Jiin Xio,Lingli Dong,Huiing Jin,c,Juncheng Zhng,Kunpu Zhng,N Liu,Xinyun Hn,Hongyun Zheng,Wenming Zheng,*,Dowen Wng,,**

aState Key Laboratory of Wheat and Maize Crop Science,College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,Henan,China

bState Key Laboratory of Plant Cell and Chromosomal Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China

cUniversity of Chinese Academy of Sciences,Beijing 100049,China

dState Key Laboratory of Wheat and Maize Crop Science,College of Agronomy,Henan Agricultural University,Zhengzhou 450002,Henan,China

Keywords:Common wheat Disease resistance Gene silencing Puccinia striiformis

A B S T R A C T Triticum urartu(AA,2n=2x=14),a wild grass endemic to the Fertile Crescent(FC),is the progenitor of the A subgenome in common wheat.It belongs to the primary gene pool for wheat improvement.Here,we evaluated the yellow rust(caused by Puccinia striiformis f.sp.tritici,Pst)reactions of 147 T.urartu accessions collected from different parts of the FC.The reactions varied from susceptibility to strong resistance.In general,there were more accessions with stronger resistance to race CYR33 than to CYR 32.In most cases the main form of defense was a moderate resistance characterized by the presence of necrotic/chlorotic lesions with fewer Pst uredinia on the leaves.Forty two accessions displayed resistance to both races.Histological analysis showed that Pst growth was abundant in the compatible interaction but significantly suppressed by the resistant response.Gene silencing mediated by Barley stripe mosaic virus was effective in two T.urartu accessions with different resistance responses,indicating that this method can expedite future functional analysis of resistance genes.Our data suggest that T.urartu is a valuable source of resistance to yellow rust,and represents a model for studying the genetic,genomic and molecular basis underlying interaction between wheat and Pst.

1.Introduction

Wheat is the most widely cultivated staple food crop in the world. Although several forms of wheat are cultivated,hexaploid common wheat(Triticum aestivum,AABBDD,2n=6x=42)accounts for most of the total wheat production[1].Wheat is threatened by several major diseases,including yellow or stripe rust caused by Puccinia striiformis f.sp.tritici(Pst)[2,3].At present,more than 80%of the global wheat production is affected by yellow rust,causing more than five million metric tons of yield loss annually [3, 4]. Use of resistant cultivars is the most effective and economic measure for preventing losses to yellow rust; however,resistance when controlled by a single gene is vulnerable to the evolution of new Pst races[2–5].Consequently,identification, characterization and deployment of new sources of resistance are a top priority in the battle against yellow rust[2–6].

Common wheat evolved in the Fertile Crescent(FC)about ten thousand years ago,with its A and D genomes donated by the diploid Triticeae species T.urartu(AA,2n=2x=14)and Aegilops tauschii (DD, 2n=2x=14), respectively [7]. The B genome originated from Aegilops speltoides or a close relative.The first two grasses belong to the primary gene pool for wheat improvement and genes from T.urartu and Ae.tauschii can be transferred to common wheat by wide hybridization[8,9].To date,more than 70 permanently designated genes(loci/alleles)have been reported to confer resistance to yellow rust[10–12],several of which(Yr10,Yr18,Yr36,and Yr46)have been molecularly cloned[13–16].No formally named yellow rust resistance gene in common wheat is derived from T.urartu,although several of them(e.g.,Yr8,Yr9,Yr28,Yr37,Yr38,Yr40,and Yr42)originated from related species[17].The yellow rust responses of the T.urartu accessions from different parts of the Fertile Crescent(FC)are still not widely reported,with only one report published 20 years ago documenting the reactions of 16 T.urartu accessions of unknown geographic origin to a single Mexican Pst isolate[18].Furthermore,genes underlying interaction between T.urartu and Pst have not been studied at the genetic and molecular levels.

