High-throughput RNA sequencing reveals differences between the transcriptomes of the five spore forms of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen

Abstract

The devastating wheat stripe (yellow) rust pathogen, Puccinia striiformis f. sp. tritici (Pst), is a macrocyclic and heteroecious fungus. Pst produces urediniospores and teliospores on its primary host, wheat, and pycniospores and aeciospores are produced on its alternate hosts, barberry (Berberis spp.) or mahonia (Mahonia spp.). Basidiospores are developed from teliospores and infect alternate hosts. These five spore forms play distinct roles in Pst infection, disease development, and fungal survival, etc. However, the specific genes and mechanisms underlying these functional differences are largely unknown. In this study, we performed, for the first time in rust fungi, the deep RNA sequencing to examine the transcriptomic shift among all five Pst spore forms. Among a total of 29,591 identified transcripts, 951 were specifically expressed in basidiospores, whereas 920, 761, 266, and 110 were specific for teliospores, pycniospores, aeciospores, and urediniospores, respectively. Additionally, transcriptomes of sexual spores, namely pycniospores and basidiospores, showed significant differences from those of asexual spores (urediniospores, teliospores, and aeciospores), and transcriptomes of urediniospores and aeciospores were more similar to each other than to the three other spore forms. Especially, the basidiospores and pycniospores which infected the berberis shows wide differences in the cell wall degrading-enzymes and mating and pheromone response genes. Besides, we also found that there are 6234 differential expressed genes between the urediniospores and pycniospores, while only have 3 genes have alternative splicing enents, suggesting that differential genes expression may make more contribution than AS. This comprehensive transcriptome profiling can substantially improve our understanding of the developmental biology of the wheat stripe rust fungus.

Keywords

Puccinia striiformis f. sp. tritici / Transcriptome / Gene expression / Rust life cycle / Spore stages

References

[1] AndersS, PylPT, HuberW. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics, 2015, 31: 166-169

[2] AniksterY, WahlI. Coevolution of the rust fungi on Gramineae and Liliaceae and their hosts. Annu Rev Phytopathol, 1979, 17: 367-403

[3] CantuD, SegoviaV, MacleanD, BaylesR, ChenX, KamounS, DubcovskyJ, SaundersDG, UauyC. Genome analyses of the wheat yellow (stripe) rust pathogen Puccinia striiformis f. sp. tritici reveal polymorphic and haustorial expressed secreted proteins as candidate effectors. BMC Genomics, 2013, 14: 270

[4] CarreiraRA, BhargavaV, HillebrandJ, KolliparaRK, RamaswamiM, BuszczakM. Repression of Pumilio protein expression by Rbfox1 promotes germ cell differentiation. Dev Cell, 2016, 36(5):562-571

[5] CesariE, LoiarroM, NaroC, PieraccioliM, FariniD, PellegriniL, PagliariniV, BielliP, SetteC. Combinatorial control of Spo11 alternative splicing by modulation of RNA polymerase II dynamics and splicing factor recruitment during meiosis. Cell Death Dis, 2020, 11(4):240

[6] ChangEC, BartholomeuszG, PimentalR, ChenJ, LaiH, WangLHL, YangP, MarcusS. Direct binding and in vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein Scd2. Mol Cell Biol, 1999, 19: 8066-8074

[7] ChenXM. Pathogens which threaten food security: Puccinia striiformis, the wheat stripe rust pathogen. Food Sec, 2020, 12(2):239-251

[8] ChenC, ChenH, ZhangY, HannahRT, MargaretHF, HeY, XiaR. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant, 2020, 13(8):1194-1202

[9] DobonA, BuntingDC, Cabrera-QuioLE, UauyC, SaundersDG. The host-pathogen interaction between wheat and yellow rust induces temporally coordinated waves of gene expression. BMC Genomics, 2016, 17: 380

[10] DuplessisS, LorrainC, PetreB, FigueroaM, DoddsPN, AimeMC. Host adaptation and virulence in heteroecious rust fungi. Annu Rev Phytopathol, 2021, 25(59):403-422

[11] FuC, HeitmanJ. PRM1 and KAR5 function in cell-cell fusion and karyogamy to drive distinct bisexual and unisexual cycles in the Cryptococcus pathogenic species complex. PLoS Genet, 2017, 13: e1007113

[12] GarnicaDP, UpadhyayaNM, DoddsPN, RathjenJP. Strategies for wheat stripe rust pathogenicity identified by transcriptome sequencing. PLoS One, 2013, 8: e67150

