1. Breeding Drought Tolerant Corn and Elucidating the Mechanisms of the Corn-Aspergillus flavus Interactions as Influenced by the Environment Baozhu Guo and Bob Kemerait University of Georgia, Department of Plant Pathology USDA-ARS, Crop Protection and Management Research Unit Tifton, GA AMCOE 2013 research project reports, February 25, 2014, Grand Hyatt, San Antonio, TX

2. Acknowledgement USDA-ARS Brian Scully Xinzhi Ni Joe Knoll Ted Webster Matt Krakowsky, (Raleigh, NC) Paul Williams (Mississippi State) Marilyn Warburton (Mississippi State) Hamas Abbas (Stoneville, MS) University and Industry Dewey Lee Anton Coy Wenwei Xu (TX) Daniel Gorman (Pioneer) Seth Murray (TX) Zhi-yuan Chen (LSU) Ashli Brown (MS) Nick Smith

3. Aflatoxin Resistance/ $Yield$ Drought tolerance/ Three factors have been primarily responsible: 1) drought/yield/quality/aflatoxin; 2) aflatoxin contamination/quality/safety/drought; 3) insect pressure/yield/aflatoxin. Genes/markers What Do You Want Us to Do? Genes/markers

4. Plant Breeding: Host Plant Resistance Reduced aflatoxin through increased resistance to Aspergillus flavus and subsequent aflatoxin contamination Reduced aflatoxin through increased tolerance to Drought Stress Understand the role of oxidative stress caused by drought and high temperature in aflatoxin production and its remediation by the host-corn and the pathogen-A. flavus. Yield Integrated Strategies to Manage Aflatoxin Contamiantion Improve agronomic performance (including earliness as an avoidance mechanism) Planting date Cropping system and bio-control We need all these tactics combined together Objectives and Goals:

5. Diverse Germplasm Used in Breeding Programs Source General QualityOther General Traits. Latin-American:+Tropical, Late, photoperiod, Insect Resistance (BZ) CIMMYT-Mexico+++Tropical, Agronomics, Drought, Insect Resistance South African+Drought, White flints, Subtropical Mediterranean +++Drought, Agronomics, Yield China++Agronomics, Yield & Aflatoxin (TUN & CY) GEM Materials+++Agronomics, Yield & Aflatoxin X-PVPs+++Agronomics, Yield Holden’s Foundation+++Agronomics, Yield Hawaiian+/++Disease Resistance & Aflatoxin ARS-NC+++Agronomics, Yield ARS-MS/LA+++Yield, Insect Resistance & Aflatoxin ARS-Tifton++Insect Resistance & Aflatoxin (CEW,FAW & GT- MAS)

6. InbredYear OriginPedigree GT-601 2007 TiftonGT-MAS:gk GT-602 2007 TiftonGT-MAS:gk GT-603* 2011 Tifton GT-MAS:gk GT-8882014 Tifton DK 888:N11 (GEM) * Indicates “best” in group compared to the Research Standard: ‘MP 313E ‘ Plus 3 Inbreds from Texas A & M: Tx736, Tx739, Tx740 6 Inbreds from ARS New Orleans & IITA: TZAR 101 –TZAR 106 2 Inbreds from ARS Mississippi State: MP718 & MP 719 Recent Inbred Releases

7. GT Series GT Series – Tifton, GA HBA-1 X GT-603 GT-603 GT-601, GT-602 and GT-603 all derived from same population GT-MAS:gk GT-603 appears to be the “better” of the three, but all have similar levels of resistance. GT-603 was comparable to MP 313E for aflatoxin resistance but not superior. All three Yellow dent, but GT-603 has dark cob and deeper yellow kernels and tassels 10 days earlier than MP 313E. GT-603 appeared to show better heterosis when test crossed to Stiff Stalk inbred B73 rather than Non Stiff Stalk inbred Mo17, suggesting it fits in NSS group. HybridInbred

8. DK888:N11-002-034-B-002-001-B*3 Raleigh 2014 release: “GT 888”- Tifton/ Raleigh DK888 Hybrids Derived from GEM Program, Yellow Dent, Originally from Thailand Improved Aflatoxin resistance, but not superior to MP 313E (96 ppb) Tassel time earlier than Mp313E and nearly equivalent to GT-603. Fits better in Non Stiff Stalk (NSS) Group ( LH51, Mo17, Oh43). Heterotic with “Stiff Stalk Group” (SS) (B14, B73, LH132). Hybrids Commercial Hybrids Inbred