We previously determined the draft genome sequence of T.urartu[19],and found that this species is an efficient model for analyzing the molecular basis of resistance to the wheat powdery mildew fungus at the genome-wide level[20].These findings,together with little knowledge of the interaction between T.urartu and Pst,prompted us to evaluate if T.urartu can be developed as an effective model for studying the genetics of stripe rust resistance in wheat.Towards this end we evaluated the reactions of 147 T.urartu accessions from six countries in the FC to major Chinese Pst races CYR32 and CYR33[2].The infection processes in two T.urartu accessions with different reactions were examined.Finally,we tested if the gene silencing approach mediated by Barley stripe mosaic virus(BSMV)might be effective in different T.urartu accessions in order to employ this strategy in future genetic analyses of interaction between T.urartu and Pst.

2.Materials and methods

2.1.Plant materials,inoculation conditions and phenotyping

The 147 T.urartu accessions and their geographic origins are listed in Table S1.They were grown in a growth chamber at a day/night temperature cycle of 22/20°C,16 h light/8 h darkness photoperiod,and 70%relative humidity.Pst races CYR32 and CYR33 were maintained in the wheat disease assessment facility of the Institute of Genetics and Developmental Biology,Chinese Academy of Sciences.For each accession and each Pst race,10 uniformly developed seedlings were inoculated at the two-leaf stage.Briefly,the seedlings were sprayed with 0.02%Tween 20 in distilled water(v/v);the fresh Pst spores diluted with talcum powder were then applied to the surface of the leaves using a larynx atomizer. The inoculated seedlings were immediately placed in darkness for 24 h at 16°C with 90%moisture[21].Afterwards,the seedlings were returned to the above growth conditions,and reactions were recorded at 14 days post inoculation(dpi).

Phenotype scoring followed a 0–4 scale[21]with some modifications.Specifically,we classified the reactions into five types, viz., IT0 (no visible symptoms; immune), IT1(necrotic/chlorotic spots without uredinia;high resistance),IT2(necrotic/chlorotic spots with moderate uredinia;moderate resistance), IT3 (moderate to abundant uredinia with chlorosis; moderate susceptibility), and IT4 (abundant uredinia without necrosis and chlorosis;high susceptibility)(Fig.1).Tests were repeated to check for reproducibility of reactions.

2.2.Histological staining of Pst growth

Fig.1–Representative yellow rust reactions of T.urartu accessions.Accessions exhibiting these infection types(ITs)were regarded as being immune(IT0),highly resistant(IT1),moderately resistant(IT2),moderately susceptible(IT3),and highly susceptible(IT4).The arrow indicates a chlorotic spot.These reactions were obtained with Pst race CYR33.

Histological staining was carried out to examine the development of Pst structures in the leaves of inoculated T.urartu seedlings as described previously[22,23].Accessions G1812 and PI 428322,which were susceptible and resistant to CYR33,respectively(Table S1),were used in this experiment.Leaves collected from CYR33-inoculated seedlings at 24,72,and 120 h post inoculation (hpi) were bleached in saturated chloral hydrate,then washed with 50 mmol L?1Tris-HCl(pH 7.5),and stained using 20 μg mL?1WGA-Alexa-488(WGA-Alexa Fluor 488;Invitrogen,Corp.,Carlsbad,CA,USA).The stained leaves were washed with 50 mmol L?1Tris-HCl(pH 7.5),and examined under a DMRE microscope(Leica,Microsystems GmbH,Wetzlar, Germany). Images were captured using a digital camera(EXi Aqua,QImaging,BC,Canada).The histological staining experiment was repeated three times.

