*

State Key Laboratory of Crop Stress Biology for Arid Areas,College of Plant Protection,Northwest A&F University,Yangling 712100,Shaanxi,China

1.Introduction

Wheat,the staple food crop for more than 50%of the world population[1],is threatened by fungal diseases.Two major groups of destructive biotrophic fungi parasitizing wheat are the rusts and powdery mildew fungi that are Basidiomycetes and Ascomycetes,respectively.The rust pathogen species are Puccinia striiformis f.sp.tritici(Pst),Puccinia graminis f.sp.tritici(Pgt),and Puccinia triticina(Pt)that cause stripe rust,stem rust and leaf rust,respectively.These Puccinia species have different sexual and asexual propagation styles[2],whereas the powdery mildew pathogen Blumeria graminis f.sp.tritici(Bgt)infects wheat primarily by means of asexual(haploid)conidiospores[3].

The rusts and powdery mildew are the most widespread damaging diseases on wheat worldwide,and cause significant losses in yield and reductions in grain quality.Much effort over many years has been made to control them.The most economic and environmentally friendly means of control is resistant varieties.However,most sources of resistance remain effective for relatively short periods of time following deployment as single genes alone in cultivated varieties.This is mainly caused by genetic changes in the pathogens.It is therefore an urgent challenge to develop wheat varieties with more durable resistance.Toward this end,it is important to understand biotrophic features and pathogenicity mechanisms of these wheat pathogens.Up to now,most knowledge about the genetics of wheat biotrophic fungi is related to the extensive studies of pathogen interactions with host resistance genes.The molecular basis underlying pathogenicity and the evolution of virulence is largely unknown.At the phenotypic level,one limitation is that these organisms cannot be grown on artificial culture media and are difficult to study in a controlled manner[4].

The rapid advance in high-throughput sequencing technology hasfacilitated genome analyses of many plant pathogens.The genomic sequences of the wheat biotrophic fungi Pst,Pgt,Pt,and Bgt have become available.Features such as loss of genes and hence enzymes involved in nitrate and sulfate assimilation and expansion in numbers of sugar and amino acid transporters are providing insights on the adaptive lifestyles of obligate biotrophic pathogens.They are also useful in understanding the dynamic evolution of plant-pathogen interaction[5,6]that goes far beyond traditional genetic analyses.This review summarizes the recent progress in genomics of wheat biotrophic pathogens.We believe that the future development and application of genomics will help in breeding wheat varieties with more durable resistances than at present and may eventually win the ever-lasting arms race between hosts and their pathogens.

2.Genome sequences provide insights into the lifestyles of wheat biotrophic pathogens

2.1.Sequencing the genomes

Developments in next generation sequencing technology(NGS)have significantly reduced sequencing costs and made available a number of genomes of wheat pathogens.Among wheat rust fungi,Pgt was first to be sequenced and found to be 88.6 Mb in size[5].Whole genome sequencing of Pt and Pst followed shortly thereafter.The first sequenced Pst genome was an isolate of US Pst race PST-130;the assembled genome size was 64.8 Mb[6](Table 1).The second Pst genome sequence was derived from an isolate of the predominant Chinese race CYR32;it was 110 Mb and had a high sequencing depth[7].A third high quality genome assembly was from an isolate of US races PST-78 and was 117.31 Mb in size[8].For Pt three draft genome sequences were reported,one from an isolate of the widely virulent race 77[9],another from an isolate of a less widely avirulent race 106[9],and the third from an isolate of race 1(BBBD),the earliest race identified in North America[8](Table 1).

Five Pgt isolates from Australia[10]and 10 Pst isolates from different countries were subsequently sequenced [7,11].Sequence analyses uncovered high genome heterozygosity of the dikaryotic rust fungi and rapid changes in genome sequences.A number of candidate avirulence genes were predicted.Transcriptome analyses of six Pt isolates were undertaken and effective secretomes were identified for 120 Pt isolates from North America and Europe following sequencing[12,13].

