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Biotecnologie ambientali aa

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1 Biotecnologie ambientali aa 2012-2013

2 Le piante coltivate e la sindrome da domesticazione: shattering e dormienza
Rischi e benefici ambientali delle piante transgeniche in paragone a quelle convenzionali. Convenzione di Rio, Protocollo di Cartagena e normativa sulle piante create tramite ingegneria genetica Piante per una maggiore sostenibilità ambientale (es. plastiche biodegradabili), per il risanamento (fitodepurazione) e come biosensori di contaminazione. Interazione pianta-microrganismo: le risposte di difesa delle piante e generazione di specie resistenti. Interazione simbiotiche pianta-microrganismo: fissazione dell’azoto (batteri azoto fissatori)

3 Le piante sono cibo per:
Uomo Animali Insetti Nematodi Microorganismi Batteri Funghi ... Virus

4 Perdite colturali Fattori biotici Fattori abiotici
Infestanti -Monocot. -Dicot. -Erbe parassite Animali -Insetti -Acari -Nematodi -Lumache -Mammiferi -Uccelli Patogeni -Funghi -Batteri (fitoplasmi) Virus/ viroidi Ferite Luce -Carenza -Eccesso Sostanze (nutrienti) -Carenza -Eccesso (tossiche) -Osmosi -Inibizione Acqua -Siccità -Allagamento Temperatura -Caldo -Freddo -Gelo Determinano una perdita quantitativa e talvolta anche qualitativa Fattori biotici ed abiotici di perdita di produzione

5 http://www. forestryimages. org/support/lightbox
Light box to register Corn rootworm Mais convenzionale (a sinistra) con evidenti rosure da piralide e infestazioni di Fusarium. Il mais Bt (a destra) risulta più sano. Perdita quantitativa (minor resa) e qualitativa (contaminazione da micotossine) 5

6 Patogeni Cause di tipo biotico
Perdite di produzione potenziale ed attuale cumulative per le principali colture (frumento, riso mais, orzo, patata, soia, barbabietola e cotone) nel periodo per le sole cause di tipo biotico. Efficacia = perdita potenziale evitata. Cause di tipo biotico Perdite di produzione potenziale ed attuale ( ) per le principali colture 2 6

7 Patate trattate e non trattate con fungicidi (contro la Phytophtora)
Crop losses caused by plant pathogens, insect pests, and weeds account for $500 billion worth of damage. Worldwide, pesticide applications costing $26 billion dollars annually are applied to manage pest losses. A major contribution of biotechnology to increase environmental sustainability of agriculture is the development of pest resistant varieties  Reduction of pesticide treatment & yield increases

8 Biotechnological approaches
- Enhancing resistance with plant genes Exploiting the plant defence mechanisms: Hypersensitive Response (HR) and Systemic Acquired Resistance (SAR) - Pathogen derived resistance For example, some viral genes can protect plants from infection by the virus from which the gene was derived - Antimicrobial proteins Fungi, insects, animals, and humans contain genes encoding antimicrobial compounds which can be used to improve plant resistance - Plantibodies Plants can be engineered to express an antibody against a protein crucial for pathogenesis resulting in a level of immunity or resistance to the pathogen

9 Plant defense systems - Physical barriers
Thrichomes (leaf hairs), wax, cuticle, cell walls... - Chemical defenses Preformed: antimicrobial compouns such as phytoalexins Induced: PR (Pathogenesis Related) protein, e.g ROS produced at infection sites and leading to HR (Hypersensitive response - Immune system Active recognition of pathogens through: - conserved microbial molecules (PAMPs) - microbial virulence effectors (avr proteins) AIM: understand & exploit natural defense mechanisms to maximize yield and quality with minimum amount of pesticides

10 pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), through PRRs, leading to PAMP-triggered immunity (PTI). ─ Non-host resistance ─ Basal ─ Broad spectrum Pathogens recognized through: - conserved microbial molecules (PAMPs) - microbial virulence effectors (avr proteins) The majority of plants are immune against the majority of microbes with pathogenic potential microbial virulence effectors, usually through intracellular resistance proteins (R proteins), causing effector-triggered immunity (ETI). ─ Host resistance ─ R-gene mediated ─ Race specific ETI classically referred to as gene-for-gene (vertical or race-specific) resistance. It generally occurs between cultivars of a given plant species bearing a particular R gene and a limited number of pathogenic strains carrying the matching virulence effector. R gene–mediated resistance is widely used in breeding programs to control plant diseases. Usually NOT a broad-spectrum disease resistance. It is often rapidly overcome by evolving pathogens that lose or mutate the nonessential recognized effector or that produce new effectors to counteract ETI

