycosylphosphatidylinositol) anchor.
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The two LysMe proteins had a single LysM domain and they are localized in intracellular or
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extracellular matrix. LysMn protein harbored single LysM and F box-like domain with
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intracellular localization. Exon and intron structures were varied among the LysM-RLK members (Table 2).
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However, four genes (BdLYK3, BdLYK4, BdLysMe1, and BdLysMe2) are composed of single
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exon, seems to escape alternative splicing. Subcellular localization of LysM-RLK proteins
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were predicted with CELLO program and revealed membrane, chloroplast, cytoplasm,
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nucleus, and extracellular resident proteins. This observation agrees with the previous
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observation by Gust et al. (2012) who depicted that LysM-RLK members localize according
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their signal peptides. Also, transmembrane helices (TMH) of the LysM proteins were
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predicted using PRED-TMR algorithm which supports the cellular localizations of LysM
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proteins (Table 2). In general, functionally characterized LysMe proteins reside inside or
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outside of the cell. However, BdLysMe1 and BdLysMe2 were predicted to include one
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transmembrane helix and may potentially localize in the cell membrane.
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All BdLysM-RLK family proteins have at least one LysM domain with changing
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amino acid length (29 to 43) (Table 1). In addition to LysM domain, LYP members include
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additional LysM domain. Also, F-box-like, protein tyrosine kinase and protein kinase
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domains were the characteristic modules for LysMn, and LYK, respectively. LysMe members
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were comprised of single LysM domain (Table 1; Fig. 1). The number of LysM domains
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within individual protein range from one to twelve across kingdoms (Zhang et al 2009). This
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number can confer flexibility for binding and target selection. In addition, multiple LysM
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modules (1 to 6) increase the binding to peptidoglycan (Mesnage et al. 2014). In rice,
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OsCERK1 contains only one conserved LysM domain and does not bind to
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chitooligosaccharides. Instead, heterodimerization with OsCEBiP (containing two LysM
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domains) is required for chitin binding and innate immunity response (Shimizu et al. 2010).
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ACCEPTED MANUSCRIPT Moreover, the number of LysM in plants is variable for LYK proteins, which ranges from one
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to three (Zhang et al. 2009). Chitin binding site of AtLYK5 was formed by three LysM
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modules with a binding energy of −8.9 kcal mol−1 (Cao et al. 2014). On the other hand, it was
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reported that the second LysM of AtCERK1 is essential for chitin binding (Liu et al. 2012).
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Similarity, the second LysM of OsCEBiP is essential for chitin binding (Hayafune et al.
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2014). However, in Brachypodium, all BdLYK members composed of single LysM module.
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Further studies are required to understand LYK interaction model in Brachypodium during
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the activation of immune system.
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ACCEPTED MANUSCRIPT Fig 2. Chromosome distribution of LysM-RLK genes in Brachypodium genome. Tandem
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duplicated genes were labelled with blue rectangular. Position of the genes were stated
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with blue and red arrows representing (–) or (+) strand, respectively. The chromosome
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size was shown at the bottom of each representative chromosome as mega base (Mb)
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pair.
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3.2. Characteristics of LysM-RLKs in Brachypodium
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The information regarding protein characteristics (pI, GRAVY score, MW, and length) of
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each BdLysM-RLK members were retrieved from SwissProt and Expasy’s ProtParam
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(Gasteiger, 2005) databases and listed in Table 2. Analysis showed that they are varied and
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well-separated between each subgroup (LysMe, LYK, LysMn, and LYP). For instance, LYK
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proteins (LYK1 to LYK4) were relatively large proteins than the members from other
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subgroups in terms of amino acid length (average is 658 aa) and molecular weight (average is
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70 kDa). LysMe subgroup harbored small sized protein members with an average protein
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length of 96 aa. The isoelectric points (pI) are varied between 8.39 (BdLYP1) to 4.55
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(BdLYP4). According to pI value, most of the proteins (eight members, 72.7%) showed acidic
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character. Hydropathicity (GRAVY) index ranged between -0.4 (BdLysMn) and 0.4
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(BdLysMe1). Two of the BdLysMe members (BdLysMe1 and BdLysMe2) have the highest
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GRAVY score showing hydrophobic character. Additionally, most of the LysM-RLK proteins
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(eight members, 72.7%) exhibited hydrophobic nature.
