A Disulphide Isomerase Gene(PDI-V)from Haynaldia Villosa Contributes To Powdery Mildew Resistance In Common Wheat

Every year plant diseases are responsible for billions of dollar worth of crop losses worldwide.Powdery mildew,caused by the fungus Blumeria graminis(DC)f.sp.tritici(Bgt),is one of the most important foliar diseases of wheat around the globe that cause reduction in yield upto 40%.Most of powdery mildew resistant genes often act only against specific races of the pathogen and their resistances are easily lost when new fungal strains develop.However,Pm21 from Haynaldia villosa(Syn.Dasypyrum villosum,2n=2x=14,VV),confers durable and broad-spectrum resistance(BSR)to Bgt.Efforts have been made previously to clarify the resistance signaling pathways of Pm21-mediated BSR which resulted in the identification of two genes,a serine/threonine kinase gene Stpk-V and an E3 ligase gene CMPG1-V,both positively contribute to BSR.The previous study showed that CMPG1-V has ubiquitination activity in vitro and is proved to be a positive regulator of the powdery mildew resistance.The CMPG1-V mediated resistance is related to ROS and the SA pathways.To further elucidate the molecular pathway of the CMPG1-V mediated powdery mildew resistance,a yeast two hybrid cDNA library of H.villosa leaves was constructed and the interacting cDNAs were screened.Among these cDNAs,one positive clone was identified to encode protein disulphide isomerase(PDI)-like protein.So,this study reported the contribution of PDI-like gene from wheat wild relative H.villosa in combating against powdery mildew(Bgt).The main results obtained are as follows:1.Cloning of PDI-Vgene and its salient featuresPDI-V was identified by screening the Y2H cDNA library using CMPG1-V as bait.Based on the sequence of interactor clone number CMIN 5,the 1762 bp full-length PDI gene was cloned from H.villosa,having an ORF of 1323 bp which encoded 440 amino acids.The BLASTp analysis showed that PDI-V is the member of thioredoxin super family and belonged to PDI-a-P5 sub-family which has typical three domains(a,a' and b).Hence it was designated as PDI-V.The genomic sequence of PDI-V was cloned to know the gene structure.Sequence analysis revealed the size of cloned genomic sequence was 5728 bp which had nine exons and eight introns that showed high homology with that of wheat TaPDIL-5 in terms of exon size and number of exons/introns.The phylogenetic analysis of PDI-V with orthologue sequences from A,B,D genome of wheat,barley and rice clearly demonstrated that it was highly conserved across the grass family.The alignment of PDI protein sequences from grass family also depicted high amino acid similarity,conserved domains and architecture with that of PDI-V.Phylogenetic analysis based on protein sequence showed that PDI-V homologs from monocots clustered into one clade,and PDI-V was closest to PDIs from H.vulgare,T.aestivum,T.urartu and Ae.tauschii.Using a set of wheat-H.villosa alien chromosome lines,PDI-V was located on long arm of 5V.PDI-V is predicted to localize in endoplasmic reticulum membranes due to the presence of NDEL signal at its C-terminal.To confirm the subcellular localization of PDI-V,vectors of PDI-V-GFP and ER marker gene(RFP)were transient co-expressed in epidermal cells of onion.Complete overlap of GFP and RFP signals were observed,indicating PDI-V-GFP was localized to the lumen of endoplasmic reticulum.2.Interaction of PDI-V with CMPG1-VY2H confirmed the interaction of PDI-V with CMPG1-V in yeast.To test their physical interaction in vivo,bimolecular fluorescence complementation(BiFC)with split yellow fluorescent protein(YFP)was employed.Co-expression of nYFP-PDI-V and cYFP-CMPG1-V in onion epidermal cells resulted in the complementation of YFP in the cytoplasm,proving their interaction in vivo.In order to confirm the data obtained by Y2H and BiFC assays,in vitro interaction between CMPG1-V and PDI-V was proved using pull-down assay.In vitro ubiquitination assay showed that in the presence of all the requisite components for ubiquitination,GST-PDI-V was monoubiquitinated by MBP-CMPG1-V.Cell free degradation assay showed that regardless of the presence or absence of the 26S proteasome inhibitor reagent MG132,the amount of GST-tagged PDI-V showed no significant change when co-incubated with MBP-tagged CMPG1-V,indicating GST-tagged PDI-V could not be degraded by CMPG1-V and hence should not be its degradation target.3.Functional analysis of PDI-Vin powdery mildew disease responseqRT-PCR analysis showed that in different