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12 - EDS1: enhanced disease susceptibility
T-B6: Arabidopsis transgenica con il gene RPW8 che conferisce resistenza all’oidio - EDS1: enhanced disease susceptibility The gene is an essential component of R gene-mediated disease resistance in Arabidopsis and has homology to eukaryotic lipases - NDR1 is a pathogen-induced component required for disease resistance and is an integrin-like protein with a role in fluid loss and plasma membrane-cell wall adhesion - NahG transgene encodes salicylate hydroxylase (destroys saliylic acid)

13 T-B6 riconosce e blocca il patogeno al sito di infezione
Northern analysis indicated that defense gene PR-1 was induced in T-B6 (B) leaves 48 hours after inoculation with E. cichoracearumUCSC1, but not in Col-0 (C) leaves. T-B6 induce prima e molto più fortemente i geni PR (Pathogenesis related)

14 PAMPs are conserved across a wide range of microbes, which may or may not be pathogenic.
Essential for viability or lifestyle, therefore microbes are less likely to evade host immunity through mutation or deletion of PAMPs, compared with virulence effectors. PTI constitutes an important aspect of non-host resistance, which accounts for why most plants are resistant to the majority of pathogens they encounter It is multilayered and has received less attention Plant cells detect microbes through Pattern-recognition receptors (PRRs) which recognize conserved pathogen-associated molecular patterns (PAMPs). Typically membrane-bound receptor-like kinases with extracellular domains for MAMP detection/binding (e.g. leucine rich repeats or carbohydrate-binding LysM domains). A few plant PRRs have been identified: Flagellin, EF-Tu… Plant mutants in which PAMP recognition is affected are more susceptible to adapted pathogens (reflecting defects in basal resistance) and allow some degree of disease progression by non-adapted pathogens (reflecting defects in non-host resistance)

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16 Pathogens Biotrophic (co-survive) Necrotrophic (kill)
Hemi-biotrophic (let alive and then kill) A virulent pathogen Is one that a plant has little specific defense against An avirulent pathogen Is one that may harm but not kill the host plant  Recognition of specific pathogen-produced molecules (Avr) by the corresponding plant receptors encoded by disease resistance (R) genes

17 Plant cell is resistant
Avirulent pathogen A pathogen is avirulent if it has a specific Avr gene corresponding to a particular R allele in the host plant Receptor coded by R allele (a) If an Avr allele in the pathogen corresponds to an R allele in the host plant, the host plant will have resistance, making the pathogen avirulent. R alleles probably code for receptors in the plasma membranes of host plant cells. Avr alleles produce compounds that can act as ligands, binding to receptors in host plant cells. Signal molecule (ligand) from Avr gene product R Avr allele Avirulent pathogen Plant cell is resistant

18 Virulent pathogen If the plant host lacks the R gene that counteracts the pathogen’s Avr gene  the pathogen can invade and kill the plant No Avr allele; virulent pathogen Plant cell becomes diseased Avr allele No R allele; plant cell becomes diseased Virulent pathogen R If there is no gene-for-gene recognition because of one of the above three conditions, the pathogen will be virulent, causing disease to develop. R-gene mediated resistance Activated upon recognition of Avr Consists of local resistance and SAR Often associated with HR (in case of biotrophs) and SAR Extensively studied

19 Plant Responses to Pathogen Invasions
A hypersensitive response against an avirulent pathogen seals off the infection and kills both pathogen and host cells in the region of the infection 4 Before they die, infected cells release a chemical signal, probably salicylic acid. 3 In a hypersensitive response (HR), plant cells produce anti- microbial molecules, seal off infected areas by modifying their walls, and then destroy themselves. This localized response produces lesions and protects other parts of an infected leaf. Signal 5 The signal is distributed to the rest of the plant. 4 5 Hypersensitive response Signal transduction pathway 6 3 6 In cells remote from the infection site, the chemical initiates a signal transduction pathway. Signal transduction pathway 2 Acquired resistance 7 2 This identification step triggers a signal transduction pathway. 1 7 Systemic acquired resistance is activated: the production of molecules that help protect the cell against a diversity of pathogens for several days. Avirulent pathogen 1 Specific resistance is based on the binding of ligands from the pathogen to receptors in plant cells. R-Avr recognition and hypersensitive response Systemic acquired resistance

20 Hypersensitive Response (HR)
Burst of oxygen reactive species around infection site Synthesis of antimicrobial phytoalexins Accumulation of Salicylic Acid (SA) Directly kill and damage pathogens Strengthen cell walls, and triggers apoptosis Restrict pathogen from spreading Rapid and local

21 Systemic Acquired Resistance
Systemic acquired resistance (SAR) Is a set of generalized defense responses in organs distant from the original site of infection Is triggered by the signal molecule salicylic acid (which activates plant defenses throughout the plant before infection spreads)