The reference genome of Bgt isolate 96,224 was sequenced[14];the genome size was 82 Mb(Table 1).Three additional Bgt isolates(JIW2,70,and 94,202)from England,Israel and Switzerland were also sequenced.The results indicated that the diversity in modern populations was not reduced,and the diversity of haplotypes provides ample genetic potential for adaption and variation.The genome of Bgt has thus maintained high levels of adaptability andflexibility indicating a unique evolution of this biotrophic pathogen[14].

Table 1–Genomic features of wheat biotrophic fungi.

2.2.Transposable elements(TEs)contributed to genome expansion of biotrophic pathogens

Compared to the genome size of other basidiomycete fungi,which are mostly below 50 Mb[13],the average genome size of rust fungi is much larger,often between 100 and 200 Mb[8].The genomes of rust fungi contain high amounts of repetitive DNA made up of transposable elements(TEs),which are likely responsible for the expanded genome size.Sequence analysis of a Pst race PST-130 genome showed a TE content of 17.8%whereas the corresponding content in the genome of the Chinese isolate of CYR32 contained more than 50%TEs,making the genome size of the latter almost double of that of the former[7,8].A higher repeat content was found in Pt,with an average of more than 51%[8](Table 1).The estimated TE content for the Bgt genome was even higher,up to 90%[14].TEs are regarded as an important factor in accelerating evolutionary changes in genes related to pathogenicity and survival ability[15].They also cause chromosomal rearrangements,gene inactivation and loss,and gene duplication[16].The abundant TEs in wheat biotrophic fungi may be responsible for their rapid genomic change,expansion and evolution[17].

2.3.Biotrophic features of rust and powdery mildew fungi

Wheat rust and powdery mildew fungi are strict biotrophic pathogens that complete their uredinial and conidial life cycles on living host plants.Little is known about the molecular mechanisms underlying biotrophic lifestyles.Genome analyses have already revealed extensive features associated with their obligate biotrophic lifestyles.Many specific subfamilies of transporters were present in the genomes of rust and powdery mildew pathogens.For example,the Pst genome contains 12 amino acid and 7 sugar transporter genes,including two hexose transporters that are homologous to the HXT1 gene in U.fabae(the faba bean rust pathogen)[7].In addition,there are 21 oligopeptide transporter(OPT)genes in Pgt,more than the 5 to 16 found in other basidiomycete fungi[5],suggesting increased potential for peptide uptake in rust fungi.The extended transporter families in rust fungi and up-regulated expression imply important roles in accumulation of essential nutrients.In contrast,rust fungal genomes lack genes typically present in saprotrophic basidiomycetes,such as nitrate/nitrite transporters and nitrate reductases needed in the NH4+assimilation pathway [5,7].This implies inability of nitrate assimilation and dependence on the uptake amino acids from host cells.The absence of a sulfite reductase in Pgt indicates the absence of a sulfate assimilation pathway in development of obligate biotrophy as these functions are supplied by the host[5].

2.4.Genomic information reveals an arsenal of secreted proteins

It is well known that biotrophic fungi produce many,small secreted proteins(SPs)that are likely involved in subverting host immune responses.Indeed,many species-specific genes encoding SPs were identified in each of the rust and powdery mildew genomes.A total of 2092 SPs were predicted in the Pst genome,accounting for 8.3%of the total number of predicted protein genes[7](Table 1).Pgt was estimated to produce 1459 SPs[7],and more than 660 SPs were identified in isolates of two Pt races[9](Table 1).More SPs were found in a widely virulent Pt race than in an isolate of a race with a more narrow virulence range.It is interesting that many effectors are specific to the individual pathogen species,perhaps revealing a relatively rapid evolution of these proteins.Among the three rust fungi,62%of effectors in Pst are specific to that species.Only 5%of SPs are conserved between the rust and powdery mildew fungi[18,19],indicating species-specific evolution in each pathogen.

