Why to study plant-pathogen interaction ?

1. A detailed study of plant- microbe interaction should provide sustainable practical solutions for the control of plant diseases in agricultural crops

2. Such studies will help in elucidate the signaling mechanism by which plant cells cope with a stress situation

3. Study can lead us to discover how organisms from different kingdom communicate with one another

Mode of infection-related terms• Mechanical pressure- enzymic attack• Natural opening- stomata or lenticles• Invading- wounded tissue only• Necrotrophy- killing of plant cells• Biotrophy- plant cells remain alive• Hemibiotrophy –pathogen initially keeps cells alive but

killing them later• Susceptible/Compatible & Resistant/Incompatible• Supressors (ellicitors)&Toxins (phytotoxins)• Pathogenesis –Pathogen reproduction• Virulent - Pathogen strain that causes disease

Plant -pathogen interaction & disease development

• Plant pathology (also phytopathology)- scientific study of plant diseases caused by pathogens and environmental conditions • Pathogens-Organisms that cause infectious disease include fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, nematodes and parasitic plants. Not included are ectoparasites like insects, mites, vertebrate or other pests that affect plant health by consumption of plant tissues.

• Plant pathology also involves the study of pathogen identification, disease etiology, disease cycles, economic impact, plant disease epidemiology, plant disease resistance, how plant diseases affect humans and animals, pathosystem genetics, and management of plant diseases.•

Plant pathogens

• FungiThe majority of phytopathogenic fungi belong to the Ascomycetes and the Basidiomycetes.The fungi reproduce both sexually and asexually via the production of spores. These spores may be spread long distances by air or water, or they may be soil borne. Many soil borne spores, normally zoospores, are capable of living saprotrophically, carrying out the first part of their lifecycle in the soil.Fungal diseases can be controlled through the use of fungicides in agriculture, however new races of fungi often evolve that are resistant to various fungicides

Fungi

Rice blast, a necrotrophic fungus

Powdery mildew, a Biotrophic Fungus

Biotrophic fungal pathogens colonize living plant tissue and obtain nutrients from living host cells. Necrotrophic fungal pathogens infect and kill host tissue and extract nutrients from the dead host cells. See Powdery Mildew and Rice Blast images below.

Significant fungal plant pathogens1.Ascomycetes• Fusarium spp. (causal agents of Fusarium wilt disease)• Thielaviopsis spp. (causal agents of: canker rot, black root rot,

Thielaviopsis root rot)• Verticillium spp.• Magnaporthe grisea (causal agent of blast of rice and gray leaf spot

in turfgrasses)

2.Basidiomycetes• Rhizoctonia spp.• Phakospora pachyrhizi (causal agent of soybean rust)• Puccinia spp. (causal agents of severe rusts of virtually all cereal

grains and cultivated grasses)• Cont……………………

Cont…………………………………3. Oomycetes• The oomycetes are not true fungi but are fungal-like

organisms. They include some of the most destructive plant pathogens including the genus Phytophthora which includes the causal agents of potato late blight and sudden oak death.

• Despite not being closely related to the fungi, the oomycetes have developed very similar infection strategies and so many plant pathologists group them with fungal pathogens.

• Significant oomycete plant pathogens• Pythium spp.• Phytophthora spp.; including the causal agent of the

Great Irish Famine (1845–1849) late blight disease in potato-an epidemic in Ireland-leads to emigration of more than one million people to US and other countries

Bacteria

Crown gall disease caused by Agrobacterium

Most bacteria that are associated with plants are actually saprotrophic, and do no harm to the plant itself. However, a small number, around 100 species, are able to cause disease.

Bacterial diseases are much more prevalent in sub-tropical and tropical regions of the world.

Most plant pathogenic bacteria are rod shaped (bacilli). In order to be able to colonize the plant they have specific pathogenicity factors. Five main types of bacterial pathogenicity factors are known.

