Group Meeting Andrew Diener 12-17-02 natural resistance to Fusarium oxysporum cloning eds3 genetic screens for Fusarium mutants

(1) natural resistance to Fusarium oxysporum RFO1 confers resistance to all crucifer F. oxysporum pathogens and is located at the end of chromosome 1. RFO2 confers resistance to F. oxysporum f. sp. matthioli and is located in the middle of chromosome 5. Does a RFO3 exist? If so, RFO3 confers resistance to F. oxysporum f. sp. conglutinans and is located on chromosome 1 cetromeric to RFO1.

Fusarium oxysporum Crucifer Pathogens HOST forma specialis cabbage conglutinans, race 1 conglutinans, race 2 radish raphani stock matthioli, race 1 matthioli, race 2

Distribution of PKS sequences + = presence of homologous sequence x = presence of heterologous sequence –– = absent

Fusarium resistance CABBAGE Resistance to F. oxysporum f. sp. conglutinans race 1 is a single dominant Mendelian trait. J Agric Res (1930) 41: 1 TOMATO Each of three races of F. oxysporum f. sp. lycospersici is recognized by a dominant resistance gene I-1, I-2 or I-3. I-2 encodes a LZ-NBS-LRR protein. Plant Cell (1997) 9: 521 Plant Cell (1998) 10: 1055

pellets are watered with Peat Pellet Dip Inoculation pellets are watered with bud cell (conidia) suspension 2 1/2 weeks 2 1/2 weeks sow inoculate score

Disease Index 1 2 3 4 5 5 = unaffected 4 = stunted petioles 1 2 3 4 5 5 = unaffected 4 = stunted petioles 3 = vascular chlorosis in older leaves 2 = yellows and death in older leaves 1 = restricted growth of young leaves 0 = death

} Arabidopsis genetics: mapping population “F1-selfed population” Parents: Col-0 X Ty-0 Only the genotype of plants with a recessive phenotype are unambiguous. F1 “F1-selfed population” 1/4 of F2 plants are homozygous recessive. (self) All F2 chromosomes are recombinant. F2 1/4 of F2 chromosomes give informative recombination. } 1 C/C : 2 C/T : 1 T/T

Arabidopsis genetics: mapping population Parents: Col-0 X Ty-0 The genotype of plants is unambiguous from both dominant and recessive phenotypes. F1 X Ty-0 “F1-backcrossed population” 1/2 of F2 chromosomes are recombinant. F2 1/2 of F2 chromosomes give informative recombination. 1 C/T : 1 T/T

Parental Contribution to F2 genotype 50% Col-0 25% Col-0 50% Ty-0 75% Ty-0 recessive contribution from both parents recessive contribution from Ty-0 parent only

Polygenic Traits mapping population 1 gene 2 genes 3 genes F1 selfed 1/4 1/16 1/64 F1 backcrossed 1/2 1/4 1/8

Resistance to Fusarium oxysporum f. sp. matthioli Unlike f. sp. conglutinans and f. sp. raphani, most ecotypes are completely resistant to f. sp. matthioli. Ty-0 is a susceptible ecotype Col-0 is completely resistant Col-0 X Ty-0 mapping population 8 in 128 F2 plants from F1 selfed population were susceptible = 1/16 = 1/4 X 1/4 = two genes. 14 in 60 F2 plants from F1 backcrossed population were susceptible = 1/4 = 1/2 X 1/2 = two genes.

F2 : (F1:Col-0 X Ty-0) X Ty-0 X X X Col-0 Ty-0 Flat #1

F2 : (F1:Col-0 X Ty-0) X Ty-0 X X X Col-0 Ty-0 Flat #1

F2 : (F1:Col-0 X Ty-0) X Ty-0 Col-0 Ty-0 Flat #2

F2 : (F1:Col-0 X Ty-0) X Ty-0 Col-0 Ty-0 Flat #2

“F1 backcross” Col-0 Ty-0 F2 (F1 Col-0 X Ty-0) X Ty-0 number of plants disease index

5 1 Genetic Linkage to loci on chromosome 1 and 5 C : T C : T PHYC ciw9 ciw8 CTR1 nga280 nga111 1 F21M12 C : T C : T sensitive resistent sensitive resistent 4 : 7 7 : 7 7 : 7 10 : 3 3 : 11 8 : 6 3 : 9 10 : 2 5 : 8 4 : 9 7 : 7 9 : 5 2 : 12 11 : 3

