Genome Dynamics of a Cereal Killer: Strategies for Bangladeshi fields with 100% loss. Controlling Ancient and Emerging Crop Diseases

Magnaporthe oryzae causes: Ancient disease: rice blast Emerging disease: wheat blast

Ministry of Agriculture ordered infected fields burned

2007 Photo: Valent 2009 Photo: von Tiedemann & Duveiller Rice pathotype Wheat pathotype

Barbara Valent Dept. of Plant Pathology, Kansas State University Nature 532, 421-422 (2016)

Plant Diseases Threaten our Food Wheat blast caused by the fungus Magnaporthe oryzae

Potato Late Blight – Phytophthora infestans Banana Fusarium Wilt – F. oxysporum cubense Triticum pathotype

Hard to control: One spore can kill the entire wheat head. One useful resistance gene gives partial head blast control. Irish Potato Famine New Race TR4 Favorable environment Wheat Stem Rust – Puccinia graminis tritici Rice Blast – Magnaporthe oryzae oryza overwhelms resistance plus fungicides. Seed-borne fungus with potential huge impact on global grain trade.

New Race UG99 Super Hybrid Rice in China Warm rainy weather at heading results in 100% empty heads.

Wheat blast Presentation Outline

Wheat blast identified in Brazil in 1985; spread to other countries in South America; jumped to Bangladesh in 2016. • M. oryzae is highly evolved for pathogenicity on grasses Effectors: proteins specifically expressed and secreted in the host

Spore Appressorium Biotrophic Interfacial Complex

Brazil, 2012 Invasive Hyphae

Cytoplasmic Effectors • Evolutionary Bangladesh, 2016 Paritosh Malaker – Genome dynamics with supernumerary mini-chromosomes CIMMYT – Mobile EFFECTOR genes impact blast disease control Wheat blast affected ~15% of total wheat area in Bangladesh in the first year

1 Presentation Outline Effectors are generally small unique proteins only expressed during plant invasion

• M. oryzae is highly evolved for pathogenicity on grasses Avirulence (AVR) Effectors trigger resistance Resistance Gene Products: NBS-LRR Resistance Receptors Root Clade

Blast Clade

Wood Clade

Other Fungi AVR Effectors • Evolutionary genomics – Genome dynamics with supernumerary mini-chromosomes – Mobile EFFECTOR genes impact blast disease control Zhang et al., 2018, Sci. Rep.

The blast fungus needs to invade living host cells so M. oryzae: a sophisticated cereal killer hypersensitive host-cell death blocks infection Susceptible interaction - rice without resistance gene Pita Conidium with Spore Tip Mucilage Glues the spore to the hydrophobic rice Fungus with Avirulence surface through rain or dew, which are Effector AVR-Pita critical for penetration.

Hypersensitive resistance on rice with resistance gene Pita

Fungus with Avirulence Effector AVR-Pita

2 μm Micrographs - Rick Howard, DuPont-Pioneer

Appressoria generate high pressure to puncture the host Blast Clade diverged ca. 21 million years ago surface and enter the plant

Fungal Order Magnaporthales Saprobe on submerged wood

Leaf and Seed Head Penetration Peg 24 mya • APPRESSORIA to penetrate cuticle • 21 mya Hundreds of small Appressorium 5 μm unique EFFECTOR proteins to hijack plant processes Extremely high turgor pressure (80 times atmospheric pressure) Root Pathogens and powerful adhesives critical for penetration Zhang et al., 2018, Sci. Rep. Rick Howard, DuPont

2 At Least 100 Pathogenicity Genes Characterized: Many Effectors are key to understanding blast disease impact appressorium differentiation and function 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 / Resistance Video from Nick Talbot, Sainsbury Lab Kershaw et al, , 2019

At Least 100 Pathogenicity Genes Characterized: Many After penetration: the blast fungus delivers 100’s of impact appressorium differentiation and function ‘cytoplasmic’ effectors inside plant cells

Critical for infection: • Hyphal Differentiation • Co-opting Plant Processes

Red Rings: Cytoplasmic effectors Green lines: (Extracellular effectors) Repeated in each newly invaded cell. Video from Nick Talbot, Sainsbury Lab Kershaw et al, Genetics, 2019 Blue: Rice Cytoplasm

Fungal invasive hyphae are enclosed in a plant-derived Effectors that Accumulate in BICs are Translocated into Extra-Invasive Hyphal Membrane (EIHM) the Rice Cytoplasm

Rice Nucleus Merge FM4-64

C.H.Khang

Green – Cytoplasmic EYFP FM4-64 channel shows • EXTRACELLULAR EFFECTOR BAS4:GFP (green) is secreted by the Red – FM4-64 EIHM in this black and Yellow – co-localization white inverse image fungus and retained within host-derived EIHM • CYTOPLASMIC EFFECTOR PWL2:mCherry (red) is secreted into the Live Cell Imaging of the fungus invading rice leaf sheath cells BIC and translocated into the rice cell Kankanala et al., Plant Cell 2007

3 How are cytoplasmic effectors secreted into BICS? Summary: Hundreds of Pathogenicity Genes and Effectors Contribute to Disease

Extracellular effectors PWL2 Cytoplasmic • Mutation of pathogenicity genes shows an impact on disease effectors BAS1 development – Use of Host Induced Gene Silencing of PTH genes to control disease?

