Functional Characterization Of Calcuim Signal Transduction Related Genes In The Broad-spectrum Powdery Mildew Resistance Pathway

Wheat powdery mildew,caused by Blumeria graminis f.sp.teitici(Bgt),is one of the most serious wheat diseases worldwide.Cultivation and utilization of disease resistance varieties became more and more effective to prevent powdery mildew.Due to the huge and complex wheat genome,the identification,location and cloning of new resistance gene resources progressed at a slow pace,and the research on the disease resistance mechanism was also relative lagged.Pm21,which was located to the short arm of chromosome 6V(6VS)of Haynaldia villosa by the development of the wheat-H.villosa translocation line T6VS.6AL showed broad-spectrum resistance to all powdery mildew isolates.The expression profile of genes in H.villosa induced by powdery mildew was detected by the Affymetrix Barley GeneChip(Cao AZ,2006),and a lot of Bgt up-regulate genes had been found.In these genes,a couple of calcium channel related genes(MPK12.CaMBP4?CBL1?CPK12)were screened and may particularly participant in the powdery mildew resistance pathway that mediated by Pm21 in H.villosa.Using the methods of phytopathology and genetic engineering,the function and underlying mechanism of these calcium pathway related genes in broad-spectrum powdery mildew resistance.1.Calcium pathway participates in powdery mildew resistant reaction mediated by Pm21Nannong 9918,carrying Pm21,with high resistance to Bgt,was treated by Ca2+channel inhibitor LaCl3 from the root absorption.By contrast,Yangmai 158,a moderate susceptible wheat variety,and Nannong 9918 were treated by water using the same method as the control groups.Then they were inoculated by Bgt using the local mixed races and the Bgt fugal development was observed in detail.The result showed that the Bgt resistance of the LaCl3 treated Nannong9918 had reduced during 12hpi(hours past inoculation)to 72hpi.But the control group showed no significant change of the levels of the Bgt resistance.Next,the ROS accumulation and the number of PCD cells in Bgt interactional epidermal cells of LaCl3 treated Nannong9918 also decreased.This might lead to the reduced Bgt resistance of LaCl3 treated Nannong9918.It's also proved that there is a close relationship between calcium pathway and ROS/HR reaction.Moreover,4 genes,MPK12,CaMBP4,CBL1 and CPK12,were selected from the Genechip,and which were significantly up-regulated by Bgt.The expression level of these genes were down-regulated by Bgt with time course in the LaCl3 treated Nannong9918.This indicated that MPK12,CaMBP4,CBL1 and CPK12 were involved in the Ca2+ signal pathway and response to calcium signal that activated by Bgt.The expression level of NADPH-OX,APX and SOD related to ROS pathway also changed notably in LaCl3 treated Nannong9918.The expression level of PR1,PR5 and PR10 related to SA pathway reduced notably in LaCl3 treated Nannong9918.A conjecture has been raised that calcium pathway participates powdery mildew resistant reaction,which mediated by Pm21,and by regulating the expression of genes related to ROS and SA pathways.2.Function analysis of MPK12,CaMBP4,CBL1 and CPK12 in powdery mildew resistant reactionThe function of the MPK12,CaMBP4,CBL1 and CPK12 were furher characterized comprehensively by testing the expression pattern in different wheat varieties inoculated by Bgt.The result showed that the expression level of these genes were up-regulated quickly by Bgt in Nanong 9918 which carries Pm21.The development of Bgt spores was regular in Nannong9918 after the MPK12,CaMBP4,CBL1 and CPK12 were silenced by the BSMV-VIGS technique.This indicated that Bgt resistance of Nannong9918 had reduced by silencing MPK12,CaMBP4,CBL1 and CPK12.But the obvious colony couldn't be observed in the gene silencing leaves inoculated with Bgt.This result indicated that silencing single gene couldn't make Nannong9918 to be susceptible to Bgt.In conclusion,these four calcium pathway genes may particularly participant in the early powdery mildew resistant reaction,which mediated by Pm21.The expression levels of MPK12,CaMBP4,CBL1 and CPK12 were up-regulated by Bgt in later time course in Yangmai 158(without Pm21),Yangmai11(with Pm4a),IGVI465(with Pm6)than in Nanong 9918.It is deduced that MPK12,CaMBP4,CBL1 and CPK12 participate in the ETI stage mediated by different Pm genes.3.Positive regulations between MPK12,CaMBP4,CBL1,CPK12 and ROS pathwayThe expression levels of genes involved in ROS pathway were regulated by the MPK12,CaMBP4,CBL1 and CPK12 in those gene silenced plants.The expression level of NADPH-OX,which is the key enzyme for producing ROS,was down-regulated by Bgt.At the same time,the expression levels of APX and CAT,which are the key enzymes for purging H2O2,were up-regulated by Bgt.The expression levels of PR3 and PR9 were down-regulated by Bgt.The result proved that MPK12,CaMBP4,CBL1 and CPK12 regulate ROS pathway positively.Moreover,the expression level of MPK12,CaMBP4,CBLI and CPK12 were up-regulated by Bgt notably in the H2O2 treated H.villosa,and were down-regulated by Bgt in DMTU treated H.villosa simultaneously.The results indicated that ROS can feedback to MPK12,CaMBP4,CBL1 and CPK12 positively.4.Over-expressing of Hv-MPK12 increases the powdery mildew resistance of Yangmai 158The Hv-PMK12 gene was transformed into the callus of a moderate susceptible variety Yangmai158 by the biolistic bombardment method.Totally180 regenerated plants were obtained,in which 17 were identified as the positive transgenic plants.The preliminary evaluation result of plants at the T0 generation shows that the over expression of Hv-MPK12 can increase the powdery mildew resistance of Yangmai 158.17 positive transgenic plants had different resistance phenotype.Some reached 0 level;one had HR reaction;eight had 4-5 level.