Plant Biotechnology: Biotic and Abiotic Stresses

Plant Biotechnology: Unit IV - Biotic and Abiotic Stresses of Plants

Introduction to Plant Stresses

Plants face various stresses that impact their growth and development. These stresses are broadly categorized into:

  • Biotic Stresses: Caused by living organisms.

  • Abiotic Stresses: Caused by non-living environmental factors.

Biotic Stresses

Plant-Pathogen Interactions

  • Involve prokaryotes, fungi, and viruses.

  • Lead to diseases, but plants have natural resistance mechanisms.

  • Disease resistance can be enhanced through biotechnological approaches, such as over-expression of PR-proteins.

  • Herbicides also act as biotic stress factors.

Disease Resistance

  • Most plants are resistant to most pathogens; disease is the exception, not the rule.

  • The interaction between plants and pathogens drives diversity in defense and pathogenicity genes.

Natural Disease Resistance in Plants

  • Plants possess pre-existing anatomical defenses like cuticle, bark, and wax.

  • They also have pre-existing antimicrobial proteins and phytoanticipins.

Biotechnological Approaches

  • Over-expression of PR-proteins.

  • Transgenic approaches for improving tolerance to herbicides.

  • Plant-based detoxification mechanisms.

Pathogens and Disease

  • Plant pathogens are infectious agents that cause disease in plants, resulting in significant yield losses (10-30% annually).

  • Up to 25% of plant genes respond to pathogen infection.

  • Examples of organisms causing plant diseases include bacteria, oomycetes, and fungi.

Germ Theory and Koch's Postulates

  • Louis Pasteur and Robert Koch established the germ theory, demonstrating that animal diseases are caused by microbes.

  • Koch’s postulates to establish a microbe as the causal agent of a disease (1880s):

    • The microbe is always associated and isolated from the patient with the disease.

    • The microbe must be grown in pure culture.

    • The microbe can be injected or inoculated into an animal (plant) and cause disease.

    • The microbe can be re-isolated in pure culture.

Disease Triangle

  • Disease development depends on the interaction between host, pathogen, and environment.

  • The pathogen must overcome plant defenses.

  • The environment must favor the pathogen.

  • The host plant must be susceptible.

Human Influence on Diseases

  • Monoculture, introduced pathogens and vectors, and growing practices contribute to disease spread.

Strategies of Pathogenicity

  • A successful pathogen must find and attach to the host, gain entry, avoid defenses, grow, reproduce, and spread.

Pathogen Arrival and Attachment

  • Wind, water, insects, and chemotaxis help pathogens reach their hosts.

  • Some pathogens use extracellular polysaccharides or hydrophobin proteins to attach to the plant surface.

Penetration and Circumvention of Physical Barriers

  • Pathogens penetrate through stomata or directly puncture the cell wall using appressoria.

  • Some pathogens produce melanized appressoria to build up high pressure and puncture the cell wall.

Pathogen Lifestyle: Biotrophs, Necrotrophs, and Hemibiotrophs

  • Biotrophs: Live within host tissue without causing cell death.

  • Necrotrophs: Kill cells and consume their contents.

  • Hemibiotrophs: Switch between biotroph and necrotroph lifestyles.

Biotroph Interactions

  • Fungal and oomycete biotrophs make haustoria for nutrient and signal exchange.

  • Haustoria remain outside the plant plasma membrane.

Plant Defense Mechanisms

  • Plants employ anatomical defenses (cuticle, bark, wax).

  • They have pre-existing antimicrobial proteins and phytoanticipins.

  • Inducible systems are triggered by elicitors, effectors, or cell wall fragments.

  • Systemic responses include systemic acquired resistance (SAR) and induced systemic resistance (ISR).

Plant Immune Responses

  • Plants resist pathogens through recognition and defense responses.

  • Pathogen recognition involves:

    • MAMPs/PAMPs (Microbe/Pathogen-Associated Molecular Patterns)

    • Effectors.

  • Plant recognition involves:

    • Pathogen recognition receptors (PRRs)

    • Resistance (R) proteins.