Earlier work showed that haustoria are crucial infection structures in effector delivery and nutrient uptake[20].Transcriptome analysis of RNAs isolated from Pgt haustoria and germinated urediniospores revealed genes related to the processes of cell proliferation,including cell wall biogenesis and DNA replication that were upregulated in germinated urediniosporess[10].In Pst,up-regulated genes are related to the production of ATP and TCA indicating that haustoria are crucial for biotrophic colonization[21].For Pgt,520 secreted proteins(HSPs)were identified in the haustorial transcriptome, including 430 haustorially upregulated secreted proteins and 90 genes that were expressed in germinated spores as well as haustoria[8].Discovery and characterization of relevant effectors will be useful for identifying specific avirulence alleles whose transcripts interact with corresponding products of host resistance genes[22,23].Eventually,tests for specific secreted proteins(effectors)will replace current methods of race determination.Thus the genomics of wheat biotrophic fungi are rapidly expanding our knowledge of avirulence/virulence,pathogenicity and evolution,and may help in developing breeding strategies for producing varieties with durable resistance.

3.Progress in functional genomics of wheat biotrophic pathogens

3.1.Cloning and functional characterization of avirulence genes

Cloning of the Avr genes and uncovering the molecular mechanisms underlying recognition of Avr and R gene products are essential for developing new strategies to improve disease resistance in wheat.In barley,the powdery mildew fungus Blumeria graminis f.sp.hordei(Bgh)Avr genes Avra10 and Avrk1 were isolated by map-based cloning and found to encode unusual avirulence proteins lacking signal peptides[24].In Melampsora lini, five avirulence genes(AvrL567,AvrM,AvrP,AvrP123,and AvrP4)were identified at four loci[25–27],among which the AvrL567 and AvrM alleles were obtained by map-based cloning and the other three were obtained from a haustorial-specific cDNA-library through functionalverification [26,27].Recent completion of an improved M.lini genome,together with a high-density genetic map,facilitated cloning of Avr genes AvrL2 and AvrM14[28].In Bgt,Avr genes AvrPm3a2/f2and AvrPm2a were identified by genome-wide association analysis and map-based cloning[29,30](Table 2).Avirulence for resistance gene Pm2a was controlled by a single locus,and three alleles for AvrPm3a2/f2were cloned from a second locus that was recognized by Pm3a[29]and Pm3f[29].Transient expression of AvrPm2a and AvrPm3a2/f2in wheat plants carrying corresponding R gene alleles and co-expression of AvrPm2a-Pm2a and AvrPm3a2/f2-Pm3a/Pm3f confirmed that specific induction of HR is allele-dependent.

Among the cereal rust fungi,two avirulence effectors from Pgt have been verified.These effectors are recognized by the barley resistance protein RPG1 to activate an RPG1-mediated hypersensitive response [31](Table 2).Although few avirulence genes have been identified in wheat rust fungi,great efforts have been made.During wheat-Pt interaction,10 pairs of Australian Pt isolates that differ in avirulence/virulence to Lr20 were sequenced.Using the Pt Race 1 isolate reference genome,the genetic variation associated with pathogenicity to Lr20 was analyzed across the 20 Pt genomes.More importantly,a series of candidate effectors related to Lr20-mediated resistance were identified that were highly variable between virulent and avirulent isolates[32].The candidate avirulence effectors that could be recognized by Lr20 to trigger Lr20-specific resistance will be further dissected by functional studies.

Pathogen effectoromics takes advantage of high-throughput test systems to characterize functional Avr genes.In the oomycete Phytophthora infestans,several Avr genes(Avr-blb1,Avrblb2,Avvnt1)were identified through functional screening of the RxLR effectorome[33–35].Three M.lini Avr genes were also identified from candidate effectors derived from haustorial transciptome data[26].One approach for avirulence effector screening developed in recent years is use of the bacterial type III secretion system to deliver the fungal effectors into wheat leaf cells of R gene near-isogenic lines to test for induction of HR and defense response.Effector genomics integrated with functional screening will accelerate cloning of Avr genes and further decipher Avr-R gene product recognition mechanisms underlying the interactions of rust and powdery mildew pathogens with their host plants[32].

3.2.Mining effectors in the genomes of rust pathogens

Waves of effectors are secreted by pathogenic fungi to modulate host physiology and suppress host immunity[36].The availability of fungal genome sequences allows integration of genomics, transcriptomics and effectoromics to develop a framework for functional effector mining in closely related isolates and to correlate them to avirulence/virulence profiles.For example,comparative analyses of four sequenced genomes of US and UK Pst isolates identified sets of effector candidates related to their different avirulence/virulence profiles[11].RNA-seq and polymorphism analyses highlighted variants that were enriched in haustoria and discovered five effectors in two Pst isolates.The specific expression of these polymorphic effectors in haustoria may suggest that their allelic variants are essential for virulence/avirulence and should be functionally studied with priority.

