Biotecnologie ambientali aa 2013-2014

1. Biotecnologie ambientaliaa 2013-2014

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)

4. Le piante sono cibo per: Uomo Animali Insetti Nematodi Microorganismi Batteri Funghi (Virus) PARASSITA: organismo che vive a spese di un altro traendone nutrimento. SAPROFITA: si nutre di materia organica morta. PATOGENO VEGETALE: organismo che per completare il ciclo vitale cresce dentro la pianta e così facendo ha effetti negativi sulla pianta. Virulento: patogeno che causa malattia di un determinato patogeno.

5. Fattori biotici ed abiotici di perdita di produzione 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 spesso anche qualitativa Sono un problema per la sicurezza alimentare, la salute e l’ambiente

6.  Salute 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)

7. Infection by Aspergillus Infection by Fusarium

8. Major mycotoxins and their effects Fumonisin B1, B2 and B3 are 1. Carcinogenic (liver, kidney, esophagus) • Teratogenic (neural tube defects) • Stunting agents Aflatoxin B1 and G1 are • Mutagenic (chromosomal aberrations) • Carcinogenic (liver, kidney) Aflatoxin B1 Fumonisin B1 Aflatoxin is a known human carcinogen Strict contamination limits apply Fumonisin is a known teratogen

9. Quetzaltenango has a mostly indigenous population that consumes high amounts of maize as their staple food   Alcune popolazioni presentano un’alta incidenza di difetti del tubo neurale (NTD per 10000 nati vivi) General U.S. popul.: <3 Quetzaltenango: 106 US Mean incidence and range in incidence of various locations within the regions or countries are shown; the bar for Limpopo represents one data point. In Italy the incidence of spina bifida is 3.3/10,000 in the 1992-1999 period. Total NTDs: 6.8 Marasas et al. (2004)

10. Spina bifida (a common form of NTD) Spina bifida (Latin: "split spine") is a developmental birth defect caused by the incomplete closure of the embryonic neural tube. Some vertebrae overlying the spinal cord are not fully formed and remain unfused and open. If the opening is large enough, this allows a portion of the spinal cord to protrude through the opening in the bones. There may or may not be a fluid-filled sac surrounding the spinal cord. Other neural tube defects include anencephaly, a condition in which the portion of the neural tube which will become the cerebrum does not close, and encephalocele, which results when other parts of the brain remain unfused. http://en.wikipedia.org/wiki/Spina_bifida Spina bifida http://www.scienceclarified.com/images/uesc_02_img0090.jpg

11. 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. Cause di tipo biotico Perdite di produzione potenziale ed attuale (2001-2003) per le principali colture  Ambiente: 1) occorre coltivare più terra e 2) i trattamenti hanno effetti ambientali

12.  Economia 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 potential contribution of biotechnology to improve the sustainability of agriculture is the development of pest resistant varieties  Reduction of pesticide treatment & yield increases

13. Conosci il tuo nemico: caratteristiche dei patogeni virulenti Cell wall degrading enzymes – especially necrotrophic fungi with broad host range Toxins (both host selective and non selective) – es. HC-toxin from Cochliobolus carbonum or Fusicoccin from Fusicoccum amygdalii Stimola la H+-ATPasi della membrana plasmatica di tutte le piante  stomi aperti Inhibits maize histone deacetylase (HD) at 2 μM (host selective)

14. Haustoria – specialized feeding structures (especially biotrophic fungi) Austoriodi oidio (Erisiphe graminis) Gli austori implicano una grande superficie di contatto tra le membrane plasmatiche del patogeno e dell’ospite: sono funzionali allo scambio di nutrienti

15. Classificazione: -Necrotrofi -Biotrofi -Emibiotrofi  Conosci le tue difese

16. Plant defense systems Secondary metabolites: not directly involved in growth or reproduction. Often involved in plant defense. Divided into 3 large chemical classes: terpenoids, phenolics, and alkaloids. - Physical barriers Thrichomes (leaf hairs), wax, cuticle, cell walls... - Chemical defenses (metabolites and proteins) Preformed: antimicrobial compouns such as phytoalexins, gluosinolates, cyanogenic glucosides…) Terpenoids (basati sull’isoprene) Esempi di metaboliti secondari che rendono la pianta resistente alle pesti Gossypol Digoxin Azadirachtin

17. Medicarpin, a phytoalexin Nicotine Alkaloids Psoralen, a furanocoumarin Caffeine Phenylpropanoids Theobromine

18. - Chemical defense Induced: PR (Pathogenesis Related) protein pathogen-degrading enzymes (chitinases, glucanases, protease inhibitors…) e.g ROS produced at infection sites ROS: highly reactive oxygen species (molecules) capable of damaging the cells of invading organisms; also called oxidative burst - Cell suicide (Apoptosis) One form of inducible defense is the HR (Hypersensitive response) triggered by the presence of the pathogen Plant cells also respond to microbial attack by rapidly synthesizing and depositing callose between the cell wall and cell membrane adjacent to the invading pathogen