Bacterial pathogenicity factors• 1. Cell wall degrading enzymes – used to break down the plant

cell wall in order to release the nutrients inside. Used by pathogens such as Erwinia to cause soft rot.

• 2. Toxins These can be non-host specific, and damage all plants, or host specific and only cause damage on a host plant.

• 3. Effector proteins These can be secreted into the extracellular environment or directly into the host cell, often via the Type three secretion system. Some effectors are known to suppress host defense processes.

• 4. Phytohormones – for example Agrobacterium changes the level of auxins to cause tumours.

• 5. Exopolysaccharides – these are produced by bacteria and block xylem vessels, often leading to the death of the plant.

Phytoplasmas ('Mycoplasma-like organisms') and spiroplasmas

Vitis vinifera with "Ca. Phytoplasma vitis" infection

Phytoplasma and Spiroplasma are a genre of bacteria that lack cell walls, and are related to the mycoplasmas which are human pathogens. Together they are referred to as the mollicutes. They also tend to have smaller genomes than true bacteria. They are normally transmitted by sap-sucking insects, being transferred into the plants phloem where it reproduces.

Viruses, viroids and virus-like organisms

• There are many types of plant virus, and some are even asymptomatic. • Normally plant viruses only cause a loss of crop yield. Therefore it is not

economically viable to try to control them, the exception being when they infect perennial species, such as fruit trees.

• Most plant viruses have small, single stranded RNA genomes. These genomes may only encode three or four proteins: a replicase, a coat protein, a movement protein to allow cell to cell movement though plasmodesmata and sometimes a protein that allows transmission by a vector.

• Plant viruses must be transmitted from plant to plant by a vector. This is often by an insect (for example, aphids), but some fungi, nematodes and protozoa have been shown to be viral vectors.

•

Nematodes

Root-knot nematode galls

Nematodes are small, multicellular wormlike creatures. Many live freely in the soil, but there are some species which parasitize plant roots. They are a problem in tropical and subtropical regions of the world, where they may infect crops. Potato cyst nematodes (Globodera pallida and G. rostochiensis) are widely distributed in Europe and North and South America and cause $300 million worth of damage in Europe every year. Root knot nematodes have quite a large host range, whereas cyst nematodes tend to only be able to infect a few species. Nematodes are able to cause radical changes in root cells in order to facilitate their lifestyle.

Protozoa

• There are a few examples of plant diseases caused by protozoa. They are transmitted as zoospores which are very durable, and may be able to survive in a resting state in the soil for many years. They have also been shown to transmit plant viruses.

• When the motile zoospores come into contact with a root hair they produce a plasmodium and invade the roots.

Parasitic plants

• Parasitic plants such as mistletoe and dodder are included in the study of phytopathology. Dodder, for example, is used as a conduit for the transmission of viruses or virus-like agents from a host plant to either a plant that is not typically a host or for an agent that is not graft-transmissible.

Physiological plant disorders

• Significant abiotic disorders can be caused by:1.Natural & 2. Man-made1.Natural• Drought• Frost damage, and breakage by snow and hail• Flooding and poor drainage• Nutrient deficiency• Salt deposition and other soluble mineral excesses (e.g.

gypsum)• Wind (windburn, and breakage by hurricanes and

tornadoes)• Lightning and wildfire (also often man-made)

Man-made (arguably not abiotic, but usually regarded as such

• Soil compaction• Pollution of air, soil, or both• Salt from winter road salt application or

irrigation• Herbicide over-application• Poor education and training of people working

with plants (e.g. lawnmower damage to trees)• Vandalism

Epidemiology-Disease resistance

• Plant disease resistance• Management1.Quarantine2.Cultural3.Plant resistance4.Chemical5.Biological6.Integrated

Quarantine

• Wherein a diseased patch of vegetation or individual plants are isolated from other, healthy growth. Specimens may be destroyed or relocated into a greenhouse for treatment/study. Another option is to avoid introduction of harmful non-native organisms by controlling all human traffic and activity although legislation and enforcement are key in order to ensure lasting effectiveness.