    • Effector targets/susceptibility factors.

Zig-Zag Model of Plant-Pathogen Interactions

  • Describes the dynamic interplay between plant immunity and pathogen virulence.

  • Pattern Triggered Immunity (PTI): triggered when the Pathogen is recognized.

  • Effector Triggered Immunity (ETI): triggered when the Effector is recognized.

  • Effector Triggered Susceptibility: Pathogen effectors suppress defense response.

Plant Defense Responses

  • Include increased synthesis of stress hormones, up-regulation of pathogenesis-related (PR) genes, synthesis of antimicrobial compounds (phytoalexins), production of reactive oxygen species (ROS), and production of callose.

Pathogen Recognition Receptors (PRRs)

  • Recognize conserved microbial elements via extracellular leucine-rich repeat domains and initiate defense responses through intracellular kinase domains.

  • Plants also respond to their own cellular damage through DAMPs (Damage-Associated Molecular Patterns).

PAMPs and PRRs

  • PRRs recognize PAMPs (pathogen-associated molecular patterns).

  • Examples include:

    • FLS2 recognizing flagellin.

    • CERK1 recognizing chitin.

Pattern Triggered Immunity (PTI)

  • Activated by PAMP/PRR interactions.

  • Involves kinase cascades and calcium ion influx triggering transcriptional responses.

Reactive Oxygen Species (ROS) in Immune Signaling

  • PAMP binding triggers phosphorylation of BIK1, activating NADPH oxidase RBOHD and producing ROS.

Phytoalexins and Phytoanticipins

  • Antimicrobial compounds that ward off pathogens; can be preformed (phytoanticipins) or induced (phytoalexins).

Defense Responses: Callose, ROS, and Phytoalexins

  • Callose acts as a barrier to pathogen attack.

  • ROS and phytoalexins are toxic to pathogens.

Pathogen Effectors

  • Enhance virulence by suppressing plant immune responses or contributing to pathogen viability.

Effector Action

  • Act outside the pathogen, in the plant cell, or in the apoplast.

  • Delivered via Type-III secretion systems (T3SS) in bacteria or secreted from haustoria/hyphae in fungi and oomycetes.

Resistance (R) Proteins

  • Intracellular immune receptors that recognize effectors.

  • Activation leads to enhanced defense responses.

R Protein Classes

  • Include NB-LRRs, Receptor-Like Proteins (RLPs), and Receptor-Like Kinases (RLKs).

R Protein Recognition of Effectors

  • Can bind directly, recognize effector complexes, or recognize effector-modified host proteins.

Effector Triggered Immunity (ETI)

  • Faster, stronger, and more prolonged than PTI.

  • Involves production of salicylic acid (SA), ROS, hypersensitive cell death response (HR), and expression of pathogenesis-related (PR) proteins.

Hypersensitive Response (HR)

  • Induced by R protein activation, leading to cell death at the site of infection.

  • ROS is generated in the apoplast, chloroplasts, and mitochondria.

Pathogenesis-Related (PR) Proteins

  • Includes B-1,3-glucanases, chitinases, and thaumatin-like proteins.

  • Known families are listed using gene symbols ypr1, ypr2, etc.

Systemic Acquired Resistance (SAR)

  • Uninfected tissues show enhanced resistance to subsequent pathogen challenge.

  • Mobile signals mediate this systemic response.

Summary of Plant Immunity

  • PTI is suppressed by effectors, which can trigger ETI.

  • R proteins recognize effectors and induce ETI.

Plant Responses to Necrotrophic Pathogens

  • Few known R genes confer resistance to necrotrophs, making breeding for resistance more challenging.

Salicylate and Jasmonate Antagonism

  • Salicylates are associated with biotroph defense, while jasmonates are associated with necrotroph defense.

Strategies to Prevent and Manage Disease

  • Avoid or eliminate the pathogen.

  • Manipulate the environment to favor the plant.

  • Make the plant resistant through genetic or other methods.

Prevention

  • Agricultural inspectors check imported plants for pests and pathogens.