Col-0 X Sg-1 Genotyping susceptible F2 plants from F1-backcross chromosome 1 plant nga111 nga280 A3 S S A9 S C C5 C S C7 C C D2 S C D3 S S D6 S S D7 S S D8 S C D9 C C E2 S C E3 S S S 9 6 C 3 6 Col-0 X Sg-1 Fusarium oxysporum f. sp. conglutinans

Ty-0

Number of Plants Disease Index

Future Plans Just inoculated 240 Col-0 X Ty-0 F2 plants from F1-backcross. Should obtain about 60 susceptible F2 plants and 60 strongly resistant F2 plants.

enhanced disease susceptibility mutants pad4 eds5/sid1 eds16/sid2 eds12 npr1 eds10 eds4 eds11 eds6 eds9 eds3 eds-N26 Mutants obtained from direct genetic screens for increased susceptibility to either Pseudomonas syringae or Erysiphe orontii Genetics (1996) 143: 973 Genetics (1998) 149: 537 Plant J (2000) 24: 205

Susceptibility to Fusarium oxysporum Col-0 (wild type) R pad4 S eds5/sid1 S eds16/sid2 S eds12 R npr1 R eds10 S eds4 S eds11 R eds6 R eds9 R eds3 S eds-N26 S R = resistance S = sensitive

Mutants with defective accumulation of salicylic acid are required for Fusarium resistance. Col-0 (wild type) R pad4 S eds5/sid1 S eds16/sid2 S eds12 R npr1 R eds10 S eds4 S eds11 R eds6 R eds9 R eds3 S eds-N26 S } known to be required for inducible SA accumulation

Mutants with defective Systemic Acquired Resistance are not required for Fusarium resistance. Col-0 (wild type) R pad4 S eds5/sid1 S eds16/sid2 S eds12 R npr1 R eds10 S eds4 S eds11 R eds6 R eds9 R eds3 S eds-N26 S SA-dependent PR-1 induction not required for resistance } Plant J (2002) 29: 11

Mutants with defective Induced Systemic Resistance are required for Fusarium resistance. Col-0 (wild type) R pad4 S eds5/sid1 S eds16/sid2 S eds12 R npr1 R eds10 S eds4 S eds11 R eds6 R eds9 R eds3 S eds-N26 S resistance requires ISR-dependent genes } Plant J (2002) 29: 11

Undefined eds mutants with enhanced susceptibility to Fusarium Col-0 (wild type) R pad4 S eds5/sid1 S eds16/sid2 S eds12 R npr1 R eds10 S eds4 S eds11 R eds6 R eds9 R eds3 S eds-N26 S

Cloning eds3 Previous work with eds3: (Kristen’s Work) isolated for Eds to Pseudomonas syringae segregated in F2 150 Eds+ : 51 Eds– in outcross to Ler Genetics (1996) 143: 973. claimed to be 14 cM telomeric from LFY3 TAIR database, from Meinke?

Eds to Pseudomonas syringae 3 days post inoculation: O.D. 0.0002 MOCK Col-0 Ler eds3 (Col-0)

Eds to Fusarium oxysporum wild type pad4 esd3 edsN26 1 X 106 1 X 105 1 X 105 conidia / mL

Genotyping of Eds– plants from a F1 selfed population C = Col-0/Col-0 H = Col-0/Ler L = Ler/Ler

Candidate gene approach: WRKY51 is located exactly where eds3 is claimed to be: about 14 cM telomeric from LFY3. WRKYs are transcriptional regulators which are regulated by pathogen recognition and regulate other disease-related genes. WRKY51 is one of three unusual WRKYs and lacks conservation at amino acids residues important for DNA binding. We have PCR amplified the WRKY51 sequence from wildtype and eds3 DNA as two overlapping fragments.

Fusarium oxysporum haploid genome 40-50 Megabasepairs 8,000-13,000 genes sequenced genome (Fusarium graminearum) simple transformation targeted knockouts

Fusarium mutants have reduced virulence. pks8 pacC fmk2 fmk1 WT 3106 1106 Susceptible Sg-1 ecotype inoculated with dilutions of f. conglutinans, race 2 mutants 3105 conidia/mL 1105 3104 1104 mock

(2) genetic screens for Fusarium mutants Loss of virulence Loss of root adhesion Plate assays

Loss of virulence inoculation OR

Loss of root adhesion 15-30’ mutant: {fmk1}

Acknowledgements Fred Ausubel Corby Kistler Julia Dewdney Simone Ferrari Kristin Hladun Sachiko Miyata Joulia Plotnikova Mary Wildermuth Corby Kistler USDA Cereal Disease Lab University of Minnesota Support is provided by a grant from the National Institutes of Health. This