GENOME WIDE ANALYSIS OF DISEASE RESISTANCE MLO GENE FAMILY IN SORGHUM [Sorghum bicolor (L.) MOENCH]

SINGH V.K.¹, SINGH A.K.², CHAND R.³, SINGH B.D.⁴*
¹Centre for Bioinformatics, Faculty of Science, Banaras Hindu University, Varanasi- 221 005, UP, India
²Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi- 221 005, UP, India
³Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi- 221 005, UP, India
⁴School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi- 221 005, UP, India
* Corresponding Author : brahmadsingh@gmail.com

Received : 12-06-2012     Accepted : 18-07-2012     Published : 03-09-2012
Volume : 2     Issue : 1       Pages : 18 - 27
J Plant Genom 2.1 (2012):18-27
DOI : http://dx.doi.org/10.9735/0976-8823.2.1.18-27

Powdery mildew of sorghum (Erysiphe pisi var. pisi) is of worldwide occurrence and causes substantial yield losses. As far as resistance against powdery mildew is concerned, the presence of powdery mildew-resistance gene O (Mlo) plays a key role. The Mlo gene was first identified in barley, and the Mlo protein was found to be an integral plasma membrane-localized protein that possesses seven transmembrane regions. In the present work a total of 12 well reported Mlo genes from Oryza sativa, 15 well characterized Mlo genes from Arabidopsis thaliana genome were taken for comparative studies in sorghum (Sorghum bicolor). Comparative analysis of Mlo proteins from S. bicolor genome revealed the presence of 13 hypothetical genes within the genome. Map viewer analysis indicates that the predicted S. bicolor SbMlo genes are distributed on eight of the ten chromosomes. Sorghum chromosome 9 has 3 genes; chromosome 10 and chromosome 1 have 2 genes each, while chromosomes 2, 3, 4 and 5 have 1 gene each. Sub-cellular localization of identified Mlo proteins encoded by genes SbMlo1, SbMlo2, SbMlo4, SbMlo5, SbMlo6, SbMlo9, SbMlo10, SbMlo11, SbMlo12 and SbMlo13 are present in plasma membrane; SbMlo3 product is located in endoplasmic reticulum, SbMlo7encoded protein is located in vacuolar membrane and SbMlo8 product is present in the nuclear region. In silico characterization (using phylogenetic classification, motif analysis and cis-acting elements studies) suggested its diverse function associated with disease resistance based on specific expression containing fungal elicitor responsive elements. Gene specific primers, expression primers and universal primer were designed to check the expression and availability of SbMlo genes through wet lab experimentation.

Mildew resistance, Sorghum bicolor, comparative analysis, SbMlo, disease resistance, powdery mildew resistance.

The powdery mildew disease in cereals is caused by an obligate biotrophic ascomycete fungus belonging to order Erysiphales. The Mlo (powdery-mildew-resistance gene o) gene was first identified in barley [6] and it constituted the largest family of seven-transmembrane (7TM) domain proteins found in both monocot and dicot plants [12] . Many Mlo homologs have been identified in various plants; for example, thale cress (Arabidopsis thaliana) and rice (Oryza sativa), whose full genome sequences are available, contain 15 AtMlo [11] and 12 OsMlo [22] genes, respectively. Similarly, multiple Mlo genes have been identified in othe species, e.g., 17 VvMlos in grapes [16] , 7 TaMlos in wheat [21] , 2 BrMlos in brassica [25] , 2 LjMlos in lotus [25] , 2 CaMlos in capsicum [25] , and 3 LeMlo in tomato [25] . Mlo gene family members are reported to play crucial roles in modulating defense responses and cell death [19,25,26,38] .
Cereals and millets belong to family Poaceae and provide most of our calorie and protein requirements [35] . Disease resistance gene cluster synteny and genome wide gene family study can give a better understanding of the gene organization at genome level, which may facilitate genetic improvement of crops. Sorghum is the fifth most important ‘cereal’ crop in the world based on total grain production [15] . Sorghum has a relatively small genome (750 Mbp) and it has diverged from maize and rice; this would greatly aid the discovery and analysis of disease resistance genes through comparative genomics. Sorghum is a C4 plant that exhibits drought tolerance, stalk reserve retention capacity and potential to produce more grain per unit photosynthetic area [24] . Expression of Mlo genes can be affected by biotic and abiotic stimuli and the inducibility of Mlo expression under a range of conditions suggested a broad role of Mlo genes [26] . Identification of Mlo genes in other crops would be helpful in understanding the evolution and functional divergence at this locus, and it would also contribute to breeding of powdery mildew resistant cereal varieties.
This paper reports genome wide in silico identification of putative SbMlo gene family of S. bicolor (L.) Moench using the recently available whole genome shotgun sequence of Sorghum for annotation, chromosomal localization, gene organization and phylogenetic tree inferences based on function motifs. Further, comparative phylogeny of O. sativa, A. thaliana and Sorghum Mlo gene families has been attempted, and the putative functions of the predicted SbMlo genes were investigated by analyzing the cis-regulatory elements and transcription factors associated with these genes in the promoter and transcribed regions.