[13] Gómez-RedondoI, PlanellsB, NavarreteP, Gutiérrez-AdánA. Role of alternative splicing in sex determination in vertebrates. Sex Dev, 2021, 15(5–6):381-391

[14] HacquardS, DelaruelleC, FreyP, TisserantE, KohlerA, DuplessisS. Transcriptome analysis of poplar rust telia reveals overwintering adaptation and tightly coordinated karyogamy and meiosis processes. Front Plant Sci, 2013, 4: 456

[15] HennessyCMR, SackstonWE. Inheritance of spore colour in Puccinia helianthi. Can J Genet Cytol, 1972, 14: 271-278

[16] HuangXL, ChenXM, CoramT, WangMN, KangZS. Gene expression profiling of Puccinia striiformis f. sp. tritici during development reveals a highly dynamic transcriptome. J Genet Genom, 2011, 38: 357-371

[17] JinY, SzaboLJ, CarsonM. Century-old mystery of Puccinia striiformis life history solved with the identification of Berberis as an alternate host. Phytopathology, 2010, 100: 432-435

[18] KimMS, PintoSM, GetnetD, NirujogiRS, MandaSS, ChaerkadyR, MadugunduAK, KelkarDS, IsserlinR, ShobhitJ. A draft map of the human proteome. Nature, 2014, 509(7502):575-581

[19] KimD, LangmeadB, SalzbergSL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods, 2015, 12: 357-360

[20] LingP, WangMN, ChenXM, CampbellKG. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici). BMC Genomics, 2007, 8: 145-158

[21] LorrainC, MarchalC, HacquardS, DelaruelleC, PetrowskiJ, PetreB, HeckerA, FreyP, DuplessisS. The rust fungus Melampsora larici-populina expresses a conserved genetic program and distinct sets of secreted protein genes during infection of its two host plants, larch and poplar. Mol Plant-Microbe Interact, 2017, 31: 695-706

[22] MerliniL, DudinO, MartinSG. Mate and fuse: how yeast cells do it. Open Biol, 2013, 3: 130008

[23] NiuH, LiX, JobE, ParkC, MoazedD, GygiSP, HollingsworthNM. Mek1 kinase is regulated to suppress double-strand break repair between sister chromatids during budding yeast meiosis. Mol Cell Biol, 2007, 27: 5456-5467

[24] OlmoVN, GroteE. Prm1 targeting to contact sites enhances fusion during mating in Saccharomyces cerevisiae. Eukaryot Cell, 2010, 9: 1538-1548

[25] PengH, LiuY, XiaMH, BaiXX, GuoJ, HuangLL, KangZS, ZhanGM. Sexual selfing and genetic analysis of virulent and avirulent genes of isolate CYR29 of Puccinia striiformis f. sp. tritici. Mycosystema, 2021, 40(4):759-769 in Chinese

[26] RaudaskoskiM, KotheE. Basidiomycete mating type genes and pheromone signaling. Eukaryot Cell, 2010, 9: 847-859

[27] Roelfs AP (1982) Effects of barberry eradication on stem rust in the United States. Plant Dis 66(2):177–181. https://doi.org/10.1094/PD-66-177

[28] RogersJV, RoseMD. Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae. G3: Genes. Genome, Genet, 2015, 5: 111-121

[29] Schafer W (1994) Molecular mechanisms of fungal pathogenicity to plants. Annu Rev Phytopathol 32:461–477. https://doi.org/10.1146/annurev.py.32.090194.002333

[30] Shen SH, Park JW, Lu ZX, Lin L, Henry MD, Wu YN, Zhou Q, Xing Y (2014) rMATS: Robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. PNAS 111:e5593–5601. https://doi.org/10.1073/pnas.1419161111

[31] TangheA, Van DijckP, DumortierF, TeunissenA, HohmannS, TheveleinJM. Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains. Appl Environ Microbiol, 2002, 68: 5981-5989

[32] TangheA, Van DijckP, TheveleinJM. Why do microorganisms have aquaporins?. Trends Microbiol, 2006, 14: 78-85

[33] TaricaniL, FeilotterHE, WeaverC, YoungPG. Expression of hsp16 in response to nucleotide depletion is regulated via the spc1 MAPK pathway in Schizosaccharomyces pombe. Nucl Acids Res, 2001, 29: 3030-3040

[34] TianY, ZhanGM, ChenXM, TungruentragoonA, LuX, HuangZJ, LL, Kang ZS, . Virulence and simple sequence repeat marker segregation in a Puccinia striiformis f. sp. tritici population produced by selfing a Chinese isolate on Berberis shensiana. Phytopathology, 2016, 106: 185-191

