Genome-wide characterization and comparative analysis of the MLO gene family in cotton

Accepted Manuscript Genome-wide characterization and comparative analysis of the MLO gene family in cotton Xiaoyan Wang, Qifeng Ma, Lingling Dou, Zhen Liu, Renhai Peng, Shuxun Yu PII:

S0981-9428(16)30056-0

DOI:

10.1016/j.plaphy.2016.02.031

Reference:

PLAPHY 4429

To appear in:

Plant Physiology and Biochemistry

Received Date: 12 December 2015 Revised Date:

1 February 2016

Accepted Date: 23 February 2016

Please cite this article as: X. Wang, Q. Ma, L. Dou, Z. Liu, R. Peng, S. Yu, Genome-wide characterization and comparative analysis of the MLO gene family in cotton, Plant Physiology et Biochemistry (2016), doi: 10.1016/j.plaphy.2016.02.031. 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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Research Article Genome-wide characterization and comparative analysis of the

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MLO gene family in cotton

Xiaoyan Wang1+, Qifeng Ma2+, Lingling Dou2, Zhen Liu1, Renhai Peng1* and Shuxun Yu2*

Anyang Institute of Technology, College of Biology and Food Engineering, Anyang, Henan, 455000, China; 2 State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, Henan, 455000, China

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Xiaoyan Wang: [email protected] Qifeng Ma: [email protected] Lingling Dou: [email protected] Zhen Liu: [email protected] Renhai Peng: [email protected] Shuxun Yu: [email protected]

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These authors contributed equally to this work

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Corresponding author: Renhai Peng, [email protected] Anyang Institute of Technology, College of Biology and Food Engineering, Anyang, Henan, 455000, P. R. China

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Tel: +86-372-2909876

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Fax: +86-372-2525377

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Corresponding author: Shuxun Yu, [email protected] State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, Henan 455000, P. R. China

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Tel: +86-372-2525365

Fax: +86-372-2525363

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Abstract In plants, MLO (Mildew Locus O) gene encodes a plant-specific seven transmembrane (TM) domain protein involved in

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several cellular processes, including susceptibility to powdery mildew (PM). In this study, a genome-wide characterization of

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the MLO gene family in G. raimondii L., G. arboreum L. and G. hirsutum L. was performed. In total, 22, 17 and 38

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homologous sequences were identified for each species, respectively. Gene organization, including chromosomal location,

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gene clustering and gene duplication, was investigated. Homologues related to PM susceptibility in upland cotton were

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inferred by phylogenetic relationships with functionally characterized MLO proteins. To conduct a comparative analysis

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between MLO candidate genes from G. raimondii L., G. arboreum L. and G. hirsutum L., orthologous relationships and

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conserved synteny blocks were constructed. The transcriptional variation of 38 GhMLO genes in response to exogenous

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application of salt, mannitol (Man), abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA) and salicylic acid (SA) was

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monitored. Further studies should be conducted to elucidate the functions of MLO genes in PM susceptibility and

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phytohormone signalling pathways.

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Keywords: Gossypium, MLO, Gene family, Synteny blocks, Abiotic Stress, Phytohormone

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1. Introduction

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Powdery mildew (PM) is an obligate fungal pathogen that causes PM disease in a broad range of plants, including important crops such as pepper, tomato, apple, strawberry, and cotton [1]. It is difficult to diagnose at the early stages of the

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disease, and it can easily spread unnoticed. According to previous observations, PM disease primarily affects the leaves of

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sea-island cotton and upland cotton. In general, PM presents similar symptoms in cotton: white or brown spots on leaf tissues,

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particularly at the bottom of the plant, whereas upper leaves exert some resistance. Afterwards, tissue death of diseased spots

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causes infected leaves to crinkle, curl, and prematurely drop. Although blossoms and fruits are not the initial PM fungal

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targets, they can also become infected.

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Mildew locus O (MLO) proteins belong to a plant-specific protein family containing seven transmembrane (TM) domains

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[2, 3]. In addition, a C-terminal calmodulin-binding domain (CaMBD) and an extracellular N-terminus [3, 4] have been

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identified in this family. PM resistance was first characterized in barley plants in 1942 and the immunity was acquired

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because of the absence of a susceptibility gene which was named Mildew Locus O (MLO). Recessive MLO gene mutations

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confer durable broad-spectrum resistance to all discovered isolates of barley powdery mildew fungus Blumeria graminis f. sp

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hordei (Bgh) [2, 3]. Then, the discovery and identification of PM disease resistance in other plant species, such as 1

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Arabidopsis [5], pea [6] and tomato [7], has confirmed that PM resistance deriving from loss-of-function mutations in MLO

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functional orthologue is a common phenomenon. Therefore, broad-spectrum PM resistance in plants could be introduced by

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silencing of MLO gene [8]. Calmodulin-binding of MLO proteins promotes PM susceptibility in barley [9]. Moreover, pharmacological studies have

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suggested that the influx of Ca2+ ions is important for MLO protein function [4]. Therefore, Ca2+ may be a candidate signal

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because plant cells generate a transient Ca2+ signal in response to pathogen attack [10]. In addition, a complex mechanism

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may exist during the interaction between MLO genes and PM. Nevertheless, there is limited information on the precise

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mechanism of MLO proteins. It has been revealed that PM fungi target MLO proteins as an access to trigger pathogenesis

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because vesicle-associated and actin-dependent defence pathways are negatively regulated by functional MLO proteins in

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the circumstance of attempted PM penetration. Studies in tomato [7], barley [11], pepper [12], and grape [13] confirmed that

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early stages of PM infection are associated with up-regulated expression of MLO susceptibility-related gene, with a peak at

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six hours after inoculation.

