Study On The Mechanism Of Calcium Signaling In Pear Scab Resistance

Scab is one of the main diseases in China's pear producing areas,and it harms the leaves and fruits of pears and causes huge losses to the fruit farmers' economy.In production,it was found that ‘Huangguan'pear showed high resistance to scab while ‘Xuehua' pear was a susceptible variety.Currently,the mechanism of scab-resistance in pears is still unclear and Ca2+,NO and ROS are often involved as cell messengers or signaling molecules in plant resistance to stress.Therefore,in this study,we selected‘Huangguan' pear and ‘Xuehua' pear as plant materials to analyze DEGs of pears before and after infection by scab utilizing high-throughput sequencing.Besides,we studied at the cellular and molecular levels the differences of intracellular Ca2+,NO and ROS of two different resistant pear varieties after infection by V.nashicola and their intrinsic relationship with salicylic acid signaling pathway.Finally,we researched the response of pear defense enzymes to V.nashicola and the alleviation effect of exogenous Ca2+ and NO.These will provide a theoretical basis for elucidating the signal transduction mechanism of pear scab-resistance.The main research results are shown as follow:1.High-throughput sequencing was conducted on the ‘Huangguan' pear leaves infected by V.nashicola for 72 h,a total of 3,609 DEGs were obtained,among which 1,692 were up-regulated and 1,917 were down-regulated,mainly involving plant pathogen interaction,endogenous hormone signal transduction,substance synthesis and metabolism,and plant resistance.Furthermore,the signal network of plant-pathogen interaction was analyzed and it was found that pears might be linked to SA signaling pathway through Ca2+,NO and ROS signals to activate pathogenesis-related proteins and improve disease resistance.2.The temporal and spatial variation characteristics of protoplast Ca2+ in ‘Xuehua' pear and‘Huangguan' pear were studied.Fluo-3 AM esterification probe was used to analyze the intracellular Ca2+fluorescence intensity of pears.It was found that V.nashicola could cause the increase of intracellular Ca2+levels in a short time,but there were differences between the different varieties.Meanwhile,the study of Ca2+ subcellular localization also shown an increase of intracellular calcium precipitation during infectionterm,and studies on the co-treatment calcium inhibitors(EGTA,La Cl3,and BAPTA)with V.nashicola showed that intracellular Ca2+ levels were inhibited significantly by La Cl3 and EGTA,while BAPTA had no significant inhibitory effect.Therefore,the increase of intracellular Ca2+ levels in pears may be mainly due to intercellular Ca2+ influx.In addition,q RT-PCR analysis of calcium sensors genes in pears showed that scab induced up-regulated expression of related genes in scab resistant varieties ‘Huangguan' pear,while the induction effect was not significant in ‘Xuehua' pear,which may be caused by different calcium signals in the two pear varieties.3.The results of DAF-FM DA and DCFH-DA fluorescent probes showed that the infection of V.nashicola induced the increase of NO and ROS fluorescence intensity in ‘Xuehua' and ‘Huangguan' pears protoplasts,among which,the change of NO in ‘Huangguan' pear cells began earlier but decreased in the late stage of treatment,while the level of NO in ‘Xuehua' cells gradually increased.Similar results were obtained in the detection of ROS fluorescence intensity.Then we discussed the source of NO in pear cells,and concluded that the NO of the ‘Xuehua' is mainly regulated by nitrate reductase(NR),while the NO of‘Huangguan' may be catalyzed by NOS and NR.In addition,we found that NO and ROS levels in pear cells could be significantly inhibited by calcium inhibitors.NO inhibitors(L-NAME and Na N3)had an inhibitory effect on ROS accumulation but no significant effect on Ca2+,while intracellular NO and Ca2+were not inhibited by ROS scavengers.And CAT had a certain inhibitory effect on cellular NO content.Therefore,we believed that Ca2+ was upstream of NO and ROS,NO was upstream of ROS,and NO production was regulated by H2O2.4.We analyzed the effects of scab on SA synthesis and the expression of regulatory factors in two pear cultivars,and explored the relationship between Ca2+,NO and SA.The results showed that the induction effect of SA by V.nashicola on ‘Huangguan' pear was much more significant than that on ‘Xuehua' pear;the expression levels of disease-related genes(PR1 and PR5)were increased by NPR1 and MAPK.In addition,Ca2+ and NO had an activation effect on SA signaling pathway in pears.5.After the leaves of two pear cultivars were infected by the scab,study on the physiological,biochemical changes related to disease resistance and the expression of corresponding genes were conducted.Besides,we carried out research on the mitigative effect of exogenous Ca Cl2 and SNP treatment in pears against scab infection and damage.It was found that V.nashicola caused different degrees ofdamage to the chloroplasts of the two varieties and the content of chlorophyll was reduced,the accumulation of MDA was increased,and the damage to the susceptible variety ‘Xuehua' pear was more serious;the activity of various protective enzymes(SOD,POD,CAT,APX and chitinase)and the expression levels of the corresponding genes were promoted,especially in the scab resistant variety‘Huangguan';Exogenous Ca2+ and NO enhance the resistance to scab by increasing pear chlorophyll,protective enzyme activity and expression level.V.Nashicola infected and opened the plasma membrane calcium channel,increased the intracellular Ca2+ level through the action of ion influx.Free Ca2+ combined with various calcium receptors(proteins)to produce intracellular specific calcium signals and induced the production of NO and ROS signaling molecules;the early signals of three kinds of cells work together to activate the plant-associated disease-resistant signal transduction pathway,promote SA synthesis and simultaneously induce the expression of key regulatory factors NPR1 and MAPK,thereby improving the disease-related genes(PR1and PR5)expression levels,PRs protein accumulation;pear cells Ca2+ and NO may reduce MDA accumulation by enhancing cell chlorophyll content,and improve disease-related protective enzyme activity,protect plant photosynthesis while maintaining normal redox state in cells,thereby causing the pear to exhibit resistance to the infection of V.nashicola.