In search of function for hypothetical proteins encoded by genes of SA-JA pathways in Oryza sativa by in silico comparison and structural modeling.

Knowledge of rice genome brings new dimensions to the management of abiotic stresses; however, gene sequences in the rice genome are yet to be assigned structure and function. Hydrogen peroxide, salicylates and jasmonates act as signal molecules in plants employing common machinery to manage abiotic stress. The present work is primarily focused to assign a structurefunction relationship by modeling of the hypothetical proteins of SA-JA signaling pathway known in Arabidopsis thaliana and compare them with corresponding proteins in rice in silico. Thirteen known gene sequences with their encoded proteins for SA/JA pathway in model plant A. thaliana were obtained and similar gene sequences from rice were retrieved at NCBI. Five rice gene sequences Os09g0392100, Os03g0233200, OsJ_33269, OsJ_23610 and Os01g0194300 resulted in hypothetical protein products with unknown structure and function. Modeling and comparison of 5 proteins from rice and Arabidopsis showed 73 - 98% identity with acceptable RMSD values of 0.6 - 1.7 upon superimposition. Results suggest conserved nature of these proteins during evolution. The hypothetical protein from rice contains similar functional protein domain as that in A. thaliana and therefore are likely to perform similar functions in rice. There is a cross talk between the genes in SA/JA pathway wherein Os09g0392100 or EDS1, Os03g0233200 or PR5, OsJ_33269 or PAD4 and OsJ_23610 or SFD-1 activates the pathway and Os01g0194300 or NPR1 inhibit the pathway. Further investigation through wet-lab experiments are in progress to look into suppression/activation of the genes of SAJA signaling in rice plants exposed to abiotic stress.

Background

With the availability of rice genomic information [1] it is possible to exploit gene sequences from rice varieties, for stress management and enhanced crop yield [2]. Rice plants respond to various abiotic and biotic stresses via a number of mechanisms [3]. Salicylic acid and Jasmonic acid have been reported to act as signal molecules in abiotic stress amelioration in plants [4-9]. Protective action of SA in the presence of abiotic stress is tied with cell membrane stabilization [7] and suppression of antioxidant enzyme activities [9]. Jasmonic acid a derivative of linolenic acid [10] is involved in the plant defense mechanism through a family of peptide ligands [11-13] resulting from enhanced antioxidant levels [3]. Since plants response to abiotic stress, salicylic acid and jasmonic acid is characterized by the altered formation of reactive oxygen species, therefore it is quite likely that genes involved in SA and JA pathways collide in plants under abiotic stress. The authors used the gene sequences reported in SA/JA pathway from model plant Arabidopsis thaliana and searched for similar genes in rice genome. Surprisingly most of the resultant sequences for SA/JA pathway obtained upon similarity search from rice were unannotated. The present study therefore aims to annotate the selected unknown gene sequences of rice having similarity with A. thaliana genes involved in SA/JA pathway by using gene ontology, homology search, structural modeling and superimposition tools in silico, so as to better understand role of these genes in SA/JA pathway in rice plants.

Methodology

Sequence retrieval and data analysis:

Nucleotide sequences were obtained from National Centre for Biotechnology Information (http://ncbi.nlm.nih.gov). Full genome of model plant Arabidopsis thaliana (Arabidopsis Genome Initiative 2000) [14] was initially chosen and retrieved from the nucleotide database at NCBI and the protein coding genes identified in the cross linking pathway of salicylate and jasmonate were retrieved from NCBI's database [15]. The corresponding proteins of the genes retrieved from Arabidopsis were checked for the presence of functional domains and protein signatures using InterPro Scan sequence search [16] of EMBI (European Bioinformatics Institute).

Prediction of Gene Ontology:

The ontology of the genes involved in SA/JA pathway was decided using TAIR (The Arabidopsis Information Resource), a database of genetic and molecular biology data for the model higher plant Arabidopsis thaliana [14]. Data available from TAIR includes the complete genome sequence along with gene structure, gene product information etc which is updated every two weeks from the latest published research literature and community data submissions. Protein products for the thirteen genes were obtained and their sequences were retrieved. Shown in SA/JA pathway Figure 1 and listed in Table A (see supplementary material).

Figure 1

Genes involved in signaling via salicylate and jasmonate derivatives in plants exposed to abiotic/biotic stress and plant defense. EDS-1: Enhanced Disease Susceptibility1; CPR-6: Constitutive expressor of PR genes 6; PAD-4: Phytoalexin Deficient 4; EDS-5: Enhanced Disease Susceptibility 5; SID-2: Salicylic-acid-Induction Deficient 2; TGA-2: TGAelement binding protein 2; SFD-1: Suppressor of Fatty-aciddesaturase Deficiency 1; PR: Pathogenesis-Related gene.; CPR- 5:Constitutive expressor of PR genes 5; NPR-1: Non-expressor of PR1; SNI-1: Transcriptional repressor of SAR genes; SSI-2: Suppressor of SA-Insensitivity.

