Studies On The Mechanism Of GhmiR7814/GhNBS-LRR And GhmiR8687/GhWATs Participating In Cotton Verticillium Wilt Resistance

Cotton verticillium wilt,as a soil-borne fungal vascular disease,seriously decreases cotton fiber quality and yield.The strains of verticillium wilt pathogens in the field is complex and highly variable,so field management and chemical control are not always effective or efficient to control its damage.It is the most usful measure to breed novel cottoncultivars with resistance to Verticillium dahliae.However,due to the shortage of germplasm resources for verticillium wilt resistance,the progress of traditional disease resistance breeding is very slow.With the development of genetic engineering and molecular biology,it plays important roles in cotton disease resistance breeding by isolation and identification of defence-related genes.MicroRNAs（miRNAs）,a class of non-coding RNA,participates in plant growth and development and biotic/abiotic stress response by regulating post-transcriptional level of target genes.Dissection of the mechanism of miRNA and its corresponding target genes functioning in plant disease resistance are crucial to breed cotton disease resistance cultivars.In this study,based on the sRNA and degradeomic database of Gossypium hirsutum roots infected by V.dahliae previously reseached,two miRNAs with significantly different expressions and their target genes were identified,named GhmiR7814 and GhmiR8687,GhNBS-LRR and GhWATs,respectively.G.hirsutum cv.BD18 was used to analyze and identify the disease-resistant functions of the two miRNAs and corresponding target genes.The main research results are as follows:1.Based on the analysis of qRT-PCR,miR7814 was constitutive expression in root,stem,leaf,and cotyledon,with the lowest expression in root,while GhNBS-LRR was predominant expression in root.The expression of miR7814 and GhNBS-LRR were induced by V.dahliae,miR7814 showing down-regulated expression,while GhNBS-LRR doing significantly up-regulated expression,which suggest that GhNBS-LRR can be post-transcriptionally regulated by miR7814.2.The equally mixture Agrobaterium cells containing GhNBS-LRR^WT-GUS and Pre-miR7814,or GhNBS-LRR^m-GUS and Pre-miR7814 were injected in tobacco leaves to perform transient expression in plants cells.Then,the results of GUS staining analysis showed that miR7814 inhibited the expression of GhNBS-LRR.And by qPCR analysis,miR7814 can specifily cleave the target gene GhNBS-LRR.3.The GhNBS-LRR-silenced plants by VIGS significantly decreased the resistance to V.dahliae infection,the STTM7814-silenced plants by VbMS increased resistance,and the OE-miR7814 overexpression plants obtained by VbMO also decreased resistance.These results suggest that GhNBS-LRR acts as a positive regulator in plants resistance to V.dahliae infection,while miR7814 played a role of negative regulation.4.According to the degradaemic data,miR8687 was identified to target the GhWATs in cotton.GhWATs had three homologous genes,designated as GhWAT1,GhWAT2,and GhWAT3,respectively.The tissue expression patterns of GhWATs were similar,predominantly expressing in the roots,stems,and hypocotyls,which are highly lignified tissue,indicating that GhWATs was involved in lignification.The expression of GhWAT1 and GhWAT3 were up-regulated and GhWAT2 was down-regulated when plants were induced by V.dahliae.The expression level of IAA-and SA-treatment showed opposite trends,that is,GhWAT1,GhWAT2,and GhWAT3 were up-regulated by IAA application,while those were down-regulated by SA.5.The GhWATs genes single-and triple-silenced plants were obtained using VIGS technology for characterizing GhWATs.The phenotype analysis showed that single-silenced plants had no obvious growth change compared with the control,while the triple-silenced plants showed a dwarfing phenotype.The results of antonomic analysis showed that xylem development in triple-silenced plants was inhibited,while lignin deposition in the xylem increased compared with single-silenced plants and the control.Based on qPCR analysis,the expression levels of lignin synthesis-and lignifications-related genes were higher than the control.6.The results of flurenscence analysis in protoplast showed that GhWAT1,GhWAT2 and GhWAT3 were localized on tonoplast.In the triple-silenced plants,the SA signaling response-related genes were activated,leading to SA accumulation,possibly due to the auxin transport disruption.7.When GhWAT1,GhWAT2 or GhWAT3 was silenced,the single-silenced plants showed a comparable phenotype and disease resistance with control plant.However,GhWATs triple-silencing increased plant resistance to V.dahliae infection,indicating that the three homologus genes were functionally redundant.Collectly,GhWATs in triple-silenced plants acts as negative regulators in plant resistance against V.dahliae infection possibly through the SA signalling pathway and lignin deposition in the xylem.To sum up,miRNA can participate in the resistance response of plants by regulating the expression of target genes.In cotton,GhmiR7814/GhNBS-LRR and GhmiR8687/GhWATs can regulate cotton plant resistance against V.dahliae infection,and regard as candidate for disease resistance breeding.