Magnaporthe oryzae (synonym Pyricularia oryzae)

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1 Magnaporthe oryzae (synonym Pyricularia oryzae) Wheat Blast in Bangladesh, Feb Image from Dr. Paritosh Malaker, BARI Barbara Valent Dept. of Plant Pathology, Kansas State University

2 Wheat Genetics Resource Center Kansas State University Wheat in Kansas Dr. Bikram Gill On average, Kansas is the largest wheat producing state Kansas produces 3 of the six classes of wheat grown in the US Hard Red Winter (95%) Soft Red Winter (1%) Hard White (4%) Kansas Wheat Commission very active in promoting wheat research

3 Wheat Blast Research in the U.S AFRI Competitive Grant from USDA-NIFA Brazil Bolivia Paraguay Blast Integrated Project (BIP): AFRI Competitive Grant from the USDA- NIFA

4 Host-specific pathotypes of Magnaporthe oryzae (synonym Pyricularia oryzae) Ancient disease: rice blast Emerging disease: wheat blast Found in all rice growing regions ~100 major resistance (R) genes identified (>25 identified) R genes are defeated in 2 to 3 years Still often controlled by fungicides 2007 Photo: Valent 2009 Photo: von Tiedemann & Duveiller M. oryzae Oryza pathotype (MoO) M. oryzae Triticum pathotype (MoT) Identified in Brazil in 1985, spread in South America, and now South Asia One partial resistance gene: (2NS) translocation from Ae. ventricosa Control with fungicides unreliable

5 Two recently emerged diseases caused by closely related M. oryzae populations Wheat blast Gray leaf Spot M. oryzae Triticum pathotype (MoT) Identified in Brazil in 1985, spread in South America, and now South Asia One partial resistance gene: (2NS) translocation from Ae. ventricosa Control with fungicides unreliable M. oryzae Lolium pathotype (MoL) First identified in Pennsylvania in 1992 Now an important problem for golf courses in the U.S. Some MoL strains already infect wheat, although not as aggressively as MoT

6 M. oryzae, a single species with host-adapted pathotypes infecting diverse cereal crops Ryegrass Family tree based on whole genome sequencing (SNPs/Mb uniquely aligned DNA) Wheat Rice And on analysis of 2682 single copy genes 76 strains analyzed Gladieux et al mbio 2018

7 Key Questions How does M. oryzae infect grasses? What does M. oryzae genomics tell us about pathogen variability? (Effectors are mobile genes) Given experience with many rice blast R genes, why do we find so few R genes in wheat? How can we control wheat blast?

8 Presentation Outline A hemibiotrophic pathogen that colonizes living host cells New reference MoT genome and pathogen comparative genomics Progress toward finding new resistance to combine with the 2NS resistance Potential biotech strategies for controlling wheat blast

9 Magnaporthe oryzae (syn. of Pyricularia oryzae) causes ancient and emerging diseases Rice Blast Caused by M. oryzae Oryzae pathotype (MoO) Wheat Blast Caused by M. oryzae Triticum pathotype (MoT) Asexual reproduction in the field Much research on pathogenicity mechanisms ~100 major rice resistance genes known, >25 identified 13 MoO avirulence (AVR) genes cloned (11 pairs) Evidence for both sexual and asexual reproduction Pathogenicity mechanisms appear conserved So far, one resistance (the 2NS resistance) known Many cloned MoO AVR genes are conserved in MoT

10 Magnaporthe oryzae (syn. of Pyricularia oryzae) causes ancient and emerging diseases Rice Blast Caused by M. oryzae Oryzae pathotype (MoO) Wheat Blast Caused by M. oryzae Triticum pathotype (MoT) Asexual reproduction in the field Much research on pathogenicity mechanisms ~100 major rice resistance genes known, >25 identified 13 MoO avirulence (AVR) genes cloned (11 pairs) Evidence for both sexual and asexual reproduction Pathogenicity mechanisms appear conserved So far, one resistance (the 2NS resistance) known Many cloned MoO AVR genes are conserved in MoT

