Mechanism Analysis Of Powdery Mildew Resistance Of CMPG1-V From Haynaldia Villosa L.

Wheat powdery mildew,caused by Blumeria graminis f.sp.tritici(Bgt),is one of the most destructive wheat diseases worldwide.Powdery mildew resistance gene Pm21,which confers durable and broad-spectrum resistance(BSR)to Bgt,is from Haynaldia villosa L.,(2n=2x=14,VV genome)and being widely used in wheat breeding program in China.However,little is known about the mechanism of Pm21-mediated BSR.Exploring disease resistance genes and better understanding of their mechanisms at the molecular level are of great significance in wheat improvement for disease resistance.In previous research,by Microarray analysis using Barley 1 Genechip(Affymetrix),an E3 ligase coding gene CMPG1-V has been cloned from H.villosa.Transgenic wheat expressing CMPG1-V showed improved broad spectrum powdery mildew resistance at seedling and adult stages.In this study,homologous cloning,expression analysis and RNAi were conducted to further elucidate the resistance pathway and mechanism mediated by CMPG1-V.The main results obtained were as follows:1.Thirty-eight CMPG orthologs were cloned from 14 common wheat varieties and wheat related species,including six different genomes(A,B,D,H,V and R,respectively).The amino acid sequence alignments showed that CMPG were highly conserved among grasses,they shared 91.41%-98.46%identity with CMPG1-V.2.Based on nucleotide sequence alignments between CMPG1-V and TaCMPGs,a primer pair specific for CMPG1-V,CMPG-NP-F and CMPG-NP-R,were designed.PCR and subsequent sequencing of specific amplicons from T.durum-H.villosa amphiploid(genome AABBVV),a complete set of Chinese spring-H.villosa alien addition lines(DA1V-7V),two translocation lines T6VS·6AL and T6AS·6VL showed CMPG1-V was mapped to the 6VL of H.villosa.BLASTn showed CMPG1-V orthologs in Hordeum vulgare,Oryza sativa and Brachypodium distachyon were located on 6HL,R2 and long arm of Bd3,respectively.Both R2 and Bd3L are syntenic to the long arm of homologous group 6 chromosomes of wheat;wheat TaCMPGs were physically mapped to the bins of 6AL4-0.55-0.90,6BL5-0.40-1.00 and 6DL12-0.68-0.74.The results indicate that the CMPG1-V orthologs in grass species are well conserved and are located in the syntenic regions of the long arm of homologous group 6 chromosomes.3.To determine the subcellular localization of CMPG-V,YFP-CMPG1-V fusion vector were constructed and transformed into the protoplasts of Yangmail58.The results indicated that YFP-CMPG1-V was located in the nucleus,plasma membrane,Endoplasmic Reticulum(ER)and partially in the Trans-Golgi Network/Early Endosome(TGN/EE).4.The plants of T2-26 and T2-46 were further evaluated for their resistance to 23 different Bgt isolates at the seedling stage.Both lines were highly resistant(Infection Type as grade 0 and 0;)to 18 isolates.The expression levels of PR genes related to SA phytohormone pathway(PR1,PR2 and PR10),Chitinasel and Chitinase2 were much higher in T4-26 and T4-46 than those in Yangmai158 after Bgt treatment.Quantitative analysis showed that overexpression of CMPG1-V in the transgenic plants may lead to the increase of H2O2 contents when infected by Bgt.The activities of ascorbate peroxidase(APX)and guaiacol peroxidase(POD)were significantly increased in the transgenic plants after Bgt infection.It was found that more program cell death(PCD)were occurred in transgenic plants(13.66%?33.77%)than that in Yangmai158(3.33%?3.82%)at 24 h after inoculation(hai)and 48 hai.These results indicated that CMPG1-V mediates the defense response by blocking the growth of Bgt,which associated with the increase of ROS accumulation and PCD.5.RNAi was used to silence CMPGs in powdery resistant wheat variety NAU9918,which carries the Pm21,by bombardment method,and 396 regenerated plants were obtained.In which,nine plants were identified to be positive transgenic.qRT-PCR analysis showed that CMPG expression was significantly decreased in five positive transgenic plants compared with NAU9918.Evaluation of powdery mildew resistance and detection of hyphae growth of Bgt showed that the proportion of secondary hyphae(SH,17.54%?20.79)and conidiophores(CH,1.96%?4.48%)formed in these five transgenic plants were higher than that in NAU9918(SH,9.52%and CH,0.67%).The expression levels of PR genes related to SA phytohormone pathway(PR1,PR2 and PR10)in the five transgenic plants were significantly lower than that in NAU9918.These results indicated the involvement of CMPGs in defense response to Bgt in wheat,particularly in broad spectrum disease resistance,and suggested the association of the ROS and SA pathways with the CMPG mediated powdery mildew resistance.6.One of or both the conserved amino acids cysteine(C53)and tryptophan(W80)within the U-box motif were mutated to alanine(A),resulting in three mutant variants,CMPG1-VC53A,CMPG1-VW80A and CMPG1-VC53AW80A and were used to test their ubiquitination activity in vitro and in vivo.The results showed that either single or double mutations in CMPG1-V led to the loss of E3 ligase activity,confirming that these two amino acids are critical for its E3 ligase activity.Co-expression of GFP-CMPG1-VC53AW80A and YFP-CMPG1-V in protoplasts of Yangmai158 indicated that loss of E3 ubiquitin ligase activity did not affect the subcellular localization of CMPG1-V.7.The CMPG1-VC53AW80A was transformed into Yangmai 158 by bombardment,and 141 To regenerate plants were obtained.Western blot identified that CMPG1?VC53AW80A-HA accumulated in Ti-CMPGC53AW80A-78 and was proved to be positive transgenic plant.At seedling stage,the derived T1 transgenic plants were all susceptible to Bgt infection,indicating the direct link of the E3 ubquitin ligase activity of CMPG1-V with its role in powdery mildew resistance.8.CMPG1-V mono-antibodies,199461R1-1/3N3 and 199461R1-1/4D18 were obtained.The antibodies are important for further research involved in CMPG protein accumulation,modification and identification of its interaction proteins.9.One of the antibody,Anti-CMPG 4D18 was used to test the CMPG accumulation in Yangmai 158 and H.villosa.The CMPG protein could not detected in samples without any treatment.After treatment with the 26S-proteasome inhibitor AM114,the abundance of the CMPG protein in Yangmai 158 was obviously increased,as shown by the appearance of a clear band.After treatment with a Bgt isolate E26,the abundance of CMPG1-V in H.villosa was also obviously increased.These data suggest that Bgt infection induced CMPG accumulation and instability,which may regulted by the 26S-proteasome pathway.