Identification Of A Hv-CMPG1 Interaction Protein HvHIPP1 From Haynaldia Villosa L.and Its Functional Analysis In Powdery Mildew Resistance

Powdery mildew,caused by Bgt（Blumeria graminis f.sp.tritici,Bgt）,is one of the most serious fungal diseases of wheat.The powdery mildew resistance gene Pm21 is introduced from a diploid wheat relative Haynaldia villosa L..Pm21 shows broad spectrum resistanee to all the current Bgt isolates and is now being widely used in wheat breeding program in China.The cloning of genes related to powdery mildew resistance from H.villosa and elucidate the molecular machemnism underlying Pm21 mediated powdery mildew resistance will provide gene resouces for wheat breeding for disease resistance.In previous research,by Microarray analysis using Barley GeneChip,an E3 ligase coding gene Hv-CMPG1 has been cloned from H.villosa and proved positively regulating powdery mildew resistance of wheat by when transformed into wheat variety Yangmai 158 by bombardment.To identify the interaction protein and elucidate the resistance pathway mediated by Hv-CMPG1,in this study,a yeast two-hybrid（Y2H）library was constructed,Hv-CMPG1’s interact proteins were screened,cloned and their function in powdery mildew resistance was characterized.The main results obtained were as follows:1.A nucleus system Y2H cDNA library of H.villosa leaves before and after Bgt inoculation was constructed.The library includes more than 0.5 million clones,and the average length of insert sizes is more than 200 bp.2.By screening the Y2H library using Hv-CMPG1 as a prey,17 candidate interaction clones were identified.Based on gene annotation and interaction intensity with Hv-CMPG1 of these clones,one clone named as CMIN2 was selected for further study.Clone CMIN2 contains the full length gene,with an ORF of 456 bp,encoding a protein with 151 amino acids.BLASTp results showed that the predicted protein is a farnesylation protein,belonging to HIPP（Heavy metal associated isoprenylated plant protein）type.The protein has a HMA domain and a fameyslation motif in the C terminal.The gene is designated HvHIPP1.3.Phylogenetic analysis showed that HvHIPP1 has more than 80%similarity with its homologs of several grass species.HvHIPP1 was most close to EMT21257 from Ae.tauschii indicated by their 98%similarity.Using a set of wheat-H.villosa alien chromosome lines,HvHIPP1 was locate on the chromosome region of 3VL FL:0.78-1,00 in H.villosa.BLASTn analysis showed that HIPP1 homologs were located on chromosome 3H（HvFP3,303070633-303071577）of H.vulgare,Bd3（Bradi3g04270.1,2936502-2937263）of B.distachyon,6AS1-0.35-0.65（EMS63565）of T.urartu,R1（19382611-19414396）of rice and chromosome 7（XP002444859,170550-171704）of S.bicolor.4.In the epidermal cells of onion,the transcient overexpressed fusion protein GFP-HvHIPP1 was subcellular localized in the plasma membrane and nucleus.To analyze the effect of the fanesylation domain on the HvHIPP1 localization,the HvHIPP1 was point mutated.In the epidermal cells of onion,the mutant GFP-HvHIPP1m failed to localize to the plasma membrane,green GFP signal spots were found to be distributed in the cytoplasm,indicating the fameyslation motifi of HvHIPP1 is critical for its membrane localization.5.To test the interaction of Hv-CMPG1 and HvHIPP1 and the ubiquitination of HvHIPP1 by Hv-CMPG1,purified MBP-Hv-CMPG1 and GST-HvHIPP1 fusion proteins were used for Pull-down and in vitro ubiquitination assays.The results showed that the recombinant GST-HvHIPP1 could be detected only when GST-HvHIPP1 was incubated with MBP-Hv-CMPG1,confirming the direct interaction of HvHIPP1 with Hv-CMPG1.When all the essential components（E1,E2,ubiquitin,Hv-CMPG1）for ubiquitination were present,the ubiquitinated GST-HvHIPP1 could be detected by Western blotting using anti-GST,indicating HvHIPP1 can be ubiquitinated by Hv-CMPG1 in vitro.A cell-free degradation assay showed that,when co-incubated with Hv-CMPG1 in vitro,there was no obvious reduction of HvHIPP1 at different timepoints either with or without MG 132 treatments,indicating that the GST-HvHIPP1 was not degraded by the ubiquitination system.6.qRT-PCR showed that the expression of HvHIPP1 was tissue specific,the expression in the leaves and roots of H.villosa was 7-13 folds higher than those in the stems and immature spikes.In response to Bgt infection,the expression patterns of HvHIPP1 were different in different genotypes.In H.villosa,the HvHIPP1 expression was up-regulated at 45 min after Bgt inoculation,reached the peak at 2 hai and the reduced gradually to the original level before Bgt inoculation.In T.durum-H.villosa amphiploid,the HvHIPP1 expression was also up-regulated at 45 min after Bgt inoculation,but reduced to the original level before Bgt inoculation at 2 hai.In wheat-H,villosa addition line DA3V,the HvHIPP1 expression was also up-regulated but delayed to 24 hai.In wheat variety Chinese spring,the HIPP1 homologs expression was slightly down-regulated at 6 hai.In the wheat variety Nannong 9918 earrying the Pm21,no obvious HIPP1 homologs expression change was observed.These results indicated that HvHIPP1 participated the powdery mildew resistance in H.villosa,rapid upregulated expression of HvHIPP1 was critical for its function.7.The wildtype and mutant HvHIPP1 genes were transciently overexpressed in the epidermal cells of a powdery mildew susceptible wheat variety Yangmai 158.The haustorium indexes（HI）were compared to study the function of HvHIPP1 in powery mildew resistance.When co-expressing the wildtype HvHIPP1 and GUS genes,the HI was 35.0%,which was significantly lower than the 58.7%when only expressing the GUS gene.However,when co-expressing the mutant HvHIPP1 and GUS genes,the HI was 51.7%,which was not significant compared to that when only expressing the GUS gene.Silencing of the HvHIPP1 in the T.durum-H.villosa amphiploid using VIGS resulted in compromised powdery mildew resistance shown by the sporelation on the leaves.The HvHIPP1 was transformed into Yangmai 158 by bombardment,a total of 190 To plants were regenerated,and 35 were identified to be positive transgenic plants.Among them,5 plants were highly resistant to Bgt at both the seedling and adult stages.Further evaluation of the T1 lines showed that all these five lines were segregated for powdery mildew resistance,with resistance to susceptible ratios as 2:1-7:1.One of the T2 line HIPP-T2-2 was used for powdery mildew resistance evaluation to 25 different Bgt isolates,it was resistant to 14 isolates.When transformed the mutant HvHIPP1m gene,89 T0 plants were regenerated,and two were identified to be positive transgenic plants.At seedling stage,these two T1 transgenic lines were all susceptible to Bgt infection.These results indicated that the HvHIPP1 play positive role in powdery mildew resistance of wheat,also confirming the importance of the farnesylation motif for this biological role.8.HvHIPP1 expression was rapidly up-regulated in the leaves of H.villosa after treatments of ABA,ET,MeJA and H₂O₂,while no obvious expression change in response to SA treatment.The HvHIPP1 expression in the transgenic plants overexpressing HvHIPP1 and VIGS plants with HvHIPP1 silenced was analyzed.The HvHIPP1 positively regulated the gene expression related to the SA and H₂O₂ pathways.These reveal that the ABA,ET,JA and H₂O₂ may regulate HvHIPP1,HvHIPP1 contributes to powdery mildew resistance by regulating the SA and H₂O₂ pathways.