9. Near-future release: “GT A2R” and SynAMP43- Tifton Derived from same population as GT-601, GT-602 and GT-603 Improved aflatoxin resistance, but not as good as GT-603 Has broader General Combining Ability and produces hybrids with higher yields than others from GT Population Fits better in “Stiff Stalk Group” (SS) (B14, B73, LH132); Heterotic with Non Stiff Stalk (NSS) ( LH51, Mo17, Oh43). Hybrids Commercial Hybrids

10. DesignationPedigreeRepTiftonMississippi Commercial CK 1P31P414 2381846 Commercial CK 2P31G984 109558 Commercial CK 3DK6974 167105 Commercial CK 4BH8740VTTP4 75365 Commercial CK 5BH8910RR/HX4 104997 Commercial CK 6BH9051RR4 163335 GT-1Hi33 x Ni7077-64 148471 GT-2HBA x GT6034 154155 GT-3CY1 x GT6034 9589 GT-4LH132 x SynAMP434 173535 GT-5LH132 x GTA2R4 135313 GT-6Hi27 x GT6034 5364 GT-7LH51 x SynAMP434 2054476 Average128 115567 2012 SERAT (Southeast Region Aflatoxin Test) TEST-Afl (ppb)

11. 2013 Hybrid Performance Test, 8 reps x 5 ears, Tifton Hybrid Ear Length (cm) Rows Kernels Ear Worm Damage (cm) Other Damage 1 Total Wt. (g) 2 Kennel Wt. (g) Yield (bu) Total Aflatoxin (ppb) LH 132 x GT603171621969853 205 52 LH 132 x Syn Am1 (P43)2013211036882 212 79 Hi 41 x Mo 172014211069934 224 121 GT A2 R x B731718221032891 214 149 LH 51 x GT603201322930805 193 157 CY 1 x A632181422953821 197 166 LH132 x GT A2 R151721887769 183 184 Lo 964 x A632 (green)171422850713 171 193 GP282 x GT603171922832732 176 204 GT603 x A632181622990855 205 209 Syn AM1 (P43) x Mo 172212211090933 224 223 GT603 x A63816 21708608 146 229 Lo 964 x A632 (red silk)181422886761 183 230 CY 2 x A6321816221018866 208 232 Syn AM1 (P43) x A632191221915747 179 274 Lo 1016 x A63216 22764662 159 276 Grace E-5 x GT603181521910779 187 336 Hi 27 x Mo 17211421889745 179 362 LH 51 x Syn AM1 (P43)211221942793 190 367 DKC 69-4318 22996876 210 489 Lo 1016 x A638151722806703 169 496 GT A2 R x Mo 17181422849717 172 511 GT A 2R x A632161722848718 172 525 Tun 18-2 x GT6031915221031908 218 555 LH 51 x GT603201322930818 196 597 CY 1 x A638161522828697 196 848 Lo 964 x A638161422797676 162 991 P2023HR191522993872 209 1158 GP 280 x GT603171622924778 187 1314 CY 2 xMo 17181422878749 180 1942