tissues of H.villosa,the expression of PDI-V was 19 and 22 times higher in root and leaf than that in stem.However when two-week-old H.villosa seedlings were challenged with Bgt mixture,PDI-V expression in the leaves increased slightly at 30min post inoculation,and reached a level of six folds higher than the control at 24 h post inoculation(hpi).In Chinese Spring the PDI remained down regulated after Bgt inoculation while in amphiploid AABBVV and DA5V the PDI-V expression triggered at 30 mpi.At 24 hpi the PDI-V expression was two folds higher both in amphiploid AABBVV and DA5V than 0 hpi but significantly much lesser than H.villosa.Single cell transient over-expression assay(TOA)results depicted that the haustorium index(HI)of moderately susceptible variety Yangmai 158 reduced from 59.80%(GUS only)to 38.40%when co-transformed with GUS and pBI220-PDI-V.Statistically same results were obtained for the construct having the domain a(pBI220-PDI-Va)of PDI-V.This proved that PDI-V plays positive role in powdery mildew resistance of wheat and its domain a was involved in restraining the haustorium formation of Bgt and restrict their further penetration.Silencing of PDIs in T.durum-H.villosa amphiploid and Nannong 9918 by virus induced gene silencing(VIGS)predisposed to Bgt penetration of host plants in epidermal cells as the results indicated that the percentage of Bgt conidia producing secondary hyphae and the number of SH per conidium were significantly high in PDI silenced plants as compared to control.On other hand stable transgenic plants overexpressing PDI-V in receptor variety Yangmai 158 were generated by particle bombardment.The transgenic plants from T0 to T2 generations were characterized.Overall,56 T0 regenerated plants were obtained among which 11 were PCR positive.Based on PCR analysis for positive transgenic detection and seedling stage disease evaluation for T1 transgenic lines,the segregation ratio between resistant to susceptible ranged from 0.2:1(T1-5)to 7:1(T1-1).Moreover two stable transgenic line,i.e.PDI-V-T2-1 and PDI-V-T2-2,showed enhanced resistance against local Bgt mixture at seedling and adult stage than Yangmai 158.To confirm the essential role of domain a of PDI-V for its chaperon activity involved in Bgt resistance,a point mutation PDI-VC57A was created in catalytic motif of domain a.The PDI-VC57A was transformed into Yangmai 158 and 13 positive T0 transformants overexpressing PDI-V were identified and all showed similar level of powdery mildew susceptibility as that of Yangmai 158,confirming the essential role of domain a for the function of PDI-V in powdery mildew resistance of wheat.4.Hormone based disease response mechanism of PDI-VThe expression pattern of PDI-V in response to different phytohormones(MethJA,ethylene,ABA,SA)treatments in H.leaves suggested that SA treatment could up-regulate the PDI-V expression after 24 h while PDI-V expression varied significantly with respect to all the tested times in response of MeJA treatment.On other hand Ethylene and ABA treatments could not induce any significant change in PDI-V expression level at all the tested time intervals.Application of H2O2 rapidly triggered the expression of PDI-V in H.villosa.In accordance to this increased level of H2O2 was observed in PDI-V over-expressed transgenic lines(T2)while expression of SA marker genes i.e.PR1,PR5 and PR10 were up-regulated in these lines after Bgt inoculation.From these results we speculated that increase of H2O2 production in PDI-V transgenic plants due to SA signaling pathway might contribute to the inhibition of fungal growth which resulted in improved powdery mildew resistance.5.Cloning promoter region of PDI-VThe promoter sequence of PDI-V was cloned to figure out the important cis-regulating motifs involved in pathogen defense.Sequence analysis revealed the size of cloned promoter region was 2086 bp.Expression of GUS in wheat cells proved that PDI-V promoter has the functional activity.In silico analysis of the PDI-V promoter depicted a large number of elements responsible not only for endosperm or embryo specific expression but also for defense or stress responses against pathogens including fungi and other abiotic stresses like high or low temperature and drought.More over up-regulation of PDI-V expression in response to different abiotic stresses also supported the in silico analysis.As the results depicted that salt,cold and heat shock stresses induced the expression changes of PDI-V in the leaves of H.villosa,while drought could not do so.