International Collaboration, Open Science and Open Data Sharing to Manage Worrisome Wheat Blast in South Asia by Genomics and Genome Editing

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1 Department of Biotechnology Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Bangladesh International Collaboration, Open Science and Open Data Sharing to Manage Worrisome Wheat Blast in South Asia by Genomics and Genome Editing Approaches Tofazzal Islam yahoo.com 6 th Annual South Asia Biosafety Conference September 2018, Westin Hotel, Dhaka

2 Contents Wheat blast: a killer of wheat in Bangladesh and beyond; Our story of open data sharing and open science for rapid determination of the origin first outbreak of wheat blast in Bangladesh; Novel approaches to tackle the dangerous enemy of our food security through international collaboration.

3 Emergence of fungal diseases are increasing (Fisher et al. 2012) Despite modern agricultural practices, diseases of the major food crops cause up to 15% pre-harvest yield loss. Fisher et al. 2012, Nature 484:

4 Emerging fungal diseases and food security World population is projected to reach around 9.7 billion by To keep up with the pace of population growth, food production will need to increase by % by 2050 for a well-fed world population Emerging fungal diseases are increasingly recognized as presenting a worldwide threat to food security. In the nineteenth century, late blight led to starvation, economic ruin and the downfall of the English government during the Irish potato famine. In the twentieth century, Dutch elm blight and chestnut blight laid bare urban and forest landscapes Trade and transport promotes globalization of fungi and thus creating new opportunities for evolution. Late blight of potato Phytophthora infestans Rice blast Magnaporthe oryzae Oryzae Wheat rust (Ug99) Puccinia graminis f. tritici

5 Hymenoscyphus fraxineus, the fungus which causes Chalara dieback of ash, is treated as a quarantine organism under national emergency measures.

6 The news report, which first drew our attention to wheat blast in Bangladesh

7 Symptoms of fearsome wheat blast in Meherpur in February 2016 which caused up to 100% yield loss!

8 Clearing pathogen by burning of blast affected wheat field by the decision of Ministry of Agriculture of Bangladesh (video clip at

9 World wheat production. (A) World wheat production by continents. Average values during were used for the graph. (B) Top ten wheat producing countries. All data were obtained from FAOSTAT (FAOSTAT, 2016)

10 Importance of wheat as food crops Wheat is a critical staple providing 20% of the calories and over 25% of the protein consumed by humans (FAO, In Bangladesh, wheat is the second important cereal crop after rice Production: 1.35 m t from m ha at 3.1 t/ha Present demand of wheat is approx. 7 m t Demand is increasing every year with change in dietary habit and population increase

11 Wheat blast Wheat blast is a fungal disease caused by a filamentous fungus, Magnaporthe oryzae (anamorph Pyricularia oryzae) Emerged for the first time in Paraná state of Brazil in 1985 and then spread in neighboring countries, Bolivia, Argentina, and Paraguay. Yield loss can be up to 100%

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14 WHO WE ARE? Wheat blast research is a collaborative efforts of researchers from- - Bangabandhu Sheikh Mujibur Rahman Agricultural University led by Tofazzal Islam -The Sainsbury Laboratory led by Sophien Kamoun, and - The Genome Analysis Centre (TGAC) led by Diane Saunders Tofazzal Sophien Diane

15 Hypothesis Wheat blast in Bangladesh may be caused by a native blast fungus from rice or other grasses through host jump (mutation). Or, Wheat blast in Bangladesh may be imported from South America through seeds. Approach Field pathogenomics approach for determination of genetic identity and origin of wheat blast in Bangladesh

16 Field pathogenomics Field pathogenomics is new method to analyze fungal diseases from field samples and pinpoint the exact genotype in a faster way. Current techniques rely on time-consuming phenotypic characterization checking the response of different plant varieties to infection by the pathogen or costly in-lab processes. These methods can only sample a relatively small proportion of the fungal population.

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18 Collection of sample Extraction of mrna

19 Flow chart of Field Pathogenomics Approach

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24 OPEN WHEAT BLAST news in weekly Science and Nature The Open Wheat Blast website might help. Rapid data sharing is becoming more common in health emergencies, such as the outbreak of Zika virus in the Americas. The plant-pathology community has a responsibility to allow data to be used to combat diseases that are happening now, and not worry too much about whether they may or may not get a Nature paper out of it, says Talbot.

25 Enemy of wheat blast detected a news report published in number 1 highly Circulated national daily the Prothom Alo on April 20, 2016

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27 Pierre Gladieux et al from INRA, France PHYLOGENOMIC ANALYSES REVEAL THAT THE 2016 OUTBREAK OF WHEAT BLAST IN BANGLADESH IS CAUSED BY A SOUTH AMERICAN WHEAT-INFECTING LINEAGE OF MAGNAPORTHE ORYZAE

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30 - More than 15,000 ha of wheat crops in 8 districts devastated by wheat blast. - Yield loss was up to 100% -Rainfall coupled with higher temperature during flowering or heading stage in February 2016 were conducive to disease. - Late sowing of wheat facilitates infestation Geographical distribution and severity of the wheat blast outbreak in eight southwestern districts of Bangladesh. The map depicts the intensity of the 2016 wheat blast outbreak across Bangladesh. The percentage of affected area and the total area (hectares) under cultivation are shown for each district based on the color chart (Islam et al. 2016, BMC Biology 14:84)