Plant resistance

• Sophisticated agricultural developments now allow growers to choose from among systematically cross-bred species to ensure the greatest hardiness in their crops, as suited for a particular region's pathological profile. Breeding practices have been perfected over centuries, but with the advent of genetic manipulation even finer control of a crop's immunity traits is possible. The engineering of foodplants may be less rewarding however, as higher output is frequently offset by popular suspicion and negative opinion about this "tampering" with nature

Chemical

• Many natural and synthetic compounds exist that could be employed to combat the above threats. This method works by directly eliminating disease-causing organisms or curbing their spread; however it has been shown to have too broad an effect, typically, to be good for the local ecosystem. From an economic standpoint all but the simplest natural additives may disqualify a product from "organic" status, potentially reducing the value of the yield.

Biological & Integrated

• Crop rotation may be an effective means to prevent a parasitic population from becoming well established, as an organism affecting leaves would be starved when the leafy crop is replaced by a tuberous type, etc. Other means to undermine parasites without attacking them directly may exist

• The use of two or more of these methods in combination offers a higher chance of effectiveness.

Disease development• Fungal plant pathogens use a wide range of pathogenesis

strategies-Nechrotrophic, enzymic attack, toxin productione.g.Pythium,Botrytis, &Cochliobolus carbonum(maize fungal pathogen)HC-toxin inhibits histone

deacetylase (activates plant defense gene)AAL toxin(PCD in tomato plantsFusicoccin toxin targets PM_H+-ATPase leads to irreversible stomata opening

& plant wilting followed by PCD and necrosisBiotrophic-haustorial penetartione.g.downy and powdery mildew ,Cladosporium fulum(-nce of haustoria)-grow

out side the PCW(apoplast) survive on leaked nutrientsHemitrophic-phytophtera infestans(late blight disease in potato)

Survival – promoting characteristics of virulent pathogens

• High rate of reproduction during growing season of plant• A very effective mode of dispersal (by wind, water,rain

splash, vector)• A high capacity to generate genetic diversity—haploidy

permits mutations of functional importance to give an immediate selective advantage with in the pathogen population.

• Followed by sexual reproduction produce a novel pool of recombinant genotype from which new epidemics can arise.

• Monoculture of crop plants & well adapted pathogen genotype

Disease development- a disease cycle

• The synchronous interaction between host, pathogen, and environment governs the development of disease.

• Plant disease cycles represent pathogen biology as a series of interconnected stages of development including dormancy, reproduction, dispersal, and pathogenesis.

• The stages of the disease cycle form the basis of many plant disease prediction models.

&• The relationship of temperature and moisture to disease

development and pathogen reproduction serve as the basis for most contemporary plant disease prediction systems

• Cont………………………..

Cont………………………………….• These interactions can be conceptualized as a

continuous sequence or cycle of biological events including dormancy, reproduction, dispersal, and pathogenesis.

• Although plant pathologists have long realized the importance of these events, Gaumann was among the first to critically evaluate the progression of events leading to disease .Gaumann called this unchanging sequence of events the “infection chain,” but more recently the terms “disease cycle” or “infection cycle” have come into common usage.

Plant disease prediction models• There are numerous ways to organize a presentation of

plant disease prediction models. • Previously, models considered information on the host,

pathogen, environment, disease, or combinations of these factors , timing of predictions relative to crop establishment, and specific attributes of the epidemic that were modeled.

• Recently, addressing models for plant disease prediction have focused on the conceptual approach to model development and provide numerous examples of disease prediction systems .

• Here we use the disease cycle as a conceptual framework for our discussion of disease prediction models.

