A journey to understand wheat Fusarium head blight resistance in the Chinese wheat landrace Wangshuibai

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The Applied Plant Genomics Laboratory,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China

1.Introduction

Fusarium head blight(FHB),caused by Fusarium graminearum,is arguably the most destructive disease in wheat.It greatly reduces yield and kernel quality in epidemic years and is almost not curable due to lack of immune germplasm[1–2].More than that,the fungal mycotoxin contaminated kernels render the grains unsuitable for food or feed[3].It has been a great challenge for wheat producers to avoid consequent economic damage.Among the measures adopted to curb this disease,deployment of scab-resistant varieties is the most preferred strategy for its economy and environmental friendliness.

Resistance to FHB in wheat is controlled by polygenes that usually have small effects and are vulnerable to environmental influences[4,5].Resistance is further complicated by its different manifestations,such as type I resistance against initial penetration,type II against fungal spread within spikes[6],type III for toxin decomposition[7],and less kernel infection and yield tolerance[8].Because of these complexities,understanding of the resistance mechanisms was very limited and progress in scab resistance breeding was slow and far from meeting our needs.

Since the end of last century,the application of molecular genetics to crops has greatly speeded up FHB resistance research in wheat.Buerstmayr et al.[9]summarized findings from 52 studies on FHB resistance QTL mapping using various germplasm accessions.Currently,more than 250 QTL distributed on all 21 chromosomes have been documented[10–28].As expected,most QTL have small effects and are yet to be verified.The effort is ongoing to cloning the FHB resistance QTL.Besides fine mapping of four major-effect QTL identified in common wheat,Fhb1[29,30],Fhb2[31],Fhb4[32]and Fhb5[33],a gene encoding a chimeric lectin on chromosome 3BS was reported to be responsible for FHB resistance conferred by Fhb1[34],which albeit is still under dispute.

In order to decipher the mechanisms underlying FHB resistance,our group has been conducting wheat FHB resistance research since 1998,mainly by forward genetics approach.Here we review the progress made in QTL mapping,cloning,functional gene discovery,and marker-assisted FHB resistance improvement.

2.Chromosome regions affecting FHB resistance

Many of the FHB resistance germplasms characterized and used in breeding programs worldwide can be traced back to the Chinese cultivar Sumai 3,developed by the Taihu Regional Institute of Agricultural Sciences,located in the Lower Yangtz River region where FHB occurs frequently and severely because of the warm and humid conditions during flowering.Cultivar Wangshuibai (WSB), indigenous to this region, displays excellent FHB resistance(Fig.1)that is at least as resistant as Sumai 3,but has not been successfully used in breeding programs.To determine the chromosomal regions conferring FHB resistance in WSB, we generated a recombinant inbred line(RIL)population from a cross of WSB with Nanda 2419[35]and constructed a molecular marker map of 4223.1 cM,comprising 887 loci[36].Nanda 2419 is a cultivar with moderate FHB resistance,compared to the most susceptible cultivars.Four field trials were conducted to evaluate the type I resistance of the RILs,which was measured as percentage of infected spikes(PIS)15 days post anthesis,after spray-inoculation at anthesis and scattering scabby wheat grains on the soil surface before anthesis[37].Seven QTL repeatedly detected in more than two trials were identified(Table 1).The WSB alleles of five of these QTL,including Qfhi.nau-4B and Qfhi.nau-5A the two with the largest effects,contributed to better resistance.

To detect type II resistance QTL,we evaluated the number of diseased spikelets(NDS)and the length of diseased rachis(LDR)of 10–20 spikes per line 20 days after single floret inoculation at anthesis in three trials.Three type II resistance QTL in WSB and one in Nanda 2419 were detected.Moreover,there was a minor type II resistance QTL from WSB repeatedly detected through composite interval mapping in the Xwmc419–Xbarc181 interval on chromosome 1B[35].Among the FHB resistance QTLfound in Nanda2419,the Qfhi.nau-2BandQfhi.nau-2Bintervalsoverlapped and were collectively referred to as Qfh.nau-2B thereafter.

