Julian Avila 5/20/2014 PLANT PATHOGEN INTERACTIONS IN TODAYS LECTURE: A few types of plant pathogens INNATE IMMUNITY Plant's tools for preventing disease Melotto et al., Stomata in PTI. Plant disease Pseudomonas as a model for studying plant- pathogen interactions

Why should we care? Liz West

THE STRATEGIES PATHOGENS USED ARE DIVERSE Biotrophic HOST SPECIALIZED Necrotrophic HOST SPECIALIZED NON-HOST SPECIALIZED Hemitrophic Phytophtora infestans Powdery mildew on cabbage (Oomycetes) Botrytis cinerea Pseudomonas syringae

Plant pathogens in the news Liz West

LIMEPOCALYPSE

Retrieved on 5/13/14

SAP

Huanglongbing on mandarin orangesmandarin oranges 黃龍病 -Insect transmitted -Bacterial (Candidatus liberibacter) -Phloem restricted -Originally found in Asia -Appeared in Florida in Reported in Mexico in No treatment or resistant variety known CITRUS GREENING DISEASE (Huanglongbing)

PATHOGENS PLANTS Using what you have learned in your lifetime, compile a list of all the strategies you can think of that pathogens and plants have developed to cause and prevent disease respectively. For example The pathogen Candidatus liberibacter enters plant tissue in wounds caused by plant-feeding insects. Plants release chemical compounds to warn other plants of insect attacks. vs.

PATHOGENS PLANTS vs. COLONIZE -ENTRY MECHANISMS THRIVE -REPLICATE -DIVIDE -REPRODUCE

DETECTION SYSTEMS CHEMICAL WEAPONS PHYSICAL BARRIERS SABOTEURS DECOYS

Pseudomonas syringae as pathogenesis/resistance model Bacterial Speck Disease Caused by Pseudomonas syringae pathovar tomato (Pst)

by Tom Zitter and R. Thilmony Pseudomonas syringae as a model Conveniently, Pst can also infect Arabidopsis thaliana PNAS 102(31): 11064–11069 Conveniently, different P. syringae strains have been sequenced P. syringae pv. syringae B728a vs. P. syringae pv. Tomato DC300

Pst colonization mechanism INFECTION OF SUSCEPTIBLE PLANTS -Pst can survive on the leaf surface. -Pst enters the apoplast through stomata or wounds Melotto et al EPIPHYTIC GROWTH APOPLASTIC GROWTH STOMATA

A) Open (top and middle panels) and closed stomata (bottom panel). (B) Stomatal aperture in intact leaves (left panel) or epidermal peels (right panel) of Col-0 plants exposed to water (white bars) or Pst DC3000 (gray bars). In this and all other figures, results are shown as mean (n = 60 stomata) ± SEM unless otherwise noted. Melotto et al Colonization

PLANT RESPONDS TO THIS NON-SPECIFICALLY Pst USED A WEAPON 2h 4h Melotto et al Stomatal aperture in epidermal peels of Col-0 plants exposed to water (white bars) or E. coli O157:H7 (gray bars). Colonization Pst DC3000E. coli O157:H7

What do plants sense in pathogens? (E) Effect of the Nitric Oxide Synthase (NOS) inhibitor L-NNA (0.2 mM) on stomatal closure when coincubated with PAMPs (5 mM flg22 or 100 ng/ml LPS) THIS FIGRE GIVES US INFORMATION ON: -EXAMPLES OF WHAT PLANTS SENSE -A GLIMPSE INTO THE MECHANISM BY WHICH STOMATA CLOSE IN RESPONSE TO SENSING BACTERIA

FLAGELLIN ELONGATION FACTOR Tu PEPTIDOGLICAN LIPOPOLYSACCHARIDE CHITIN XYLANASE HEPTAGLUCAN (HG) PATHOGEN/MICROBIAL ASSOCIATED MOLECULAR PATTERNS (PAMPs/MAMPS) *flg22 is a short immunogenic fragment of flagellin

FLAGELLIN QRLSTGSRINSAKDDAAGLQIA Flg22: The Plant Journal (1999) 18(3), 265–276

PAMP TRIGGERED (INNATE) IMMUNITY - PTI The perception of PAMPS triggers a set of defense responses. In the next couple of slides we will explore some of them.