The Arabidopsis (A. thaliana) genome contains 15 Mlo genes [7] designated as AtMlos [11] . The Rice (O. sativa) genome contains 12 genes encoding homologs of Mlo protein; these genes were designated as OsMlos [29] . Based on the information provided by Chen et al. (2006), the Arabidopsis Mlo sequences were retrieved from TAIR web site [http://www.arabidopsis.org/] and reported rice Mlos genes were retrieved from NCBI database [27] . The retrieved sequences were subjected to homology search with the available sequence information at NCBI database using BLASTn, BLASTx and tBLASTx [1,2] tools. Upstream and downstream sequences of Mlo domain homologs were retrieved from the whole genome shotgun sequence of S. bicolor for fishing out putative SbMlo genes. The annotated sequences were further subjected to bioinformatics tools and software, namely, FGENESH [30] for prediction of full length genes with putative full CDS and protein sequences.

Each of the SbMlo genes was positioned on Sorghum chromosomes by the BLASTn search with NCBI genomes (chromosome) database. The structures of predicted SbMlo genes were determined using FGENESH server and intron/exon boundaries were manually identified.

The putative Mlo protein sequences of S. bicolor were subjected to protein functional analysis using PFAM version 24.0 [17] and MOTIFSCAN [14] databases. These sequences were then submitted to PSHORT [http://wolfpsort.seq.cbrc.jp/] server for identification of localization signals. For identification of transmembrane helices, HMMTOP [http://www.enzim.hu/hmmtop/html/submit.html] was used for finding the topology of proteins. For amino acid composition and physico-chemical properties, PROTPARAM [http://expasy.org/tools/protparam.html] was used.

Identified putative Mlos from S. bicolor were used for phylogenetic classification of Mlos from O. sativa and A. thaliana. The sequences from different species were aligned using Clustalw [33] and phylogenetic inferences were constructed using MEGA5.0 [32] .

All Mlo proteins from Arabidopsis, rice and Sorghum were used for conserved motif study using MEME version 4.4.0 [4,5] in Mlo protein domain (Functional Signature Sequences) part. Mlo protein sequences were subjected to INTERPROSCAN version 4.4 [28] for protein functional analysis.

Identified complete Mlo genomic sequences from S. bicolor were used to design primers for complete gene amplification. Expression primers to study the expression of identified genes during pathogenesis were designed using primer3 [31] based on hypothetical mRNA obtained from gene prediction tool.

Domain based homology search using tBLASTn txid4558 of Whole-Genome-Shotgun Sequences (WGS) database identified 13 genes, designated as SbMlo1 to SbMlo13, out of 14 hits form S. bicolor genome [Table-1] .

Using gene prediction tool, 12 full length genes were successfully predicted out of the 13 identified genes, and their organization is summarized in [Table-2] . Chromosome 9 has three of these genes, chromosomes 1, 6 and 10 have two genes each, while chromosome 2, 3, 4 and 5 have one gene each. The number of exons per gene ranged from 8 to 15, but most of the genes had 11-14 exons. Thus, Mlo genes were distributed on eight out of the 10 chromosomes of S. bicolor.

