Study On Cotton MiR319b-TCP4-like Participating In Regulation Of Plant Defense Against Verticillium Dahliae

Interaction of plant and microorganism promotes biological evolution,which is one of the driving forces of biodiversity.Therefore,studying on plant defense against pathogens can reveal plant immunity mechanisms,which also paves a way for plant disease resistance breeding.Cotton is an important cash crop in the world.It can provide natural fiber,edible oil and protein feed,which is closely related to our daily life.However,cotton Verticillium wilt seriously restricts cotton production.Identification of cotton disease related genes and their application in disease resistant variety breeding are required to further strengthen.Micro RNA(miRNA)plays important roles in plant growth and development and defense,More and more studies show that miRNA is involved in plant defense against pathogens invasion.Based on our previous studies,a differentially expressed miRNA and its response target gene were screened and identified from Upland Cotton miRNA transcriptome database and degradation database,namely ghr-miR319 b and GhTCP4-like.Then,ghrmiR319 b and GhTCP4-like were characterized to participate in cotton plant resistance to Verticillium dahliae infection.And according to omics,molecular genetics and biochemistry,the molecular mechanism of ghr-miR319b-GhTCP4-like module regulating cotton resistance was revealed.The research results mainly include:1.Bioinformatics analysis showed that ghr-miR319 b had a typical conserved seed sequence in plant mir319 family.The target gene GhTCP4-like encodes a CIN type TCP transcription factor.The expression levels of ghr-miR319 b GhTCP4-like significantly changed in plants at 7 and 10 days after V.dahiae infection compared to the mock treated plants.As expected,the expression level change of GhTCP4-like showed the opposite trend under pathogen induction.According to degradation omics analysis and transient expression GUS reporter gene analysis,ghr-miR319 b guided the cleavage of GhTCP4-like transcript to reduce its expression level.2.To evaluate GhTCP4-like and ghr-miR319 b function in plant defense,the GhTCP4-like silencing material TRV: GhTCP4-like was first developed by VIGS technology,which then were suffered to V.dahliae infection.The results showed the GhTCP4-like silenced plants showed more serious disease symptom than the control(TRV:00).The disease plant rate and the disease index of GhTCP4-like silencing plants were significantly higher than those of the control.The analysis of stem fungi recovery culture showed that the hypha content in silenced plants was significantly higher than that of control.Therefore,GhTCP4-like was a positive transcription factor for cotton plant resistance to V.dahliae infection.At the same time,the ghr-miR319 b silenced plants was generated by combination of VIGS and STTM technologies.qPCR analysis showed that the expression of ghr-miR319 b in silenced plants decreased by 50%compared with the control,while the expression of GhTCP4-like increased by about70%.When the VIGS plants were inoculated with V.dahliae,ghr-miR319 b silenced plants showed higher resistance than the control.Compared with the control,the leaves were slightly chlorotic,the color of stem sections was lighter,and the amount of stem fungi was lower in ghr-miR319 b silenced plants.These data suggest that ghr-miR319 b is a negative regulator of cotton plant resistance to V.dahliae.In conclusion,ghrmiR319b-GhTCP4-like module is involved in cotton resistance to V.dahliae.3.Bioinformatics analysis showed that GhTCP4-like was a typical transcription factor,which was predicated to locate in the cell nucleus.The GhTCP4-like promoter region was predicated to contain light response element,drought response element,SA response element,etc.Previous studies have shown that some TCPs in plants are involved in the expression of genes related to SA synthesis pathway.In this study,the expression of GhICS1 in GhTCP4-like and ghr-miR319 b silenced plants is significantly changes compared to the control.And the promoter region of GhICS1 contained a cis element GTGGCACC,which is a typical CIN type TCPs binding site.Analysis of electrophoretic mobile shift assay(EMSA)and tobacco transient expression GUS reporter gene showed that GhTCP4-like could bind to GhICS1 promoter cis element and promote GhICS1 expression in cells and in vitro.HPLC-MS analysis confirmed that under V.dahliae inoculation,the SA content in GhTCP4-like silenced plants was significantly lower than that in the control,suggesting that GhTCP4-like can directly activate the expression of GhICS1,promote SA accumulation and improve plant disease resistance.4.In order to further explore the molecular mechanism of ghr-miR319b-GhTCP4-like function in plant defense,co-expression analysis predicted that GhTCP4-like may interact with GhNPR1.The interaction of GhTCP4-like and GhNPR1 was proved by yeast two hybrid(Y2H)and luciferase complementation assay.The GUS reporter gene analysis showed that GhNPR1 could promote the transcriptional activation of GhTCP4-like to GhICS1.These results suggest that GhNPR1,as a receptor of SA,can promote the transcriptional activity of GhTCP4-like to GhICS1,improve the level of SA,which forms a positive regulatory cycle to accelerate the response of plants to pathogen invasion.In conclusion,the results of this study show that cotton miR319b-GhTCP4-likeICS1 pathway is involved in plant resistance to V.dahliae.And GhNPR1 can interact with GhTCP4-like,which cooperates with miR319b-GhTCP4-like-ICS1 pathway to regulate SA synthesis rate and promote plant defense to pathogens infection.