Cloning And Function Analysis Of LecRK-V Gene In Haynaldia Villosa

Powdery mildew,caused by Blumeria graminis f.sp.tritici(Bgt),is a worldwide wheat disease presenting an increasingly serious situation and becomes one of the major constraints of yield and qulity.Therefore,exploring new powdery mildew resist gene is of important significance.Haynaldia villosa original from the Mediterranean and the Near East Transcaucasia areas is an annual self-pollinated diploid herb.H.villosa possesses high level of resistance against key biotic factors like powdery mildew,rusts,wheat yellow mosaic virus(WYMV)and has recognized as a key potential genetic resource to common wheat.Recently we identified some key genes involved in the resistance to powdery mildew in H.villosa by Barley genechip microarray analysis.Among these an E3 ligase HvCMPG1 was cloned and confered resistance to Bgt by positively regulating the powdery mildew resistance(Fei,2012;Wang,2010).To elucidate the reistance pathway mediated by HvCMPG1,a yeast two-hybrid(Y2H)library was constructed to identify the interacting proteins following the protocol suggested by Li et al.,(2014)a lectin receptor kinase was detected and designed as LecRK-V.Studies have shown that LecRK involved in plant defense responses.In this work,single-cell transient expression,transgenic technology,virus induced gene silence were used to highlight the function of LecRK-V in powdery mildew resistance.The main results obtained were as follows:1.Cloning of LecRK-V:HvCMPGl was used as a prey to screening the Y2H library,a lectin receptor kinase was identified.After sequencing and searching in NCBI database using BLAST tool revealed that the sequence has the highest homology with lectin receptor kinase in barley(AK367307.1).Primers were designed according to the sequence AK367307.1,cDNA of H.villosa leaves that inoculated with Bgt for 24 hours was used as a template for PCR amplification,and a 1716bp full length sequence encoding 572 amion acids was amplified It contained celluar lectin domain,a transmembrane domain and an intercellular kinase domain,belonging to L-type lectin receptor kinase,and we designed this gene as LecRK-V.2.Phylogenetic analysis of LecRK:All the protein sequence containing L-type lectin was selected from wheat and its relative species,to construct the phylogenetic tree.On the basis of their sequence similarity,we divide LecRK into 8 groups;lectin domain and kinase domain were used as a query respectively to analysis the conserved motif by MEME online tools.The results showed that in group 1,2,3,4,5 lectin domain was more conserved,as compated to group 6,7 and 8 which had higher conservation for kinase domain..3.The subcellular localization of LecRK-V:TMHMM software analysis predicted transmembrane domain of LecRK-V ranging from 190 to 212 amino acids,hence we hypothesized LecRK-V associated with a membrane.Vector pAN580-LecRK-V was constructed and transformed into Yangmai 158 protoplasts.GFP signal was detected on the membrane of the protoplast which implied the membrane protein of LecRK-V.4.Expression analysis of LecRK-V:qRT-PCR was used to analysis the expression of LecRK-V after Bgt,chitin,NaCl,PEG and 4℃ treatment.LecRK-Vup-regulated 4.25 folds after 45 min Bgt treatment in the leaves of H.villosa;LecRK-V up-regulated 7.98 folds under chitin treatment;LecRK-V could also response to NaCl treatment in 1 hour with 67.2 up-regulation;LecRK-V did not response to PEG until 12 hours after treatment only up-regulate 3.21 folds;after 4℃ treatment,no obvious expression was observed during 0-12 hours,and in 24 hours LecRK-V up-regulated.These results implied that the expression of LecRK-V could be regulated by Bgt,chitin and three abiotic stresses with different expression pattern.5.Function analysis of LecRK-V in powdery mildew resistance:5.1 A single-cell transient over-expression assay analysis of LecRK-V:LecRK-V was overexpressed in Yangmai 158 epidemical cells by a single-cell transient over-expression assay.After inoculated mix Bgt,the haustorium index(HI)level of Yangmai1 58 was 60.68%,while transformed pAHC25 only carrying GUS gene as a control.However,while co-transformed with GUS and pBI220-LecRK-V the HI decreased to 38.10%.As a result LecRK-V improved the resistance before the information of haustorium.5.2 VIGS analysis of LecRK-V:LecRK-V was silenced in T.durum-H.villosa amphiploid(AABBVV)by virus induced gene silencing assay.y empty vector and BSMV:PDS used as controls,qPT-PCR analysis reveal LecRK-V in T.durum-H.villosa amphiploid was down-regulated to 0.26,which implied LecRK-V was efficiently silenced.After onset of chlorotic phenotype,the leaves were inoculated with E26 and E31 respectively.The result turned out that though LecRK-V was efficiently silenced in BSMV:LecRK-V,the plants did not compromise resistance to infection by Bgt isolates E36 and E31.Microscopic observation showed no secondary hyphae could form in both BSMV:LecRK and controls.Thus we speculated that there were some other powdery mildew resistance genes that only silenced LecRK-V could not cause susceptible.5.3 Genetic transformation:pBI220-LecRK-V was constructed with CAMV35S promoter,and was co-transformed into a moderate variety Yangmai158 young embryo with pAHC20 by genegun bombardment method.280 T0 plants were obtained.Using PCR selection,9 positive transgenic plants were identified that showed high resistance to Bgt mix in Nanjing.All the 9 positive transgenic plants expressed higher LecRK-V compared to Yangmai 158 and negative transgenic control.Leaves detached from the 9 positive transgenic plants were inoculated with mix Bgt,presenting powdery mildew resistance.We can conclude that over-expression of LecRK-V in Yangmai 158 improved resistance to Bgt.Plants derived from LecRK-V-T0-37-1 and LecRK-V-T0-40-4,which expressed high LecRK-V expression,named as LecRK-V-T1-7 and LecRK-V-T1-9 to do further study.The detached leaves from LecRK-V-T1-7 and LecRK-V-T1-9 showed high resistance to mix Bgt.The T2 plants derived from LecRK-V-T1-7 were all positive plants identified by PCR,and expressed higher LecRK-V than Yangmai 158 and negative transgenic plants.The T2 plants derived from LecRK-V-T1-9 exhibited segregation,and the segregation ratio was 3:1.Line LecRK-V-T2-7 was chosen and evaluated with 23 different Bgt isolates in seedling stage.In Comparison to transgenic receptor Yangmai 158,LecRK-V-T2-7 showed a broad resistance to 18 Bgt isolates,and exhibited HR to 22 Bgt isolates.6.Analysis of the relationship between ROS and LecRK-V mediated powdery mildew resistance:DAB staining was used to evaluat the H2O2 accumulation in Yangmai 158,Nannoong 9918,LecRK-V-T2-7 and negative transgenic line LecRK-V-T2-1 respectively.The results indicated that 24 hours after E26 inoculation,cells H2c2 only detected at the Bgt infection site were 3.25%,20.27%,19.9%and 3.07%,while cells H2—2 accumulation distributed in the whole cell were 2.99%,14.26%,7.73%and 2.92%;24 hours after E31 inoculation,cells H2—2 only detected at the Bgt infection site were 4.37%,12.21%,28.48%and 6.47%,while cells H2O2 accumulation distributed in the whole cell were 2.38%,22.52%,7.08%and 3.79%.These results provided that 24 hours after inoculation,the oxidative burst in LecRK-V-T2-7 was higher than Yangmai 158 and negative transgenic line LecRK-V-T2-1.