12. Hybrid Ear Length (cm) Rows Kernels Plant Stand Ear Worm Damage Other Damage Total Wt. (g) Kennel Wt. (g) Yield (Bu) GT A2 R x CY117.916.218.01.91.31486.51286.0 303 LH 132 x Mo 1722.414.211.01.11.41399.01219.5 293 CY2 x B7318.619.019.51.51.31383.01202.0 289 GP 280 x Syn AM1 (P43)19.814.313.51.41.71350.51136.0 273 Lo 964 x GP 28020.015.019.51.11.41307.01128.0 271 GP 280 x GT 603 (P50)18.816.015.51.61.51241.01119.5 269 GT 603 (P50) x DK 888N19.116.219.51.31.71312.01104.5 265 GT 603 (P50) x DK 888N20.015.317.01.21.81325.51093.5 262 Syn AM 1 (P43) x B7320.114.616.01.31.41265.51087.5 261 Lo 1016 x CY317.216.019.51.61.21305.51079.0 259 DKC 69-4318.2 18.81.41.91216.11074.0 258 GT 601 x AT 70915.717.019.51.51.01203.01067.5 256 Lo 1016 x CY217.318.019.51.51.41239.01067.0 256 LH 51 x Syn AM1 (P43)22.612.418.01.11.21209.51057.0 254 LH 132 x GT A2 R17.816.815.01.71.41216.01048.0 252 Lo 964 x CY119.513.820.01.81.91221.01044.5 251 GP 280 x Syn AM1 (P43)18.414.218.01.21.81230.51042.5 250 GT 603 (P50) x GP 28218.517.019.01.8 1187.01041.5 250 CY1 x GT 603 (P50)17.216.419.51.3 1202.01034.5 248 Syn AM 1 (P43) x LH 5122.512.818.01.31.21211.51032.5 248 Lo 1016 x CY115.917.219.51.71.11205.01029.0 247 LH 51 x B-7320.915.010.51.91.41196.51028.0 247 CY1 x B7316.318.219.51.51.91153.51019.0 245 Lo 964 x A632 RED SILK19.515.219.52.21.51192.01017.5 244 Syn AM 1 (P43) x Mo 1722.112.617.51.41.31197.51011.5 243 LH 132 x Syn AM1 (P43)19.514.418.51.31.51192.51010.5 242 GP 282 x Syn AM1 (P43)19.914.413.01.21.41197.51009.0 242 P2023 HR20.715.419.01.91.61234.6993.0 238 Syn AM 1 (P43) x LH13218.814.419.01.01.31155.0989.0 237 LH 51 x GT A2 R19.714.217.01.11.81162.5985.5 236 Hi 41 x Mo 1719.613.818.01.81.31126.0980.0 235 GT 603 x LH 13217.115.818.01.51.21141.5978.5 235 LH 51 x GT 60321.513.611.51.51.61110.0973.0 234 Lo 964 x Syn AM1 (P43)20.314.819.51.21.91161.0972.5 233 GT A2 R x Mo 1718.514.419.01.71.91083.5961.5 231 GP 282 x Mo 1722.912.811.01.21.61108.5960.5 230 CY1 x GT A2 R15.516.218.01.6 1106.5958.5 230 GT 603 (P50) x CY418.014.818.01.91.51128.0955.5 239 2013 Hybrid Performance Test, 4 reps x 5 ears, Tifton

13. iTRAQ-based proteomics of corn lines tolerant and sensitive to drought stress isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology

14. Figure 1. Experimental design and iTRAQ-based workflow showing different steps describing corn kernels response to drought stress treatments. Maize drought tolerant line Lo964 Maize drought sensitive line B73 ControlDroughtControlDrought Protein extraction (Trichloroacetic acid/Acetone) Solubilization (6M urea, 50mM TEAB, 2% CHAPS) Reduction with DTT and Alkylation with IAA Trypsin digestion Peptide iTRAQ Labeling Peptide mixture SCX seperation and fractionation nLC-MS/MS and Data analysis Quantification: Bradford method SDS-PAGE Two biological replicates 113 117 114 118 115 119 116 121 Two biological replicates and four technical replicates Analysis of differential proteome patterns and functional annotation Drought treatment for 7 days at 20 DAP

15. Lo964B73 Protein-protein interaction analysis: Transcription and protein folding Carbohydratemetabolism Proteindegradation Redoxhomeostasis ABA and Lea protein Signal transduction and protein metabolism Carbohydratemetabolism

16. DNARNA Protein Modified protein Transcription AP2-EREBP-type transcription factor; fibrillarin-2 Protein synthesis ribosomal protein; QM-like protein; histone deacetylase 6 Protein folding, assembly HSPs; TCP-1; histone deacetylase 6 Protein degradation proteasome subunit alpha type; proteasome subunit alpha type 1 Figure 7. transcription and protein metabolism B73 Lo964 B73 Degradation

18. why does Aspergillus flavus produce aflatoxins”? Hypothesis: given the role of oxidative stress (drought) in the promotion of aflatoxin biosynthesis, the hypothesis has been proposed that aflatoxin may function as a form of antioxidant protection to the fungus from the oxidative stress caused by the “Hot” and “Dry” environment. Jake Fountain, Ph.D. Student

19. Increasing H 2 O 2 Concentration Stimulates Aflatoxin Production 0 mM0.1 mM5 mM10 mM AFB 1 A. Flavus Strain 3557 (Toxigenic) Non-Concentrated YES Culture Media – 7 Days Hydrogen Peroxide Concentration Gradient = 0, 0.1, 5, 10, 50, 100mM 50mM and 100mM concentrations were lethal. 3% H 2 O 2 : 0mL 0.1mL/1000mL 5.7mL/1000mL 11.3mL/1000mL

20. Fungal Biomass is Affected by High H 2 O 2 Concentration and Aflatoxin Production n = 6 Error Bars ± SD ** p < 0.0001 ** A. flavus strains A1 (AF -) and A9 (AF +) were cultured on PDB with H 2 O 2 concentration gradient from 0 to 10mM for 3 Days. Both strains at 25mM incubated for 6 days due to slow growth.

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