The percentage of Fusarium-damaged kernels(FDK)directly reflects the damage level caused by FHB on wheat grains and its variation represents the so-called type IV scab resistance.To identify chromosomal regions governing type IV resistance and investigate its relationship with other scab resistance types,QTL associated with percent FDK were mapped[38].TypeI and typeII resistance QTL were found to be the major factors governing type IV resistance.In addition,the interval linked to Xcfd14 on chromosome 7D was associated with FDK and the number of spikelets per spike and compactness[38,39].The 2AS Xgwm328–Xgwm425interval was associated with FDK[38]and DON content[40].

The association of these identified intervals with FHB resistance was supported in other studies that used either different resistant lines or different mapping populations involving WSB[9].A few minor QTL for FHB resistance in WSB were detected using alternative susceptible mapping parents;for example,a QTL linked to Xgwm2 on chromosome 3AS[41],and a 7AL QTL linked to Xgwm276[42,43].Other major findings on chromosome locations of wheat FHB resistance QTLs are summarized in Table 2.As shown in the table,WSB carries the highest number of repeatedly identified FHB resistance QTL,in consistence with its performance against FHB.This implies the importance of QTL pyramiding for resistance enhancement.Next to WSB in terms of number of repeatedly identified QTL is Frontana with five QTL.

3.Fine mapping and cloning of FHB resistance genes

Because of limitations in population size and marker density of genetic maps in most primary mapping studies,and the dependence of QTL effects on environment,verification and marker saturation of detected QTL are always necessary for breeding and QTL cloning.To achieve this goal,we developed NILs with Qfh.nau-2B,Qfhs.nau-3B,Qfhi.nau-4B,Qfhi.nau-5A,and Qfhs.nau-6B through maker-assisted backcrossing using susceptible lines PH691(PH)and MY99-323(MY)as recurrent parents.Compared with the recurrent parents,the introduction of Qfhs.nau-3B and Qfhs.nau-6B substantially increased type II resistance,and introduction of Qfhi.nau-4B and Qfhi.nau-5A enhanced type I resistance[44](Fig.2).The improvements were statistically significant in field trials(Fig.3).NILs containing Qfh.nau-2B performed significantly better than the recurrent parents in both type I and type II responses.

Fig.1–Spikes of Wangshuibai in a field(left,photo taken on 24th of May 2016)compared with the spikes of a susceptible line in the same field(right,photo taken on the 20th of May 2016).Both cultivars have similar heading dates,but WSB still showed resistance even when allowing more time for disease development.

In order to reduce the genetic background effects in fine mapping,the NILs were used to develop secondary F2populations by crossing them with the respective recurrent parents.Recombinants covering the QTL intervals were identified through marker genotyping of populations each up to 20,000 plants.Evaluation of the derived homozygous recombinants in multiple trials showed that there were clear differences in FHB resistance between lines with the added QTLs and those without them[32,33].This allowed us to treat the QTL as single genes,a prerequisite for finemapping. After genotyping the QTL intervals in the recombinants with markers developed using comparative genomics,BACs for chromosome walking,and publicly available genomic and EST sequences,Qfhs.nau-3B(Fhb1),Qfhs.nau-6B(Fhb2),Qfhi.nau-4B(Fhb4),and Qfhi.nau-5A(Fhb5)were successfully fine mapped(Fig.4).

Qfhs.nau-3B was delimited to the Xwgrb597–Xmag9404 interval through genetic and BAC-physical mapping.Fhb1 in Sumai 3 was also placed in the same interval. There were three SNPs that distinguished WSB from Sumai 3 at the sequence level,but they appeared to have no genetic consequence.A candidate gene for Fhb1 was identified and when expressed in multiple transgenic lines it significantly enhanced FHB resistance(unpublished results).

A gene encoding a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain(PFT)on chromosome 3BS was recently reported to be responsible for the Fhb1-conferring resistance[34].This conclusion was mainly based on association mapping studies with 40 cultivars,RNA interference experiments,mutant analysis and transgenic plant evaluations.However,we were unable to confirm that the resistance in WSB and Sumai 3was due to the existence of this gene.Firstly,in ourwork the PFT gene sequence of PH691 was identical to that of WSB and Sumai 3,but PH691 was highly susceptible to F.graminearum.Secondly,in the RIL population derived from Nanda 2419×WSB,a recombinant with the WSB PFT allele alone was susceptible in multiple trials(Fig.5-A).Recombinants with the WSB PFT allele alone obtained in secondary F2populations derived from the NILs showed similar results.Thirdly,among 151 cultivars used in an association analysis 44 had the WSB/Sumai 3 PFT allele,but only 12 of them were resistant(Fig.5-B).Fourthly,the expression levels of PFT in WSB,and particularly inNanda 2419,without F.graminearum infection were extremely low and became negligible after infection(Fig.5-C).We conclude that association of PFT with FHB resistance insome germplasms is due to its tight linkage to the resistance gene.