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Since the HvMLO gene was first identified in barley [2], MLO genes have been discovered in Arabidopsis thaliana [14],

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Oryza sativa [15], Vitis vinifera [16], Triticum aestivum [17], Glycine max [18], Cucumis sativus [19], Malus domestica [20]

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and Solanum lycopersicum [21]. More detailed studies have uncovered that medium-sized gene family of MLO is

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plant-specific and MLO-based PM resistance is not confined to monocotyledones, but is also discovered in distantly related

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dicotyledones [22]. For example, mutant alleles of AtMLO2, one of 15 MLO genes present in Arabidopsis thaliana, caused

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partial resistance to the adapted strains such as Golovinomyces orontii and G. cichoracearum. Complete PM resistance was

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produced when two other homologous genes AtMLO6 and AtMLO12 were also mutated [5]. Subsequently, two studies

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showed that loss-of-function of the SlMLO1 gene was the cause of resistance to PM disease in tomato [7, 23]. It was

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demonstrated that pea PM resistance was associated with loss-of-function mutations in an MLO-homologous locus [6]. The

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results of virus-induced gene silencing suggested that both CaMLO1 and CaMLO2 are involved in the susceptibility of

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pepper to the PM fungus Leveillula taurica [23]. Recently, NtMLO1, which is predicted to be an orthologue of tomato

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SlMLO1 and pepper CaMLO2, was shown to be involved in PM susceptibility [24].

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Apart from susceptibility/resistance to PM disease in both monocotyledonous and dicotyledonous plants, increasing reports

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have suggested that MLO may be involved in a variety of developmental processes. Leaf mesophyll cells in MLO barley

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mutants have been shown to undergo spontaneous cell death, which is an indication of accelerated leaf senescence [2, 11].

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MLO family members in Arabidopsis presented tissue-specific expression patterns and silencing of AtMLO7 involved in

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pollen tube reception by the embryo sac led to decreased fertility [25]. Two additional Arabidopsis genes, AtMLO4 and 2

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AtMLO11, control root architecture, as null mutants generate asymmetrical root growth and exaggerated curvature [26]. More

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results have revealed that MLO family members are involved in diverse abiotic stresses for Capsicum annuum CaMLO2

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intensely induced upon exogenous treatment of pepper leaves with the phytohormone abscisic acid (ABA) and drought stress,

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is shown to act as a suppressor of ABA signalling to prevent water loss from leaves under drought conditions [27]. MLO genes have been intensively studied in many monocots and dicots, but very little research has focused on cotton.

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Published genomic data on G. raimondii (DD; 2n = 26) [28], and G. arboretum (AA; 2n = 26) [29] as well as G. hirsutum

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(AADD; 2n = 52) [30] provide an opportunity to conduct a comprehensive overview of the MLO gene family in diploid and

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tetraploid cotton species. In this study, we characterized the MLO gene family in these three species with respect to their

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structural, genomic and gene-expression features. Moreover, we assessed the orthologous relationships between the G. raimondii L., G. arboreum L. and G. hirsutum L. genomes.

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2. Materials and methods

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2.1. In silico identification and annotation

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Genomic databases of G. raimondii L. (D5, JGI__v2.1), G. arboreum L. (A2, BGI _v1.0) and G. hirsutum L. (AD1, BGI _v1.0), available at the CottonGen website (https://www.cottongen.org/) [31], were downloaded for the identification of

15

MLO homologue nucleotide and protein sequences. Then, several local BLAST searches using the Arabidopsis AtMLO1

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amino acid sequence as a query were performed. Candidates with an E-value less than 1.0e-20 were estimated to be MLO

17

homologs, and their gene coding regions, genomic DNA and deduced amino acid sequences were acquired. Conserved MLO

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domains within the acquired MLO sequences were confirmed by searching NCBI's conserved domain database

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(http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and Pfam's protein domain families (http://pfam.xfam.org/). The

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presence and number of TM helices in the proteins of interest were predicted using the online software TMHMM

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(http://www.cbs.dtu.dk/services/TMHMM-2.0/).

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2.2. Gene organization

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The chromosomal localization of each MLO gene in G. raimondii L., G. arboreum L. and G. hirsutum L. was deduced

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based on the available genomic information at the CottonGen database. Mapchart 2.2 software was used to visualize the

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distribution of MLO genes on the chromosomes, with the exception of a small portion of genes that have not been localized to

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a chromosome [32]. Introns and exons of each MLO gene were determined by comparing the cDNAs with their

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corresponding genomic DNA sequences. Intron/exon composition and position were analysed by the Gene Structure Display

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Server (GSDS) tool (http://gsds.cbi.pku.edu.cn/). 3

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Gene duplication events were identified when the following conditions were fulfilled: (1) the alignment covered more than

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80% of the longer gene, (2) the identity of the aligned regions was greater than 80% of the alienable region, and (3) only one

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duplication event was taken into account for tightly linked genes [33].

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2.3. Phylogenetic analysis

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A total of 38 GhMLO amino acid sequences together with 36 other MLO homologues from 9 dicot and monocot species were used to construct phylogenetic trees. Amino acid sequences of 36 known MLOs from Arabidopsis thaliana [14],

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Hordeum vulgare [2], Oryza sativa [15], Zea mays [14], Solanum lycopersicum [21], Pisum sativum [6], Vitis vinifera [16],

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Malus domestica [20] and Capsicum annuum [23] were obtained based on the published information. A total of 74 MLO

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protein sequences were included to perform multiple alignments using ClustalW [34] with the default parameters. A

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neighbour-joining phylogenetic tree was constructed by MEGA 6.0 software [35] with the pairwise deletion option and

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Poisson correction model. Bootstrapping (1000 replicates) was used to evaluate the degree of support for a particular

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grouping pattern in the phylogenetic tree.

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2.4. Detection of synteny blocks

Conserved synteny blocks between MLO candidate genes from G. raimondii L., G. arboreum L. and G. hirsutum L. were

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inferred by running the OrthoClusterDB tool available at GDR (http://genome.sfu.ca/cgi-bin/orthoclusterdb/runortho.cgi)

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[36]. To run OrthoCluster, the user must provide n (n ≥ 2) genome files and one correspondence file with their corresponding

18

orthologous relationships as input. Thus, orthologous groups of the MLO family among the three Gossypium species were

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previously generated using the OrthoMCL database (http://orthomcl.org/orthomcl/) [37].

