Defense Mechanism Of Rose Against The Infection Of Marssonina Rosae

Modern roses are among the most important ornamental plants in the worldwide.However,they are very susceptible to black spot disease during cultivation,which can cause leaf black spots or even leaf shedding to affect the ornamental value of rose.The pathogen of rose black spot disease is Marssonina rosae,a hemibiotrophic fungus,and according to previous researches,at least 11 races have been discovered.There is no rose cultivar which is completely immune to the M.rosae till now.In our research,the pathogen of rose black spot disease was isolated and identified,and was used to evaluate the black spot disease resistance of Rosa hybirda ‘Porcelina'×R.rugosa ‘Dahong Zizhi'population.Differential transcriptome response to M.rosae between the resistant strain and the susceptible strain was analyzed to predict the effective resistance induction mechanism.At the same time,the effects of salicylic acid,jasmonic acid and auxin on effective resistance induction of rose black spot disease was discussed through exogenous interference with the synthesis and transportation of three hormones.The function of 7 hormone-related genes and 4 cell-wall-related genes obtained by transcriptome analysis on the resistance of rose black spot disease were verified by virus-induced-gene-selience technique.The regulatory mechanism of these genes on the resistance induction of rose black spot disease was analyzed.The key factors that induce or hinder the effective defense mechanism against rose black spot disease were screened,and the induction mechanism of broad-spectrum resistance of rose black spot disease was initially clarified.The main results are as follows:1.Marssonina rosae was isolated,purified and identified from leaves infected by rose leaf spot in Beijing.The isolated strain was used to evaluate the resistance to black spot disease of the population of Rosa 'Pocelina' × R.rugosa 'Dahong Zizhi',and 6 black spot disease resistant lines and 12 susceptible strains were obtained.2.Inoculate the black spot disease resistant strain R13-54 and susceptible strain R12-26 to analyze the induction of the activity of disease-related proteins and the systemic acquired resistance during the infection process.The defense responses of both R13-54 and R12-26 were activated,the activity-related protein activity and the expression of NPR1 were both twice induced,and the two peaks occur in 1-2dpi and 10-12 dpi and the bottom occurs at 4-6dpi.However,the strength of resistance induction induced in the R13-54 was significantly stronger than that in R12-26.3.Comparing transcriptomes of the two lines in response to the invasion of M.rosae at 0hpi,36 hpi,6dpi,and 12 dpi,which was showed that a large number of different genes were induced by M.rosae both in susceptible and resistant lines.The differential specificity genes induced by pathogenic invasion in the two strains were significantly enriched in the pathways related to hormones and cell walls.Contrast with the control group and 0hpi group,significantly up-regulation genes enriched in the auxin-related pathway,jasmonic acid-related pathway and cell wall metabolism synthesis pathway,which may participate in negative regulation of the effective resistance to M.rosae in R12-26;however these genes are down-regulated during the early 36 hpi in R13-54.4.The influences of three hormones' synthesis affected by exogenous interference on the resistance to black spot disease of R12-26 and R13-54 were analyzed;the content of endogenous hormones,the synthesis and signal transduction of three hormones were analyzed to predict the regulation mechanism.The high levels of constitutive accumulation of jasmonic acid can cooperate with auxin to negative regulation of the induction of the resistance to black spot disease by inhibiting the activation of salicylic acid signaling pathway to the resistance to black spot disease.Auxin can inhibit the defense mechanism induced by salicylic acid by further promoting the accumulation and synthesis of jasmonic acid.However,when the higher level of jasmonic acid was suppressed,auxin will not take participate in the negative regulation of resistance to black spot disease.5.A TRV-mediated VIGS technology system which can be used in rose leaflets was developed to analyze the function of 7 hormone-related and 4 cell-wall-related candidate genes during the induction of the regulation of effective disease response to M.rosae in rose.The inhibition of the auxin glycosylation gene Rc UGT74E1 and auxin response gene Rc AX15 A promoted the synthesis of salicylic acid and improved the resistance of rose spot disease;the up-regulated jasmonic acid accumulation after inhibiting Rc PIN1 affected the induction of effective resistance to M.rosae regulated by salicylic acid;the silencing of Rc SAMT indicated that inoculation of M.rosae could induce the up-regulation of Rc SAMT in R12-26 to promote the conversion of SA to Me SA.However,the function of jasmonic acid anabolic related genes needed further confirmation.These results further verified that jasmonic acid and auxin work together to inhibit the induction of effective resistance to M.rosae by salicylic acid signaling.6.Using VIGS technology down-regulated 4 cell-wall-related genes that were different significantly up-regulated in R12-26 compared with R13-54.After inoculation with M.rosae,the down-regulation of cell-wall degradation-related genes Rc PLY18,Rc BXL1 and Rc XYNC and cell wall reinforcement-related gene Rc EXP15 induced the higher resistance to black spot disease of R12-26.This indicated that the change of cell-wall induced by the invasion of M.rosae negatively regulated the effective resistance induction in R12-26.7.The analysis of changes in endogenous hormone content,hormone-related signal transduction after silencing the four cell wall-related genes(Rc BXL1,Rc PLY18,Rc XYNC and Rc EXP15)and the responsible of these four genes after exogenous hormone spraying showed that the early accumulation of jasmonic acid inhibited the synthesis of salicylic acid and induced the strong expression of Rc PLY18,leading to the degradation of pectin and promoting the diseases;Jasmonic acid could also promote the accumulation of salicylic acid,which further strengthen the inhibition of Rc XYNC and Rc EXP15,and improved the effective defense against pathogenic invasion;however,in the early stage of response to the invasion of black spot disease,the accumulation of salicylic acid did not induced at the same time as the accumulation of jasmonic acid,which stimulated the expression of Rc XYNC and Rc EXP15,which promoted the development of disease;the expression of Rc XYNC may depend on the accumulation of auxin to participate in the negative regulation of disease resistance,and the effect of Rc EXP15 on disease may not through auxin.In summary,high levels of constitutive accumulation of jasmonic acid and auxin together inhibited the activation of the salicylic acid signaling pathway to reduce the resistance to black spot disease;the crosstalk between cell wall degradation and auxin signaling further promoted the synthesis of jasmonic acid,weaken the inhibition of salicylic acid to jasmonic acid,to reduce the resistance of M.rosae.The above results provided a theoretical basis for further research on the induction mechanism of the effective defense of rose against black spot disease,and provided a basis for further breeding of black spot disease resistant rose cultivars.