Table 1–QTL repeatedly detected in the WSB×Nanda 2419 population in different trials.(Excerpted from Lin et al.[35,37]and Ma et al.[38].)

Table 2 – Distribution of repeatedly detected QTL in wheat germplasms a. +, QTL present; ?, QTL not detected.

Fig.2–Numbers of diseased spikelets(NDS)(measures of type II resistance)20 days after single floret inoculation(A)and percent diseased spikelets(PDS)(measures of type I resistance)15 days after spray-inoculation of NILs with different FHB resistance QTL(B).The field trials were carried out in a randomized complete block design in two replications for each type of the inoculations at Jiangpu in Jiangsu province(2013JP,2014JP)and at Fengyang in Anhui province(2013FY).NIL-3B has Qfhs.nau-3B,NIL-6B has Qfhs.nau-6B,NIL-4B has Qfhi.nau-4B,and NIL-5A has Qfhi.nau-5A.

Qfhs.nau-6B was mapped to a 2.2 cM interval,in which Fhb2 was previously mapped[31].Qfhi.nau-4B was designated as Fhb4 and Qfhi.nau-5A as Fhb5[32,33](Fig.4).Fhb5 is located in an～50 Mb interval very close to the centromere of chromosome5A,a region of low recombination that makes map-based cloning a challenging task.However,the different mapping populations could help to solve some of the recombination suppression problems.For example,the recombination in the Fhb1 interval was severely suppressed in the combination ofFhb1 NIL×Mianyang 99-323,but more relaxed in the combination of Fhb1 NIL×PH691(unpublished results).

4.Discovery of genes related to FHB response by reverse genetics

Fig.3–Effect of Qfhs-nau-3B when transferred to PH(PH691)and MY(MY99-323)on FHB resistance 20 days after single floret inoculation.A and B,close-up views;C.field view of the MY NIL with Qfhs-nau-3B compared to MY.

Fig.4–Marker maps surrounding individual FHB resistance QTL.The numbers to the left of each map indicate the genetic distances between adjoining markers.N,population size used in recombinant screening for each QTL.The grey bars indicate the QTL regions.

Plant resistance to pathogen attack is a finely regulated process associated with a number of cellular events,such as oxidative burst induction[45],hypersensitive response[46],accumulation of toxic compounds[47],and fortification of cell walls[48].It is conceivable that a large gene network regulates resistance responses.Accordingly,a common approach in identification of FHB resistance-related genes is through reverse genetics.Following early studies that demonstrated the induction of defense-related genes by F.graminearum infection[49–51],numerous studies have tried to identify FHB resistance-related genes through high-throughput omics[52–67].

Fig.5–Relevancy of the WSB/S3 PFT allele with FHB resistance.(A)Genotypes and phenotypes of key recombinants identified in 530 RILs derived from Nanda 2419×WSB.WGRB956 is a PFT-specific marker.Black rectangles,WSB chromatin;open rectangles,Nanda 2419 chromatin.R1 and S1,resistant control and susceptible controls,respectively.*Significantly different from the susceptible control at P=0.05.(B)NDS(averaged over three separate trials in different years)distribution in 44 cultivars with the WSB/Sumai 3 PFTallele.(C)Expression levelsmeasured in FPKM(fragments per kilobasemillion) of the PFT gene after inoculation with F.graminearum in WSB and Nanda 2419.Spikelets were collected for RNA extraction and RNA-seq data generation at 0,12,and 24 h after inoculation with F.graminearum.