  • Cultural practices, such as maintaining healthy soil, can protect plants.

Chemical Controls

  • Fungicides are used to eradicate pathogens.

Biocontrol Agents

  • Attack or compete with pathogens.

  • Enhance plant defenses through induced systemic resistance (ISR).

Genetic Approaches to Disease Resistance

  • Introgression of R genes.

  • Quantitative disease resistance breeding.

  • Biotechnological approaches to improve pathogen recognition, increase defense signaling, interfere with pathogen virulence, and prime defense responses.

Classical Approaches: R Genes and Quantitative Disease Resistance

  • R gene introgression can confer complete resistance.

  • Quantitative disease loci (QDL) promote partial disease resistance.

Exploiting R Proteins and PRRs

  • Stacking/pyramiding R genes in one cultivar.

  • Growing multiple transgenic lines, each with a different R gene.

Biotechnological Approaches: Candidate Genes for Resistance

  • Introducing pattern recognition receptors (PRRs) to improve pathogen recognition.

Biotech Approaches to Enhancing Immunity

  • Introduce or modify PRRs.

  • Overexpress or modify signaling intermediates.

  • Detoxify or sequester effectors.

  • Use biological controls or chemical induction of priming.

Effector Manipulation of Host Susceptibility Factors

  • Pathogens like Xanthomonas oryzae pv. oryzae (Xoo) secrete TAL effectors that manipulate host gene expression.

  • CRISPR-Cas9 editing can be used to mutate effector binding elements (EBEs) in susceptibility genes, resulting in resistance.

Host-Induced Gene Silencing (HIGS)

  • Transgenic plants expressing shRNA can silence pathogen genes, preventing multiplication and pathogenicity.

Viral Disease Control

  • Control the vector.

  • Use chemical controls.

  • Produce virus-free plant stocks.

  • Introgression R genes.

  • Cross protection (inoculation with a mild virus strain).

Herbs as Biotic Stress Factors

  • Herbicides are used to control weeds and can be classified by their mode of action.

Herbicide Tolerance

  • Transgenic approaches are used to improve tolerance.

  • Plant-based detoxification mechanisms are utilized.

Commercial Success of Herbicide Tolerance

  • Traits include herbicide tolerance and insect resistance.

  • Herbicide tolerance can be achieved through various mechanisms, including:

    • Agrobacterium CP4-resistant gene.

    • Maize resistant gene.

    • Oxidoreductase detoxification.

    • Acetylation detoxification.

    • Mutant plant acetolactate synthase.

Reasons for Herbicide Tolerance Success

  • Pre-existing knowledge about herbicide mode of action.

  • Availability of herbicide-resistant microorganisms and tolerant plants.

Strategies to Obtain Herbicide Tolerance

  • Overexpression of the target enzyme.

  • Mutation of the target enzyme.

  • Detoxification of the herbicide.

  • Enhanced plant detoxification.

Glyphosate (Roundup) Tolerance

  • Glyphosate inhibits EPSP synthase, disrupting aromatic amino acid biosynthesis.

Strategies for Glyphosate Tolerance

  • Overexpress wildtype EPSP synthase.

  • Overexpress mutated EPSP synthase.

  • Introduce glyphosate detoxification enzymes.

Liberty Link® / Glufosinate

  • Glufosinate inhibits glutamine synthase, leading to ammonia buildup.

  • Resistance is achieved through the bar or pat genes from Streptomyces, which break down glufosinate.

Abiotic Stresses

Nature and Plant Responses

  • Water deficit stress

  • Salt stress

  • Cold and heat stress

Abiotic Stress Definition

  • The combined effect of suboptimal abiotic environmental conditions on plant performance, reducing yield.

  • Abiotic stress tolerance is the ability to withstand these conditions better than the average plant.

Main Abiotic Stresses

  • Drought: Caused by low or irregular rainfall.

  • Salt: Occurs in coastal and irrigated regions.

  • Cold: Includes chilling and freezing.

  • Mineral Imbalance: Involves macro- and micro-element deficiencies or excesses.