Extracellular • Mutation of effector genes in laboratory studies rarely impacts effectors: secreted disease development, suggesting functional redundancy for by conventional, effectors golgi-dependent secretion Cytoplasmic effectors: targeted Therefore, the fungus can lose an individual effector to BICs by a nonconventional gene and still cause disease. secretion system that bypasses the Golgi

How do cytoplasmic effectors cross the rice plasma membrane? Presentation Outline

• M. oryzae is highly evolved for pathogenicity on grasses Avirulence (AVR) Effectors trigger resistance Resistance Gene Products: NBS-LRR Resistance Receptors

PWL2:mRFP BAS4:eGFP

Effector Co-localization Studies Co-localization with Inhibition of plant AVR Effectors fluorescent plant endocytosis by VIGS and components (plasma chemicals • Evolutionary genomics By co-opting host membrane) clathrin-mediated – Genome dynamics with supernumerary mini-chromosomes endocytosis – Mobile EFFECTOR genes impact blast disease control Ely Oliveira-Garcia

Where do cytoplasmic effectors go and what do they do? Avirulence (AVR) effectors trigger resistance

R R R R R R R R Resistance Resistance

• 13 AVR genes cloned (11 correspond to identified rice R genes) nucleus • >100 major R genes identified in rice; many more AVR genes Pwl2:mCherry:NLS (red) Red fluorescent cytoplasmic effector remain to be identified Bas4:EGFP (green) PWL2 - black and white inverse image • The rice blast fungus is able to overcome/break individual Many cytoplasmic effectors move through resistance genes within 2 to 3 years of their deployment by host plasmodesmata into neighboring cells mutation/loss of effectors Khang et al., Plant Cell (2010)

4 Recognition of an AVR effector by a corresponding rice Host Jump to Wheat – Loss of an AVR/R Gene Interaction resistance receptor triggers resistance PWT3

“Breakdown” of R genes in the field comes • Lolium strains with the from mutation, mainly deletion, of the PWT3 AVR gene don’t corresponding AVR gene in the pathogen Wide cultivation of infect wheat with the R rwt3 wheat in Brazil Host genotypes gene Rwt3. in the 1980s Rwt3 Wheat rwt3 Wheat Pita_ r/r • This model implies that Population of fungus with PWT3 rwt3 cultivars served as increases Pathogen springboards for the host AVR-Pita_ S Emergence of mutant jump of fungal strains genotypes fungus (Infects the entire with PWT3 in Brazil. wheat population avr/avr

Rwt3 Wheat rwt3 Wheat Resistance and avirulence are generally inherited as single dominant genes Yukio Tosa Kobe University Inoue et al., Science, 2017

Telomere-to-telomere genome assembly for Pathogen AVR effector genes are key to M. oryzae biology aggressive wheat blast strain B71

Scaf. 1-5 • M. oryzae is a haploid Ascomycete with ~40 Mb 1 • Determine rice cultivar specificity (e.g. AVR-Pita1) 7 genome with ~13,000 genes • Wheat pathogen B71 has 7 core chromosomes that are highly • Highly mutable AVR genes (frequencies of 10-3, conserved relative to rice pathogens often by gene deletion) responsible for the rapid 6 2 • Scaffolds 1-5 are not anchored ability of the fungus to overcome deployed R to chromosomes 1 – 7 in the rice genes reference genome

5 • Determine host species specificity 3 4 Sanzhen Liu Bolivian strain B71 isolated in 2012 is closely related to the Bangladeshi strains Peng et al, bioRxiv, 2018

M. oryzae, a single species with crop-adapted pathotypes M. oryzae dispensable mini-chromosomes identified in the 1990s

S. cerevisiae 1992 chrom. sizes Eleusine pth. (Finger Millet ) 1,640 – • Core chromosomes are >5 Mb 2,500 kb in size

Lolium pth. • Many strains contain mini- (Ryegrass) 980 kb chromosomes generally <3 Mb Setaria pth. in size (Foxtail Millet ) • CHEF Gel 240 kb Found in 0, 1, 2 or more copies in different strains

Triticum pth. OryzaRice pth. • Mini-chromosomes don’t (Wheat) (Rice) ~7000 segregate normally in meiosis and can be missing in asexual 1985 years ago spores Family tree based on whole genome sequencing (SNPs/Mb uniquely aligned DNA) Gladieux et al. mBio 2018 Southern Blot Orbach et al. Mol. Plant-Microbe Interact., 1996

