Screening And Identification Of Maize MiRNA-Mediated Resistance To Curvularia Lunata

Curvularia leaf spot caused by Curvularia lunata（Walk）Boed,which caused serious yield loss in the corn production area in northern China,is an important disease in corn production.However,there are few reports about the molecular regulation mechanisms of miRNA-mediated resistance to C.lunata.In this study,the related miRNA resistance to C.lunata were screened and identified by s RNA high-throughput sequencing,miRNA microarray,and stem-loop RT-PCR techniques.Target genes of some miRNAs were identified using the degradome sequencing technology.The possible pathways of miRNA-mediated disease resistance were analyzed,which laid a foundation for further study on the molecular regulation mechanism of maize miRNA-mediated resistance to Curvularia leaf spot.The main findings are as follows:1.Using miRNA high-throughput sequencing technology to identify the miRNAs in the interactions between maize and C.lunata,the maize disease resistance inbred line Luyuan and the sensitive inbred line Huangzao4 were used as materials.2004 and 1897 miRNAs were identified in the control groups and inoculation groups of susceptible responses,respectively.The inoculation groups had 123 miRNAs less than the control groups,indicating that the expression of some miRNAs was suppressed in the susceptible response.1304 and 2199 miRNAs were identified in the control groups and inoculation groups of disease-resistant responses,respectively.The inoculation groups had 895 miRNAs more than the control groups,indicating that miRNA expression was promoted during the disease-responsive reaction,implying that the increase of miRNA expression abundance was related to the disease resistance of maize.2.Through bioinformatics analysis,443 conserved miRNAs of 120 families,11non-conserved miRNAs of 4 families and 72 new miRNAs of 32 families were identified.Most miRNA families contain only one member,but some miRNA families,such as miRNA 156,contain 35 members,miRNA169,miRNA166,miRNA167,miRNA159/171,and miRNA395 have 32,29,26,25,and 20 members,respectively.These findings indicate that the diversity of maize miRNA family after infection by C.lunata.3.Using miRNA microarray technology,237 differentially expressed miRNAs were screened in Huangzao4（infectious disease）,there were 76,90,and 71 differentially expressed miRNAs at 3 hpi,9 hpi,and 15 hpi,respectively,and the number of differentially expressed miRNAs increased first and then decreased after inoculation.A total of 247 differentially expressed miRNAs were identified in Luyuan（resistance disease）.There were 55,72,and 120 differentially expressed miRNAs at 3 hpi,9 hpi,and 15 hpi,respectively,and the number of differentially expressed miRNAs showed an increasing trend after inoculation.Therefore,it is speculated that the increasing number of differentially expressed miRNAs in response to disease may promote maize resistance to C.lunata.4.The target genes of differential miRNAs were analyzed using the degradome sequencing and bioinformatics techniques.A total of 13062 target genes of 1013 miRNAs were identified.By analyzing the results of the microarray,combined with the degradome data and the biological function of target genes,a total of 46 disease-related miRNAs were obtained.The target gene of zma-MIR159e-p3₁ss17CA is the VQ motif-containing protein,which is involved in the regulation of WRKY transcription factor.The target gene of bdi-mi R5054₁ss10TA is BAX inhibitor1,which regulates cell death.ath-mi R159 b target gene is myb transcription factor 65,and ppt-mi R894_R-3 target gene is rubopenoxin,the target gene of zma-mi R164h-5p_R-4 is cinnamic acid-4-hydroxylase,PC732 and metacaspase protein（AMC1）,PC169 and thioredoxin family protein（Trx）and the miRNA are all related to disease resistance.5.Fluorescence quantitative PCR technique was used to verify the relationship and regulation pathway of disease resistance miRNA and target genes,such as zma-mi R169 and nuclear factor Y（NY）,zma-MIR408 and tetracycline tripeptide protein（DG1）,zma-mi R393 and auxin signaling F-box2（AFB2）,zma-mi R164 and NAC 80（NAC）,PC732 and metacaspase proteins（AMC1）,PC169 and thioredoxin（Trx）.The results showed that most of the target genes showed a negative correlation with their miRNA expression patterns.The experiments further analyzed the expression of PC732 and PC169 and their predicted target genes at 0,0.5,1,3,9,15,24,and 36 hpi in Luyuan and Huangzao4.The results showed that PC732 had two peak expressions at 3hpi and 15 hpi in Luyuan,and peaked earlier than Huangzao4.The target gene of PC732 also showed a down-regulation pattern with PC732 arrival of peak values.However,PC732 peaked at 15 hpi in Huangzao4,but its target gene was not down-regulated.It was speculated that PC732 may not have a regulatory effect on the target gene AMC1 in Huangzao4.The PC169 expression level showed a downward trend after 9hpi reached the peak in Luyuan and fluctuates slightly at 24 hpi.The target gene of PC169 has been slowly rising after 3hpi to the highest expression level at 24 hpi,and 36 hpi began to decline.In Huangzao4,PC169 peaked at1 hpi,but its target gene did not change much more at all time points.It is speculated that PC169 regulates thioredoxin to scavenge reactive oxygen species in the middle and later stages of resistant varieties,thereby mediating resistance to C.lunata.Therefore,maize may regulating the metacaspase and thioredoxin by PC732 and PC169 to resist the infection of C.lunata.