[35] TianY, ZhanGM, LuX, ZhaoJ, HuangLL, KangZS. Determination of heterozygosity for avirulence/virulence loci through sexual hybridization of Puccinia striiformis f. sp. tritici. Front Agric Sci Eng, 2017, 4: 48-58

[36] WangMN, ChenXM. First report of Oregon grape (Mahonia aquifolium) as an alternate host for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) under artificial inoculation. Plant Dis, 2013, 97: 839-839

[37] WangLK, FengZX, WangX, WangXW, ZhangXG. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics, 2010, 26: 136-138

[38] WangL, ZhengD, ZuoSX, ChenXM, ZhuangH, HuangLL, KangZS, ZhaoJ. Inheritance and linkage of virulence genes in Chinese predominant race CYR32 of the wheat stripe rust pathogen Puccinia striiformis f. sp. tritici. Front Plant Sci, 2018, 9: 120

[39] XiaCJ, WangMN, CornejoOE, JiwanDA, SeeDR, ChenXM. Secretome characterization and correlation analysis reveals putative pathogenicity mechanisms and identify candidate avirulence genes in the wheat stripe rust fungus Puccinia striiformis f. sp. tritici. Front Microbiol, 2017, 8: 2394

[40] XiaCJ, WangMN, YinCT, CornejoOE, HulbertSH, ChenXM. Genome sequence resources for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) and the barley stripe rust pathogen (Puccinia striiformis f. sp. hordei). Mol Plant-Microbe Interact, 2018, 31: 1117-1120

[41] XiaCJ, WangMN, YinCT, CornejoOE, HulbertSH, ChenXM. Genomic insights into host adaptation between the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) and the barley stripe rust pathogen (Puccinia striiformis f. sp. hordei). BMC Genomics, 2018, 19: 664

[42] XiaCJ, LeiY, WangMN, ChenWQ, ChenXM. An avirulence gene cluster in the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) identified through genetic mapping and whole-genome sequencing of a sexual population. mSphere., 2020, 5(3):e00128-20

[43] XiaCJ, QiuA, WangMN, LiuT, ChenWQ, ChenXM. Current status and future perspectives of genomics research in the rust fungi. Int J Mol Sci, 2022, 23(17):9629

[44] XuJH, LinningR, FellersJ, DickinsonM, ZhuWH, AntonovI, JolyDL, DonaldsonME, EilamT, AniksterY. Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi. BMC Genomics, 2011, 12: 161

[45] YinCT, ChenXM, WangXJ, HanQM, KangZS, HulbertS. Generation and analysis of expression sequence tags from haustoria of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici. BMC Genomics, 2009, 10: 626

[46] Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Geneontology analysis for RNA-seq: Accounting for selection bias. Genome Biol 11:R14. https://doi.org/10.1186/gb-2010-11-2-r14

[47] YuLP, ZhangHR, GuanXB, QinDD, ZhouJ, WuX. Loss of ESRP1 blocks mouse oocyte development and leads to female infertility. Development, 2021, 148(2):dev196931

[48] ZhaoJ, KangZS. Fighting wheat rusts in China: a look back and into the future. Phytopathology Res, 2023, 5(1):6

[49] ZhaoJ, WangL, WangZY, ChenXM, ZhangHC, YaoJN, ZhanGM, ChenW, HuangLL, KangZS. Identification of eighteen Berberis species as alternate hosts of Puccinia striiformis f. sp. tritici and virulence variation in the pathogen isolates from natural infection of barberry plants in China. Phytopathology, 2013, 103: 927-934

[50] ZhaoJ, WangMN, ChenXM, KangZS. Role of alternate hosts in epidemiology and pathogen variation of cereal rusts. Annu Rev Phytopathol, 2016, 54: 207-228

[51] ZhengWM, HuangLL, HuangJQ, WangXJ, ChenXM, ZhaoJ, GuoJ, ZhuangH, QiuCZ, LiuJ, LiuHQ, HuangXL, PeiGL, ZhanGM, TangCL, ChengYL, LiuMJ, ZhangJS, ZhaoZT, ZhangSJ, HanQM, HanDJ, ZhangHC, ZhaoJ, GaoXN, WangJF, NiPX, DongW, YangLF, YangHM, XuJR, ZhangGY, KangZS. High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus. Nat Commun, 2013, 4: 2673

Funding

National Key Research and Development Program of China(2021YFD1401000); National Natural Science Foundation of China(32102175); the 111 Project of the Ministry of Education of China(B0719026); the Open Project Program of State Key Laboratory of Crop Stress Biology for Arid Areas(CSBAAKF2021013)