Abiotic Stress Response in Plants

  • Involves C-repeat (CRT)-binding factor/dehydration-responsive element (DRE) binding protein 1 (CBF/ DREB1) transcription factors and ABA-responsive elements (ABREs).

Abiotic Stress and Tolerance Mechanisms

Drought Tolerance

  • Drought escape: Short life cycle and growth in moist places.

  • Drought avoidance: Reducing water loss and increasing water supply.

  • Drought tolerance: Adapting metabolism to water loss.

Osmoprotectants/Osmolytes

  • Maintain a water shell around macromolecules.

  • Compatible solutes do not disturb the water shell.

Desiccation Tolerance

  • Some plants and tissues are desiccation-tolerant, including seeds, pollen, bryophytes, and some vascular plants.

Cellular Responses to Desiccation

  • Prevent irreversible damage by stabilizing membranes and proteins.

LEA Proteins

  • Intrinsically-disordered proteins that accumulate during dehydration and protect cell structures.

Cell Wall Structure and Desiccation Tolerance

  • Orderly folding helps membranes and walls survive desiccation.

Antioxidant Systems

  • Induced by dehydration stress to detoxify reactive oxygen species (ROS).

Reactive Oxygen Species (ROS)

  • Superoxide, hydroxyl radical, and hydrogen peroxide.

Enzymatic and Non-Enzymatic Antioxidants

  • Detoxify ROS and can be up-regulated under water stress conditions.

Plant Response to Water Deficit

  • All plants respond to mild water deficit, while desiccation-tolerant plants take it further.

Perception of Water Deficit

  • May occur at the cell membrane through pressure sensors.

Water Deficit Signaling

  • Involves ABA, ethylene, hydraulic signals, and transcriptional cascades.

Abscisic Acid (ABA)

  • Accumulation is a rapid response to water deficit.

  • Levels are tightly controlled by synthesis, conjugation, and degradation.

Transcriptional Responses to Water Deficit

  • Well-characterized, involving thousands of genes regulated by ABA and other factors.

Water-Optimized, Drought-Tolerant Agricultural Plants

  • New technologies enable prevention, diagnosis, and elimination of water deficit effects.

Breeding Programs

  • Depend on high-throughput phenomics tools.

Yield Under Drought Stress

  • The product of water uptake (WU), water use efficiency (WUE), and harvest index (conversion of biomass to grain).

Genetic Engineering Strategies

  • Prevent stress by growing large root systems, preventing entry of toxic minerals, or attracting plant growth-promoting rhizobacteria.

  • Reduce the natural stress response.

  • Detoxify ROS.

  • Modify stress signaling response.

Target Genes for Genetic Engineering

  • Enzymes for producing protecting metabolites.

  • Transporters.

  • Chaperones.

  • Signaling proteins.

    Transcription factors.

Breeding for Drought Tolerance: Candidate Gene Approach

  • Involves genes related to transport proteins, transcription factors, osmotic adjustment, protective proteins, and signaling molecules.

Regulatory Controls on Genetically-Engineered Plants

  • Plants made using genetic engineering face additional regulatory hurdles.

Promising Genes for Drought Tolerance

  • Transcription factors, membrane transporters, and hormone synthesis genes.

The Cold Response and Signal Transduction

Cold exposure causes various physiochemical disturbances, leading to growth inhibition. Cold stress response involves signal transduction that activates transcription factors and cold-responsive genes for tolerance.

The Cold Response and Transcription

  • During cold stress, various transcription factors, including C-repeat binding factors, are involved in activating cold-responsive genes.

  • DREB1/2 regulation of drought/cold/salt stress.

The salt stress and Plant responses: SOS Pathway for Salinity Tolerance

  • Salt stress leads to an increase in Ca^{2+}, which activates SOS3. SOS3 activates SOS2. SOS2 activates SOS1 via phosphorylation.

Plant responses to heat stress

Heat stress in plants leads to ROS production, increased membrane fluidity, protein denaturation, and activation of chaperonin. These components are vital for heat stress tolerance.