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2.5. Plant materials, stress and phytohormone treatments

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Seeds of cotton plants from the CCRI 36 cultivar (G. hirsutum L.) were surface-sterilized in 75% (v/v) absolute alcohol for

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30 s and in 0.1% HgCl2 (m/v) for 3 min. After washing them in sterilized double-distilled water (ddH2O), seeds were sown

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onto sterilized Murashige & Skoog (MS) solid medium (pH 5.8) containing 1.5% sucrose and 0.7% agar. The inoculated

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culture tubes were placed in a growth chamber with 150 µmol m-2 s-1 fluorescent light at 25–26°C. Seven-day-old seedlings

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with expanding cotyledon were transplanted into new culture tubes supplemented with 200 µM sodium chloride (NaCl), 200

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mM mannitol (Man), 50 µM ABA, 200 µM ethylene (ETH), 100 µM jasmonic acid (JA) or 1 mM salicylic acid (SA).

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Treated and control plants were grown under the above conditions, and leaf samples were collected after three weeks. Three

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biological replicates were included, and each sample contained three young leaves collected from a single plant. Collected 4

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leaves were frozen in liquid nitrogen, and stored at -80°C until RNA extraction.

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2.6. Real-time quantitative RT-PCR

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Total RNA was extracted using the EASY spin plus RNA reagent kit RN38 (AIDLAB, Beijing, China) according to the manufacturer's instructions. Poly (dT) cDNA was synthesized using the Superscript III First-Strand Synthesis System

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(Invitrogen, USA). Primers (Table S1) for transcript analysis were designed with Premier 6.0 of Primer Designing Tool. The

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Histone 3 (AF024716) gene was used as an internal control. Quantitative Real Time-PCR (qPCR) was performed on an ABI

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7500 system (Applied Biosystems, USA) using SYBR Green I (with Rox) reagents to detect target products.

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The running programs were as follows: holding stage at 50°C for 2 min, 94°C for 10 min, followed by 40 cycles at 95°C for

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15 s, 60°C for 1 min. Then a melting curve was generated from 65 to 95°C to examine the specificity of target sequences.

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To measure differential expression of MLO genes, QRT-PCR data was processed by 2-

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analysed by Student's t test.

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Ethics statement

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method [38] and statistically

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We did not make use of human or vertebrate animal subjects and/or tissue in our research.

3. Results and discussion

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3.1. In silico characterization of Gossypium MLO homologues

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Conditional searches for Gossypium MLO homologues produced 38 significant matches in G. hirsutum L., 22 in G.

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raimondii L. and 17 in G. arboreum L. (Table 1-3). Predicted MLO genes GhMLO1–GhMLO38, GrMLO1–GrMLO22 and

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GaMLO1–GaMLO17 were numbered depending on their chromosomal location. We concluded that more than 90 percent of

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the 77 MLOs encoded proteins ranging from 400 to 600 amino acids, whereas six (one from G. raimondii, one from G.

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arboreum and four from G. hirsutum) had markedly different lengths as compared to MLO homologues reported in the

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genomes of Arabidopsis [14], Vitis vinifera [13] and Cucumis sativus [19], i.e., they were less than 300 or more than 600

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amino acids. The TMHMM2 programme predicted different orientations and numbers of transmembrane (TM) helices in the

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polypeptides. The number of TM domains varied from two in GhMLO8 and GaMLO4 to eight in ten of the 77 MLOs (Table

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1–3). However, 28 of the 77 MLO proteins had seven TM domains, which are conserved with respect to MLO family

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members in monocot and dicot plant species [3]. Comparison of the 77 MLO cDNAs to their genomic DNA sequences

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revealed that the number of exons varied from 8 (GhMLO8) to 18 (GhMLO22). Nearly half of the MLO genes (32 out of 77)

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contained 15 exons, which seems to be a common feature of the MLO gene family [14]. Details including the length of the

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77 sequences, the location of MLO domains and Accession Numbers are provided in Tables 1–3.

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Table 1 Members of GrMLO gene family as predicted in G. raimondii cv. Shixiya1 genome Name

Accession NO.a

Chr.

CDS

AA

Exons

length

length

TMb

MLO domain

MLO domain

c

location

length

Gorai.001G130800.1

1

1755

584

15

7

6-499

494

GrMLO2

Gorai.001G200000.1

1

1857

618

14

8

29-553

525

GrMLO3

Gorai.002G072500.1

2

1602

533

15

4

66-460

395

GrMLO4

Gorai.002G113800.1

2

1644

547

14

6

GrMLO5

Gorai.004G029300.1

4

1737

578

15

7

GrMLO6

Gorai.004G106900.1

4

1314

437

14

8

GrMLO7

Gorai.005G074900.1

5

1494

497

14

6

GrMLO8

Gorai.005G241700.1

5

1710

569

15

6

GrMLO9

Gorai.006G088500.1

6

1314

437

11

4

GrMLO10

Gorai.007G193200.1

7

1683

560

15

GrMLO11

Gorai.007G250700.1

7

1392

463

13

GrMLO12

Gorai.009G078600.1

9

1764

587

15

GrMLO13

Gorai.009G078700.1

9

1596

531

GrMLO14

Gorai.009G118700.1

9

1455

GrMLO15

Gorai.010G000800.1

10

1530

GrMLO16

Gorai.010G205000.1

10

1707

GrMLO17

Gorai.010G205100.1

10

1239

GrMLO18

Gorai.011G030800.1

11

1545

GrMLO19

Gorai.011G089600.1

11

1437

GrMLO20

Gorai.011G240700.1

11

1731

GrMLO21

Gorai.012G004100.1

12

GrMLO22

Gorai.013G197800.1

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GrMLO1

481

9-497

489

4-420

417

9-470

462

11-507

497

7-404

398

9-456

448

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5-485

6

6-419

414

7

11-505

495

15

7

11-485

475

484

14

7

6-450

445

509

15

7

8-454

447

568

15

8

18-475

458

412

12

5

6-337

332

514

14

7

20-460

441

478

14

6

6-425

420

576

15

8

6-495

490

1737

578

15

7

9-494

486

1515

504

13

7

7-455

449

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a

Available at https://www.cottongen.org/data/download/genome_JGI.

b

Presence and number of transmembrane (TM) helices in the proteins was predicted using the online software of TMHMM

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Table 2 Members of GaMLO gene family as predicted in G. arboretum genome

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(http://www.cbs.dtu.dk/services/TMHMM-2.0/). c

Presence of conserved MLO domains within the acquired MLO sequences was confirmed by searching in NCBI's conserved domain database

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(http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi).

Name

Accession NO.a

Chr.

