CRISPR/Cas9‐mediated simultaneous mutation of three salicylic acid 5‐hydroxylase (OsS5H) genes confers broad‐spectrum disease resistance in rice

Salicylic acid (SA) is an essential plant hormone that plays critical roles in basal defence and amplification of local immune responses and establishes resistance against various pathogens. However, the comprehensive knowledge of the salicylic acid 5-hydroxylase (S5H) in rice-pathogen interaction is still elusive. Here, we reported that three OsS5H homologues displayed salicylic acid 5-hydroxylase activity, converting SA into 2,5-dihydroxybenzoic acid (2,5-DHBA). OsS5H1, OsS5H2, and OsS5H3 were preferentially expressed in rice leaves at heading stage and responded quickly to exogenous SA treatment. We found that bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) strongly induced the expression of OsS5H1, OsS5H2, and OsS5H3. Rice plants overexpressing OsS5H1, OsS5H2, and OsS5H3 showed significantly decreased SA contents and increased 2,5-DHBA levels, and were more susceptible to bacterial blight and rice blast. A simple single guide RNA (sgRNA) was designed to create oss5h1oss5h2oss5h3 triple mutants through CRISPR/Cas9-mediated gene mutagenesis. The oss5h1oss5h2oss5h3 exhibited stronger resistance to Xoo than single oss5h mutants. And oss5h1oss5h2oss5h3 plants displayed enhanced rice blast resistance. The conferred pathogen resistance in oss5h1oss5h2oss5h3 was attributed to the significantly upregulation of OsWRKY45 and pathogenesis-related (PR) genes. Besides, flg22-induced reactive oxygen species (ROS) burst was enhanced in oss5h1oss5h2oss5h3. Collectively, our study provides a fast and effective approach to generate rice varieties with broad-spectrum disease resistance through OsS5H gene editing.

[1]  Yanpeng Wang,et al.  Genome-edited powdery mildew resistance in wheat without growth penalties , 2022, Nature.

[2]  Xinhua Ding,et al.  Tal2b targets and activates the expression of OsF3H03g to hijack OsUGT74H4 and synergistically interfere with rice immunity. , 2021, The New phytologist.

[3]  Guo‐Liang Wang,et al.  Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector , 2021, Cell.

[4]  B. Staskawicz,et al.  Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance , 2021, Proceedings of the National Academy of Sciences.

[5]  M. Tian,et al.  CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance , 2021, PloS one.

[6]  Jaindra Nath Tripathi,et al.  CRISPR/Cas9‐mediated editing of DMR6 orthologue in banana (Musa spp.) confers enhanced resistance to bacterial disease , 2021, Plant biotechnology journal.

[7]  Xuewei Chen,et al.  Fighting the enemy: How rice survives the blast pathogen’s attack , 2021, The Crop Journal.

[8]  Xin Li,et al.  Salicylic Acid: Biosynthesis and Signaling. , 2021, Annual review of plant biology.

[9]  W. Shen,et al.  APICAL SPIKELET ABORTION (ASA) Controls Apical Panicle Development in Rice by Regulating Salicylic Acid Biosynthesis , 2021, Frontiers in Plant Science.

[10]  M. Lenman,et al.  Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes , 2021, Scientific Reports.

[11]  A. Olea,et al.  Plant Growth-Defense Trade-Offs: Molecular Processes Leading to Physiological Changes , 2021, International journal of molecular sciences.

[12]  Xin Li,et al.  Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity , 2020, Plant Cell.

[13]  Conrad C. Huang,et al.  UCSF ChimeraX: Structure visualization for researchers, educators, and developers , 2020, Protein science : a publication of the Protein Society.

[14]  F. White,et al.  Xanthomonas diversity, virulence and plant–pathogen interactions , 2020, Nature Reviews Microbiology.

[15]  F. Meng,et al.  A bHLH transcription activator regulates defense signaling by nucleo-cytosolic trafficking in rice. , 2020, Journal of integrative plant biology.

[16]  Yuqi Feng,et al.  Two dioxygenases, SLC1 and SLC2, play essential roles in shoot development of rice. , 2019, Journal of experimental botany.

[17]  Wen-xiu Ma,et al.  Engineering Broad-Spectrum Bacterial Blight Resistance by Simultaneously Disrupting Variable TALE-binding Elements of Multiple Susceptibility Genes in Rice. , 2019, Molecular plant.

[18]  Yuelin Zhang,et al.  Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid , 2019, Science.