As an initial attempt,a proteomics-based search of proteins differentially expressed in resistantWSB 6–24 h afterF.graminearum infection was conducted in 2001 in our laboratory.Proteins associated with defense reactions were activated or translated shortly after inoculation[68].Later,by comparing the protein profiles of WSB and the susceptible WSB mutant Meh0106 created by EMS treatment,in spikes at 12 h after inoculation,we identified four jasmonic acid(JA)/ethylene(ET)biosynthesis proteins,one antimicrobial compound synthesis protein,one cell-wall formation-related protein,and three defense-related proteins,which were all differentially up-regulated in WSB[59].These results suggested the association of these proteins with FHB responses.To complement the proteomics approach,a comparative transcriptomic analysis of genes expressed in infected spikes and in spikes without pathogen infection was conducted.One hundred and sixty-three up-regulated defense-related unigenes were identified and classified into ten functional categories based on their putative functions(Table 3).Most of the up-regulated genes were involved in basal resistance or defense[52,56].Moreover,of the identified genes,nearly 50%encoded proteins for synthesis of antimicrobial compounds,modulation of the oxidative state,and resistance gene analogs(RGAs)or kinase proteins.The involvement of these genesinFHB resistance or defense was confirmed through expression profiling of WSB and the susceptible mutant,and in other studies[60–62].It is intriguing that some RGAs were found only in spikes under pathogen attack.This may imply unique roles in defense against F.graminearum. RGAs and kinases are important in regulation of crosstalk between signaling pathways mediated by phytohormones in response to pathogen infection[69].

Lignin is a major component of plant cell walls and is involved in defense against pathogen attack.Many genes,related to lignin biosynthesis,were up-regulated by F.graminearum infection in resistant lines[59,60];differences in lignin composition and structure were also observed inresistant and susceptible lines[70].Cell wall fortification could make the wall more resistant to cell wall degrading enzymes produced by pathogens and prevent the diffusion of pathogen-produced toxins[71].

Table 3–Functional classification of genes differentially expressed after F.graminearum infection.(Excerpted from Ding et al.[60].)

Jacalin-related lectins(JRLs)are a group of plant lectins with at least one domain homologous to jacalin protein first isolated from Artocarpus integrifolia[72].Most of the functionally characterized JRL genes in plants are associated with disease resistance,abiotic stress tolerance,wounding or insect damage,or even responses to multiple stresses[73,74].Interestingly,in mining JRL genes present in the wheat genome,at least 12 were inducible by F.graminearum infection[73].Over-expression of mannose-specific JRL-like gene TaJRL1 in Arabidopsis thaliana led to increased resistance to F.graminearum and Botrytis cinerea and to elevation of JA and SA levels,whereas attenuation of TaJRLL1 expression through virus-induced gene silencing increased susceptibility to F.graminearum and Blumeria graminis[75].Over-expression of TaJRL2.1in wheat up-regulated lignin synthesis-related genes and led to better FHB resistance(unpublished data).

Thus far, a great number of genes inducible by F. graminearum infection have been identified through reverse genetics in different laboratories.However,it is worth noting that substantial differences in identified genes and their expression profiles occur between studies,probably due to the differences in wheat genotypes,experimental procedures and technologies.The big challenge facing us now is to functionally verify the association of these genes with FHB response.A few wheat genes that were either differentially expressed after F. graminearum inoculation or mycotoxin treatment,or cloned based on the association of homologs in other plant species with defense responses,have been used in wheat transformation for functional analysis and practical application[76–80].Their effects on FHB responses were mostly observed in greenhouse conditions.Large-scale field evaluations are still needed to justify their use in breeding programs[76,81].

5.The role of hormones in FHB response

The mechanisms underlying resistance to necrotrophic and hemi-biotrophic pathogens,such as F.graminearum,are complicated.Many studies demonstrated that the JA and ET signaling pathways play important roles[82–86].However,there are conflicting findings for the association of the SA signal-mediated defense pathway,the central player in resistance to biotrophic pathogens [87], with resistance to necrotrophic and hemi-biotrophic pathogens[88–92].A transcriptome analysis showed that defense pathways mediated by calcium ions,SA,and PA,as well as JA and SA,are all required for resistance responses to infection by F.graminearum[59],in accordance with many other studies[53,55,58,62,63,67,92].A time-course expression profile comparison between WSB and its susceptible mutant,of a selected set of genes related to these pathways,revealed a biphasic regulation of defense signaling.This,together with observation of a sequential increase in endogenous SA and JA contents occurring in spikes after infection,points to a hypothesized model of the early cellular events,in which the early defense reactions to F.graminearum infection start with the initiation of Ca2+and SA signaling before JA signaling[59].Plants can synthesize SA via PAL or ICS1[93,94],but only PAL was inducible by F. graminearum infection. Similarly,Arabidopsis plants synthesize SA via the PAL pathway in response to the necrotrophic pathogen Botrytis cinerea[95].Based on the altered expression profiles in the mutant,it was postulated that timely and orderly activation of the SA and JA defense pathways would be critical for them to coordinately regulate resistance[60].