CDS length

AA length

Exons

TMb

MLO domain locationc

MLO domain length

GaMLO1

Cotton_A_06751

3

1671

556

15

7

9-475

467

GaMLO2

Cotton_A_19376

4

1596

532

15

6

5-470

466

GaMLO3

Cotton_A_00762

5

1710

569

15

6

11-507

497

GaMLO4

Cotton_A_08798

5

1224

407

11

2

4-389

386

GaMLO5

Cotton_A_36285

6

1302

433

13

5

4-423

420

GaMLO6

Cotton_A_12678

7

1791

596

14

6

5-536

532

GaMLO7

Cotton_A_06313

7

1692

563

15

7

50-490

441

GaMLO8

Cotton_A_26172

8

1488

495

13

7

18-402

385

GaMLO9

Cotton_A_07533

9

1467

488

14 6

6

6-464

459

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514

14

7

20-460

441

GaMLO11

Cotton_A_23415

9

1704

567

15

7

6-484

479

GaMLO12

Cotton_A_11046

10

1902

633

15

7

1-551

551

GaMLO13

Cotton_A_11047

10

1602

533

15

8

11-486

476

GaMLO14

Cotton_A_15078

10

1395

464

12

8

1-462

462

GaMLO15

Cotton_A_20369

12

1680

559

15

5

7-475

469

GaMLO16

Cotton_A_22367

13

1518

505

13

7

7-455

449

GaMLO17

Cotton_A_39162

Ca1

1674

557

15

8

14-494

481

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a

Available at https://www.cottongen.org/data/download/genome_BGI_A2.

b

Presence and number of transmembrane (TM) helices in the proteins was predicted using the online software of TMHMM

(http://www.cbs.dtu.dk/services/TMHMM-2.0/). c

Presence of conserved MLO domains within the acquired MLO sequences was confirmed by searching in NCBI's conserved domain database

SC

(http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi).

Table 3 Members of the GhMLO gene family as predicted in G. hirsutum cv. TM-1 genome Accession NO.a

Chr.

GhMLO1

CotAD_67162

1

1332

GhMLO2

CotAD_67021

1

1377

GhMLO3

CotAD_28782

2

1569

GhMLO4

CotAD_16944

2

1545

GhMLO5

CotAD_56441

2

1842

GhMLO6

CotAD_36554

3

1416

GhMLO7

CotAD_62838

4

GhMLO8

CotAD_31993

6

GhMLO9

CotAD_04379

8

GhMLO10

CotAD_12423

9

GhMLO11

CotAD_12424

9

GhMLO12

CotAD_63577

11

GhMLO13

CotAD_64129

GhMLO14

CotAD_30773

GhMLO15

CotAD_75839

GhMLO16

length

AA

Exons

TMb

MLO domain

MLO domain

locationc

length

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Name

length

13

5

6-399

394

458

11

5

9-352

344

522

15

7

9-449

441

514

15

6

9-432

424

613

15

7

5-552

548

471

14

5

4-453

450

1302

433

13

5

4-423

420

768

255

8

2

7-253

247

1281

426

13

7

7-481

475

1347

448

11

6

11-401

391

1695

564

15

7

11-482

472

TE D

443

1470

489

14

6

6-465

460

12

1089

362

12

4

60-358

299

13

1491

496

12

6

7-446

440

14

1674

557

15

8

14-494

481

CotAD_36153

14

1767

588

15

7

6-503

498

GhMLO17

CotAD_29126

15

1845

614

15

7

5-552

548

GhMLO18

CotAD_45096

17

1266

421

14

7

4-404

401

GhMLO19

CotAD_31735

18

1407

468

11

6

11-406

396

GhMLO20

CotAD_05932

18

1416

471

14

5

4-444

441

GhMLO21

CotAD_31530

19

1524

507

14

7

14-414

401

GhMLO22

CotAD_20256

19

2079

692

18

8

7-463

457

GhMLO23

CotAD_09253

20

1740

579

16

6

9-492

484

GhMLO24

CotAD_72948

20

1236

411

11

4

44-367

324

GhMLO25

CotAD_08062

22

1662

553

14

7

7-469

463

GhMLO26

CotAD_01552

22

1212

403

12

5

2-369

368

EP

6 7

Cotton_A_03932

AC C

1 2 3 4 5

GaMLO10

7

ACCEPTED MANUSCRIPT CotAD_54310

22

1596

531

15

7

11-485

475

GhMLO28

CotAD_54311

22

1725

574

15

6

11-492

482

GhMLO29

CotAD_15574

24

1470

489

14

6

6-436

431

GhMLO30

CotAD_06176

26

1692

563

15

5

9-482

474

GhMLO31

CotAD_00651

scaffold26.1

1305

434

14

7

20-433

414

GhMLO32

CotAD_07261

scaffold72.1

1437

478

13

4

9-397

389

GhMLO33

CotAD_08394

scaffold190.1

1407

468

11

6

11-406

396

GhMLO34

CotAD_49330

scaffold1917.1

1227

408

9

4

7-338

332

GhMLO35

CotAD_39473

scaffold2046.1

1407

468

13

7

8-438

431

GhMLO36

CotAD_71931

scaffold3483.1

1272

423

11

5

14-330

317

GhMLO37

CotAD_75625

scaffold3566.1

1725

574

15

8

29-509

481

GhMLO38

CotAD_74071

scaffold4982.1

1659

552

15

7

6-469

464

RI PT

GhMLO27

Available at https://www.cottongen.org/data/download/genome_BGI_AD1.

b

Presence and number of transmembrane (TM) helices in the proteins was predicted using the online software of TMHMM

6

3.2. Genomic organization of Gossypium MLO homologues

SC

1 2 3 4 5

a

(http://www.cbs.dtu.dk/services/TMHMM-2.0/).

Presence of conserved MLO domains within the acquired MLO sequences was confirmed by searching in NCBI's conserved domain database

(http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi).

M AN U

c

According to the report by Kohel [39], chromosomes 1 to 13 of tetraploid cotton were derived from the A subgenome,

8

and chromosomes 14 to 26 originated from the D subgenome. We were able to map 68 out of 77 MLOs onto chromosomes

9

in G. raimondii, G. arboreum, or G. hirsutum (Fig. 1). Generally, one or two members were located on most chromosomes,

10

with the exception of G. raimondii and G. arboretum, in which chromosomes 9 and 10 contained three genes each.

