Characterization Of Gbmpk3, A Mitogen-Activated Protein Kinase Gene From Cotton

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules found in all eukaryotes, and they play essential roles in cell signal transduction. A mitogen-activated protein kinase gene, GbMPK3, was induced upon Verticillium dahliae infection. In this work, GbMPK3was isolated from sea-island cotton Hai7124and its functional roles in response to different abiotic and biotic stress conditions were analyzed in both tobacco (Nicotiana benthamiana) and upland cotton YZ1. The main results of this work were as follows:1. Cloning and characterization of GbMPK3An EST (CLlContig1773) putatively encoded a MAPK, GbMPK3, was isolated based on the results of RNA-seq of sea-island cotton Hai7124inoculation with V. dahliae. The full-length of cDNA and DNA sequences were obtained through RACE and Genome-walking. Sequence similarity search and phylogenetic analysis revealed that GbMPK3was classified as a group A MAPK. The35S-GFP::GbMPK3constructs were completed and introduced into tobacco leaves to investigate the subcellular localization of GbMPK3, and the result indicated that GbMPK3protein mainly accumulated in the nucleus.2. Expression profile of GbMPK3The expression level of GbMPK3in cotton tissues under normal condition was evaluated by RT-PCR, and the results showed that GbMPK3expression level was low in all tissues and preferentially expressed in roots, petals, anthers and fibers. qPCR was employed to determine whether the expression of GbMPK3was affected by hormones and stresses, and GbMPK3was up-regulated upon cold (4℃), heat (42℃),10μmol/L MV,200mmol/L NaCl, dehydration, hormones (such as1mmol/L SA) and V. dahliae treatments in our study.3. Overexpression of GbMPK3enhances drought and oxidative stress tolerance in tobaccoGbMPK3was introduced into tobacco and cotton to generate transgenic plants with constitutively higher expression of GbMPK3. Homozygous (T2) transgenic tobacco were conferred with enhanced drought tolerance, reduced water loss during drought treatment, improved plant height and survival rates after re-watering. Additionally, the gene expression levels and enzymatic activity of antioxidant enzymes were more strongly induced with depressed hydrogen peroxide accumulation in GbMPK3-overexpressing tobacco compared with wild-type under drought condition. Furthermore, observation of seed germination and leaf morphology showed that tolerance of transgenic plants to MV was improved due to increased antioxidant enzyme expression, suggesting that GbMPK3may positively regulate drought tolerance through enhanced reactive oxygen species scavenging ability.4. Overexpression of GbMPK3affects tobacco disease resistance to Tobacco mosaic virus (TMV) and Botrytis cinereaWilt-type and transgenic tobacco were inoculated with different types of plant pathogens. Transgenic tobacco enhances resistance against TMV with reduced spreading rate and reproduction of TMV. Yet overexpression of GbMPK3significantly reduces tobacco resistance to the fungal pathogen Botrytis cinerea.5. Overexpression of GbMPK3reduces V. dahliae resistance in tobacco and cottonWilt-type and transgenic plants were inoculated with V. dahliae to investigate the involvement of GbMPK3in plant defence. GbMPK3overexpression tobacco and cotton shows enhanced disease susceptiblity to V. dahliae strain V991. Both wilt-type and transgenic plants shows typical symptoms of verticillium wilt several days post V. dahliae infection, including wilting and yellowing leaves. Whereas the disease symptoms appeared early in GbMPK3-overexpression plants, resulting in elevated rate of diseased plants and disease index compared to wilt-type. The V. dahliae resistance was also analyzed in VIGS built GbMPK3-silencing plants. Silence of GbMPK3in cotton did not affect pathogen invasion, but slightly reduces yellowing and wilting leaves of diseased plants, as show by the decreased disease index. Therefore, while the effect of GbMPK3-silencing on cotton resistance to V. dahliae turns out to be weak, GbMPK3overexpression could significantly enhances V. dahliae susceptibility in both tobacco and cotton.6. GbMPK3affects SA-related gene expression in cottonqPCR analysis showed that the transcripts of SA-related genes (including the transcription factor WRKY70and two maker genes PR1and PR5) were accumulated in both wild-type, GbMPK3-silencing and overexpression cotton roots at day5post V. dahliae inoculation, suggested an up-regulation role of SA signaling in susceptible cotton cultivar YZ1. The elevated level of PR1and PR5was almost the same in wild-type and GbMPK3-silencing plants, yet transcripts of WRKY70, PR1and PR5were higher in GbMPK3overexpression cotton, indicating that GbMPK3overexpression may enhances V. dahliae induced SA signaling transduction. In previous studies, researches suggested JA signaling acted as a positively regulator in plants defense to V. dahliae. SA signaling is usually known antagonistic to JA signaling. Many important genes in regulating SA and JA signaling were reported, such as WRKY70and NPR1. Thus, SA may also participate in the complex signal networks of V. dahliae resistance in plants. But the mechanism of SA involved in cotton defense and the relation between elevated SA-related defence gene expression and reduced V. dahliae resistance still need to be studied further.