6.Marker-assisted improvement of FHB resistance

In theNIL evaluations, we showed the effectiveness of individual QTL from WSB in conferring FHB resistance.To accelerate their employment in wheat production, we designed a backcross-based QTL pyramiding strategy.Starting by crossing WSB with susceptible elite lines,plants in each BCF1that carried all target QTL were selected for further backcrossing.In each generation of backcrossing selection was firstly performed for the target QTL with markers linked to them(foreground selection),and then against the donor backgrounds using markers dispersed across the whole genome(background selection).After three generations of backcross,plants homozygous for the QTL and with over 95%reconstitution of the recurrent parental genotype were selected for phenotype evaluation.QTL pyramiding was also performed by crossing different NILs.Atotal of 19 lines with different QTL combinations in the PH691 and MY backgrounds were obtained.These lines were evaluated atFengyang inAnhui province,and atJiangpu in Jiangsu in randomized complete block design trials in 2013 and 2014.The type I and type II resistance levels in PH691 were increased by over 80%by introduction of Fhb4+Fhb5 and Fhb1+Fhb2,respectively(Fig.6-A,B).Additive effects were particularly significant for Fhb4+Fhb5.Differences in disease symptoms of recurrent parents and lines with combined QTL were very clear after F. graminearum inoculation(Fig.6-C).Similar results were obtained for the MY-derived lines regardless of locations and years.The experiments have now been expanded to more elite lines from Henan,Shandong,Sichuan and Jiangsu.Preliminary results obtained so far are very promising.Hopefully,the developed lines will be promoted to production trials and used as breeding parents.

7.Perspectives

FHB has been documented in China for almost 90 years.We are still in the process of finding effective strategies to improve resistance due to the complexity of this disease.However,after about 20 years of intensive effort worldwide,basic understanding of FHB resistance genetics and dozens of resistance QTL have been achieved.The mechanisms underlying FHB resistance are starting to emerge through use of transcriptomics and proteomics.More importantly,practices in our laboratory and others have demonstrated the feasibility of breeding resistant cultivars.The use of marker-assisted selection will make the breeding process more effective and cost-efficient.Breeders can now achieve breeding goals much easier than previously.

Fig.6–Phenotypes of 13 developed lines with different combinations of QTL,compared with the susceptible control.(A)PDS 15 days after spray-inoculation for assessment of type I resistance.(B)NDS 20 days after single floret inoculation,for assessment type II resistance(2013,Jiangpu).Multiple comparisons were made by Duncan's test(P=0.01).(C)Comparison of the susceptible response of PH691 and a derived line with Fhb1,Fhb2,Fhb4 and Fhb5,20 days after single floret inoculation.

Many questions still remain.Most of the identified QTL are yet to be fully validated,the number of ready-to use QTL is limited,the resistance mechanisms are far from clear,and reasons for significant differences in genetic background effects are still unknown.Moreover,comprehensive evaluation of newly bred candidate resistant lines is still required for application in production.Since FHB resistance,agronomical and quality traits are all controlled by polygenes,selection for all traits simultaneously is still difficult.Currently,we suggest prioritizing selection of the major resistance QTL in early generations by MAS before selection ofothertraits.Genome-wide selection could be a promising approach for multiple trait improvement in the future.

Acknowledgments

We are very grateful to staff and many previous students who are not listed as co-authors but have made significant contribution to FHB research.We are also grateful to many colleagues,nationally and internationally,whose support made our work possible andmeaningful.We apologize for not citing all excellent researches on wheat FHB resistance due to limited scope of this paper.The project was mainly supported by National Key Research and Development Program (2016YFD0101802,2016YFD0101004),National Basic Research Program of China(2004CB117205,2012CB125902),National Key Technology R&D Program of China(2002AA224161),National Science and Technology Major Project(2009ZX08009-049B,2012ZX08009003),Jiangsu Collaborative Innovation Initiative for Modern Crop Production,‘111'project B08025,Natural Science Foundation of Jiangsu Province(BK20131316),Innovation Team Program for Jiangsu Universities(2014),and particularly,the long-term funding from the National Science Foundation ofChina(30025030,30430440,30721140555,31030054,30671295).

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