Upadhyaya et al.[37]built a draft genome of an isolate of Australian Pgt race 21-0 by integrating the reference-genome based assembly and de novo assembly.With the new reference genome,a pan-genome was built to visualize the different effector genes in the genomes of isolates of five other Australian Pgt races and two mutation-derived isolates.Comparison of gene expression profiles in transcriptomes of germinated spores and haustoria identified 520 SPs that were specifically expressed or induced in haustoria.By pangenome analyses,25 haustorially expressed effector variants in the two mutant pairs were identified as potentially responsible for virulence to one of four resistance genes(Sr5,Sr11,Sr27,or SrSatu)[37].On the other hand,genome-wide secretome analyses of Pt reveals that the widely virulent race 77 had more virulence factor homologs than that of the avirulent race 106.The expanded effectorome in race 77 may contribute to its greater degree of adaption and virulence variation[9].The transcriptomes of six Pt isolates were sequenced and aligned to the reference genome to identify the effective secretome during host infection.Five hundred and fifty two SPs were predicted,of which 456 were present in all races.SNP analyses identified fifteen unique polymorphic SPs as candidate avirulence effectors potentially recognized by 11 different leaf rust resistance genes[12].Table 2 provides the detailed information of identified effectors and Avr genes in the wheat rust and powdery mildew pathogens.

Table 2–Effectors and Avr genes identified in wheat biotrophic fungi.

3.3.High throughput systems for functional characterization of effectors

The large number of effectors requires efficient high-throughput heterologous systems to characterize their specificity to corresponding resistance genes.For example,a modified strain EtHAn(for Effector-to-Host Analyzer)from Pseudomonas fluorescens(Pf)has been widely used to screen and characterize fungal effectors by delivering them into host plant cells[38].Using this system,Pst effectors PEC6[39]and PSTha5a23[40]were shown to inhibit wheat pattern-triggered immunity(PTI)by delivering them into wheat cells via the type three secretion system(TTSS)(Table 2).A recently developed novel effectoromics pipeline in a heterologous Nicotiana benthamiana system was developed and proved to be powerful means of assaying pathogen effectors[41].Eight M.larici-populina candidate effectors were discovered to have distinct host subcellular compartment localizations and/or were specifically associated with N.benthamiana proteins.This system was used to investigate Pst candidate effectors,with nine effectors being shown to accumulate in certain plant subcellular compartments and to be involved in specific protein complexes.Processing body(P-body)-targeted effector PST02549 possibly associated with protein complexes that were involved in mRNA processing[42](Table 2).

Direct evidence for the role of effectors in pathogenicity of rust fungi has been evasive for a long time.This was mainly due to the unavailability of knock-out mutants in rust pathogens.Recently,it was shown that small interfering RNAs(siRNAs)derived from the expressed pathogen dsRNA in plants can enter pathogen cells and silence targeted pathogen transcripts,so called host-induced gene silencing(HIGS).This phenomenon was shown to occur for fungi,oomycetes,and insects.Using this mechanism,a Barley stripe mosaic virus(BSMV)-mediated HIGS system was developed to express dsRNAs of Puccinia genes in wheat so as to silence Pst transcripts[43].BSMV-HIGS was particularly effective in silencing haustoria-specific genes[43].The two in planta induced effectors(PEC6 and PSTha5a23)were effectively silenced compromising the Pst infection[39,40].This transient silencing approach provides a potential method for functional examination of rust pathogen effectors.

4.Genomics-based population genetics and evolutionary studies

Control of the rust and powdery mildew pathogens predominantly relies on breeding and deployment of resistant wheat varieties.However,unless efforts are made to pyramid several effective resistance genes,resistance is often ephemeral due to emergence and/or rapid increase of virulent races.Thus,the extant and potential pathogen variability should be considered when deploying resistant lines to grow for a specific season.The availability of whole genome sequences of major wheat biotrophic pathogens provides opportunities to develop molecular methods for population surveillance and diagnosis.