2.3.BSMV induced gene silencing

BSMV is a tripartite RNA virus,whose infectious cDNA has been converted into a high throughput gene silencing vector composed of three plasmids(pCaBS-α,pCaBS-β,and pCa-γb-LIC)[24]. In this work, we tested the silencing of a phytoene desaturase(PDS)gene using the BSMV vector in two different T.urartu accessions(PI 428226 and PI 428322,Table S1).A cDNA fragment (200 bp)was amplified by RT-PCR using primers designed according to the coding sequence of TuPDS(TRIUR3_18416,http://gigadb.org/dataset/100050).The synthesis of cDNA and execution of RT-PCR were conducted as reported previously[24].The two primers specific for TuPDS were 5′-AAGGAAGTTTAATTTCTCCAGGAGAA-3′ (forward) and 5′-AACCACCACCACCGTTCTCCAGTTATT TG-3′(reverse).The resultant cDNA fragment was cloned into the plasmid pCa-γb-LIC,yielding pCa-γb-TuPDSas.This construct and the three original plasmids were individually transformed into Agrobacterium tumefaciens strain EHA105, with the resulting recombinant strains used for infiltrating the leaves of Nicotiana benthamiana as detailed previously[24].Leaves infiltrated with Agrobacterium cells carrying pCaBS-α,pCaBS-β,or pCa-γb-LIC represented the wild-type empty vector control(BSMV:EVC),whereas those treated by Agrobacterium cells harboring pCaBSα, pCaBS-β, or pCa-γb-LIC-TuPDS formed the silencinginducing virus (BSMV:TuPDSas). N. benthamiana leaves exhibiting BSMV infection symptoms were harvested at around 12 dpi,and ground in Na-phosphate buffer(containing 1%celite,pH 7.2).The resulting extracts(carrying BSMV:EVC or BSMV:TuPDSas)were inoculated onto T.urartu seedlings at the two-leaf stage.TuPDS silenced phenotypes were monitored for six weeks.For each recombinant virus and each T.urartu accession,at least 30 seedlings were inoculated,with each inoculation experiment repeated three times.

3.Results

3.1.Yellow rust reactions of T.urartu accessions

The 147 T.urartu accessions exhibited a wide range of yellow rust reactions(Fig.1,Table S1).In the case of CYR32,46 accessions,including one showing IT1 and 45 exhibiting IT2,were resistant;101 accessions,including 42 showing IT3 and 59 exhibiting IT4,were susceptible(Table 1,Table S1).No accession was immune to this race(Table 1,Table S1).With CYR33,3,10,and 57 accessions responded with IT0,IT1,and IT2,respectively,and 15 and 62 exhibited IT3 or IT4(Table 1,Table S1).

Table 1–Numbers of T.urartu accessions with different infection types when inoculated with two Pst races.

Among accessions showing resistance to CYR32(46)or CYR33(70),42 were resistant to both Pst races(Fig.2,Table S1).Seventy three accessions were susceptible to both races(Fig.2, Table S1). PI428327, the only accession showing high resistance to CYR32,was also moderately resistant to CYR33(Table S1).Among the three accessions(PI428257,PI428297,and PI487268) immune to CYR33, PI428257, and PI487268 showed moderate resistance to CYR32 and PI428297 was susceptible (Table S1). Among 10 accessions (PI428199,PI428201, PI428210, PI428213, PI428214, PI428226, PI428260,PI428275,PI428294,and PI487270)showing high resistance to CYR33, five (PI428214, PI428226, PI428260, PI428294, and PI487270)were moderately resistant to CYR32(Table S1).

Among the 68 accessions from Lebanon,54%(37/68)and 78%(53/68)were resistant to CYR32 and CYR33,respectively;for the 66 accessions from Turkey,the corresponding values were 8%(5/66)and 17%(11/66),respectively(Table S2).The numbers of accessions from Syria,Armenia,Iran and Iraq were quite small(＜10,Table S2).Nevertheless,43%(3/7)and 71%(5/7)of the accessions from Syria were resistant to CYR32 and CYR33,respectively(Table S2).