The complete catalog of Mlo proteins in a single plant species is useful for viewing the existing structural and functional diversity associated with their diverse role in plants. The evolutionary relationships among the 13 SbMlo proteins were analyzed by subjecting the amino acid sequences deduced from the identified 13 SbMlo genes for multiple sequence alignment. These proteins formed two main groups: SbMlo6 and SbMlo8 formed one group (group B), and the remaining 11SbMlos formed the group A, which consisted of two subgroups (subgroups I and II had 8 and 3 proteins, respectively).
The putative amino acid sequences of the 13 SbMlo were subjected to multiple sequence alignment analysis using clustalW. Four motifs, having consensus sequence and FWF residues, are totally conserved among the 13 SbMlo proteins [Fig-1b] . Evolutionary study suggests that SbMlo10 has 80.2% and 73.7% similarity with SbMlo9 and SbMlo11, respectively, SbMlo4 is 65.1% similar to SbMlo9, SbMlo3 has 62.3% identity with SbMlo5, and SbMlo2 and SbMlo1 are 58.8% and 50.9%, respectively, identical with SbMlo13. Similarly, SbMlo7 is 50.1 and 50.2% similar to SbMlo9 and SbMlo10, respectively, SbMlo12 is 47.4% identical with SbMlo9, and SbMlo6 has 42.6% identity with SbMlo8. Over this entire alignment pattern it was found that SbMlo10, SbMlo9 and SbMlo11 showed a high degree of similarity with each other, while SbMlo6 showed the lowest percentage of homology.

The organization of the predicted SbMlo genes in terms of intron/exon distribution pattern shown in [Table-3] and [Fig-2a] . The minimum genomic gene size was 2207 bp for SbMlo5, while the maximum size was 6733 bp for SbMlo12. In terms of proteins, the minimum size was 250 aa (28.55 kDa) for SbMlo5, and the maximum size of 654 aa (74.47 kDa) was for SbMlo8. Compostional study of the indentified SbMlo proteins revealed that in most of the, Leucine was the most preponderant amino acid, followed by Val, Ala, Ser and Ile [Fig-2b] .
Transmembrane helix prediction and toplology determination of the13 SbMlo proteins were done using HMMTOP [34] . The number of transmembrane helices ranged from 5 for SbMlo10 to 9 for SbMlo1 and SbMlo 9 [Table-4] .

The predicted 13 SbMlo proteins were subjected to multiple sequence alignment along with the 15 Arabidopsis AtMlo and 12 Oryza OsMlo proteins, and a phylogenetic tree was constructed by software ClustalX2.0.10 with UPGMA method and bootstrap analysis (1,000 reiterations) using MEGA5.0 [Fig-3] .
The 40 Mlo proteins formed three groups: Group I is the largest with 17 Mlo proteins (9 Arabidopsis, 5 Oryza and 3 Sorghum proteins), group II is almost equally large with 16 proteins (3 Arabidopsis, 5 Oryza and 8 Sorghum proteins), and group III is the smallest with only 7 proteins (3 Arabidopsis, 2 Oryza and 2 Sorghum proteins). Thus group I is dominated by AtMlos, group II has predominantly Sbmlos, while group III has almost equal representation from the three species. Further, group III is the most homogeneous of the three groups, while Group I and II show considerable sequence heterogeneity. The group I has AtMlo 2, 3, 5, 6,7, 8, 9, 10 and 12, OsMlo 1, 2, 3, 6 and 12, and SbMlo 1, 2 and 13. The members of group II were Atmlo 1, 13 and 15, Osmlo 5, 7, 8, 9 and 10, and Sbmlo 4, 5, 3, 7, 9, 10, 11and 12. The group III has Atmlo 4, 11, and 14, Osmlo 4 and 11, and Sbmlo 6 and 8.

The 13 SbMlo proteins along with 15 AtMlo and 12 OsMlo proteins were analyzed for the presence of conserved motifs using MEME software. A total of 26 conserved motifs were observed in the 40 proteins using 30 number of motifs with minimum width 50 and maximum width 100 parameter [Fig-4a] [Fig-4b] .
Out of the 26 predicted motifs, Motif 01 was the most conserved pattern found in all the Sorghum, rice and Arabidopsis Mlo proteins. Motif 26 (in SbMlo13 and SbMlo8), Motif 24 (in SbMlo11 and OsMlo11), Motif 23 (in SbMlo12 and AtMlo7), Motif 20 (in AtMlo15 and SbMlo7), Motif 17 (in SbMlo8 and OsMlo6), Motif 16 (in SbMlo7 and OsMlo5), Motif 14 (in SbMlo8 and OsMlo4) and Motif 12 (in SbMlo2 and OsMlo1) were the least conserved and were observed in only two MLO proteins each. Motif 11 was present in three proteins (SbMlo6, OsMlo11 and AtMlo4), Motif 10 was detected in four MLO proteins (SbMlo8, OsMlo4, AtMlo14 and AtMlo11), Motif 13 was found in five proteins (SbMlo11, SbMlo10, SbMlo4, OsMlo9 and OsMlo2), Motif 09 (SbMlo1, OsMlo6, OsMlo3, AtMlo12, AtMlo6 and AtMlo2) and Motif 08 (Osmlo7, OsMlo11, SbMlo5, SbMlo6, SbMlo8 and SbMlo12) occurred in six Mlo proteins each, while Motif 07 was present in seven proteins (OsMlo11, SbMlo8, SbMlo4, SbMlo5, OsMlo7, SbMlo12 and OsMlo8). Thus Motif 01 was present in all the 40 Mlo proteins, and Motifs07, 08 and 09 were the next most frequent (found in 7 and 6 Mlo proteins); the remaining motifs occurred in 5 or less number of Mlo proteins.