19. PR proteins • Le proteine relative alla patogenesi (PR) sono proteine tossiche per il patogeno prodotte in grande quantita quando la pianta viene attaccata • Si trovano sia a livello intra che intercellulare a livello di parete vegetale • I diversi gruppi di PRP sono stati classificati in base alla funzione svolta, alle relazioni sierologiche, alle sequenze AA, al MW e ad altre proprieta’ • Le PRP possono esssere o estremamente acide o basiche e quindi facilmente solubili e reattive • Si conoscono almeno 14 classi di PRP, tra queste le PR1, (anti-oomiceti e anti-fungine), le PR2 (b-1,3-glucanasi), le PR3 (chitinasi), le PR4 (antifungine), le PR6 (inibitori di proteasi), le defensine, le thionine, I lisozimi, le proteine simili all’osmotina, le LOX, le proteine ricche in cys, le proteinasi, le chitosanasi e le perossidasi

20. - Immune system Active recognition of pathogens through: - conserved microbial molecules (PAMPs)  basal resistance - isolate-specific pathogen / virulence effectors (avr proteins) Quando la pianta riconosce la presenza del patogeno e riesce ad attivare i sistemi di difesa, ne blocca la crescita e il danno è piccolo o trascurabile. Se il patogeno elude o inattiva i sistemi di difesa la malattia procede, con danno più o meno grave sulla produzione Understand & exploit natural defense mechanisms to maximize yield and quality with minimum amount of pesticides

21. Plant defenses • Organisms not causing disease on any plant species, (e.g. bacterial species Pseudomonas putida), are referred to as non-pathogens. • A plant species that does not show disease when infected with a pathogen is referred to as a non-host plant species for that pathogen. When a pathogen is capable of causing disease on a particular host species, two outcomes are possible: a compatible response is an interaction that results in disease, while an incompatible response is an interaction that results in little or no disease at all.

22. Foglie di due varietà di orzo [APEX (mlo), DIAMANT (Mlo)] inoculate con tre diverse isolati di oidio (mildew). Ceppo avirulento Ceppo virulento Ceppo medio-virulento Reazione o risposta compatibile: DIAMANT con T465 Reazione o risposta incompatibile: APEX con T465 compatible - incompatible responses: what is the difference? Some individuals may harbor genes that help recognize the presence of the pathogen and activate defenses

23. Key terms • Race - strains of a species or pathovar with specificity for particular plant varieties (e.g., “cultivars”) • Resistance gene - dominant gene in the plant that mediates recognition and defense response to specific pathogen race • Avirulence gene - bacterial gene required to elicit R-gene-mediated defense; usually encodes secreted effector protein • HR - hypersensitive reaction; localized cell death; resistance marker

24. PAMPs (or MAMPs, microbe-associated), induce resistance through Pattern Recognition Receptors (PRRs)  PAMP / MAMP-triggered immunity (P/MTI). ─ Non-host resistance ─ Basal ─ Broad spectrum The majority of plants are immune against the majority of microbes with pathogenic potential Pathogens recognized through: - conserved microbial molecules (PAMPs) - microbial virulence effectors (avr proteins) ─ Host resistance ─ R-gene mediated ─ Race specific microbial virulence effectors, usually through intracellular resistance proteins (R proteins), causing effector-triggered immunity (ETI). ETI invokes intracellular NB-LRR proteins that detect either the actions or structures of pathogen effectors

25. The innate immune system detects potentially dangerous microbes through PAMPS The recognition is mediated by pattern recognition receptors (PRRs) Part of non-host resistance: it is the first layer of the plant immune system Could be target of pathogen effectors (can be blocked by succesful pathogens) May share signaling components with R-gene resistance

26. 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 their ligands (PAMPs) in the apoplastic space. Typically PRRs are 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: the paradigm is bacterial flagellin 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)

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

28. Flagellin, N-terminal QRLSTGSRINSAKDDAAGLQIA ... up to now, the best "general elicitor" in plants ... "flg22": highly conserved domain in N-terminus (Georg Felix, J. Duran, Sigrid Volko & Thomas Boller, Plant J. 18, 265-276, 1999)

29. flg22 FLS2 ion flux ROS PR gene expression callose deposition growth inhibition ethylene FLS2, a receptor-like kinase, recognizes flg22 Also mammaliansa use flagellin as a PAMP. TLR5, a Toll-like receptor, recognizes flagellin in mammals (Lourdes Gómez Gómez & Thomas Boller, Mol. Cell 5, 1003-1011, 2000)

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

31. 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. Another PAMP: EF-Tu receptor 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 (EF-Tu Receptor), 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.