1.Model application• Plant disease prediction models have great

potential to help meet the need for new management strategies and thus aid in maintaining global food supplies. However, the value of disease prediction models can only be fully realized when these systems are used to make decisions concerning plant disease management in production fields . Most past reviews of plant disease prediction models indicate that far more models are developed than are applied as part of “operational disease management systems”

2.Application of prediction models

• Information technology has fueled tremendous innovations in methods used to deploy plant disease prediction models. If plant pathologists can keep pace with these technological developments by establishing multi-disciplinary teams with meteorologists and computer information technology specialists, the future of plant disease prediction will remain bright.

Cont…………………

• Forty-eight percent of the manuscripts considered for review evaluated the deployment of prediction models as plant disease management tools. We found that the research effort expended on evaluation and application of these models is currently much greater than just a few decades ago.

Bayesian decision theory• In recent years, Bayesian decision theory has been

used to evaluate the performance of disease prediction models.

• The Bayesian approach provides a framework for making disease-management decisions objectively and for evaluating past decisions . More specifically, this approach seeks to make explicit statements about the probability of making the correct disease management decision with or without the information gained by using a predictive model. This approach was demonstrated by Madden for a model of wheat Fusarium head blight.

Cell-wall composition• The materials in a cell wall vary between species, and in

plants and fungi also differ between cell types and developmental stages. In plants, the strongest component of the complex cell wall is a carbohydrate called cellulose, which is a polymer of glucose.

• In bacteria, peptidoglycan forms the cell wall. • Archaean cell walls have various compositions, and may be

formed of glycoprotein S-layers, pseudopeptidoglycan, or polysaccharides.

• Fungi possess cell walls made of the glucosamine polymer chitin, and algae typically possess walls made of glycoproteins and polysaccharides.

• Unusually, diatoms have a cell wall composed of silicic acid. Often, other accessory molecules are found anchored to the cell wall

cell wall-structure

Plant cell walls-LayersMolecular structure of the primary cell wall in plants

Up to three strata or layers may be found in plant cell walls: The middle lamella, a layer rich in pectins. This outermost layer forming the interface between adjacent plant cells and glues them together.The primary cell wall, generally a thin, flexible and extensible layer formed while the cell is growing.The secondary cell wall, a thick layer formed inside the primary cell wall after the cell is fully grown. It is not found in all cell types. In some cells, such as found xylem, the secondary wall contains lignin, which strengthens and waterproofs the wall.

Composition of different layers• In the primary (growing)) plant cell wall, the major

carbohydrates are cellulose, hemicellulose and pectin. The cellulose microfibrils are linked via hemicellulosic tethers to form the cellulose-hemicellulose network, which is embedded in the pectin matrix. The most common hemicellulose in the primary cell wall is xyloglucan. In grass cell walls, xyloglucan and pectin are reduced in abundance and partially replaced by glucuronarabinoxylan, a hemicellulose. Primary cell walls characteristically extend (grow) by a mechanism called acid growth, which involves turgor-driven movement of the strong cellulose microfibrils within the weaker hemicellulose/pectin matrix, catalyzed by expansin proteins. The outer part of the primary cell wall of the plant epidermis is usually impregnated with cutin and wax, forming a permeability barrier known as the plant cuticle.-----------------------------cont.

….composition• Secondary cell walls contain a wide range of additional compounds

that modify their mechanical properties and permeability. The major polymers that make up wood (largely secondary cell walls) include:

1. cellulose, 35-50%2. xylan, 20-35%, a type of hemicellulose3. lignin, 10-25%, a complex phenolic polymer that penetrates the

spaces in the cell wall between cellulose, hemicellulose and pectin components, driving out water and strengthening the wall.

• Additionally, structural proteins (1-5%) are found in most plant cell walls; they are classified as hydroxyproline-rich glycoproteins (HRGP), arabinogalactan proteins (AGP), glycine-rich proteins (GRPs), and proline-rich proteins (PRPs).

Cell wall-function

• The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism.

• A major function of the cell wall is to act as a pressure vessel, preventing over-expansion when water enters the cell. They are found in plants, bacteria, fungi, algae, and some archaea. Animals and protozoa do not have cell walls.