[19]  Yongbo Hong,et al.  The OsMPK15 Negatively Regulates Magnaporthe oryza and Xoo Disease Resistance via SA and JA Signaling Pathway in Rice , 2019, Front. Plant Sci..

[20]  Q. Qian,et al.  Inducible overexpression of Ideal Plant Architecture1 improves both yield and disease resistance in rice , 2019, Nature Plants.

[21]  Zhenying Shi,et al.  The OsmiR396–OsGRF8–OsF3H‐flavonoid pathway mediates resistance to the brown planthopper in rice (Oryza sativa) , 2019, Plant biotechnology journal.

[22]  M. S. Mukhtar,et al.  Genome Editing: Targeting Susceptibility Genes for Plant Disease Resistance. , 2018, Trends in biotechnology.

[23]  Sudhir Kumar,et al.  MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. , 2018, Molecular biology and evolution.

[24]  Xin Li,et al.  Opposite Roles of Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Transcriptional Regulation of Plant Immunity , 2018, Cell.

[25]  Yuqi Feng,et al.  The dioxygenase GIM2 functions in seed germination by altering gibberellin production in Arabidopsis. , 2018, Journal of integrative plant biology.

[26]  Yuelin Zhang,et al.  Convergent and Divergent Signaling in PAMP-Triggered Immunity and Effector-Triggered Immunity. , 2017, Molecular plant-microbe interactions : MPMI.

[27]  Xingliang Ma,et al.  CRISPR-GE: A Convenient Software Toolkit for CRISPR-Based Genome Editing. , 2017, Molecular plant.

[28]  S. Gan,et al.  S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis1[OPEN] , 2017, Plant Physiology.

[29]  Jian-Min Zhou,et al.  Apoplastic ROS signaling in plant immunity. , 2017, Current opinion in plant biology.

[30]  Ping Li,et al.  A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance , 2017, Cell.

[31]  Xiuping Zou,et al.  Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus , 2017, Plant biotechnology journal.

[32]  Congmao Wang,et al.  Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion , 2017, Scientific Reports.

[33]  Jinrong Peng,et al.  ABNORMAL INFLORESCENCE MERISTEM1 Functions in Salicylic Acid Biosynthesis to Maintain Proper Reactive Oxygen Species Levels for Root Meristem Activity in Rice , 2017, Plant Cell.

[34]  Guo‐Liang Wang,et al.  OsCUL3a Negatively Regulates Cell Death and Immunity by Degrading OsNPR1 in Rice[OPEN] , 2017, Plant Cell.

[35]  C. Zipfel,et al.  Regulation of pattern recognition receptor signalling in plants , 2016, Nature Reviews Immunology.

[36]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[37]  W. Frommer,et al.  Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. , 2015, The Plant journal : for cell and molecular biology.

[38]  J. Parker,et al.  Effector-triggered immunity: from pathogen perception to robust defense. , 2015, Annual review of plant biology.

[39]  Hidemasa Bono,et al.  CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites , 2014, Bioinform..

[40]  Li Yang,et al.  Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life. , 2014, Cell host & microbe.

[41]  J. Leach,et al.  Novel insights into rice innate immunity against bacterial and fungal pathogens. , 2014, Annual review of phytopathology.

[42]  Yanpeng Wang,et al.  Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew , 2014, Nature Biotechnology.

[43]  Guoping Wang,et al.  A potential disease susceptibility gene CsLOB of citrus is targeted by a major virulence effector PthA of Xanthomonas citri subsp. citri. , 2014, Molecular plant.

[44]  E. Ono,et al.  Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants. , 2014, The Plant journal : for cell and molecular biology.

[45]  W. Frommer,et al.  Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease , 2014, Proceedings of the National Academy of Sciences.

[46]  Xiaofei Han,et al.  The Function and Catalysis of 2-Oxoglutarate-Dependent Oxygenases Involved in Plant Flavonoid Biosynthesis , 2014, International journal of molecular sciences.

[47]  S. Gan,et al.  Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism , 2013, Proceedings of the National Academy of Sciences.

[48]  Jun Li,et al.  Targeted genome modification of crop plants using a CRISPR-Cas system , 2013, Nature Biotechnology.

[49]  Bifeng Yuan,et al.  Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry , 2013, PloS one.

[50]  Qian Qian,et al.  DWARF AND LOW-TILLERING Acts as a Direct Downstream Target of a GSK3/SHAGGY-Like Kinase to Mediate Brassinosteroid Responses in Rice[W][OA] , 2012, Plant Cell.

[51]  Yuge Li,et al.  A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes , 2011, Plant Methods.