The diverse functions attributed to different Mlo genes involve interactions of transcription factors with different conserved sequences in the promoter regions of the respective genes. Cis-regulatory element analysis was carried out with a view to understand the regulatory aspects of the different SbMLO genes. This was achieved by retrieving 500bp upstream sequences from the initiation codon of 12 putative SbMlo genes since the transcription start site (TSS) and initiation codon region are missing from the SbMlo10 sequence due to non-completion of sequence. TATA and CAAT boxes were frequently located within -1 to -200bp. G-Box was the most frequent of the cis-elements; it was found in all the Mlo genes, except SbMlo5 and SbMlo10. ABRE was observed in five genes (SbMlo1, SbMlo3, SbMlo7, SbMlo9, SbMlo12), A-box was found in seven genes (SbMlo2, SbMlo4, SbMlo6, SbMlo8, SbMlo9, SbMlo11, SbMlo13), ARE was present in four SbMLO genes (SbMlo1, SbMlo7, SbMlo8,SbMlo9), ACE in five genes (SbMlo4, SbMlo5, SbMlo6, SbMlo9, SbMlo11); AAGAA in two genes (SbMlo5, SbMlo8), AT-rich sequence in two genes (SbMlo6, SbMlo7), Box-3 and 4 in two genes (SbMlo2, SbMlo7), CCAAT in five genes (SbMlo1, SbMlo7, SbMlo8, SbMlo11, SbMlo12), CGTCA in six genes (SbMlo1, SbMlo4, SbMlo5, SbMlo8, SbMlo10, SbMlo13), CCGTCC in six genes (SbMlo2, SbMlo4, SbMlo8, SbMlo9, SbMlo11, SbMlo13), CAT element in two genes (SbMlo6, SbMlo7), CATT in only SbMlo, CTAG in SbMlo11, CG-motif in SbMlo12, GATA in SbMlo9, GA-motif in SbMlo11 and SbMlo13, GT-1 motif in SbMlo6 and SbMlo12, GAG-motif in SbMlo8 and SbMlo13, GCC in SbMlo2, GARE and GTGGC motifs in SbMlo6, I-box in four genes (SbMlo5, SbMlo9, SbMlo12, SbMlo13), MBS in three genes (SbMlo4, SbMlo5, SbMlo12), O2-site in three genes (SbMlo6, SbMlo12, SbMlo13), Sp1 in eight genes (SbMlo1, SbMlo2, SbMlo5, SbMlo6, SbMlo8, SbMlo9 SbMlo12¸SbMlo13), Skn-1 in SbMlo4-9 and SbMlo13, LTR in three genes (SbMlo2, SbMlo4, SbMlo11), MNF-1 in SbMlo4, TGACG in five genes (SbMlo1, SbMlo4-6, SbMlo8, SbMlo10, SbMlo13), TATC in SbMlo11, TCCACCT-motif in four genes (SbMlo2, SbMlo3, SbMlo11, SbMlo13), TCA-element in three genes (SbMlo1, SbMlo5, SbMlo12), TATCCAT/C in SbMlo2, TGA element in SbMlo4, TCT in SbMlo5, Motif IIb in three genes (SbMlo4, SbMlo6, SbMlo12), plant AP2-like in SbMlo4, and circadian related element in three of the Mlo genes (SbMlo6, SbMlo7,SbMlo9).
Thus G-Box was the most frequent cis-elements found in 10 of the Mlo genes; Sp1, was the next most abundant element occurring in eight genes, while CGTCA, CCGTCC were present in six genes each. Other elements were found in the upstream sequences of five or less number of the Mlo genes.
Since all the 15 A. thaliana Mlo genes are fully characterized, we retrieved -1000 upstream regions of these genes to find out their putative cis-acting elements and compared them with the cis-elements identified in the upstream regions of the 12 putative Mlo genes of S. bicolor. The predicted cis-acting elements for AtMlos and their expression data retrieved using TAIR database [http://www.arabidopsis.org/] are summarized in [Table-5] . The SbMlos showing sequence homology to the AtMlos are also listed.