Fig. 2 – Numbers of T. urartu accessions resistant or susceptible to Pst races CYR32 or CYR33. Forty two accessions were resistant to both races, and 73 accessions were susceptible to both. The accessions showing IT0, IT1, or IT2 were collectively considered as being resistant, while those exhibiting IT3 or IT4 were treated as being susceptible.

3.2.Histological analysis of Pst infection in T.urartu

Pst infects wheat mainly by urediniospores falling onto the leaf surface[2,3,6].The infection process in T.urartu by Pst race CYR33 was studied following inoculation of one susceptible accession(G1812)and one resistant accession(PI428322)of T. urartu and examination by histological staining. At 24 hpi,Pst structures,including the substomatal vesicle(SV),infection hypha(IH)and haustorial mother cell(HMC),were clearly visible in G1812,whereas only SV and IH were seen in PI428322 leaf tissues(Fig.3-A,top panel).By 72 hpi,branched secondary hyphae(SH)developed from IH were present in in G1812,but not in PI428322(Fig.3-A,middle panel).At 120 hpi,branching mycelia in G1812 were much more abundant than in PI428322 (Fig. 3-A, lower panel). Consistent with the cytological observations,substantially more Pst sori appeared on the leaves of G1812 than on PI428322 by 14 dpi(Fig.3-B).Thus the infection process was slower and less expansive in the resistant than in the susceptible accession.

3.3. Effectiveness of BSMV-mediated gene silencing in T.urartu

To test the effectiveness of BSMV-induced gene silencing for studying T.urartu gene function in Pst and T.urartu interactions,a recombinant BSMV (BSMV:TuPDSas) was developed for silencing TuPDS in PI428226 and PI428322. PI428226 was moderately and highly resistant to CYR32 and CYR33,respectively,whereas PI428322 was moderately resistant to both races(Table S1).Concomitant to the inoculation with BSMV:TuPDSas at the two-leaf stage,BSMV:EVC was used as an empty vector control.At 14 dpi,conspicuous photo-bleaching was observed on the third leaf of both accessions inoculated with BSMV:TuPDSas,whereas no such phenotype was found with inoculation by BSMV:EVC(Fig.4).The photo-bleaching phenotype of TuPDS silencing persisted for more 40 days in the plants inoculated with BSMV:TuPDSas.

4.Discussion

Fig.3–Comparative analysis of the growth of Pst structures in susceptible(G1812)and resistant(PI428322)T.urartu accessions seen by histological staining(a)and Pst uredinia on the leaves(b).HMC,haustorial mother cell;IH,initial hypha;SH,secondary hypha;SV,substomatal vesicle.The data shown were representative of three independent experiments.

Fig.4–Suppression of TuPDS expression in T.urartu accessions(PI428226 and PI428322)using Barley stripe mosaic virus(BSMV)mediated gene silencing.BSMV:EVC was the empty vector control,whereas BSMV:TuPDSas carried the silencing-inducing fragment for TuPDS.Conspicuous photobleaching resulting from silencing of TuPDS was observed only on leaves infected by BSMV:TuPDSas.The symptoms were produced in three independent experiments.

Prior to this work,little was known about the yellow rust responses of T.urartu[18].An updated and more comprehensive evaluation of T.urartu accessions to yellow rust was considered desirable given that recent studies found that T.urartu was a good model for understanding and improving resistance to wheat powdery mildew[8,20,25].Here,we undertook a more comprehensive study of the seedling reactions of 147 T.urartu accessions from several countries within the FC to two Pst races.The data obtained permit us to make the following suggestions.Firstly,T.urartu accessions differed extensively in reaction to yellow rust. However,unlike many wheat relatives few accessions gave immune or highly resistant reactions(Table 1).No accession was immune to CYR32,and only one accession was highly resistant.The numbers of accessions of showing immune (3) or highly resistant (10) responses to CYR33 were higher, but still represented less than 9%of the tested lines.On the other hand, substantial numbers were moderately resistant to CYR32(30.61%)or CYR33(38.78%)(Table 1).These findings may not be surprising,because extreme or high pathogen resistance may cost too much energy,and is not beneficial for an optimal balance of plant defense and growth processes[26,27].As a wild species in nature,T.urartu is not grown in high density,and the pressure for evolving extreme or high Pst resistance is unlikely to be strong. Therefore, moderate resistance is probably the main form of defense of T.urartu to Pst infection.A previous study involving 16 accessions also showed that T.urartu mainly shows moderate resistance to yellow rust[18].