22 Systemic Acquire Resistance (SAR)
Secondary response Systemic Broad-range resistance Leads to Pathogenesis-Related (PR) gene expression Signals spreading the message: SA, JA, ethylene

23  Systemic Acquired Resistance
Salicylic Acid (SA) COOH OH Accumulates in both local and systemic tissues (not the systemic signal) Removal of SA (as in nahG plants) prevents induction of SAR Analogs: INA or BTH  Systemic Acquired Resistance

24 Mutants affecting SA synthesis
Elevated SA accumulation dnd1 (defense, no death 1): increased SA, but reduced HR, DND1 gene encodes cyclic-nucleotide-gated ion channel mpk4: constitutive SA accumulation edr1 (enhanced disease resistance 1): defective MAPKKK

25 Mutants affecting SA synthesis
reduced SA accumulation eds1 (enhanced disease susceptibility 1): lipase homolog pad4 (phytoalexin deficient 4): another lipase homolog sid1 and sid2 (salicylic acid induction-deficient): defects in chorismate pathway

26 general elicitors = PAMPs The paradigm: bacterial flagellin
... will be understood even by scientists in the animal/medical field! The paradigm: bacterial flagellin

27 Flagellin is the main building block of the flagellum
Flagellin structure (Illustration by Georg Felix & Thomas Boller)

28 "flg22": highly conserved domain in N-terminus
Flagellin, N-terminal ... up to now, the best "general elicitor" in plants ... QRLSTGSRINSAKDDAAGLQIA (Georg Felix, J. Duran, Sigrid Volko & Thomas Boller, Plant J. 18, , 1999)

29 FLS2, a receptor-like kinase, recognizes flg22
ion flux ethylene PR gene expression callose deposition growth inhibition ROS FLS2 (Lourdes Gómez Gómez & Thomas Boller, Mol. Cell 5, , 2000)

30 Flagellin sensing in human monocytes
TLR5, a Toll-like receptor, recognizes flagellin in mammals Flagellin sensing in human monocytes P.F. McDermott et al., Infect Immun. 68, , 2000: High-affinity interaction between flagellin and a cell surface polypeptide results in human monocyte activation F. Hayashi et al., Nature 410, , 2001: The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5.

31 Location of epitope recognized in plants and animals
Smith et al., Nature Immunol. 2003

32 Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) receptors are typically not variable within species and thus have not contributed widely to traditional breeding efforts. The transfer of these receptors among species has tremendous potential to deliver durable resistance, as the recognition components are highly conserved among pathogens. Although pathogens that are adapted to a particular host plant may be adept at suppressing the pattern recognition receptors (PRRs) of that host, their effectors might not recognize PRRs from other host plants. For instance, the Arabidopsis thaliana EF-Tu receptor occurs only in the Brassicaceae family, and transfer of this gene into tomato provided good resistance against various bacterial pathogens. EF-Tu (or its eliciting epitope elf18) are recognized naturally by members of the Brassicaceae through the leucine-rich repeat receptor kinases EFR. Expression of EFR, a PRR from the cruciferous plant Arabidopsis thaliana, confers responsiveness to bacterial elongation factor Tu in the solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum), making them more resistant to a range of phytopathogenic bacteria from different genera.

33 N-terminal fragment of the bacterial elongation factor Tu (EF-Tu)
EF-Tu fragments elicit response in A. thaliana. (a) Alignment of elf18 regions from selected bacteria. Capital letters on the right indicate the subgroups of elf18 peptides. Accession numbers are from UniProtKB. XANAC, X. axonopodis pv. citri 306; XANCM, X. campestris pv. musacearum 4381; XANOR, X. oryzae pv. oryzae KXO85; XANOM, X. oryzae pv. oryzae MAFF ; XANC5, X. campestris pv. vesicatoria 85-10; XANC8, X. campestris pv. campestris 8004; XANCB, X. campestris pv. campestris B100; AGRT5, A. tumefaciens C58; AGRTU, A. tumefaciens; AGRRK, Agrobacterium radiobacter K84; AGRVS, Agrobacterium vitis S4; ERWCT, E. carotovora ssp. atroseptica/P. atrosepticum SCRI1043; DIDIA, Dickeya dianthicola/E. chrysanthemi SCRI3534; ECOLI, E. coli K12; RALSO, R. solanacearum GMI1000; PSE14, P. syringae pv. phaseolicola 1448A/Race 6; PSEU2, Pss B728a; PSEPF, Pseudomonas fluorescens Pf0-1; PSESM, Pto DC3000. (b) WebLogo representation of the elf18 consensus sequence. (c) Oxidative burst triggered by elf18 peptides from different subgroups as defined in a. We calculated the eliciting activity as the amount of relative light units (RLU) produced in response to 1 μM elf18 peptide minus the amount of ROS produced in response to water in wild-type (Col-0 ecotype) A. thaliana leaf discs. Results are averages ± s.e.m. (n = 12). Lacombe et al., (2010)

