The Molecular Basis of Bacterial Innate Immunity in Arabidopsis thaliana

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1 The Molecular Basis of Bacterial Innate Immunity in Arabidopsis thaliana Brian Staskawicz Department of Plant and Microbial Biology University of California, Berkeley

2 Rice Model Plant-Pathogen Systems Arabidopsis

3 Breeding for Plant Disease Resistance and Selection for New Virulent Strains in the Field resistant germplasm identified at geographical origin of species (co-evolution of host and pathogen) interspecific hybrids and recurrent selection resistance is usually controlled by single dominant loci planting resistant variety selects for virulent pathogen breeding cycle is repeated RESULT: Natural genetic variation occurs in both the host (R gene) and the pathogen (effector gene).

4 Features of Innate Immunity Plants No mobile cells, no specialized immune cells No adaptive immunity Innate immunity using germ-line encoded pathogen receptors (150+ Arabidopsis) Innate immunity recognizes mainly a diversity of Type III effector proteins Mammals Mobile, specialized surveillance system Adaptive immunity (>10 8 potential antigen binding sites) Innate immunity recognizes a small set of conserved pathogen molecular patterns

5 Arabidopsis thaliana as a Model Host Plant

6 Functional Pathogenomics Genomic sequences of both Arabidopsis and Pseudomonas syringae have been completed. Arabidopsis thaliana Mb; 25,498 genes in 11,000 families. Pseudomonas syringae DC Mb in size.

7 Forward and Reverse Genetic Approaches Forward genetic plant screens ;chemical and radiation mutagensis. Arabidopsis - T-DNA mutagenesis, transposon mutagensis and RNAi. Pseudomonas - site-directed gene replacement. DNA (Affymetrix) microarrays available for Arabidopsis and Pseudomonas syringae. Proteomic approaches being developed in both organisms.

8 Pseudomonas syringae infection of Arabidopsis thaliana

9 plant apoplast host target colonization and disease plant cytoplasm effector proteins R gene host recognition resistance

10 Phenotypes Molecular Basis of Bacterial of Plant-Pathogen Disease Interactions Resistance resistance susceptibility resistance at cellular level cells die to prevent pathogen spread

11 Pseudomonas syringae effectors and Arabidopsis resistance proteins Susceptibility Resistance Apoplast Cell Wall Cytosol Pseudomonas syringae Pseudomonas syringae Secretion of 40+ effectors Suppression of host defenses Increased pathogen proliferation Disease Recognition of effectors Localized cell death Systemic resistance Arrest of pathogen growth

12 Conservation of Type III Secretion Loci plant pathogens Pseudomonas syringae Ralstonia solanacearum Xanthomonas spp. Erwinia spp. plant symbiont Rhizobium spp. animal pathogens Yersinia spp. (2) Salmonella spp. (SPI1, SPI2) Shigella spp. enteropathogenic E. coli enterohemorrhagic E. coli Pseudomonas aeruginosa Chlamydia spp. Bordetella spp. Burkholderia pseudomallei

13 Arabidopsis Disease Resistance Proteins LRR-TM kinase class total # = 174 L R R apoplast PM cytoplasm TIR CC TIR kinase NBS NBS NBS NBS-LRR class total # = 149 L R R L R R

14 Arabidopsis Disease Resistance Genes Genes

15 Innate Immune Receptors in Plants and Animals Innate Immune Receptors in Plants and Mammals Mammals Plants Peptidoglycan LPS Flagellin AvrXa21 Avr9 TLR2 CD14 TLR4 TLR5 FLS2 Xa21 Cf9 TIR TIR TIR PK PK NOD1 RPS4 CARD NBS TIR NBS CARD CARD NBS NOD2 Peptidoglycan CC NBS RPS2 AvrRps4 effector AvrRpt2 effector

16 Model for RPS2-mediated Innate Immunity Apoplast Cell Wall Cytosol RIN4 RPS2 NDR RIN4 Pseudomonas syringae NDR RIN4 is a negative regulator of RPS2 and NDR1 activity. Evidence: RPS2 R I AvrRpt2 N Defense responses R I AvrRpt2 N A. Overexpression of RPS2 leads to constitutive defense response. B. Overexpression of RIN4 inhibits the activation of RPS2 resistance. C. rin4/rin4 homozygous mutants are lethal in presence of RPS2. In the presence of AvrRpt2, RIN4 is eliminated and RPS2 disease resistance is activated.

17 RPS2-Mediated Disease Resistance in Arabidopsis RPS2 1 LZ P Loop Kinase 2 3A LRR (CC-NBS-LRR Protein) RPS2 activation in the presence of AvrRpt2. rps2 - plants are susceptible to P. syringae DC3000::AvrRpt2. AvrRpt2 71 C122 H208 D (Cysteine Protease Effector) P. syringae (-AvrRpt2) cause disease on Arabidopsis. Mutations in catalytic triad are inactive. (6) VPKFGNW(12) (148) VPKFGDW (154) RIN4 NDR (RPM1-Interacting Protein-4) (A Negative Regulator of RPS2 Activation) ω-site rin4 - is lethal. RIN4 over-expression leads to enhanced disease susceptibility. Required for RPS2, RPM1 and RPS5-mediated resistance. Plasma membrane-localized, GPI anchored.

18 AvrRpt2 is delivered to the plant cell via a Type III Secretion System G71-G72 TTSS effector protease Cleavage site Processed during pathogenesis within plant cell via eukaryotic protein factor. C-terminal portion sufficient for RPS2 recognition. Enhances growth of pathogen in absence of RPS2.

19 Predicted AvrRpt2 secondary structure is similar to the cysteine protease Staphopain 3D-PSSM (Kelley, et al., 2000)

20 Purification of Recombinant AvrRpt2 Protein for in vitro Studies A. 500uM IPTG 37C His 6 -C122A-HA kda 0h 1h 2h 4h 6h B. Elution from Ni-NTA resin mM Imidazole Gradient C. Gel Filtration Chromatography D. Pure Protein ~2.5mg AvrRpt2 C122A 20 20

21 Processing of AvrRpt2 in Yeast 1. Purified AvrRpt2 N71 and full-length (lanes 1 &2), 2. Protein extracts from yeast not expressing AvrRpt2 (lanes 3 & 4) 3. Yeast expressing full-length AvrRpt2 (lane 5) 4. Extracts from yeast not expressing AvrRpt2 (lane 6) 5. Yeast expressing AvrRpt2-HA were probed with HA antisera (lane 7&8).

22 Protein Enabling AvrRpt2 Processing is Common among Eukaryotes Col-0 S. cerevisiae AvrRpt2:HA x x x x Col-0 S. cerevisiae C122A:HA x x x x αha

23 Biochemical Purification of Eukaryotic Protein Factor (EPF) from S. cerevisiae Protein Enabling AvrRpt2 Processing Chromatography & Purification Hydrophobic Interaction Ion Exchange Gel Filtration AvrRpt2 Processing Gel Filtration