11 M. oryzae: a sophisticated cereal killer Conidium with Spore Tip Mucilage Glues the spore to the hydrophobic rice surface through rain or dew, which are critical for penetration. 2 μm Micrographs - Rick Howard, DuPont-Pioneer

12 Appressoria puncture the host surface Penetration Peg Appressorium 5 μm Extremely high turgor pressure (80 times atmospheric pressure) and powerful adhesives critical for penetration Rick Howard, DuPont

13 Blast is an explosive disease when the host is susceptible and environmental conditions are right The appressorium generates hydrostatic pressure at ~80 X atmospheric pressure and mechanically punches through the plant surface Appressorium function requires long periods of leaf wetness Determines susceptibility to melanin biosynthesis inhibitor fungicides that block penetration

14 Effectors are key to understanding blast disease Effectors: pathogen proteins specifically expressed and secreted in host tissue R R R R R Two opposite roles of effectors depending on host genotypes: (1) promote host susceptibility (2) trigger host immunity

15 Effectors are key to understanding blast disease Effectors: pathogen proteins specifically expressed and secreted in host tissue R R R R R Two opposite roles of effectors depending on host genotypes: (1) promote host susceptibility (2) trigger host immunity

16 The fungus invades living plant cells and secretes 100s of effectors to control the host cells and block defenses Pwl2:mRFP:NLS + Bas4:GFP Bas2:mRFP So far, at least 30 Bas effectors are translocated to the rice cytoplasm and move into surrounding neighbor cells 7 Bas effectors localized at the rice cell wall where hyphae move to neighbor cells

17 Effectors are key to understanding host susceptibility and immunity Effectors: pathogen proteins specifically expressed and secreted in host tissue R R R R R R R R Resistance Resistance Two opposite roles of effectors depending on host genotypes: (1) promote host susceptibility (2) trigger host immunity Avirulence effectors

18 Recognition of an AVR effector by a corresponding rice resistance receptor triggers resistance Breakdown of Pita genes in the field comes from mutation, mainly deletion, of AVR-Pita gene in the pathogen Pathogen genotypes AVR-Pita_ avr/avr Host genotypes Pita_ r/r S Resistance and avirulence are generally inherited as single dominant genes

19 Pathogen avirulence (AVR) effector genes are key to Magnaporthe biology: Determine rice cultivar specificity Loss of recognition mutations responsible for the rapid ability of the fungus to overcome deployed R genes Determine host species specificity

20 Loss of a single AVR/R gene interaction responsible for host jump from Lolium spp. to wheat Lolium strains with the PWT3 AVR gene don t infect wheat with the R gene Rwt3 Wheat lacking Rwt3 was planted in Paraná in the 1980s Strains with PWT3 infected wheat and subsequently lost AVR function to gain access to all wheat lines Inoue et al, Science, 2017 Yukio Tosa University of Kobe

21 Presentation Outline A hemibiotrophic pathogen that colonizes living host cells New reference MoT genome and pathogen comparative genomics Progress toward finding new resistance to combine with the 2NS resistance Potential biotech strategies for controlling wheat blast

22 MoT Near-Finished Reference Genome Bolivian strain B71 ~13,000 genes in 7 core chromosomes plus minichromosomes Effectors genes are often located at chromosome ends or on minichromosomes Sanzhen Liu Zhao Peng Mini-chromosomes serve as effector gene reservoirs. Ely Oliveira- Garcia Peng et al, biorxiv, 2018

23 Dispensable mini-chromosomes are highly variable Found in 0, 1 or 2 copies in different strains Are 50% transposons plus effector genes and other sequences from core chomosome ends

24 Genome Variability in MoT strains high relative to MoO strains

25 The wheat blast genome comprises chromosome segments inherited from other host-specialized forms Chromosome segments were acquired from strains infecting: Brachiaria Cynodon Eleusine Leersia Oryza/Setaria Paspalum Stenotaphrum Why do we need to understand MoT strain variability and evolution?