Secondly,CYR32 appear to be more virulent than CYR33 on the tested T.urartu accessions.There were fewer accessions with immune and highly or moderately resistant reactions to CYR32 than to CYR33(Table 1).Notably,three accessions immune to CYR33 were either susceptible(PI428297)or only moderately resistant (PI428257 and PI487268) to CYR32.Among 10 accessions(PI428199,PI428201,PI428210,PI428213,PI428214, PI428226, PI428260, PI428275, PI428294, and PI487270) showing high resistance to CYR33, only five(PI428214,PI428226,PI428260,PI428294,and PI487270)were moderately resistant to CYR32,with the remaining five being susceptible(Table S1).Past studies also showed that CYR32 had higher parasitic fitness than other Pst races(including CYR33)on common wheat cultivars[28,29].This relatively high parasitic fitness (or aggressiveness) of CYR32 was retained in regard to T.urartu.

Thirdly,there is a higher proportion of lines resistant to both races among accessions collected from Lebanon than from Turkey.A notable feature of T.urartu accessions from Lebanon was that they were generally collected from high altitude locations [30]. Because the genetic diversity of Lebanese T.urartu accessions was quite high[30,31],it is possible that a number of genes for resistance to stripe rust are present.

By comparative histological analysis we observed that the resistance of T. urartu accession PI428322 to CYR33 infection was associated with significantly reduced fungal growth at the early stages of infection. This resulted in development of fewer uredinia with smaller size.We also demonstrated that BSMV-mediated gene silencing was effective in the T.urartu accessions with different levels of resistance to the tested Pst races.This,together with the recombinant BSMV (i.e., BSMV:TuPDSas) developed here,may aid future functional studies of the genes involved in response of T.urartu to Pst infection.

The points discussed above have three implications for future research. First, T. urartu accessions with known reactions to stripe rust can be used in genetic analysis.Once mapped to specific chromosome regions,the genes can be transferred to common wheat by marker-assisted selection,as demonstrated previously in the transfer of a powdery mildew(caused by Blumeria graminis f.sp.tritici)resistance gene from T. urartu to common wheat [8]. Second, the resistant accessions can be tested with newly evolved Pst races,e.g.,CYR34[32],with the aim of identifying broadspectrum resistance genes that may be more durable when exploited in wheat production.Finally,T.urartu,with a much smaller and simpler genome than common wheat,can be developed as a more efficient system for studying wheat and Pst interactions.With the availability of the genomic sequence[19],resistance genes and their interacting networks in T.urartu can be unveiled using functional genomic tools(such as transcriptomics and proteomics), followed by functional validation using BSMV-mediated gene silencing.The common wheat orthologs of the characterized T.urartu genes can be identified based on the genome synteny conserved among wheat and related species[33].

5.Conclusions

The responses of 147 T.urartu accessions to Chinese Pst races(CYR32 and CYR33)suggest that T.urartu is a valuable source of resistance to stripe rust. This diploid wheat species represents a promising model for dissecting the genetic,genomic and molecular basis of interaction between wheat and Pst.

Acknowledgments

We thank Ms. Fanyun Ling (Institute of Genetics and Developmental Biology,Chinese Academy of Sciences)for assistance with Pst inoculation.This work was supported by the National Key Research and Development Program of China(2017YFD0101000)and Key Project of Henan Province of China(161100110400).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.03.009.