Changes in Cell wall & vascular tissue composition- a pathogen attack

• Cuticle & epidermal cell-walls of plant---barrier to pathogen infection

• Invade through vectors( nematodes,angiospermic parasites),bacteria (through wounds & hydathodes)

• Magnaoprthe grisea through appressorium—melanization of it restricts the water flow to allow osmotic pressure to burst the cell-wall

• Cell-wall degradation is mendatory to pathogen penetration to this barrier whether its compatible or incompatible

Enzymes involved

• Degradation of polymers of plant cell-walls• Approx.20 CWDE are reported so far. Pathogen that

cause soft rot diseases in storage organs of carrot,potato tubers produce pectinase, endopolygalacturonase, pectic lyase & pectin methyl esterase

• There is no strong evidence that any CWDE has any role in disease specificity---they are basic compatibility factor.

Genes encoding CWDE• Cutinase , polygalacturonase , xylanase, glucanase,pectic lyase, cellulase,

cellobiohydrolase ,pectin methyl esterase, lipase & protease• There is emerging evidence that plant pathogens make different isozymes

in the plant or in response to plant extracts than they do when grown in culture

• Enzymes of the pathogen that can degrade plant cell-walls have also received attention as triggers of plant defenses

• Fungal xylanase causes necrosis, ethylene synthesis. electrolyte leakage., symptoms associated with induction of active resistance & fungal endopolygalacturonase causes necrosis and induces phytoalexin biosynthesis

• CWDE might have a dual role—A number of plants have protein that inhibit microbial CWDE

• The role of Polygalacturonase –inhibiting protein (PGIP) from bean in modulating the balance between wall degradation and phytoalexin.

Phytoalexins

• Antimicrobial low mol. wt.compounds (secondary metabolites)synthesised de novo following pathogenic attack

• Chemically diverse,but a large number of them are products of shikimic acid (phenylpropanoid) pathway from which many other plant secondary metabolites like lignin & anthocyanins are also derived

• Elicitors are compounds of microbial origin that induces phytoalexin accumulation.

Pathogen related proteins (PR)

• Pathogen related proteins (PR) are induced antimicrobial plant proteins following pathogen attack or other stresses

• Acid PR accumulated in in the cell-wall and basic ones in vacuoles

• PR proteins are general resistance factors, manipulation of their genes might have practical application for the development of new disease resistant crop.

Structural alterations of the plant cell-wall

• Formation of papillae,which are knob like structures on the inner surface of the plant CW underneath penetrating fungal spores.

• Increased lignifications of cell-wall is another response frequently associated with unsuccessful penetration and the hypersensitive response

• Lignified cells are mechanically stronger and more resistant to digestion by CWDE

• Lignification could physically restrict the pathogen while biochemical events are still not known

• Hydrogen peroxide reduction catalysed by cell-wall peroxidases generate free radicles which then spontaneously crosslink to form lignin

• Plants also contain a number of wall glycoproteins that are rich in proline, glycine & hydroxy prolines also called extensins -strengthen the cell-wall and may there fore resist pathogen invasion

Mechanism of plant-pathogen interaction

• Physiological & biochemical changes during disease development depends upon nature and location of the infection

• Root & vascular pathogens – transpiration & translocation---wilting

• Plasma-membrane -electrolyte leakage---cell death• Obligate pathogens(rusts& smuts)-redirect nutrients-• Viruses-transcriptional & translational changes

Mechanism of plant resistance• Constitutive (preformed factors)—morphological & chemical

entities + nt prior to attack- e.g.cuticle, antimicrobial secondary metabolites (saponins)

• Induced (defense processes) -phytoalexins, localised cell death (hypersensitive response), pathogenesis related proteins

