Screening And Functional Analysis Of The Interacting Protein Of The Wheat Powdery Mildew Resistance Gene Pm21

Wheat（Triticum aestivum L.）is one of the most widely grown food crops in the world.Wheat powdery mildew caused by Blumeria graminis f.sp.tritici（Bgt）infection is one of the main diseases of wheat,which seriously threatens wheat production.Pm21 is a broad-spectrum powdery mildew resistance gene that discovered from the wild wheat species Haynaldia villosa,and it was introduced into common wheat by chromosome engineering through development of the wheat-H.villosa translocation line T6VS·6AL.Previously,our laboratory cloned the wheat broad-spectrum powdery mildew resistance gene Pm21,which belongs to the typical CNL disease resistance gene.Generally,the resistance mediated by NLR-type disease resistance genes is race-specific resistance,while Pm21 mediated broad-spectrum resistance,so it has great significance to study the molecular mechanism of Pm21.In this study,we used the yeast two-hybrid technology to screen Pm21 interacting proteins and analyze the functions of the identified interacting proteins.The molecular mechanism of Pm21-mediated broad-spectrum wheat powdery mildew resistance was preliminary revealed from the perspective of signal transduction.The main results of this study are as follows:1.Construction of a yeast two-hybrid library to screen Pm21 interaction proteinsConstruction of a yeast two-hybrid library:We used mixed Bgt isolates to inoculate92R137 material,and collected samples at 0h,12h,24h,and 48h after inoculation to construct a yeast two-hybrid three-frame c DNA plasmid library.The library volume of reading frame library 1 was more than 2.8×10⁶cfu,reading frame library 2 was more than2.4×10⁶cfu,and reading frame library 3 was more than 1.9×10⁶cfu.The quality of the constructd library meets the requirements for screening the interacted proteins of Pm21.Screening of Pm21 interacting proteins:Pm21 protein was proved to have no self-activation activity,and then a total of 328 yeast monoclonals were obtained through several times screening by using Pm21 as the bait.We obtained 321 sequences by sequencing these monoclonals,then these sequences were annotated by BLAST with public databases.2.Cloning of the interaction protein gene Ta-R3H-5D and bioinformatics anlysis of the R3H gene familiesCloning of the interaction protein gene Ta-R3H-5D:The interaction ability of the screened proteins were tested by one-to-one verification system,then 2018N-C8 was choosed for the following study.The 2018N-C8 encoded a protein containing a R3H-associated N-terminal domain and R3H domain.R3H domain,with the core convervative motif Rxxx H,has the ability to bind with single-stranded DNA and RNA.Using the 2018N-C8 sequence as the reference sequence,the full-length sequence of the gene was cloned from the 92R137 material.The CDS sequence is 756 bp in length and the encoded protein length is 252 aa.The sequence similarity to Traes CS5D02G141400.1,a R3H gene located on 5D chromosome of Chinese Spring is 100%,so this gene was named as Ta-R3H-5D.Bioinformatics analysis of R3H family genes:We identified 130 R3H family genes in 14 plant species.All the identified R3H proteins include the R3H domain,in which contains the most conserved site Arginine and Histidine.By constructing a phylogenetic tree,R3H family genes can be divided into four types:Clade1,Clade2,Clade3,and Clade4.The Clade1 mainly contains the R3H-SUZ domain,the Clade2 mainly includes the R3H,DEAD,Ank,Helicase＿C,HA2 and OB＿NTP＿bind domains,the Clade3 mainly includes R3H and two G-patch domains,and the Clade4 contains the R3Hassoc domain and the R3H structure area.In wheat,the R3H family genes are mainly distributed in the 1,3,4,5homeologous groups,and their evolutionary relationship is relatively conservative.3.Analyze the mechanism of interaction protein gene Ta-R3H-5DSubcellular localization of Ta-R3H-5D:The CDS of Ta-R3H-5D was constructed into the Agrobacterium expression vector p Cambia1305:Ta-R3H-5D:GFP,and we transiently expressed the fusion protein in Nicotiana benthamiana.The results showed that Ta-R3H-5D:GFP overlapped with the membrane location markers（SYP122-m Cherry）and the nuclear location markers（D53-m Cherry）,indicating that Ta-R3H-5D is mainly located on the cell membrane and nucleus in Nicotiana benthamiana.Analysis of the disease resistance function of Ta-R3H-5D:Using VIGS technology to make Ta-R3H-5D silence in 92R137,the number of hyphae per unit area of leaves increased significantly,indicating that Pm21-mediated powdery mildew resistance was compromised partially.It was speculated that Ta-R3H-5D may be involved in the broad-spectrum resistance against powdery mildew mediated by Pm21.Development of the Ta-R3H-5D overexpression and gene editing plants:The recombinant vector p BI220:Ta-R3H-5D for overexpression of Ta-R3H-5D was constructed and transformed into susceptible recipient Yangmai158,meanwhile,the recombinant vector p ICH22005:Ta U6:sg RNA_Ta-R3H-5Dfor editing of Ta-R3H-5D was constructed and transformed into resistant recipient Nannong9918.The T0 generation transgenic plants has been developed which provided stable genetically transformed plants for gene functional analysis in the future.