From complete genomic sequences of the 13 identified SbMlo genes, 24 primer pairs were designed to amplify full length genes from S. bicolor ( [Supplementary-1] ). Further, to study the expression of the SbMlo genes, 13 primer pairs were designed using primer3 [31] on the basis of the predicted RNA encoded by the SbMlo gene CDS ( [Supplementary-2] ).

The 13 predicted SbMlo genes are distributed on 8 chromosomes as provided in the chromosome data of. S. bicolor genome in NCBI datamodel. Sorghum chromosome 9 has 3 SbMlo genes, chromosomes 10 and chromosome 1 have 2 genes each, while chromosomes 2, 3, 4, and 5 have 1 gene each. The complete catalogue of Mlo proteins in a single plant species is useful for viewing the existing sequential, structural and functional diversity associated with its diverse roles played in plants. The evolutionary relationships between different Mlo proteins were analyzed by subjecting the deduced amino acid sequences encoded by the identified 13 SbMlo genes for multiple sequence alignment. Amino acid composition of these hypothetical Mlo proteins showed that they all are leucine-rich. It also provides evidence that Mlo gene family belongs to leucine-rich class of plant disease resistance genes. Multiple sequence alignment of these SbMlo proteins showed that they are a well conserved family divided into two sub-groups containing 3 clusters. The analysis of introns/exon gene structures revealed that most introns have conserved positions and phases, providing the evidence for the intron–early theory, and that multiple independent intron loss events are likely to have occurred during evolution of flowering plants. The hypothesis that genome wide and tandem duplication contributed to the expansion of the Mlo gene family across the plant kingdom seems to be applicable for Sorghum as well as two other diploid species, namely, Arabidopsis and Oryza, which contain 15 and 12 Mlo genes, respectively [21] .

Evolutionary relationship of the 13 Sorghum Mlo genes with the 12 O. sativa [22] , and 15 A. thaliana genes (obtained from complete genome of the species) revealed a similar classification pattern in sequence evolution in these three species. The 15 members of the Arabidopsis Mlo gene family are well characterized, and they have been shown to mainly function as modulators of plant defence and cell death. The coding regions of the 13 putative SbMlo genes show substantial coding sequence homology with one or the other member of the Arabidopsis Mlo gene family; therefore, the SbMlo genes are also expected to play an important role in the defense mechanism of Sorghum [3,8,23,36] Mlo genes are novel Calmodulin-binding Proteins [20] . Some members of Arabidopsis Mlo gene family are also reported to play an important role during leaf senescence, seedling development, stigma receptivity, pollen tube development, fruit ripening, and development of flower bud and flower abscission zone [7] .
Evolutionary study of SbMlo genes revealed two groups (A and B) of which group A could be divided into three clades. Clade III contained SbMlo1, 2 and 13, which exhibited similarity with AtMlo9, 10, 5, 7 and 8. Clade II contains SbMlo5 and 3, which showed homology with AtMlo12, 2, 6 and 3. Clade I consisted of SbMlo 7, 4, 9, 10, 11 and 12, which were grouped with AtMlo1, 13 and 15. Clade IV contained SbMlo6 and 8 that showed similarity with AtMlo4, 11 and 14. Cluster I of Group A consisted of SbMlo1, 13 and 2, which has been classified with AtMlo2, AtMlo6, AtMlo12, AtMlo3, AtMlo9, AtMlo10, AtMlo5, AtMlo7 and AtMlo8. Promoter region comparison between Arabidopsis and Sorghum Mlo genes showed possible role of the SbMlos during defense response to fungus, seedling development stage and leaf senescence with modulatory function as reported in the case of Arabidopsis genes [7,9,18] . Cluster -II group A had SbMlo10, SbMlo11, SbMlo9, SbMlo4,SbMlo5, SbMlo7, SbMlo3 and SbMlo12, which have been classified with AtMlo1, AtMlo15 and AtMlo13; thus these SbMlo genes may play a role during early seedling growth, cotyledon vascular system development, in pollen and in papillae [7,9,37] . Studies on Group B cluster-III revealed that SbMlo8 and SbMlo6 were closely similar with AtMlo4, AtMlo11 and AtMlo14. SbMlo8 and SbMlo6 may play an important role during seedling growth, flower development, fruit abscission as per phylogenetic classification and promoter comparison with Arabidopsis.