5 All five B71 scaffolds are from a mini-chromosome Illumina sequencing of eight additional strains

Pathogen Strain

B71mini Mini 2.0 Mb

Name Year isolated Host species Isolation location 1988 T25 1988 Triticum Paraná, Brazil Py5020 2005 Triticum Paraná, Brazil • Mini DNA recovered from gels Py22.1 2007 Triticum Paraná, Brazil • Illumina sequencing B2 2011 Triticum Quirusillas, Bolivia 2012 B71 2012 Triticum Okinawa, Bolivia • Alignment to the B71 genome 2012 P3 2012 Triticum Canindeyú,Paraguay • Genomic coverage and depth of P28 2014 Bromus Paraguay uniquely mapped reads P29 2014 Bromus Paraguay B51 2012 Eleusine Quirusillas, Bolivia

Two known effector genes, PWL2 and BAS1, are adjacently CHEF gels confirm prediction that T25 has no mini, localized on the B71 mini-chromosome B71 has one and P3 has two

T25 vs B71 P3 vs B71 Strain PWL2 BAS1

RNA Seq Reads

• PWL2 and BAS1 are located on different core chromosomes of rice

isolates perMillion • PWL2 and BAS1 are ONLY located on

the B71 mini-chromosome Reads Reads • PWL2 and BAS1 on minis are specifically expressed in planta Read depths to infer copy number variation red: two copies relative to B71 green: conserved blue: no copies relative to B71

Sequences in the B71 mini-chromosome Different minis carry distinct sequences Strains ~390 kb • B71 mini-chromosome i-chr min 0 1

2 C 4 HR 3 1 contains large duplications 7 3 R H 4 C 2 5 1 from core chromosome 6 large 0 6 0 ends 1 5 P3 mini P3 mini 2 4 small 6 large small R 3 • Transposable element (TE) H 3 C C

H

4 R

2 2 content is 53.4% in the 5 1 B71 mini-chromosome, 6 0 7 compared to 9.8% in the 4

3 0 core chromosome

C H R 2 5 1 Contains PWL2, Contains AVR-Pik, and 5 1 2 • TE’s in Mini do not show 0 3 BAS1, and AVR-Pib novel putative effector genes

5 4 R3 5 H 4 C signs of “Repeat-Induced 6 3 7

2 8 C 0 H 1 R4 Point Mutation” genome Minis acquired various sequences from defense mechanism core chromosomes, particularly core ends

6 Most over-represented transposons in minis are High mobility of the AVR-Pita gene appears involved in rapid clustered at core ends pathogen breakdown of rice R gene Pita

core chromosomes Fungus has AVR gene Fungus loses AVR gene mini vs. core

core mini ends enriched enriched TEs TEs

Fungus regains AVR gene via Horizontal Transfer Working Hypothesis: Mini-chromosomes serve as effector gene reservoirs and as mediators of horizontal transfer Similarity offers the potential for ectopic recombination, providing a possible through the fungal parasexual cycle mechanism for how minis acquire sequences from core chromosomal ends. Tosa et al., 2014, J. Gen. Plant Pathol.

Mini acquires effector genes

Loss of mini can have a low fitness cost.

Mini seems to prefer exchange with core ends, facilitating translocation. Like rice pathogens, wheat blast effector genes tend to be What is Needed to Control Blast Diseases? located at the ends of core chromosomes

Rice Blast • We have >100 R genes but the fungus rapidly overcomes • Localization of the individual genes deployed in rice PWL2 host species- specificity gene on • High diversity in local pathogen populations complicates minis contributes to regional control growing evidence Local pathogen population – informed deployment that it is a mobile of sets of complementary R genes. effector gene. Wheat Blast • This implicates minis • A few R genes identified using early strains from the 1980s and in host jumps as well 1990s not effective against current populations as resistance breakdown in crops • Only one effective head blast R gene, the ‘2NS gene’, confers partial resistance that is background dependent

We need to identify effective R genes. core ends

The mobile AVR-Pita gene is on chromosomes 1, 3, 4, 5, 6, 7 or on Acknowledgements dispensible mini-chromosomes in different strains Blast Biology Wheat Blast Genomics Chr. 4 Kansas State University Kansas State University of Florida Chr. 4 Ely Oliveira Garcia* University Frank F. White Melinda Dalby Sanzhen Liu Zhao Peng Chr. 4 Many Past Lab Members David Cook Mini University of Kentucky Ely Oliveira Garcia* Mark Farman Chr. 3 Guifang Lin Chr. 6 Nick Talbot Ying Hu Fujian Agriculture and Forestry University Mini Melinda Dalby Kobe University Haibao Tang Chr. 5 Yukio Tosa *Currently, Assistant Professor, Louisiana State Univ. Chr. 6 BLAST INTEGRATED PROJECT (2013-68004-20378) The avr-Pita3 gene (not recognized by Pita) is Telomere Repeat Sequences Plant-Biotic Interactions (2017-67013-26525) stably located on chromosome 7 in all strains. Chuma et al, 2011, PLoS Pathogens

7 Effector Art by Kirk Czymmek

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