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3.3. Chromosome distribution and duplication analysis of LysM-RLK genes in
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Brachypodium
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According to the chromosomal distribution, we found that LysM-RLK genes resided in all
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chromosomes except chromosome 5. The positions of genes and strand (- or +) was shown in
ACCEPTED MANUSCRIPT Fig. 2. One tandem duplication was estimated between LysMe group members (LysMe1 and
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LysMe2) (Fig. 2; Supplementary Table 1). This duplication event contributed to the
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expansion of LysMe subgroup that may lead to neofunctionalization of these genes during
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pathogen infection. Previous reports indicate that the total number of LysM-RLK members
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varies between plant species (Table 3). So far, LysM-RLK families have been identified in
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Arabidopsis (14), Glycine max (47), Medicago truncatula (17), and Oryza sativa (20) (Zhang
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et al. 2009) (Table 3). Accordingly, Brachypodium genome harbors the smallest number of
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LysM-RLK proteins (11) among the plants identified for LysM-RLK family. The great
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difference of the number of LysM-RLK members in those species is likely due to the different
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rounds of genome duplication events occurred during the species diversification (Shin-Han
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and Bleecker 2003).
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BdLysM-RLK proteins were depicted with red circles. Functionally verified
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Arabidopsis proteins recognizing either chitin or PGN were labelled with blue and red
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colored triangle, respectively.
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3.4. Phylogenetic analysis of BdLysM-RLK proteins
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Brachypodium orthologs of functionally characterized LysM-RLK proteins from different
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plants can give insights about their potential roles. For this purpose, we compared the critical
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residues and domain sequences of known LysM-RLK family members with currently
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identified Brachypodium ones. In Fig. 3, phylogenetic tree shows the evolutionary
ACCEPTED MANUSCRIPT relationship between Arabidopsis and Brachypodium LysM-RLK proteins. Subgroups were
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well separated from each other. Functionally validated Arabidopsis LysM-RLK members are
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shown in the tree, which can provide information about the possible functions of BdLysM-
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RLKs targeting PGN or chitin. Accordingly, two functionally characterized proteins (AtLYP1
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and AtLYP3) which recognize the PGNs were resided in the LYP subgroup. Three proteins
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recognize the chitin (AtCERK1, AtLYK4, and AtLYK5) which harbored in the LYK
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subgroup. It is likely that LYP and LYK subgroups are specialized for PGN and chitin
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targeting, respectively. This can help to identify their Brachypodium orthologs, which is
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functionalized for the pathogen recognition during infection. Additionally, a comprehensive
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phylogenetic analysis was conducted to show the relationship of BdLysM-RLK family
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proteins with the selected plant species (G. max, Arabidopsis, M. truncatula, O. sativa) (Fig.
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Fig 4. Comparative phylogenetic analyses of LysM-RLK proteins from five selected plants;
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G. max, Arabidopsis (blue) M. truncatula (yellow), O. sativa (pink) and Brachypodium
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(red). NJ (Neighbor-joining) trees were shown for each distinct group, separately: (a)
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LYK, (b) LysMe (c) LYP, and (d) LysMn.
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3.5. Expressions of BdLysM-RLK genes upon Fusarium infection and tissues
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Analysis of BdLysM-RLK gene expressions in 44 different developmental stages and tissues
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were represented in Fig. 5a. Tissue specific, spatial and temporal expression patterns were
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detected. In addition, to understand the response of LysM-RLK family members to fungal
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infection, we analyzed the GEO database revealing the gene repertoire after Fusarium
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graminearum (Fg), Fusarium head blight (FHB) causing plant fungal pathogen, infection.
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Digital expression analysis of Brachypodium spikes infected by two different Fusarium
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strains, either DON+ (Fg ph-1, produce toxin) or DON- (Fg delta tri5 mutant, unable to
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produce toxin) showed that BdLYK2, BdLYK3, and BdLYK4 were up-regulated after infection
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(Fig. 5b). Interestingly, the induction was higher in Fg DON- than the Fg DON+ samples.
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Also, BdLYK4 expression in Fusarium-infected plants was dramatically increased (4 times)
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compared to the ones from mock-inoculated plants (Fig. 5b). In the phylogenetic analyses,
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BdLYK2, BdLYK3, and BdLYK4 were resided in the same clade with AtLYK4 and AtLYK5
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(Fig. 3). Previous studies demonstrated that AtLYK4 is important for chitin recognition
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during fungal infection (Wan et al. 2012). Moreover, AtLYK5 has been proposed as a major
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chitin receptor (Cao et al. 2014). In addition to fungal responsive expressional changes of
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BdLYK4, its sequence similarity (40-45%) and phylogenetic proximity with AtLYK4 and
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AtLYK5 pointed out that BdLYK4 could be the most susceptible gene in Brachypodium,
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which may play role in chitin recognition.