4.1.Field pathogenomics to monitor shifts in pathogen population

Based on the released reference genome of the Pst isolate PST-130,Hubbard et al.[44]implemented a rapid“field pathogenomics”strategy to monitor Pst populations in the field.Transcriptome sequencing of Pst-infected wheat leaves collected from the field verified that only a single genotype was present in a single lesion,indicating accurate genotyping could be made on Pst isolates in the field.The entire genomes can also be sequenced directly.By sequencing 14 UK and 7 French Pst isolates collected between 1978 and 2011,four distinct lineages were identified in the UK Pst population and these appeared to relate to avirulence/virulence profiles.A study of 2013 samples revealed higher genetic diversity than present among historical UK isolates,indicating a dramatic change in population structure,which should be taken into consideration when developing rust resistant wheat varieties.Adaption analysis of genetic variation and differential gene expression permitted identification of polymorphic effector candidates that were related to avirulence/virulence profiles.Thus,field pathogenomics is a developing approach to integrate genetic analyses of evolving pathogen populations with high-resolution genotypic data that can be used in pathogen surveillance activities.The sequencing of nucleic acids from infected host tissue also provides a means of identifying the wheat variety,and the combined information on diversity of both host and pathogen populations may provide useful clues on future epidemics.High-throughput field pathogenomics,either using RNAseq-based or genomic DNA-based approaches,offers the possibility of rapid evaluation of pathogen and host populations at a much higher level of resolution than before.

4.2.Development of molecular markers for genetic population studies

Despite its efficiency and high throughput,direct sequencing is costly and requires specialized equipment.In this regard,traditional methods are still useful and efficient despite the rapid changes in pathogen genomes. SSR markers,for example,are more informative for genetic studies of diploid rust fungi,as they can detect heterozygosity of polymorphic loci.For Pst,SSR markers have been used as the primary marker type in genetic and population studies to examine and assess the roles of sexual and parasexual recombination[45–47],and to investigate population diversity[48,49]and structure[49].With the accumulation of sequenced genomes,many more SSR markers will be available.Twenty five SSR markers were developed by Bailey et al.from the genome sequence of the Pst race PST-130[50].Moreover,Luo et al.[51]identified 4792 SSR loci from the CYR32 isolate genome sequence by comparing its genome to those of five other Pst isolates,and established a database containing 1113 SSR markers.Among the many SSR markers 105 were polymorphic and 82 were sufficient to distinguish 21 Pst isolates and determine the genetic relationships among them.

The second promising molecular marker for population genetic study and molecular mapping is SNP,especially SP gene-derived SNP(SP-SNP)markers that were recently used to study the structure of a Pst population[52].Since SPs represent an important group of genes in biotrophic fungi,their SNP patterns may reflect pathogenic changes in pathogen populations.Recent work showed that SP-SNP genotypes were correlated with the pathotype data[52].SP-SNP data could provide insight into the Pst population structure and evolution in different epidemic areas,and may assist in global disease management.SP-SNP markers may also assist in cloning Avr genes.Xia et al.[52]genotyped a population of 352 Pst field isolates using 97 SP-SNPs markers and identified 30 SP-SNPs that were significantly associated with presence of 9 Pst Avr genes.Further study should identify the positions of Avr loci in the genome.It has to be emphasized that for association studies the pathogen isolates should be independent and the populations be large enough to avoid excessive numbers of false positives.

5.Conclusion and prospects

Pathogen populations co-evolve with variation in host genetic structure in agrosystems.The genome sequences of wheat biotrophic pathogens provide tools to study this process.Comparative genomic analyses are revealing the molecular bases of obligate biotrophy that may eventually help establish protocols for in vitro culture of biotrophic fungi by mimicking the nutrient absorption state.Integration of genomics,tran-scriptomics and effectoromics in study of effector distribution and selection patterns in international populations should provide insights into their adaption to local host populations.These insights will not only lead to a better understanding of the co-evolutionary process,but also will assist in developing sustainable management strategies for control of the cereal rusts and powdery mildew.

The authors were supported by the National Basic Research Program of China(2013CB127700),National Natural Science Foundation of China(31371882,31401693),and the 111 Project of the Ministry of Education of China(B07049).

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