Biotecnologie ambientaliaa 2012-2013

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)

Le piante sono cibo per:

Uomo

Animali

Insetti

Nematodi

Microorganismi

Batteri

Funghi

...

Virus

Fattori biotici ed abiotici di perdita di produzione

Perdite colturali

Fattori biotici Fattori abiotici

Luce

-Carenza

-Eccesso

Infestanti

-Monocot.

-Dicot.

-Erbe parassite

Animali

-Insetti

-Acari

-Nematodi

-Lumache

-Mammiferi

-Uccelli

Patogeni

-Funghi

-Batteri (fitoplasmi)

Acqua

-Siccità

-Allagamento

Temperatura

-Caldo

-Freddo

-Gelo

Sostanze

(nutrienti)

-Carenza

-Eccesso

(tossiche)

-Osmosi

-Inibizione

Virus/ viroidi

Ferite

Determinano una perdita quantitativa e talvolta anche qualitativa

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)

Patogeni

Perdite di produzione potenziale ed attuale cumulative per le principali colture (frumento, riso mais, orzo, patata, soia, barbabietola e cotone) nel periodo 1996-1998 per le sole cause di tipo biotico. Efficacia = perdita potenziale evitata.

Perdite di produzione potenziale ed attuale (2001-2003) per le principali colture

Cause di tipo biotico

Patate trattate e non trattate con fungicidi (contro la Phytophtora)

A major contribution of biotechnology to increase environmental sustainability of agriculture is the development of pest resistant varieties

Reduction of pesticide treatment & yield increases

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.

- 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

Biotechnological approaches

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

Pathogens recognized through:

- conserved microbial molecules (PAMPs)

- microbial virulence effectors (avr proteins)

pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), through PRRs, leading to PAMP-triggered immunity (PTI).

microbial virulence effectors, usually through intracellular resistance proteins (R proteins), causing effector-triggered immunity (ETI).

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

─ Non-host resistance ─ Basal─ Broad spectrum

─ Host resistance─ R-gene mediated─ Race specific

The majority of plants are immune against the majority of microbes with pathogenic potential

T-B6: Arabidopsis transgenica con il gene RPW8 che conferisce resistenza all’oidio

- EDS1: enhanced disease susceptibilityThe 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)

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)

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 encounterIt 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)

Pathogens

• 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

Biotrophic (co-survive) Necrotrophic (kill)

Hemi-biotrophic (let alive and then kill)

Recognition of specific pathogen-produced molecules (Avr) by the corresponding plant receptors encoded by disease resistance (R) genes

Receptor coded by R allele

(a) If an Avr allele in the pathogen corresponds to an R allelein the host plant, the host plant will have resistance,making the pathogen avirulent. R alleles probably code forreceptors in the plasma membranes of host plant cells. Avr allelesproduce compounds that can act as ligands, binding to receptorsin host plant cells.

Avirulent pathogen

• A pathogen is avirulent if it has a specific Avr gene corresponding to a particular R allele in the host plant

Signal molecule (ligand)from Avr gene product

Avr allele

Plant cell is resistantAvirulent pathogen

R

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 diseasedVirulent pathogen

Virulent pathogen

No R allele;plant cell becomes diseased

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 AvrConsists of local resistance and SAROften associated with HR (in case of biotrophs) and SARExtensively studied

3 In a hypersensitiveresponse (HR), plantcells produce anti-microbial molecules,seal off infectedareas by modifyingtheir walls, andthen destroythemselves. Thislocalized responseproduces lesionsand protects otherparts of an infectedleaf.

4 Before they die,infected cellsrelease a chemicalsignal, probablysalicylic acid.

6 In cells remote fromthe infection site,the chemicalinitiates a signaltransductionpathway.

5 The signal is distributed to the rest of the plant.

2 This identification step triggers a signal transduction pathway.

1 Specific resistance is based on the binding of ligands from the pathogen to receptors in plant cells.

7 Systemic acquiredresistance isactivated: theproduction ofmolecules that helpprotect the cellagainst a diversityof pathogens forseveral days.

Signal

7

6

54

3

2

1

Avirulentpathogen

Signal transductionpathway

Hypersensitiveresponse

Signaltransduction

pathway

Acquiredresistance

R-Avr recognition andhypersensitive response

Systemic acquiredresistance

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

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

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)

Systemic Acquire Resistance (SAR)

• Secondary response

• Systemic

• Broad-range resistance

• Leads to Pathogenesis-Related (PR) gene expression

• Signals spreading the message: SA, JA, ethylene

Salicylic Acid (SA)

• 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

COOHOH

Systemic Acquired Resistance

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

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

general elicitors = PAMPs

... will be understood even byscientists in the animal/medical field!

The paradigm: bacterial flagellin

Flagellin is the main building block of the flagellum

(Illustration by Georg Felix & Thomas Boller)

Flagellin structure

(Georg Felix, J. Duran, Sigrid Volko & Thomas Boller, Plant J. 18, 265-276, 1999)

"flg22": highly conserved domain in N-terminus

Flagellin, N-terminal

... up to now, the best "general elicitor" in plants ...

QRLSTGSRINSAKDDAAGLQIA

FLS2, a receptor-like kinase, recognizes flg22

flg22

ion flux

ethylene

PR gene expressioncallose depositiongrowth inhibition

ROS

FLS2

(Lourdes Gómez Gómez & Thomas Boller, Mol. Cell 5, 1003-1011, 2000)

TLR5, a Toll-like receptor, recognizes flagellin in mammals

P.F. McDermott et al., Infect Immun. 68, 5525-5529, 2000: High-affinity interaction between flagellin and a cell surface polypeptide results in human monocyte activation

F. Hayashi et al., Nature 410, 1099-1103, 2001: The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5.

EC50

FliC S. enterica 15 pM

FliC S. typhimurium 24 pM

FljB S. typhimurium 13 pM

FliC P. aeruginosa 110 pM

Fli103 3000 pM

Fli108 330 pM

Fli109 420 pM

Fli228 >30000 pM

Fli229 >30000 pM

flagellin

Flagellin sensing in human monocytes

Location of epitope

Smith et al., Nature Immunol. 2003

Location of epitope recognized in plants and animals

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.

N-terminal fragment of the bacterial elongation factor Tu (EF-Tu)

EF-Tu fragments elicit response in A. thaliana.

Lacombe et al., (2010)

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.

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)

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.

plants expressing EFR showed drastically reduced wilting symptoms

Lacombe et al., (2010)

Activation and suppression of PTI during pathogen infection: Avr genes suppress general non-host resistance mechanisms

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

MAMP perception (PRRs)

MAPK signaling

RNA metab (GRP7)

Regulators of PTI

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

Resistance genes and conserved motives identified to date

Structure of a LRR domain

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, 742-744.