34 Oxidative burst triggered by 10 μl bacterial extracts in A
Oxidative burst triggered by 10 μl bacterial extracts in A. thaliana leaf discs from wild-type (Col-0), efr, fls2 and fls2 efr plants, measured as RLU. (d) Oxidative burst triggered by 10 μl bacterial extracts from P. carotovorum 193, P. atrosepticum 1043 and D. dianthicola 3534 in A. thaliana leaf discs from wild-type (Col-0; black), efr (light gray), fls2 (gray) and fls2 efr (white) plants, measured as RLU. Results are averages ± s.e.m. (n = 12). We repeated all experiments at least three times with similar results. The response is completely abolished in efr and fls2 efr mutant leaves, revealing that the major PAMP in these extracts recognized by A. thaliana is EF-Tu. Lacombe et al., (2010)

35 transgenic EFR (right) tomato plants infected with R. solanacearum
Lacombe et al., (2010) transgenic EFR (right) tomato plants infected with R. solanacearum WT Moneymaker (MM) or VF36 and transgenic EFR- or Bs2-expressing tomato plants infected with X. perforans T4-4B. (a) Wild-type (variety Moneymaker; left) and transgenic EFR (right) tomato plants infected with R. solanacearum GMI1000. We drench-inoculated 4-week-old plants with 108 CFU ml−1 bacteria and photographed them 6 d after inoculation. (b) Disease scoring after infection with R. solanacearum GMI1000 in wild-type Moneymaker (blue) and transgenic EFR (red) tomato plants. Results are averages ± s.e.m. (n = 24). (c) Wild-type Moneymaker (MM) or VF36 and transgenic EFR- or Bs2-expressing tomato plants infected with X. perforans T4-4B. We dipped 6-week-old tomato plants in bacterial suspension (107 CFU ml−1 supplemented with 0.008% (vol/vol) Silwet-L77) and counted bacteria 14 d after inoculation. Results are averages ± s.e.m. (n = 3). plants expressing EFR showed drastically reduced wilting symptoms

36 Activation and suppression of PTI during pathogen infection:
 Avr genes suppress general non-host resistance mechanisms MAMP perception (PRRs) Regulators of PTI MAPK signaling RNA metab (GRP7) Concept of activation and suppression of PTI during pathogen infection. (A) An Arabidopsis plant showing disease symptoms (in the foreground; natural size) after infection by P. syringae bacteria (electron microscopy image in the background; magnification, 10,000×). (B) A conceptual diagram of PRR signaling and action of several P. syringae effectors for which the plant targets and immune suppression function have been characterized. Green and purple colors indicate plant targets and P. syringae effectors, respectively. Numbers in circles denote six steps targeted by effectors: MAMP perception (PRRs), the MAPK cascade (MPK3 and MPK6), RNA metabolism (GRP7), vesicle traffic (MIN7), regulators of PTI (RIN4 and RAR1), and chloroplast function (Hsp70) (22). An Arabidopsis plant infected by P. syringae with disease symptoms PRR signaling and action of several P. syringae effectors Green and purple colors indicate plant targets and P. syringae effectors, respectively

37 Resistance (R) Genes Dominant
among the most highly variable plant genes known, both within and between populations contain conserved motifs such as NBS: Nucleotide binding site Leucine-rich repeat (LRR) Leucine-zipper coiled-coil (CC) Toll/IL-1R (TIR) (Toll-interleukin-1 receptor) Protein kinase (PK), receptor-like kinase (RLKs

38 Resistance genes and conserved motives identified to date
Three novel R proteins (Xa13, Xa5 and Xa27) do not contain any conserved motifs that are known in R proteins. (NBS: nucleotide-binding site; LRR: leucine-rich repeat; TIR: Toll-interleukin-1 receptor; CC: coiled-coil; TM: transmembrane domain; PK: Protein Kinase; WRKY: WRKY domain; B-lectin: bulb-type mannose specific binding lectin domain).

39 Structure of a LRR domain
Grover and Gowthaman (2008) Strategies for development of fungus-resistant transgenic plants. Current Science 84:

40 Bibliografia Lacombe et al., (2010) Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance. Nat Biotechnol. 28:365-9. Boller & He (2009) Innate Immunity in Plants: An Arms Race Between pattern recognition receptors in plants and effectors in microbial pathogens. Science 324,


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