11

Furthermore, chromosome 22, originating from chromosome 9 of the D subgenome in G. hirsutum, contained four MLOs.

12

The majority of the 77 Gossypium MLO family members occurred as singletons, with the exception of five groups,

13

GhMLO10–GhMLO11

14

CotAD_54311),

15

(Gorai.010G205000.1–Gorai.010G205100.1) and GaMLO16–GaMLO17 (Cotton_A_11046–Cotton_A_11047), which were

16

organized as adjacent homologues with the distance ranging from 9.4 kb to 30.8 kb (Table S4). We identified two groups in

17

G. hirsutum and G. raimondii and one in G. arboretum. Interestingly, all five gene clusters were located on chromosome 9

18

or 10 (chr22 originating from subD_chr9), suggesting that there was a slight bias for the MLO homologue location.

NO.

CotAD_12423–CotAD_12424),

EP

(Accession

TE D

7

(Gorai.009G078600.1–Gorai.009G078700.1),

(CotAD_54310–

GrMLO16–GrMLO17

AC C

GrMLO12–GrMLO13

GhMLO27–GhMLO28

19

Previous studies have indicated that genomic changes, including chromosomal rearrangement, gene duplication and gene

20

expression changes, occurred during the formation of polyploid species [40]. To elucidate the expanded mechanism of the

21

MLO gene family in G. hirsutum, we investigated genomic organization of GhMLO homologues. First, we performed

22

multiple and pairwise alignments of 38 GhMLO sequences. After comprehensive analysis of pairwise alignments and the 8

ACCEPTED MANUSCRIPT physical location of each GhMLO gene, we detected 12 pairs of homologous genes and 2 tandem duplications (GhMLO1–

2

GhMLO2/GhMLO24–GhMLO23; GhMLO10–GhMLO11/GhMLO27–GhMLO28). Related information of homologous

3

genes and duplication events were presented in Table 4.

4 5 6 7

Fig. 1. Chromosomal localization of GrMLOs (A), GaMLOs (B), and GhMLOs (C). The relative sizes (unit, Mb) of G. raimondii (chr.D01–D13), G. arboreum (chr.A01–A13) and G. hirsutum (chr.AD01–AD26) chromosomes were consistent with published genomic data.

8

3.3. Phylogenetic analysis

9

AC C

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1

We performed a phylogenetic study on the newly identified GhMLO proteins. The dataset covered 38 GhMLO proteins,

10

the complete Arabidopsis thaliana MLO protein family AtMLO1–15 [14], and a series of MLO homologues that have been

11

functionally associated with PM susceptibility from grapevine (Vitis vinifera) [16], apple (Malus domestica) [20], barley 9

ACCEPTED MANUSCRIPT (Hordeum vulgare) [2], rice (Oryza sativa) [15], pepper (Capsicum annuum) [23], pea (Pisum sativum) [6], maize (Zea mays)

2

[14] and tomato (Solanum lycopersicum) [21]. Phylogenetic analysis of total 74 MLO proteins resulted in seven distinct

3

clades (Fig. 2). Clades I to VI were assigned according to a previous study of AtMLO homologues and grapevine VvMLOs

4

[14, 16]. Two additional clades (VII and VIII) included Rosaceae (P. persica, F. vesca and M. domestica) MLO homologues

5

only, as reported by Pessina et al. [20]. Four additional G. hirsutum MLO homologues GhMLO8, GhMLO13, GhMLO22

6

and GhMLO31 were grouped in clade VII with apple MdMLO18, suggesting the existence of more than six clades in the

7

plant MLO gene family. Eight G. hirsutum MLO homologues (GhMLO11, GhMLO16, GhMLO21, GhMLO25, GhMLO28,

8

GhMLO34, GhMLO36 and GhMLO38) clustered together in clade V with other MLO proteins, AtMLO2, AtMLO6,

9

AtMLO12, tomato SlMLO1, pea PsMLO1, pepper CaMLO1 and CaMLO2, which have been experimentally shown to be

SC

RI PT

1

10

required for PM susceptibility (e.g., [5, 6, 23, 41]).

11

Table 4 Homologous genes and duplication events detected in GhMLOs Pair 1

GhMLO3

522

2

GhMLO5

613

3

GhMLO6

471

4

GhMLO7

433

5

GhMLO8

255

6

GhMLO9

504

7

GhMLO12

8

GhMLO15

9

GhMLO19

10

GhMLO21

11

GhMLO25

12

GhMLO30

12 13

Identity

GhMLO4

514

95.14

GhMLO17

614

97.72

GhMLO20

471

97.03

GhMLO18

421

93.82

GhMLO22

692

95.29

GhMLO14

496

96.17

489

GhMLO29

489

97.14

557

GhMLO37

574

96.41

468

GhMLO33

468

98.29

507

GhMLO36

423

93.85

553

GhMLO34

408

90.69

563

GhMLO32

478

98.12

GhMLO1/ GhMLO2

443/458

GhMLO24/ GhMLO23

411/579

80.78/96.07

GhMLO10/ GhMLO11

448/564

GhMLO27/ GhMLO28

531/574

95.76/95.21

AC C

2

AA Length

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Event 1

SeqB

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AA Length

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SeqA

Two homologues, GhMLO5 and GhMLO17 were found to grouped in clade IV, which contained all monocot MLO

14

proteins, such as barley HvMLOs, maize ZmMLO1 and rice OsMLOs functionally acting as PM susceptibility factors [14].

15

Consistent with this finding, one MLO protein from the dicot species V. vinifera (VvMLO14) [16], one homologue from F.

16

vesca (FvMLO17) [20] and one from P. persica (PpMLO12) [20] also clustered in clade IV. Such clustering results raise the

17

question of whether exclusively monocot MLO proteins cluster in clade IV. Analysis of phylogenetic relationships revealed

18

that ten G. hirsutum MLO homologues were clustered in clade V and IV, which harboured all dicot and monocot MLO

19

proteins functionally related to PM susceptibility, thus making them susceptibility factor candidates. The phylogenetic 10

ACCEPTED MANUSCRIPT analysis performed here confirmed the presence of clade VII, first reported in Rosaceae by Pessina et al. [20]. Additional

2

studies should focus on the functional characterization of cotton MLO homologues grouped in clades IV, V and VII.