[52]  M. S. Mukhtar,et al.  Independently Evolved Virulence Effectors Converge onto Hubs in a Plant Immune System Network , 2011, Science.

[53]  G. Gheysen,et al.  The Jasmonate Pathway Is a Key Player in Systemically Induced Defense against Root Knot Nematodes in Rice1[C] , 2011, Plant Physiology.

[54]  Xianghua Li,et al.  Characterization of Xanthomonas oryzae-Responsive cis-Acting Element in the Promoter of Rice Race-Specific Susceptibility Gene Xa13 , 2011, Molecular plant.

[55]  M. T. B. Geller,et al.  Molecular , 2019, Modern Pathology.

[56]  Cai-guo Xu,et al.  A Pair of Allelic WRKY Genes Play Opposite Roles in Rice-Bacteria Interactions1[C][W][OA] , 2009, Plant Physiology.

[57]  Cyril Zipfel,et al.  News from the frontline: recent insights into PAMP-triggered immunity in plants. , 2008, Current opinion in plant biology.

[58]  S. Delaney,et al.  Metabolism of salicylic acid in wild-type, ugt74f1 and ugt74f2 glucosyltransferase mutants of Arabidopsis thaliana. , 2008, Physiologia plantarum.

[59]  J. Glazebrook,et al.  Interplay between MAMP-triggered and SA-mediated defense responses. , 2008, The Plant journal : for cell and molecular biology.

[60]  E. Abou-Mansour,et al.  Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana , 2008, FEBS letters.

[61]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[62]  Jonathan D. G. Jones,et al.  Pathological hormone imbalances. , 2007, Current opinion in plant biology.

[63]  K. Mysore,et al.  The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000. , 2007, Molecular plant-microbe interactions : MPMI.

[64]  Chang-Jie Jiang,et al.  Rice WRKY45 Plays a Crucial Role in Benzothiadiazole-Inducible Blast Resistance[W][OA] , 2007, The Plant Cell Online.

[65]  Jianjun Wang,et al.  Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility. , 2007, Plant biotechnology journal.

[66]  Xin Li,et al.  Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs. , 2006, The Plant journal : for cell and molecular biology.

[67]  F. White,et al.  Os8N3 is a host disease-susceptibility gene for bacterial blight of rice , 2006, Proceedings of the National Academy of Sciences.

[68]  Jörg Durner,et al.  Conserved requirement for a plant host cell protein in powdery mildew pathogenesis , 2006, Nature Genetics.

[69]  C. Mei,et al.  Endogenous salicylic acid protects rice plants from oxidative damage caused by aging as well as biotic and abiotic stress. , 2004, The Plant journal : for cell and molecular biology.

[70]  Xin Li,et al.  Knockout Analysis of Arabidopsis Transcription Factors TGA2, TGA5, and TGA6 Reveals Their Redundant and Essential Roles in Systemic Acquired Resistance Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.014894. , 2003, The Plant Cell Online.

[71]  S. Yoshida,et al.  Chloroisonicotinamide derivative induces a broad range of disease resistance in rice and tobacco. , 2002, Plant & cell physiology.

[72]  Frederick M. Ausubel,et al.  Isochorismate synthase is required to synthesize salicylic acid for plant defence , 2001, Nature.

[73]  L. Que,et al.  The 2-His-1-carboxylate facial triad--an emerging structural motif in mononuclear non-heme iron(II) enzymes. , 1997, European journal of biochemistry.

[74]  R. Dixon,et al.  THE OXIDATIVE BURST IN PLANT DISEASE RESISTANCE. , 1997, Annual review of plant physiology and plant molecular biology.

[75]  S. Iyer,et al.  Differential Accumulation of Salicylic Acid and Salicylic Acid-Sensitive Catalase in Different Rice Tissues , 1997, Plant physiology.

[76]  M. Wolter,et al.  The Barley Mlo Gene: A Novel Control Element of Plant Pathogen Resistance , 1997, Cell.

[77]  I. Raskin,et al.  Salicylic Acid in Rice (Biosynthesis, Conjugation, and Possible Role) , 1995, Plant physiology.

[78]  Michael F. Seidl,et al.  DOWNY MILDEW RESISTANT 6 and DMR6-LIKE OXYGENASE 1 are partially redundant but distinct suppressors of immunity in Arabidopsis. , 2015, The Plant journal : for cell and molecular biology.

[79]  H. Koga,et al.  Rice WRKY45 plays important roles in fungal and bacterial disease resistance. , 2012, Molecular plant pathology.

[80]  I. Jørgensen,et al.  Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley , 2004, Euphytica.