31 A B C D E G H F F Symptoms of wheat blast in first epidemic in Bangladesh in February 2016 Islam et al. 2016, BMC Biology 14:84

32 Morphology of wheat blast pathogen. a. Culture of isolate BTJP4-5on PDA, b. sporulation of isolates on PDA, c-d. Light microscopic photographs of conidia and conidiophore. Scale bars= 10 µm

33 MoT in PDA MoT in PDA Wheat Wheat Wheat Wheat Wheat Wheat Eleusine indica Barley

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38 India March 2017 Timeline of wheat blast emergence in South America and South Asia

39 Challenges in fighting against wheat blast Our knowledge in biology and life cycle of wheat blast are vastly unknown. Wheat blast outbreak is happened at heading stage, dominant in neck blast. Very synchronously infects the spikes up to 100% Fungicide application after the appearance of symptoms in the spike is ineffective. Resistant sources for breeding are very limited!

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41 Assuming a conservative scenario of 5 10% for blastinduced wheat production loss, this study estimated the annual potential wheat loss across the sampled countries to be million tons, equivalent to USD million. Such losses further threaten an alreadyprecarious national food security, putting pressure on wheat imports and wheat prices. The study is a call for action to tackle the real wheat-blast threat in South Asia.

42 Possible approaches for tackling wheat blast Development of blast resistant wheat Classical breeding Genetic engineering Effective surveillance and disease management A convenient and specific diagnostic tool for wheat blast detection is needed Novel non-chemical intervention/control e.g., biopesticide

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45 The MoT3 (Pick et al. 2017) assay does not distinguish between wheat and rice blast isolates of Magnaporthe oryzae

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48 Open Science and Open Data Sharing

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59 (e) Sequencing reads from selected T0 transformants showing bp chromosomal deletion in various transformants.. ( f ) Leaves of edited tomato lines (named Tomelo) infected with Oidium neolycopersici showing full resistance toward this pathogen when compared to wild-type leaves. (g) Illumina sequencing data displaying coverage of T-DNA Illumina reads in three T1 segregants line. No reads matching the T-DNA vector sequence were detected in two independent slmlo 8-2 and 8-4 mutant lines. These two tomato-edited lines are free of transgenic sequences.

60 Development of blast resistant wheat by genome editing By using CRISPR/Cas9 genome editing, we disrupt/delete S-genes in wheat genomes for development non-transgenic durable blast resistant wheat Prof. Nocolas Talbot (Exeter University), Prof. Sophien Kamoun (The Sainsbury Laboratory) and Dr. Emma Wellington (NIAB) collaborate us. BBSRC, UK funds our mission of fighting against wheat blast

61 Our collaborators for wheat and rice blast research More international collaboration is needed to tackle wheat blast before it becomes catastrophic in South Asia.

62 (b) Deletions: Two DSBs are induced at a genomic locus or chromosomal fragment leading to an excision of the targeted fragment in CRISPR-Cas9. Thorsten et al. 2018

63 Wheat Blast: Editing shma gene Objectives 1. Editing putatively important shma gene in wheat for enhanced blast resistance 2. Evaluation of blast resistance in the greenhouse Deliverables 1.Edited wheat lines showing different deletions 2.New gene targets for editing to resistance against wheat blast

64 Golden-Gate compatible Magnaporthe oryzae Agrobacterium transformation vectors We have developed the Golden Gate compatible vector pbht2g-rfp (Addgene #107162) from the pcambia-derived (Mullins et al., 2001) pbht2g vector (Khang et al, 2010). The vector was domesticated through removal of BsaI cloning sites. An RFP-marker was inserted, which is expressed in E. coli, allowing for red-white selection of transformants. The marker is lost during the Golden Gate reaction, as it is replaced by the inserted transcriptional units. Vector, sequence information and plasmid maps are available from Addgene

65 Basic workflow for the CRISPR-Cas mediated genome editing in plants

66 Microspore edited plants

67 BTS-3 BTS-4 BTS-5 BTLK6A Biological control of wheat blast Control Out of 650 plant probiotic bacteria, 4 isolates displayed strong antagonistic effects toward wheat blast fungus MoT

68 No bacteria No bacteria & No inoculation Treatment with bacterial strains 24 hrs before inoculation

69 Mechanism of biocontrol of wheat blast Chemical arsenals: diverse group of antibiotics are produced by wheat blast biocontrol Bacilli isolated from wheat and rice seeds

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72 Our wheat blast work received the Best Presenter Award from the Krishibid Institution (KIB) award from the Secretary of Ministry of Agriculture of Bangladesh International Convention of Agriculture 2016

73 Honorable Minister for Agriculture, Matia Chowdhury, MP Congratulated and Encouraged Wheat Blast Research Team

74 Won the Islamic Development Bank (IsDB) Innovation Idea Prize 2018

75 Our Team, Our Strength, Our Pride

76 Acknowledgements Organizer of the 6 th Asia Biosafety Conference 2018 Professor Sophien Kamoun, The Sainsbury Laboratory, UK Dr. Diane Saunders, John Innes Center (JIC) UK Prof. Nicolas Talbot, Exeter University, UK Dr. Daniel Croll, ETH, Switzerland Prof. Bruce McDonald, ETH, Switzerland Dr. Pierre, INRA, France My Team Members and well wishers

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