Transient ROS production in response to live Pst DC3000 wild type Arabidopsis Col-0. A: Time-course of ROS production in response to Pto DC3000 (n = 48/treatment). RLU, Relative Light Units PAMP TRIGGERED (INNATE) IMMUNITY - PTI Pst

How does this experiment work? WHY WOULD PLANTS PRODUCE H 2 O 2 ?

REACTIVE OXYGEN SPECIES CHEMICAL WEAPONS

PTI STOMATAL CLOSURE REACTIVE OXYGEN SPECIES

flg22 activates MPKs. MAPK activation with or without 100 nM flg22 for 10 min (top) and expression of each MAPK are shown (bottom). (Tagged kinases were immunoprecipitated from lysates of transfected protoplasts with the corresponding antibody and analysed with a known substrate as indicated.) Myelin basic protein (an MAPK substrate) Mitogen Activated Protein Kinase PAMP TRIGGERED (INNATE) IMMUNITY - PTI MAPKs

One of the most ubiquitous types of post-translational modifications STRUCTURE ACTIVITY LOCALIZATION PROTEIN STABILITY INTERACTION PARTNERS PROTEIN PHOSPHORYLATION

KINASE ASSAY MAPKs MAPK9 TAG Plant protoplasts Plasmid TAG-binding resin MAPK MBP γ- 32 P- ATP P The amount of incorporated phosphate shows the catalytic activity of the kinase. Which MAPK is activated by flg22? ( 32 P incorporated)

MAPKKKs MAPKKs MAPKs RESPONSE Stimuli Mitogen Activated Protein Kinase Cascade MEK1? MAPKK4/ MAPKK5 MAPK3/ MAPK6 PAMP TRIGGERED (INNATE) IMMUNITY - PTI MAPKs Response PPPPPP ( 32 P incorporated) Stimulus 1 Stimulus B RESPONSE A RESPONSE B

PTI STOMATAL CLOSURE REACTIVE OXYGEN SPECIES MAPK CASCADE ACTIVATION CALLOSE DEPOSITION Ca ++ Influx

Pst STRIKES BACK CHEMICAL WEAPONS CORONATINE A) Col-0 leaves were exposed to water (white bars), Pst DC3000 (wavy bars), or the cor mutant Pst DC3118 (gray bars). Bacterial concentration used was cfu/ml. COR+COR- TTSS- We will talk about this next week

Control of PAMP-triggered stomatal responses Salicylate Hydroxylase

PTI STOMATAL CLOSURE REACTIVE OXYGEN SPECIES MAPK CASCADE ACTIVATION CALLOSE DEPOSITION Ca ++ INFLUX SALICYLIC ACID

PAMP TRIGGERED (INNATE) IMMUNITY - PTI SYSTEMIC ACQUIRED RESISTANCE Figure 1. P. syringae-induced resistance to P. syringae. The photograph shows upper leaves of plants inoculated with either water (A) or with P. syringae (4x10 8 cells/ml) (B) on the lower leaves of the rosette. Two days after infection of the lower leaves, upper leaves were challenged with the same pathogen (10 7 cells/ml). Leaves were photographed 4 d after challenge. Summermatter, et al., Plant Physiol. (1 995) 108:

PAMP TRIGGERED (INNATE) IMMUNITY - PTI SYSTEMIC ACQUIRED RESISTANCE source: Taiz L., Zeiger E., 2010 Willow tree bark Genus: Salix Acetylsalicylic acid BAYER

How does it work? CHEMICAL WEAPONS CORONATINE “Typically, the pathogen-induced pathway relies on salicylic acid produced by the plant as a signalling molecule, whereas the herbivory-induced pathway relies on jasmonic acid as the signalling molecule.” Pst needs to shut this down! Biotrophic Necrotrophic

How does it work? CHEMICAL WEAPONS CORONATINE: A JASMONATE ANALOGUE? Nature Chemical Biology 5, (2009) SABOTEURS Pst Plant “..Jasmonates can suppress the proliferation of human cancer cells and induce their death. Methyl jasmonate induced death in breast and prostate carcinoma cells, as well as in melanoma, lymphoma, and leukemia cells ” Leukemia 2002; 16: 608–16

How does it work? CHEMICAL WEAPONS CORONATINE: A JASMONATE ANALOGUE Remember Brittney’s AUX/IAA story?

How does it work? CORONATINE: A JASMONATE ANALOGUE Remember Brittney’s AUX/IAA story? COR Robert Nordsieck Kästner et al., ICommunicative & Integrative Biology (1): e28728.

Questions?! Next week: Effector Triggered Immunity