Mechanism Analysis Of MiR164-GhNAC100 And GhWRKY22 Participating In Cotton Resistance To Verticillium Wilt

With the mounting evidences in molecular biology,there have been new research fields emerging in plant defense,such as the discovery of miRNAs.In Arabidopsis,miR164 regulates plant resistance against abiotic and biotic stresses,and leaf and lateral root development.There are recently increasing reports on the regulation of physiological and biochemical characteristics in cotton by miRNAs,but the miRNA regulation mechanisms in plant defense remain to be few.Additionally,the WRKY,one of the largest transcription factor families,occupies a pivotal position in plant regulatory networks,especially in plant defense.Although there have been several reports on the research of WRKY transcription factors in cotton disease resistance,novel WRKY proteins and their molecular mechanisms involved in disease resistance need to be further studied.In this study,mi R164 and its target gene GhNAC100 as well as Gh WRKY22 were addressed for their roles in plant defense,the main results were as follows:1.Through analysis of the sRNA and degradative group sequencing,sighificantly differentially expressed miR164 and its target gene GhNAC100 were picked out for performing function research.The expression level of miR164 and GhNAC100 showed significant changed in plants treated with Veticillium dahlia compared to the mock treatment,and both exhibited opposite expression.2.The full-length gene of GhNAC100 and the pre-miR164 precursor of miR164 were cloned.And the over-expression vectors of pBI121-NACW,pBI-121-NACM,and pBI-121-pre-mi R164 were constructed,which were then transformed into Agrobacterium GV3101.Through conducting tobacco transient expression,the results confirmed that miR164 can target GhNAC100 mRNA and degraded it,resulting in inhibiting this transcription factor expression..3.GhNAC100 expression in seedlings was significantly induced by V.dahliae inoculation.And then under treatments of Methyl Jasmonate(MeJA)and Salicylic acid(SA),GhNAC100 expression showed strong response compared to the mock treatment,indicating that GhNAC100 could be a component of SA and JA signal pathway to participate in plant disease resistance.4.To assess the GhNAC100 roles in plant defense,the virus-induced gene silencing vector pYL-156-GNAC was generated,and transferred into Agrobacterium GV3101,which used to innoclate cotton cytoledon to develop the GhNAC100-silenced plants.These silenced plants were challenged with V.dahliae for investigating the GhNAC100 funciton.The results showed that GhNAC100-silenced plants were more susceptible to disease compared to the control plants,exhibiting higher disease rates and recovering rates of pathogen growth.The expression levels of SA and JA defense marker genes,PR1 and PDF1.2,were significantly lower than those of the control plants at 72 h after V.dahliae inoculation,indicating that the GhNAC100 is involved in plant resistance to V.dahliae possibly through SA and JA signaling pathways.5.According to sequences of the disease resistance-related Arabidopsis WRKY,eight orthologous cotton WRKY genes were isolated.The expression of 8 cotton WRKY genes were analyzed under the treatment with V.dahliae.The Gh WRKY22 showed significantly up-regulated expression,which was chosen for further researching.The induction of MeJA and SA was performed on the plants and the expression of GhWRKY22 was detected.The results showed that the expression of Gh WRKY22 was regulated by SA and JA signaling pathways.6.Specific fragments of the Gh WRKY22 gene were cloned and the VIGS system was established.Subsequently,the expression of Gh WRKY22 in the cotton-silenced plants was detected.The results showed that Gh WRKY22 was strongly silenced,only 40% of the control.To investigate the GhWRKY22 function in plant defense,the silenced plants were inoculated with V.dahliae.The results showed that the silenced plants were more sensitive to the infection of pathogens,the incidence and disease values,and the recovery rate of pathogens were significantly higher than the control group.Additionally,the expression levels were analyzed.Results the expression level of disease resistance marker genes PR1,PR3,PR4,PR5,PAL and PDF1.2 was significantly lower in the Gh WRKY22-silenced plants than those of the control plants,further indicating that the Gh WRKY22 gene participates in the plant resistance to V.dahliae through SA and JA signaling pathways.