US011019776B2 ( 12 ) United States Patent ( 10 ) Patent No .: US 11,019,776 B2 Klessig et al . ( 45 ) Date of Patent : * Jun . 1 , 2021 ( 54 ) COMPOSITIONS AND METHODS FOR AOIN 43/38 ( 2006.01 ) MODULATING IMMUNITY IN PLANTS AOIN 63/10 ( 2020.01 ) ( 52 ) U.S. CI . ( 71 ) Applicant: Boyce Thompson Institute for Plant CPC A01H 3/04 ( 2013.01 ) ; A01N 43/16 Research , Inc. , Ithaca , NY ( US ) ( 2013.01 ) ; AOIN 43/38 ( 2013.01 ) ; AOIN 63/10 ( 2020.01 ) ; C12N 15/8281 ( 2013.01 ) ; ( 72 ) Inventors : Daniel Klessig , Dryden , NY ( US ) ; C12N 15/8282 ( 2013.01 ) ; C12N 15/8283 Frank Schroeder , Ithaca , NY ( US ) ; ( 2013.01 ) ; C12N 15/8285 ( 2013.01 ) Patricia Manosalva, Ithaca, NY ( US ) ( 58 ) Field of Classification Search CPC C12N 15/8282 ; C12N 15/8281 ( 73 ) Assignee : BOYCE THOMPSON INSTITUTE See application file for complete search history. FOR PLANT RESEARCH , INC . , Ithaca , NY (US ) ( 56 ) References Cited ( * ) Notice: Subject to any disclaimer , the term of this U.S. PATENT DOCUMENTS patent is extended or adjusted under 35 U.S.C. 154 ( b ) by 26 days. 8,318,146 B1 11/2012 Teal et al . This patent is subject to a terminal dis FOREIGN PATENT DOCUMENTS claimer . WO 2010/009241 A2 1/2010 WO 2010/146062 A2 12/2010 ( 21 ) Appl. No .: 16 / 161,252 WO 2012/084858 A2 6/2012 WO 2013/022985 A2 2/2013 ( 22 ) Filed : Oct. 16 , 2018 WO 2013/022997 A2 2/2013 ( Continued ) ( 65 ) Prior Publication Data US 2019/0053451 A1 Feb. 21 , 2019 OTHER PUBLICATIONS Hsueh , Y.-P. et al 2013. Nematode - trapping fungi eavesdrop on Related U.S. Application Data nematode pheromones. Current Biology, 23 ( 1 ) , pp . 83-86 ( Year: ( 63 ) Continuation of application No. 14 / 854,363 , filed on 2013 ) . * Sep. 15 , 2015 , now Pat . No. 10,136,595 , which is a (Continued ) continuation - in - part of application No. PCT /US2014 /030136 , filed on Mar. 17 , 2014 . Primary Examiner Weihua Fan ( 74 ) Attorney, Agent, or Firm Robert C. Netter, Jr .; ( 60 ) Provisional application No. 61 / 789,445 , filed on Mar. 15 , 2013 , provisional application No. 62 / 079,242 , Dann , Dorfman , Herrell & Skillman filed on Nov. 13 , 2014 , provisional application No. ( 57 ) ABSTRACT 62 / 152,570 , filed on Apr. 24 , 2015 . Compositions and methods for enhancing disease resistance ( 51 ) Int. Ci . in plants are disclosed . A01H 3/04 ( 2006.01 ) C12N 15/82 ( 2006.01 ) 39 Claims , 25 Drawing Sheets AOIN 43/16 ( 2006.01 ) Specification includes a Sequence Listing . COM OH ?? I ascr # 9 ?? . I ascr # 18 OH OH OH ?? ascr # 1 ascr # 7 HO Z HO I OH Y" OH US 11,019,776 B2 Page 2 ( 56 ) References Cited Srinivasan et al . , A Modular Library of Small Molecule Signals Regulates Social Behaviors in Caenorhabditis elegans, PLOS Biol ogy , 2012 , e10012371 , 10 ( 1 ) . FOREIGN PATENT DOCUMENTS Srinivasan et al . , A blend of small molecules regulates both mating and development in Caenorhabditis elegans, Nature , 2008 , 1115 WO WO - 2013 / 022997 2/2013 AOIN 43/16 1118 , 454 ( 7208 ) . WO WO - 2013022997 A2 * 2/2013 A61P 25/00 Hseuh et al . , Nematode - trapping fungi eavesdrop on nematode pheromones , Current Biology, 2013 , 83-86 , 23 . Bose et al ., Complex small - molecule architectures regulate pheno OTHER PUBLICATIONS typic plasticity in a nematode , Angew Chem Int Ed Engl . , 2012 , 12438-12443 , 51 ( 50 ) . Ludewig, Andreas H. , and Frank C. 