MiR319-MYB33-SPL9-DFR Pathway Participates Upland Cotton Response Against Verticillium Dahliae

MicroRNAs(miRNAs),a class of non-coding small molecule RNA,have a crucial role in the plants response to the pathogens invading.The miR319 is one of the early discovering miRNA in plants,which functions major in the development of plants leaf and flower.Regulation of miR319 to plant defense had been a very few reported,so its potential mechanisms of plant innate immunity remain to be revealed.In this study,we selected ghr-miR319,a novel miRNA,showing up-regulation expression under Verticillium dahliae(V.dahliae)investigation.In this dissertation,we employed molecular genetics,biochemistry and physiology to dissert ghr-miR319 and its target genes function in plant defense to pathogens.To establish a set of simple and highly effective approaches for analyzing cotton miRNA level,we firstly developed a Stem-loop RT-PCR to analyze the contents of miRNA.The three special primers were designed,including stem-loop primer,Forward primer and Universal reverse primer.Based on analysis of RT-qPCR Ct values of10-fold diluted cDNA,the two sets of primers possessed highly amplifying effective for monitoring miR156 and miR159 levels,reached 102% and 103.6%,respectively.The sensitivity assays of Stem-loop RT-PCR showed that the limited contents of total RNA for monitoring miR156 and miR159 were obviously different,which was 20 ng and 2pg,respectively.Moreover,the expression levels of miR156,miR159 and miR5658 in cotton root,stem and leaf could be well tested by this Stem-loop RT-PCR technique.These results suggested that Stem-loop RT-PCR is facilitate in plant gene function analysis,especially in miRNA regulation.In combination with bio-information,according to upland cotton small RNA(sRNA)libraries and degradome sequencing analyses,the interaction between the miR319 and its target gene GhMYB33 was identified including paring and cut site.Under the treatment of V.dahliae,the ghr-miR319 and its target gene GhMYB33 relative expression level showed up-regulated and down-regulated in plants by Stem-loop RT-PCR and qPCR analysis,respectively.The plants with over-expression and knock-down of ghr-miR319 as well as the silencing of target genes GhMYB33 and GhSPL9,were developed by virus-induced gene silencing(VIGS)methods,which were further subjected to challenge to V.dahliae.The results showed that the plants with over-expressed ghr-miR319,silenced-GhMYB33 and silenced-GhSPL9 increased the plants resistance to V.dahliae,additionally,the anthocyanin contents were also accumulated in these cotton.But knock-down expressed ghr-miR319 repressed the plants resistance to V.dahliae,indicating that anthocyanin could participate in plants defense.In general,the data documented that miR319-MYB33-SPL9-DFR pathway plays important roles in plant defense against V.dahliae possibly through anthocyaninaccumulation.Our results provide useful information to understand the molecular mechanism of miRNA in plant innate immunity.Moreover,ghr-miR319 and its target genes can be used as candidate genes to breed significant commercialization cultivars for disease resistance.