26 Recent MoT strains are more aggressive than early strains + 2NS Strain T-25 from 1988 Strain B71 from NS Cruz et al., 2016, Crop Science

27 Presentation Outline A hemibiotrophic pathogen that colonizes living host cells New reference MoT genome and pathogen comparative genomics Progress toward finding new resistance to combine with the 2NS resistance Potential biotech strategies for controlling wheat blast

28 Wheat blast disease reactions confirmed by field tests in Bolivia Christian Cruz Two test cycles per year: winter cycle in Santa Cruz wheat production areas and summer cycle in Quirusillas blast hot spot An efficient method for inoculation and irrigation allows good tests even in non-conducive weather Okinawa, Bolivia, June 26, 2016

29 Field testing in Bolivia - One inoculation of seedlings in spreader roles gives 100% head blast in susceptible varieties First blast symptoms about 7-10 days after inoculation Inoculation at tillering stage (about 30 days after planting)

30 o Screening for non-2ns resistance Combined data from USDA-ARS, KSU-BRI, ANAPO, Biotrigo and under field conditions (ANAPO, CIAT-Bolivia) No variety is immune to MoT o The 2NS translocation still is the major source of resistance o Potential new sources of resistance: o Mapping populations with highly resistant 2NS cultivars with potential other resistance o Aegilops tauschii o Emmer lines o Parents in the Akhunov NAM population o CIMMYT lines o And more Giovana Cruppe et al. in preparation

31 Screening for resistance in wild wheat (Ae. tauschii) Giovana Cruppe et al. in preparation

32 Biotech strategies for resistance Testing rice R genes in wheat (Harold Trick et al.) Using CRISPR-CAS to knock-out wheat susceptibility genes HIGs Now testing in Rice (Ralph Dean and Jin-rong Xu et al.)

33 Similar Symptoms Different Strategies Magnaporthe oryzae No mycotoxins known Not applicable Pressure-based penetration through outer plant surfaces Hijacks and grows in living plant cells using 100s of effector proteins Resistance (R) gene receptors recognize AVR effectors and trigger death of the plant cell, blocking the fungus Sexual cycle not important for disease Abundant transposons, has mini-chromosomes with effectors and transposons Fusarium graminearum Mycotoxin-contaminated grain a major health threat Some mycotoxins are important for infection Enters wheat head through anthers Grows outside host cells and invades them after they are dead No classic R genes; major QTLs Fusarium species independent Sexual cycle required for disease No transposons, no duplicated genes, no mini-chromosomes

34 Mixed infection: Brusone and scab in wheat fields near Londrina, Paraná, 2009 Can HIGS work to control both FHB and Brusone at the same time? Andreas von Tiedemann & Etienne Duveiller

35 Blast Integrated Project: Novel Strategies for Managing Blast Diseases on Rice and Wheat Kansas State University Christian Cruz William Bockus Erick De Wolf (Taylor Fischer) Sunghun Park Jim Stack Harold Trick Barbara Valent (Ely Oliveira-Garcia) USDA/ARS/FDWSRU, Fort Detrick, MD Gary L. Peterson Kerry F. Pedley The Ohio State University Pierce Paul (Karasi Mills) Laurence Madden Thomas Mitchell Guo-Liang Wang University of Kentucky Mark Farman EMBRAPA Wheat, Brazil José Mauricio Fernandes João L. Nunes Maciel ANAPO, Bolivia Diego Baldelomar CAPECO/INBIO, Paraguay Man Mohan Kohli Dale Bumpers National Rice Research Institute Yulin Jia University of Arkansas Yeshi Wamishe Lanier Nalley Purdue University Jin-Rong Xu North Carolina State University Ralph Dean The Pennsylvania State University Yinong Yang

36 Blast Integrated Project Team 2013 Manhattan, KS 2014 St. Louis, MO 2015 St. Louis, MO 2016 Ft. Detrick, MD