Histopathological Research And Identification & Mechanism Analysis Of MiRNAs During Grape-Botrytis Cinerea Interaction

Gray mould disease caused by Botrytis cinerea is one of the most severe fungi disease which harms the growth of grapevines and retards the development of grape industry.It reported that miRNAs play a key role in the regulation of plant-pathogen interactiona.These miRNAs affect plant resistance through inhibiting their targets.However,most researches about gray mould resistance of grapevines are mainly about finding resistance genes,uncovering metabolic pathways and their regulation on transcriptional level.Studies about miRNAs mediated resistance response of grapevine are still rare.Searching for miRNAs from grapevine involved in gray mould resistance can not only enrich our understanding about how grapevine adapt to biotic attack,but also provide new ideas for improving or breeding resistant varieties.In this research,we selected highly resistant China Vitis amurensis cv.Shuangyou and susceptible cultivars Vitis vinifera cv.Red Globe to comparatively analysis the differences of B.cinerea development and cell wall related enzyme responses in the interaction with B.cinerea.Furthermore,through small RNA high-through sequencing,find differently expressed miRNAs and biology functions involved in two grapes of different resistance at different infection stages with B.cinerea.The main results were as follows:1.In the interaction between B.cinerea and Red Globe,B.cinerea infection was completed within 24 h after inoculation accompanied by obvious disease spots.A direct and effective interaction relationship was established,and the growth and expansion of B.cinerea began rapidly and new conidia were generated.However,B.cinerea infection was extremely slow,and spore deformation did not appear until 48 h after inoculation in the unsusceptible Shuangyou,indicating that some substances may be produced or some defense reactions may be triggered upon early contact with B.cinerea.Shuangyou has thicker cutin layer and more epidermal hairs than Red Globa.And find right amoubt of reactive oxygen species accumulation near grey mould spore and hyphae on Shuangyou leaves.As such,B.cinerea infection was limited,and the whole infection process was not completed on Suangyou。2.Cellulose,pectinase,β-1,3-glucanase,and chitinase activities were more remarkable in the infected leaves compared with that in the healthy leaves in both Red Globe and Shuangyou.The cellulose and pectinase activities were significantly increased at 4 h in the leaves of Shuangyou while high enzymatic activities were observed at 12 h in the leaves of Red Globe.Although β-1,3-glucanase activity was significantly increased at 4,48,and 72 h in the leaves of Red Globe,higher β-1,3-glucanase activity was maintained from 8 h to 18 h in the leaves of Shuangyou.Chitinase activity was high from 4 h to 18 h in the leaves of Shuangyou,but the infected leaves of Red Globe had higher activity at 48 h.Thus,an earlier response of cellulase and pectinase occurred in infected leaves of Shuangyou than Red Globe.Chitinase and β-1,3-glucanase maintained relatively high enzymatic activities at the early infection stage of B.cinerea in Shuangyou leaves.3.12 131 700 and 10 959 531 clean reads were obtained from Red Globe and Shuangyou leaves.The length of small RNA mainly distributed between 18 and 27 nt,with21 nt and 24 nt as dominant length.Small RNA sequences generated from each library were independently aligned with known and experimentally validated mature miRNAs in mi RBase.After homology search,there were 258 mature miRNAs.According to whether it had been found in grape,the total 258 miRNAs divided into161 existed miRNAs and 97 known MIRNAs and them belong to 35 miRNAs families.It seemed that 79 miRNAs prefered expression in Red Globe than Shuangyou both in control and inoculation and 41 miRNAs prefered to express in Shuangyou.In Shuangyou mi R159a/b,mi R3624-3p/5p,mi R3629a-5p,mi R3636-3p,MIR1446-x,MIR1535-x,and MIR870-x expressed far higher than Red Globe..4.When compared control samples with treated samples respectively from Red Globe and Shuangyou at each time point,totally 103 miRNAs were changed at least two-fold and significant(P<0.05)different.Among them MIR160,mi R159a/b,MIR2118-y and MIR2218-y involved in grape-B.cinerea interaction,and them contributed to ‘Shuangyou’resistance to B.cinerea.403 targets were predicted for 103 differently expressed miRNAs.We found targets enriched in cell wall metabolic process in Red Glone and Shuangyou,and there were a relation between expression of these targets and enzyactivies.Moreover,it had verified that MIR858-x targeted GSVIVG01028091001(MYBF1/R2R3 MYB12)and GSVIVG010354590(myb-related protein);vv-mi R159 a targeted GSVIVG01012447001(GAMYB),through q RT-PCR and degradome sequencing.