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Fig 5. Heatmap analysis of the BdLysM-RLK genes shows tissue specific expression. Color
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legend represents the log2 calculated relative expressions (a). Response of BdLysM-
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RLK genes after Fusarium graminearum (Fg) infection (b). DON+ and DON-
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represent the Brachypodium plants infected by different Fg strains with high or reduced virulence on Brachypodium spikes, respectively. Expressions were normalized by GCRMA method.
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LysMe1 expression did not change significantly upon Fg infection or mock
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inoculation. Interestingly BdLYP1 and BdLYP4 were decreased after Fg DON+ and DON-
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infection. Recent studies demonstrated that AtLYP2 and AtLYP3 are involved in PGN
ACCEPTED MANUSCRIPT recognition and play indispensable roles for immunity to bacterial infection (Tanaka et al.
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2013). However, they are not responsive to chitin oligosaccarides (Willmann et al. 2011;
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Shinya et al. 2012). Similarly, our analysis showed that BdLYP1 and BdLYP4 expression did
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not increase after Fg infection, even their expression were found to be decreased. These
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results suggested that BdLYP proteins could be spesific to bacterial PGNs rather than fungal
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chitin.
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Fig 6. Interaction of the LysM domain of the BdLYK proteins and triacetylchitotriose
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(ZINC24425833). a. LYK1 b. LYK2 c. LYK3 d. LYK4. Yellow lines depict hydrogen
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bonds formed between ligand atoms and their corresponding residues atoms.
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3.6. Chitin and LYK-LysM interaction
ACCEPTED MANUSCRIPT LYK proteins are prone to recognition and binding of chitin elicitors secreted from pathogens
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during infection. Previous reports proved that AtCERK1, AtLYK4, and AtLYK5 can bind to
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chitin (Petutschnig et al. 2010; Shinya et al. 2012; Wan et al. 2012; Cao et al. 2014).
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However, AtLYK2, AtLYK3 and AtLYK5 do not appear to be involved in chitin elicitor
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signalling (Wan et al. 2012). To assess this interaction between BdLYKs and chitin molecules
341
(Supplementary Fig. 2), modeling and docking studies were conducted. Three-dimensional
342
structure of the proteins was computationally predicted and LysM domain was targeted by
343
triacetylchitotriose (ZINC24425833). 1-Click Docking program calculates the binding
344
affinities of -6.0, -7.8, -6.8, and -5.2 kcal/mol for BdLYK1, BdLYK2, BdLYK3, and
345
BdLYK4, respectively (Fig. 6). Among these, BdLYK2 has the highest binding affinity (-7.8
346
kcal/mol). It was -8.6 and -9.2 for AtLYK4-chitotetraose and AtCERK1-chitotetraose
347
interaction, respectively (Tanaka et al. 2013). The predicted three-dimensional models show
348
that LysM domain of BdLYK3 and BdLYK4 has very similar structures (Fig. 6). However,
349
their predicted chitin interacting residues are variable: R178, N223, and I231 for BdLYK3;
350
and H196, L198, A250, D251, and L253 for BdLYK4 (Fig. 7a, b). Also, LysM domain
351
architecture (including the first and the second LysM domains) from all identified BdLysM-
352
RLK proteins exhibits a variable sequence pattern (Fig. 7c), which enables the LysM domain
353
unique for target selection and suggesting that the proteins have been evolved to recognize
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variable targets.
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Fig 7. Alignment of LysM modules from BdLYK3, BdLYK4, AtCERK1, and AtLYK4. The
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conserved amino acid residues are labelled with blue font according to the percent
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identity score. Chitin binding residues were labelled with red rectangular (a): Docking
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model between triacetylchitotriose and proteins, BdLYK3 and BdLYK4 (b): Weblogo analysis shows the LysM domains resided in all BdLysM-RLK family members, including the first and the second LysMs for BdLYPs (c): Analysis show some
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conserved residues among the BdLYKs, which are abundant in between BdLYK3 and
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BdLYK4. However, LysM domain exhibits sequence variance within the BdLysM-
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RLK members.
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Compliance with ethical standards This article does not contain any studies with human
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participants or animals performed by any of the authors.
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Conflict of Interest All authors declare that they have no conflict of interest.