4 5 6 7 8 9 10 11 12 13 14

AC C

3

EP

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1

Fig. 2. Phylogenetic analysis of MLO proteins. The phylogenetic tree represents a consensus tree with branch lengths proportional to sequence distance. Numbers indicate bootstrap values (from 1000 replicates) that support the respective branch. The dataset includes 38 GhMLOs (GhMLO1–38) and 36 other MLO proteins from Arabidopsis thaliana, grapevine (Vitis vinifera), apple (Malus domestica), barley (Hordeum vulgare), rice (Oryza sativa), pepper (Capsicum annuum), pea (Pisum sativum), maize (Zea mays) and tomato (Solanum lycopersicum). Genbank accession numbers of translated MLO proteins used in phylogenetic analysis: AtMLO1 (Z95352); AtMLO2–15 (AF369563–AF369576); VvMLO3 (CAO18135); VvMLO4 (CAO21819); VvMLO6 (CAO66388); VvMLO9 (CAN84002); VvMLO13(CAO68971); VvMLO14(CAO66265); VvMLO17 (CAO68972); MdMLO5 (MDP0000163089); MdMLO7 (MDP0000123907); MdMLO11 (MDP0000239643); MdMLO18 (MDP0000928368); MdMLO19 (MDP0000168714); HvMLO (CAB06083); HvMLO-h1 (CAB08860); OsMLO1 (CAB08606); OsMLO3 (BAG93853); CaMLO1 (AAX31277); CaMLO2 (AFH68055); PsMLO1 (ACO07297); ZmMLO1 (AAK38337); SiMLO1 (AAX77013). 11

ACCEPTED MANUSCRIPT 1

We conducted further multiple alignments among MLO proteins in clade V to identify conserved domains (Fig. 3). Twelve proteins from 5 species presented a high degree of conservation in their seven predicted TM domains, which define this

3

protein family [14]. We also identified a calmodulin-binding domain consisting of a stretch of approximately 10–15 amino

4

acids proximal to TM domain 7 [4]. Moreover, two other conserved regions within the C-terminus of several MLO proteins

5

have been suggested to modulate PM infection [42]. Peptide domain I is characterized by the presence of serine (S),

6

threonine (T) and proline (P) residues, whereas peptide domain II contains the consensus motif D/E-F-S/T-F (Fig. 3). All of

7

the GhMLO proteins within clade V contain the two conserved domains mentioned above, except GhMLO25 contains a

8

modified motif II of I-F-S-L.

9

3.4. Synteny block detection

SC

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2

Previous studies have indicated that allotetraploid cotton species were derived from an interspecific hybridization event

11

between A and D-genome diploid species [40]. The recent availability of genome sequences for G. raimondii, G. arboreum

12

and G. hirsutum offers great potential for comparative genomics studies, which aim to provide insights into structures and

13

functions of genomic features. First, we identified a total of 517 orthologous relationships between G. raimondii, G.

14

arboreum and G. hirsutum MLO homologues (Table S2). Because of homologous genes and duplication events in the G.

15

hirsutum genome, numerous many-to-one relationships were identified. Orthologues are genes in different species that evolve

16

from one single gene in their last common ancestor. Such genes often retain identical biological roles in the present-day

17

organisms. A perfect synteny block is a conserved block of genes that share exactly the same order and strandedness and

18

contain no mismatches compared with the chromosomes of related species.

TE D

Then we predicted 25, 28 and 18 conserved non-nested synteny blocks between G. hirsutum and G. raimondii, G. hirsutum

EP

19

M AN U

10

and G. arboretum, and G. raimondii and G. arboretum, respectively (Fig. 4). Notably, 12 conserved synteny blocks were

21

discovered among the three Gossypium genomes (Fig. 5). Thirteen blocks were not included in Fig. 4 and 5 because they

22

involved genes that could not be localized to a specific chromosome. These conserved segments contain different numbers of

23

genes, ranging from 1 to 3. The size distribution of conserved non-nested blocks is shown in Fig. 6, and detailed information

24

about each block is shown in Table S3. Desirable blocks were detected because of a close evolutionary relationship among

25

these three species. In addition, because of the existence of homologous genes, some many-to-one relationships were

26

generated in some blocks.

AC C

20

12

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ACCEPTED MANUSCRIPT

1

13

ACCEPTED MANUSCRIPT Fig. 3. Multiple sequence alignment of GhMLO proteins and selected MLO proteins in clade V based on Fig. 2. Arabidopsis thaliana (AtMLO2, AtMLO6 and AtMLO12), Solanum lycopersicum SiMLO1 (AAX77013) and Capsicum annuum CaMLO2 (AFH68055)) have been functionally characterized as susceptibility genes. Vitis vinifera VvMLO3 and VvMLO13 [16] clustered in clade V. The multiple sequence alignment was generated by CLUSTALX2 using default parameters. The positions of seven TM regions (TM1–7) inferred from the experimentally determined topology of HvMLO [2] and the approximate position of the calmodulin-binding domain (CaMDB) were previously defined [4]. Two additional conserved domains I and II were previously identified [42], and the above-mentioned domains were indicated by lines above the sequences.

10

A total of 83 conserved non-nested synteny blocks were predicted after pairwise comparative analysis of MLO

11

homologues. In particular, genes situated on G. raimondii chromosomes 2, 5, 7, 9 and 11 are predicted to have

12

corresponding orthologues on G. hirsutum chromosomes 2, 20, 18, 22 and 22, respectively, whereas genes on G. arboreum

13

chromosomes 5, 7, 9 and 10 are suggested to originate from conserved blocks on G. hirsutum chromosomes 20, 2, 19 and 22,

14

respectively (Fig. 4). The corresponding chromosomes that contain the largest number and highest density of perfectly

15

conserved synteny blocks in G. hirsutum, G. raimondii and G. arboreum are chr2- chr2D- chr7A, chr20- chr5D- chr5A and

16

chr22- chr9D- chr10A. These data suggest that genes within these conserved blocks may be co-regulated by specific locus

17

control regions (LCRs), which can control the expression of a group of genes. This finding indicates conservation and

18

rearrangements of certain chromosome segments, which play an important role in the evolution or adaptive changes of these

19

three close species.