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Biol . 2012 , 961-966 , 7 ( 6 ) . * cited by examiner U.S. Patent Jun . 1 , 2021 Sheet 1 of 25 US 11,019,776 B2 OH OH HO ? ascr # 9 HOoI pmascr # 18 OM ascr # 1 ascr # 7 HO FIG . 1 Q - PR - 1 NA (UM ) NA (UM ) SA ( 50uM ) SA (500 MM ) FIG . 2 U.S. Patent Jun . 1 , 2021 Sheet 2 of 25 US 11,019,776 B2 O - PR - 1 CB .; FIG . 3A FIG . 3B 10 8 5 4 4 2 1 0 NA (Leaf ) 0 NA ( Root ) o 0 0.01 0.01 SA ( Leaf) 0 50 250 SA ( Leaf ) 0 50 50 250 250 FIG . 4A FIG . 4B U.S. Patent Jun . 1 , 2021 Sheet 3 of 25 US 11,019,776 B2 PR - 1 POF1.2 Butubulin 8 - tubulin NA ( Leaf) 0 0.3 0.01 0.3 0.01 -- NA ( Root ) o 0.01 0.3 0 0.01 SA ( Leaf) SO 50 SA ( Leaf ) 0 50 PDF1.2 W 6 - tubulin NA ( Leaf) 0 0.01 SA ( Leaf) 50 50 FIG . 5A FIG . 5B CFUK10€/ml ** FIG . 6 U.S. Patent Jun . 1 , 2021 Sheet 4 of 25 US 11,019,776 B2 LOH OH 1111 OH ?? . I ascr # 10 HO ascr # 3 OH HO asc : # 7 OH I FIG . 7 OH ***** ( CHahn HO n = 6 , ascr# 16 7, ascr # 18 n = 8 , ascr # 20 n = 11 , ascri26 FIG . 8 U.S. Patent Jun . 1 , 2021 Sheet 5 of 25 US 11,019,776 B2 OH ascr # 8 COH icas # 9 HN . HOW ? ?????OH Ho?????? OH OH mbas # 3 HO . ascr # 9 OH OH FIG . 9 2 LesionSize(cm2) 1 FIG . 10A FIG . 10B U.S. Patent Jun . 1 , 2021 Sheet 6 of 25 US 11,019,776 B2 FIG.11B . RioGrande ) cm ( Size lesion FIG.110 M82 mi/ number Sporangia FIG.11A TSIN U.S. Patent Jun . 1 , 2021 Sheet 7 of 25 US 11,019,776 B2 /cm2CFU106 11 0 $ 0 FIG . 12A 1 FIG . 12B CFUx10$/cm2 ili FIG . 12C U.S. Patent Jun . 1 , 2021 Sheet 8 of 25 US 11,019,776 B2 7 Ennammanna CFUx10"/crna 2 ascr18 FIG . 13A changedExpressionfold 10 T AtFRK1 FIG . 13B ajewanetaqunujueja/ FIG . 13C U.S. Patent Jun . 1 , 2021 Sheet 9 of 25 US 11,019,776 B2 CFUx10$/cm ? FIG . 13D Bufferonly SAonly 0.01AM0.01 uM+EO SA WTI 0.3 MM+ SA Asc3 PR1 PDF1.2 Tub FIG . 13E U.S. Patent Jun . 1 , 2021 Sheet 10 of 25 US 11,019,776 B2 BufferonlySAonly TO'O WT0.01 UM# SA 0.3 UM+ SA Asc 9 PR1 PDF1.2 Tub FIG . 13F CFUX106/cm2 1$ FIG . 136 U.S. Patent Jun . 1 , 2021 Sheet 11 of 25 US 11,019,776 B2 BufferonlySAonly 0.01 PM PR1 PDF1.2 Tub FIG . 13H BufferonlySAonly 0.01UM PR1 PDF1.2 Tub FIG . 131 U.S. Patent Jun . 1 9, 2021 Sheet 12 of 25 US 11,019,776 B2 14Mascr18 0.3 Time after treatment ( minutes ) FIG . 14A Snowwwwwwwwww Lesionsize(cm) 1 FIG . 14B Water SCF18 casus FIG . 14C U.S. Patent Jun . 1 , 2021 Sheet 13 of 25 US 11,019,776 B2 Lesionsize(cm) T ascr18 easc18 FIG . 14D Sporangoia/ml T FIG . 14E U.S. Patent Jun . 1 , 2021 Sheet 14 of 25 US 11,019,776 B2 0.25 ascr 9 ( 1 ) Oscr 9 ( 2 ) ascr 10 ( 3 ) ascr 4 ( 4 ) 0.1 basal FIG .