The cis-regulatory element analysis of the predicted SbMlo genes revealed major putative function as regulation of genes associated with abiotic and biotic stresses, photoperiod response, growth hormone and meristem specific elements. The occurrence of CAAT, TATA and G-Boxes is very high in the upstream regions of the predicted SbMlo. Genes SbMlo5, 10, 4, 5, 6 and 12 showed MYB binding site involved in flavonoid biosynthetic gene regulation. Skn-1 element required for endosperm expression was observed in SbMlo4 to SbMlo9. TTGAC (WBOXATNPR1), TCA (Salicylic acid responsive), TGAC (WBOXNTERF3), TCT (light responsive), TATCCAT/C, TGACG (methyl jasmonate), TCCACCT, CCGTCC elements were also observed in the upstream regions, which may play a role in regulation of genes involved in defense mechanism of Sorghum plants.

In this study, a comprehensive computational analysis was conducted, and 13 members of the Mlo gene family were identified in Sorghum. A complete overview of this gene family in Sorghum is presented, including the multiple sequence alignment, gene structures, phylogeny, chromosomal locations and their cis-regulatory element analysis. The comparative phylogenetic analysis with respect to the Mlo gene family clearly indicated the proximity of Sorghum Mlos with rice and Arabidopsis Mlo genes, even when Sorghum and rice are monocots, while Arabidopsis is a dicot. Further, the presence of similar groups and subgroups in comparative phylogeny of Sorghum, rice and Arabidopsis Mlo genes indicates conservation of Mlo gene sequences even in widely separated taxonomic groups of plants. The identified proteins showed similarity with signature accession PF03094 (Pfam database), IPR004326 (INTERPROSCAN), cellular component integral to membrane (GO: 0016021) and biological process with cell death (GO: 0008219) from Gene Ontology database [http://www.geneontology.org/] . The in silico investigation of putative genes from Sb Mlo gene family needs to be supported by wetlab experiments through expression profiling of respective genes by designing the molecular markers using hypothetical mRNA and amplification of full length candidate Mlo genes from S. bicolor.

The facilities provided by DBT-funded SUB-DIC, Centre for Bioinformatics, Faculty of Science, Banaras Hindu University, Varanasi are thankfully acknowledged.

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	Fig. 1a- Phylogenetic classification of the13 identified SbMlo proteins from S. bicolor
	Fig. 1b- Alignment of 13 SbMlo proteins identified from S. bicolor
	Fig. 2a- Complete gene organization including Exon-intron boundaries for each SbMLO genes
	Fig. 2b- Amino acid composition comparartive graph of the 13 identifies SbMlo proteins
	Fig. 3. Ancestral states of the phylogenetic tree were inferred using the maximum parsimony method; the total numbers of Mlo gene sequences taken were 15 from A. thaliana, 12 from O. sativa and 13 from S. bicolor.
	Fig.4a- Highly representative motif study using Sorghum, rice and Arabidopsis Mlo proteins.
	Fig.4b- Multilevel consensus sequences for the MEME defined motifs observed among Mlo proteins from Sorghum, rice and Arabidopsis.
	Fig. 5- Cis-acting elements study of 12 SbMlo genes using PLANTCARE
	Supplementary Table 1- Genomic primer pairs for amplification of identified full length genes.
	Supplementary Table 2- Primer for expression studies of SbMlo genes.
	Table 1- Genome wide identification of Mlo gene family in S. bicolor
	Table 2- Organization of the S. bicolor Mlo genes.
	Table 3- SbMlo intron-exon boundaries, CDS size, full length of the encoded hypothetical amino acid sequence with molecular weight and theoretical pI.
	Table 4- Transmembrane helices prediction and toplology of the predicted 13 SbMlo proteins from S. bicolor.
	Table 5- Study of cis-acting elements in upstream region of AtMlo genes and their function based on GUS activity patterns. The elements common in SbMlo and AtMlo promoter region are highlighted in green color.
	Table 6- Cis-acting Elements, their sequences and function