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Table 1. Domain structure of BdLysM-RLK proteins. Domain1
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113 197 200 217 127 114 107 119 111 54 48
156 240 243 244 171 160 156 165 164 97 87
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PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476 PF01476
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LysM LysM LysM LysM LysM LysM LysM LysM LysM LysM LysM
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Bradi1g11687.2 Bradi4g16350.1 Bradi3g06770.1 Bradi3g51790.1 Bradi2g40627.1 Bradi4g37090.1 Bradi1g46200.1 Bradi1g76177.2 Bradi3g57756.2 Bradi2g51930.1 Bradi2g51851.1
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BdLysMn BdLYK3 BdLYK4 BdLYK2 BdLYK1 BdYLP1 BdYLP4 BdYLP2 BdYLP3 BdLysMe1 BdLysMe2
Domain Pfam ID Start End
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Prot ID
Domain2 HMM HMM from Domain length /to 1 44 44 F-box-like 10 44 44 Protein tyrosine kinase 1 44 44 Protein kinase domain 1 29 44 Protein kinase domain 1 44 44 Protein kinase domain 1 44 44 LysM 5 44 44 LysM 1 44 44 LysM 4 44 44 LysM 3 42 44 5 39 44
Pfam ID Start End
PF12937 PF07714 PF00069 PF00069 PF00069 PF01476 PF01476 PF01476 PF01476
39 386 403 398 273 178 176 183 184
78 644 671 655 548 221 218 226 226
HMM HMM from length /to 5 39 48 47 259 260 44 258 264 13 258 264 2 259 264 1 44 44 1 44 44 1 44 44 1 44 44
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Table 2. Characteristics of LysM-RLK in Brachypodium
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Subs Gravy Gene ID Exon Localization ** TMH* pI MW (kD) Score Chr Strand Chr Location BdLYK1 Bradi2g40627.1 10 PM Chlr Cyt 2 5,74 63.60 -0.045 2 + 40700069..40703030 52555458..52559509 BdLYK2 Bradi3g51790.1 4 PM Chlr 1 6,34 73.14 0.003 3 + 17136796..17139097 BdLYK3 Bradi4g16350.1 1 PM Chlr 2 8,65 70.29 0.003 4 + BdLYK4 Bradi3g06770.1 1 PM 3 6,87 73.63 -0.001 3 + 5002793..5004886 51247464..51248121 BdLysMe1 Bradi2g51930.1 1 Extracellular 1 5,45 10.43 0.462 2 + BdLysMe2 Bradi2g51851.1 1 Extracellular 1 5,04 9.56 0.303 2 51206759..51207037 BdLysMn Bradi1g11687.2 2 Nucl 0 7,77 28.09 -0.402 1 8694155..8697817 42224657..42227694 BdLYP1 Bradi4g37090.1 2 Extracellular 1 8,39 38.44 0.354 4 + BdLYP2 Bradi1g76177.2 4 Extracellular 1 6,05 37.56 0.225 1 73079251..73081951 57189190..57192732 BdLYP3 Bradi3g57756.2 5 Extracellular 1 5,11 43.48 0.258 3 + 44732649..44736017 BdLYP4 Bradi1g46200.1 6 Extracellular 0 4,55 39.81 0.305 1 * TMH transmembrane helices were predicted using PRED-TMR algorithm (Pasquier et al. 1999) http://athina.biol.uoa.gr/PRED-TMR/
Nucl (bp) 2962 4062 2302 2094 658 279 3663 3038 2701 3643 3369
CDS (bp) Prot (aa) 1734 577 2049 682 2031 676 2094 697 308 101 279 92 768 255 1122 373 1089 362 1302 433 1194 397
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** Predicted using CELLO v.2.5: subCELlular LOcalization predictor http://cello.life.nctu.edu.tw/ (Yu et al. 2006). PM, plasma membrane; Chlr, chloroplast; Nucl, nucleus; Cyt, cytoplasm.
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Mm Type Bd At Gm Mt Os 9 LYK 4 5 21 8 6 2 LYP 4 3 4 2 6 NA LysMe 2 3 16 5 4 NA LysMn 1 3 6 2 4 11 Total 11 14 47 17 20 Lv et al. (2018) Reference This study Zhang et al. (2009) Bd, B. distacyon; At, A. thaliana; Gm, G. max; Mt, M. truncatula; Os, O. sativa; Mm, Morus multicaulis; NA, not applied.
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Table 3. Number of LysM-RLK family members from various plants.
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Highlights Brachypodium genome comprises of 11 LysM-RLK genes
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BdLysMe genes were found to be tandem duplicated
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Fungal chitin and LYK interaction was modelled by docking analyses
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BdLYK3 and BdLYK4 are the most susceptible LYKs for chitin perception
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