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20

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1 2 3 4 5 6 7 8 9

14

1 2 3 4 5 6

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ACCEPTED MANUSCRIPT

Fig. 4. Circos diagram of synteny blocks identified between G. raimondii, G. arboreum, and G. hirsutum MLOs. The chromosomes of G. raimondii (D01–D13), G. arboreum (A01–A13), A subgenome (AD01–AD13) of G. hirsutum, and D subgenome (AD14–AD26) of G. hirsutum were filled with light red, light green, dark green and dark red, respectively. A total of 61 coloured lines connecting two chromosomal regions denote syntenic regions between G. raimondii, G. arboreum, and G. hirsutum. Ten blocks were not included because involved genes were not localized to definitive chromosomes.

7 8

15

1 2 3 4 5 6 7

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ACCEPTED MANUSCRIPT

Fig.5. Circos diagrams of synteny blocks detected among GhMLOs, GrMLOs and GaMLOs. The chromosomes of G. raimondii (D01–D13), G. arboreum (A01–A13), A subgenome (AD01–AD13) of G. hirsutum, and D subgenome (AD14– AD26) of G. hirsutum were filled with light red, light green, dark green and dark red, respectively. One coloured circle with three lines indicates a synteny block among the three genomes. Three blocks were not depicted because they did not demonstrate positioning of related genes.

16

M AN U

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ACCEPTED MANUSCRIPT

Fig. 6. Size distribution of conserved non-nested synteny blocks, obtained by OrthoCluster, preserving gene order. We did not permit mismatches and did not consider strandedness. In total, 25, 28 and 18 conserved non-nested synteny blocks were predicted between G. hirsutum and G. raimondii (AADD- DD), G. hirsutum and G. arboretum (AADD- AA), G. raimondii and G. arboretum (DD- AA), respectively. Moreover, 12 conserved blocks of MLO candidate genes were found among the three genomes (AADD- DD- AA). These conserved segments contain different numbers of genes, ranging from 1 to 3 genes.

8

3.5. Gene expression analysis during leaf development

TE D

1 2 3 4 5 6 7

To estimate the temporal expression patterns of GhMLO genes during leaf development of upland cotton, we analysed

10

publicly available RNA-Seq data containing 3,624 differentially expressed genes during leaf development of 15-, 25-, 35-,

11

45-, 55-, and 65-day-old plants [43]. Four genes, GhMLO15, GhMLO21, GhMLO25 and GhMLO38, were differentially

12

expressed during the six leaf developmental stages from young and mature to senescent phases (Fig. 7). The relative

13

expression of GhMLO15 and GhMLO21 in leaves was higher than that of GhMLO25 and GhMLO38. GhMLO15 was

14

significantly up-regulated at almost all phases, although it sharply decreased at the 35-day-old stage. During leaf growth

15

stages, GhMLO21 transcripts continuously increased, especially after 35 days. GhMLO25 and GhMLO38 expression slightly

16

increased overall during leaf development. In summary, four differentially expressed genes were up-regulated during leaf

17

development, indicating that they could be involved in the regulation of leaf senescence.

18

3.6. Responses of GhMLOs to stress and phytohormonal stimuli

19 20

AC C

EP

9

To determine whether GhMLOs are involved in environmental stress or phytohormone signalling pathways, we examined gene expression of GhMLOs in response to exogenous application of salt, Mannitol, ABA, ETH, JA or SA. Experimental 17

ACCEPTED MANUSCRIPT data demonstrated that only five genes were not differentially expressed and twenty genes were sharply up-regulated or

2

down-regulated under treatments (Fig. 8). All data of gene expression were presented in Table S5. Transcripts of eleven

3

genes (Fig. 8A, B and C) were increased by more than ten-fold compared to non-treated plants, whereas six genes (Fig. 8D

4

and E) were found to be significantly suppressed in leaves under some of these conditions.

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1

6 7 8 9

Fig. 7. Transcriptional variation of four G. hirsutum MLO genes during leaf development of 15-, 25-, 35-, 45-, 55-, and 65-day-old plants. Published RNA-Seq data [43] containing 3,624 differentially expressed genes during leaf development were analysed. Four genes, GhMLO15, GhMLO21, GhMLO25 and GhMLO38 were differentially expressed during six leaf developmental stages from young and mature to senescent phases.

10

Previous analysis of phylogenetic relationships revealed that ten G. hirsutum MLO homologues were clustered in clade V

11

and IV, which harboured all dicot and monocot MLO proteins functionally related to PM susceptibility [5, 6, 14, 23, 41].

12

Experimental results showed that all of them were differentially expressed under stress or phytohormone application. Three

13

(GhMLO6, GhMLO18 and GhMLO25) out of 38 genes were intensely responsive to ABA treatment. ABA is an important

14

phytohormone that can convert the initial stress signal, such as drought or high salinity, into a cellular response [44, 45]. 18

ACCEPTED MANUSCRIPT Intense induction of these three genes by ABA suggests that they are specifically involved in the response of the ABA

2

signalling pathway. GhMLO6, GhMLO11, GhMLO17 and GhMLO28 were dramatically up-regulated in upland cotton

3

leaves cultured in the presence of 200 µM ETH. ETH is a gas phytohormone with significant functions throughout the

4

whole dicotyledon and monocotyledon life cycle, ranging from growth and development to a variety of stress responses [46,

5

47]. In addition, three genes (GhMLO9, GhMLO17 and GhMLO23) showed opposite expression pattern in response to

6

exogenous JA and SA.

RI PT

1

The functions of MLO family members in modulating plant defence responses against PM and regulating cell death has

8

been confirmed [2, 5-7, 11, 24, 41]. However, accumulating evidence has suggested that MLO may be involved in a variety

9

of abiotic stresses [11, 27, 48-49]. Four GmMLOs from soybean were responsive to various abiotic stresses and

SC

7

phytohormone treatments [48]. The results of virus-induced silencing of CaMLO2 in chili pepper and over-expression in

11

Arabidopsis support that CaMLO2 participate in drought stress regulation, acting as a suppressor of ABA signalling [27].