32. N-terminal fragment of the bacterial elongation factor Tu (EF-Tu) EF-Tu fragments elicit response in A. thaliana. Lacombe et al., (2010)

33. 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)

34. 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

35. MAMP perception (PRRs) Regulators of PTI MAPK signaling RNA metab (GRP7) 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

36. Quantitative output of the plant immune system 4 2 3 Phase 1 The ultimate amplitude of disease resistance or susceptibility is proportional to [PTI – ETS + ETI]. In phase 1, plants detect microbial/pathogen-associated molecular patterns (MAMPs/PAMPs, red diamonds) via PRRs to trigger PAMP-triggered immunity (PTI). In phase 2, successful pathogens deliver effectors that interfere with PTI, or otherwise enable pathogen nutrition and dispersal, resulting in effector-triggered susceptibility (ETS). In phase 3, one effector (indicated in red) is recognized by an NB-LRR protein, activating effector-triggered immunity (ETI), an amplified version of PTI that often passes a threshold for induction of hypersensitive cell death (HR). In phase 4, pathogen isolates are selected that have lost the red effector, and perhaps gained new effectors through horizontal gene flow (in blue)—these can help pathogens to suppress ETI. Selection favours new plant NB-LRR alleles that can recognize one of the newly acquired effectors, resulting again in ETI.

37. ETI: R-gene mediated resistance • Dominant and usually NOT broad-spectrum • Classically referred to as gene-for-gene (vertical or race-specific) resistance • Major focus (genetic simplicity and economic importance) in breeding programs to control plant diseases • 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. I geni di resistenza hanno spesso motivi conservati: il più comune è LRR Un altro dominio spesso presente è NBD (nucleotide binding site)

39. Structure of a LRR domain

40. 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 R Avr allele Avirulent pathogen Plant cell is resistant

41. R No Avr allele; virulent pathogen Plant cell becomes diseased Avr allele No R allele; plant cell becomes diseased Virulent pathogen Virulent pathogen No R allele; plant cell becomes diseased 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 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

42. 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 • Recognition induces defense responses 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. It is often rapidly overcome by evolving pathogens that lose or mutate the nonessential recognized effector or that produce new effectors to counteract ETI The R gene–mediated defenses typically involve a rapid, localized necrosis, or hypersensitive response (HR), at the site of infection, and the localized formation of antimicrobial chemicals and proteins that restrict growth of the pathogen.

43. 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 cellsrelease a chemicalsignal, probablysalicylic acid. 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. 5 The signal is distributed to the rest of the plant. Signal 5 4 Signaltransductionpathway 6 Hypersensitiveresponse 3 6 In cells remote fromthe infection site,the chemicalinitiates a signaltransductionpathway. Signal transductionpathway Acquiredresistance 2 7 2 This identification step triggers a signal transduction pathway. 7 Systemic acquired resistance isactivated: theproduction ofmolecules that helpprotect the cellagainst a diversityof pathogens forseveral days. 1 Avirulentpathogen 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

44. Cell autofluorescence Hypersensitive cell death Response of a barley epidermal cell in response to an attack by a germinating spore of barley powdery mildew.

45. 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

46. Alcune tra le ROS più comuni: H2O2 HO· (radicale ossidrile) O2- (anione superossido) NO O2* (Ossigeno singoletto) stato eccitato semi-stabile dell’ossigeno molecolare ROS are toxic by-products of aerobic metabolism, but are also signaling molecules involved in several developmental processes in all organisms. An oxidative burst often takes place at the site of attempted invasion during the early stages of most plant-pathogen interactions. A second ROS production can be observed during certain types of plant-pathogen interactions, triggering hypersensitive cell death.

47. Diversi processi producono, covertono e degradano le ROS (SOD, Catalasi…) Processi coinvolti nella risposta ai patogeni

48. Systemic Acquire Resistance (SAR) • Secondary generalized defense response • Systemic (distant from the infection site) • Broad-range resistance • Leads to Pathogenesis-Related (PR) gene expression • Triggered by the signal molecules (which activate plant defenses throughout the plant before infection spreads) • Signals involved: SA (Salicylic Acid), Methyl Salicylate, JA (Jasmonic Acid), ethylene

49. COOH COOCH3 OH OH Salicylic Acid (SA) & Methyl Salicylate • SA accumulates in both local and systemic tissues (systemic signal: MeSalycilate?) • Removal of SA (as in NahG plants) prevents induction of SAR

50. Mutanti alterati nell’Acido Salicilico • reduced SA accumulation • eds1 (enhanced disease susceptibility 1): lipase homolog • NahG (salicilato idrossilasi): gene batterico che degrada il SA • pad4 (phytoalexin deficient 4): another lipase homolog • sid1 and sid2 (salicylic acid induction-deficient): defects in chorismate pathway