Functional Characterization Of Nicotiana Tabacum MAPK Genes

Nicotiana tabacum(common tobacco) is an important research model and economic plant. Studies on tobacco disease-related genes not only improve our understanding of the plant defense mechanism, but also help to reduce production lost caused by disease. The mitogen-activated protein kinase(MAPK or MPK) cascade is an important signaling module and play critical roles in response to biotic and abiotic stresses as well as plant growth and development. However, little is known about tobacco MAPK genes. In this study, we focused on identification, evolutionary analysis and functional analysis of tobacco MAPK genes. In addition, RNA-seq technology was performed to investigate plant defense mechanism at transcriptional level. The major findings are listed as follows:① The published tobacco sequence data, such as TGI(Tobacco Genome Initiative) and ESTs, were recuited to design primers for cloning 17 tobacco MAPKs, among which 11 were newly identified. Phylogenetic analysis showed that tobacco MAPK genes were distributed into six clusters. Amino acid alignment analysis showed that all the 17 MAPKs contained 11 conserved domains and TXY/MEY motifs.② Because most of identified MAPK genes play important roles in response to biotic and abiotic stresses, we performed QRT-PCR analysis to investigate the expression pattern under treatment of SA, Me JA and drought. This investigation revealed that expression of 9 tobacco MAPKs were regulated by SA treatment, and 11 MAPKs were regulated by Me JA treatment. In addition, we also found that 6 of MAPKs were affected by drought treatment. These results suggested that tobacco MAPKs might play critical roles in plant denfese response.③ We used the published Solanaceae genomes, including pepper, tomato, potato and eggplant, to identify MAPK gene family for each of them. The evolutionary history of MAPK genes in Solanaceae was investigated using these data as well as tobacco and an outgroup Arabidopsis. The numbers of MAPK genes in each plant were very similar. Phylogenetic analysis showed that both gene gain and lost events occurred in this gene family. In addition, gene structure analysis revealed the diversity of intron numbers and length, even though the CDS were conserved. Molecular evolution analysis uncovered that a large proportion of MAPKs might undergo purifying selection. However, positive selection might drive the evolution of some MAPK sub-groups, such as MKA1, MKA8 and MKB1.④ We choose Nt MPK2, the ortholog gene of At MPK4, to investigate its function. Structure analysis revealed that this gene contained multiple disease-related transcription factor binding sites in the promoter region, suggesting that the expression of this gene might be regulated by plant defense signals. Investigation of expression pattern showd that this gene was expressed in leaves and flowers, indicating that it might function in the two organs. We also constusted 35S:Nt MPK2 overexpression vector and transferred this gene into tobacco genome. Several plants were validated as the positive transgene lines at genome level, RNA level and antibody screening.⑤ To investigate the function of Nt MPK2, wild type tobacco and Nt MPK2 transgene tobacco were treated with virulent Pst DC3000. The wild type tobacco leaves became diseased, with wet, chlorotic, spreading lesions when inoculated with virulent Pst DC3000. In constrast, the transgene lines showed less diseased symptom or no diseased symptom. We also analyzed the number of bacterial colonies recovered from infected wild type and transgene tobacco. The three transgenic lines had significantly lower bacterial counts at 24 hpi compared to the wild type plants. These results showed that Nt MPK2 transgene enhanced resistance to virulent Pst DC3000 in tobacco. We also analyzed tobacco resistance to avirulent Pst DC3000 by counting the number of bacterial colonies. Similar to virulent Pst DC3000 analysis, the transgenic lines had lower bacterial counts at 24 hpi compared to the wild type plants. However, after 24 hpi, the bacterial counts in transgenic lines dramatically increased and were significantly higher than that in wild type plants at 48 hpi. This result suggested that multiple pathways might be involved in response to avirulent Pst DC3000.⑥ To further understand the function of Nt MPK2, we use avirulent Pst DC3000, which produced hypersensitive response(HR) lesions in tobacco, to inoculate wild type and transgenic tobacco. We also analyzed the number of bacterial colonies recovered from infected wild type and transgenic tobacco at 0, 12, 24, 36 and 48 hpi. Similar to previous result, the transgenic tobacco had significantly lower bacterial counts than the wild type tobacco at 24 hpi. However, the wild type tobacco showed similar numbers of bacterial colonies to transgenic tobacco at 36 hpi. At 48 hpi, the bacterial counts in transgenic tobacco overwhelmed that in wild type plants. These results indicated that multiple pathways might involve in plant defense response to Pst DC3000.⑦ RNA-seq technology was used to investigate the differentially expressed gene in four samples(10 m M Mg Cl2 mocked wild type tobacco and Nt MPK2 transgenic tobacco, avirulent Pst DC3000 infected wild type tobacco and Nt MPK2 transgenic tobacco). De novo assembly genereated 215,050 contigs and 152,824 unigenes. We found 1,325 differentially genes regulated by avirulent Pst DC3000 and 32 genes affected by Nt MPK2 transgene. GO annotation of 1,325 Pst-regulated genes showed that function of these genes involved in binding, catalytic, transcription regulation, enzyme regulator, metabolic process, biogicial process, transporter and response to stimuli and so on. Further investigation uncovered that multiply pathways involved in tobacco response to Pst DC3000, including SA pathway, JA pathway, ethylene pathway and secondary metabolite biosynthesis pathway. Annotation of Nt MPK2-mediated genes showed that these gene were glycine-rich cell wall structural protein, abscisic acid and environmental stress-inducbile protein TAS14, asparagine synthetase and GDSL esterase/lipase and so on. In addition, non-coding RNA might be involved in Nt MPK2-mediated resistance to pathogen in tobacco.Through this study, we performed a comprehensive analysis of tobacco MAPKs, including genome-wide identification, evolutionary history, functional analysis and transcriptional changes. The information provides valuable clues to study plant defense mechanism at molecular level as well as identify new disease-related genes and non-coding RNAs.