Homology Search and Structural Modeling:

Sequences obtained above were used as query for searching homologous sequences in Oryza sativa using BLASTp [17]. Seven protein sequences (homologous) in rice that had not been assigned any function till date were obtained. Of these five proteins of O. sativa and A. thaliana were then modeled for their respective 3-dimensional structures using Geno3d [18]. Proteins of O. sativa and corresponding protein of A. thaliana were superimposed [19] for structural similarity based upon RMSD, percent identity, and other parameters. The structures were viewed using UCSF Chimera tool [20] an interactive molecular graphics program with high extensibility, interactive molecular visualization and analysis system. The sequence for each protein from O. sativa and A. thaliana for best 20 structures were obtained.

Validation of various physiological parameters:

The obtained proteins were also checked using ProtParam tool of ExPASy Proteomics Server. This allows the computation of various physical and chemical parameters like theoretical pI, hydropathicity, estimated half-life, etc. for a given protein stored in Swiss-Prot or TrEMBL or for a user entered sequence.

Result & Discussion

SA/JA pathway genes of Arabidopsis thaliana and Gene ontology prediction:

Overall 13 genes involved in SA/JA pathway were retrieved from A. thaliana database at NCBI and 13 homologous gene sequences were obtained for each gene from O. sativa genome by BLAST algorithm. Of these five gene sequences OSJ_014658, Os04g0689900, OsI_04142, OsJ_33269, OSJ_23610 and Os01g0194300 from rice having >70% identity and corresponding to genes of A. thaliana TAIR ID: AT3G48090, AT1G75040, AT3G52430, AT2G40690, AT1G64280 respectively were selected, which resulted in hypothetical proteins whose structure and functions are not known. Gene ontology study of A. thaliana genes using Interpro domain search program predicted presence of conserved gene specific domains and their function in SA-JA cross linking (Table 1) and this is still not reported in rice.

Retrieval of predicted cross linking genes from O. sativa genome:

Protein known in O. sativa to that of A. thaliana were screened using BLOSUM62 matrix. Protein sequences with more than 70% identity with Arabidopsis genome were selected in O. sativa. Of which OSJ_014658, Os04g0689900, OsI_04142, OsJ_33269, OSJ_23610 and Os01g0194300 corresponding to EDS1, PR5, PAD4, SFD1, and NPR1 genes (TAIR ID: AT3G48090, AT1G75040, AT3G52430, AT2G40690, AT1G64280 respectively) of A. thaliana respectively were obtained as hypothetical protein and are still unannotated.

Structural modeling of proteins of A. thaliana and O. sativa involved in SA/JA pathway:

Sequence similarity could be a measure for functional similarity; however for two proteins to be functionally similar it is important that the two proteins are also structurally similar. Model were created for the known proteins of SA/JA pathway retrieved for A. thaliana and corresponding proteins (BAF25000.1, NP_001049473.1, EAZ17725.1, EEE66834.1, NP_001042286) of O. sativa. Five unknown protein sequences of O. sativa were modeled for their structure using Geno3d tertiary structure prediction tool which resulted in three PDB files for each of the query protein, out of which the most appropriate structural coordinate file was chosen based on R-value, B-factor, Ramachandran plot etc. (data not included). The O. sativa proteins were aligned structurally with corresponding A. thaliana protein and their superimposed structures were evaluated Table 1 (See supplementary material). Reasonably good RMSD values were obtained that lie between 0.6-1.7 suggesting proper structural and functional similarities between the proteins of the two species. The functional similarity was further validated by Protparam (Table B, see supplementry material) that suggest the rice proteins to have similar estimated half life of >20 hrs (in yeast), a varied hydropathicity and all had a theoretical pI ranging between 4.75 and 9.83.

Conclusion

In conclusion results suggest that similar to that in Arabidopsis there exist a SA- JA cross linking pathway in O. sativa as well. In SA-JA interacting pathways 13 genes, are found to be expressed that either activate (EDS-1, R-gene, CPR-6, PAD-4, EDS-5, SID- 2, TGA-2, SFD-1, PR) or inhibit (CPR-5, NPR-1, SNI-1, SSI-2) the effect of abiotic stress in A. thaliana. Of these, the structure and function of five gene sequences from O. sativa genome are not known. In silico comparative structural modeling and comparison of physiological parameters of the proteins coded by these five genes in A. thaliana and O. sativa using different bioinformatics tools, suggest them to be conserved in nature. Results, predict that the hypothetical protein translated from these five unanotated genes in rice are similar in structure to the proteins obtained for similar genes from A. thaliana and therefore these protein probably have similar functions as that in A. thaliana. It is also likely that these genes are involved in SA- JA cross linking pathway where OsJ_014658 or EDS1, Os04g0689900 or PR4, OsI_04142 or TGA2, OsJ_33269 or PAD4 and OsI_23610 or SFD-1 might activate the SA-JA signaling pathway in rice whereas Os01g0194300 or NPR1 perhaps inhibit the pathway. Further investigation through wet-lab experiments are in progress that would provide a better insight into suppression or activation of these genes involved in SA-JA signaling in rice plants exposed to abiotic stress.