12

The expression of HbMLO1 from the rubber tree was intensely induced by diverse phytohormones (including ethephon, JA,

13

SA, ABA, indole-3-acetic acid, and gibberellic acid), H2O2, and wounding treatments, but no intense response to PM

14

infection was found [45]. In the current study, various abiotic stresses and phytohormone treatments induced or suppressed

15

the expression of 33 GhMLOs. The clear response of GhMLOs to stress conditions or phytohormone supplement suggests

16

that they may participate in the salt, Man, ABA, ETH, JA and SA responsive signalling pathways. We propose that future

17

studies should focus on elucidating the roles of the MLO gene family in response to environmental stimuli.

18

4. Conclusion

TE D

Our work led to the identification of 22 MLO homologues in G. raimondii L., 17 in G. arboreum L. and 38 in G. hirsutum

EP

19

M AN U

10

L. The majority of the 77 Gossypium MLO members were organized as singletons, with the exception of five gene clusters.

21

After comprehensive analysis of pairwise alignments and the physical location of each GhMLO gene, we detected 12 pairs

22

of homologous genes and 2 tandem duplications. Clearly, the phylogenetic analysis performed in this study confirmed the

23

presence of clade VII, which was previously reported in the Rosaceae MLO family. A total of 83 conserved non-nested

24

synteny blocks were predicted after pairwise comparative analysis of MLO homologues among the three Gossypium species.

25

Four genes (GhMLO15, GhMLO21, GhMLO25 and GhMLO38) were differentially expressed during six leaf developmental

26

stages from young and mature to senescent phases. The general and intense response of GhMLOs to stress conditions or

27

phytohormone supplement suggests that MLO gene family may participate in the salt, Man, ABA, ETH, JA and SA

28

responsive signalling pathways in upland cotton.

AC C

20

19

ACCEPTED MANUSCRIPT

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1

2 20

ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7

Fig. 8. Relative expression analysis of twenty G. hirsutum MLO genes in response to abiotic treatments and phytohormone application. Seven-day-old seedlings were cultured within MS solid medium as a control (CK) or supplemented with 50 µM ABA, 200 µM sodium chloride (NaCl), 200 µM ethylene (ETH), 100 µM JA, 1 mM SA or 200 mM mannitol (Man). Treated and control plants were grown under the same conditions, and leaf samples were collected after three weeks of treatments. Three biological repeats were performed, and each sample contained three young leaves collected from one single plant. Data in the graph were mean values with standard deviation (error bar) from three replicates. Statistical analysis was conducted by Student’s t-test (** P<0.01, * P<0.05).

9

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8

Additional files: Table S1 Quantitative PCR primers of target genes used in the study.

11

Table S2 Ortholgous relationships generated between G. raimondii, G. arboreum and G. hirsutum MLO homologs.

12

Table S3 Information of synteny blocks predicted between G. hirsutum, G. raimondii and G. arboretum MLO homologs.

13

Table S4 Gene clusters found in three cotton species of 77 MLO homologs.

14

Table S5 Relative expression analysis of 38 G. hirsutum MLO genes in response to abiotic treatments or phytohormone

15

application.

16

Contributions:

M AN U

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10

Conceived and designed the experiments: Renhai Peng, Xiaoyan Wang and Shuxun Yu. Performed the experiments:

18

Xiaoyan Wang, Qifeng Ma and Lingling Dou. Analyzed the data: Xiaoyan Wang and Qifeng Ma. Contributed

19

reagents/materials/analysis tools: Renhai Peng and Shuxun Yu. Wrote the paper: Xiaoyan Wang. Edited the manuscript:

20

Xiaoyan Wang and Zhen Liu.

21

Acknowledgements

EP

We would like to thank doctoral candidate Xihua Li for her assistance of Circos software. The work described in this paper

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22

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17

23

was supported by the National High-tech Research and Development Projects of China (2013AA102601) and funded by the

24

Major Projects of Anyang City Science and Technology Plan (ANKE20140208).

25

Reference

26 27 28 29 30 31

[1] D.A. Glawe, The powdery mildews: a review of the world's most familiar (yet poorly known) plant pathogens, Annu Rev Phytopathol, 46 (2008) 27-51. [2] R. Buschges, K. Hollricher, R. Panstruga, G. Simons, M. Wolter, A. Frijters, R. van Daelen, T. van der Lee, P. Diergaarde, J. Groenendijk, S. Topsch, P. Vos, F. Salamini, P. Schulze-Lefert, The barley Mlo gene: a novel control element of plant pathogen resistance, Cell, 88 (1997) 695-705. [3] A. Devoto, P. Piffanelli, I. Nilsson, E. Wallin, R. Panstruga, G. von Heijne, P. Schulze-Lefert, Topology, subcellular 21

ACCEPTED MANUSCRIPT localization, and sequence diversity of the Mlo family in plants, J Biol Chem, 274 (1999) 34993-35004. [4] M.C. Kim, S.H. Lee, J.K. Kim, H.J. Chun, M.S. Choi, W.S. Chung, B.C. Moon, C.H. Kang, C.Y. Park, J.H. Yoo, Y.H. Kang, S.C. Koo, Y.D. Koo, J.C. Jung, S.T. Kim, P. Schulze-Lefert, S.Y. Lee, M.J. Cho, Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue, J Biol Chem, 277 (2002) 19304-19314. [5] C. Consonni, M.E. Humphry, H.A. Hartmann, M. Livaja, J. Durner, L. Westphal, J. Vogel, V. Lipka, B. Kemmerling, P. Schulze-Lefert, S.C. Somerville, R. Panstruga, Conserved requirement for a plant host cell protein in powdery mildew

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ACCEPTED MANUSCRIPT Highlights 1. Our work led to the identification of 77 MLO homologues in three Gossypium species.

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2. A total of 83 non-nested synteny blocks were predicted among the three species.

3. Four genes were differentially expressed during six leaf developmental stages.

4. 33 genes were induced or suppressed in response to stress or phytohormone

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treatments.

ACCEPTED MANUSCRIPT

Contributions:

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Conceived and designed the experiments: Renhai Peng, Xiaoyan Wang and Shuxun Yu. Performed the experiments: Xiaoyan Wang, Qifeng Ma and Lingling Dou. Analyzed the data: Xiaoyan Wang and Qifeng Ma. Contributed reagents/materials/analysis tools: Renhai Peng and Shuxun Yu. Wrote the paper: Xiaoyan Wang. Edited the manuscript: Xiaoyan Wang and Zhen Liu.