1. hypersensitive reactions2. Phytoalexins3. Elicitors4. Pathogenesis-related proteins5. Structural alterations of the plant cell wall6. Acquired resistance7. Desease resistance genes (R genes)-transposon tagging &

chromosomal walking----positional cloning

Strategies utilized by plant pathogensNecrotrophic Biotrophic Hemibiotrophic

Attack strategy

Secreted CWDEs, host toxins, or both

Intimate intracellular contact with plant cell

Initially biotrophic then necrotrophic

Specific features of interaction

Plant tissue killed, colonized by the pathogen, extensive tissue maceration

Plant cell remains alive during infection, minimal plant cell damage

Alive only in initial stages, extensive plant cell damage in later stages

Host range Broad Narrow; often only a single species of plant is attacked

intermediate

Examples Rotting bacteria (Erwinia spp.) rotting fungi (Botrytis cinerea)

Fungal mildews and rusts, viruses & endoparasitic nematodes, pseudomonas spp. bacteria

Phytophthora infestans (causal agent of potato late blight disease)

Plant pathogens

• FungiThe majority of phytopathogenic fungi belong to the Ascomycetes and the Basidiomycetes.The fungi reproduce both sexually and asexually via the production of spores. These spores may be spread long distances by air or water, or they may be soil borne. Many soil borne spores, normally zoospores, are capable of living saprotrophically, carrying out the first part of their lifecycle in the soil.

Fungal plant pathogenesis -strategies • 70,000 - 80,000 known fungal spp. are able to• colonize plants and cause disease• Toxins have a highly specified mode of action -e.g. HC- toxin

produced by maize fungal pathogen (Cochliobolus carbonum) inhibits the activity of histone deacetylase

• Also produces non- host selective toxins-e.g.fusicoccin-targets the PM-H+-ATPase in many plant sps.-action leads the irreversible opening of stomata and plant wilting , cell death & necrotrophic colonization

• Biotrophic- haustoria formation -e.g. Downy and powdery mildew -few like Cladosporium fulvum -don’t form haustoria

Bacteria

Crown gall disease caused by Agrobacterium

Most bacteria that are associated with plants are actually saprotrophic, and do no harm to the plant itself. However, a small number, around 100 species, are able to cause disease.

Bacterial diseases are much more prevalent in sub-tropical and tropical regions of the world.

Bacterial plant pathogenesis -strategies

• Colonizing the apoplast to cause rots, spots,vascular wilts, cankers and blights

• Most are gram – negative -rod shaped --e.g. pseudomonas, xanthomonas & Ervinia

• Characteristic features are -1.during their parasitic life, most bacteria reside within the intracellular spaces of various plant organs or in the xylem,2.many cause considerable plant tissue damage by secreting either toxins, extracellular polysaccharides (EPSs) or CWDE at some stages

• The secreted EPSs –surround growing bacterial colony& by saturating intercellular spaces with water or by blocking the xylem to produce wilt symptoms. cont…….

Cont…………..

• Erwinia (have a broader host range) deploy pectic enz. Cause extensive cell death &tissue maceration---cleave plant cell-wall ploymers either by hydrolysis (polygalacturonase) or through beta- eliminations (pectate or pectin lyase)

• The multigenic nature of the gene families (hypersensitive responses & pathogenicity cluster) are required for bacterial pathogenesis.e.g. hrp gene seq. from plant bacteria show same virulence strategy for animals also.

Nematodes

Root-knot nematode galls

Nematodes are small, multicellular wormlike creatures. Many live freely in the soil, but there are some species which parasitize plant roots. They are a problem in tropical and subtropical regions of the world, where they may infect crops. Potato cyst nematodes (Globodera pallida and G. rostochiensis) are widely distributed in Europe and North and South America and cause $300 million worth of damage in Europe every year. Root knot nematodes have quite a large host range, whereas cyst nematodes tend to only be able to infect a few species. Nematodes are able to cause radical changes in root cells in order to facilitate their lifestyle.

Viral plant pathogenesis -strategies • Plant pathogenic viruses are biotrophs move by way of

plasmodesmata and phloem• Symptoms -tissue yellowing (chlorosis),or browning

(necrosis), mosaic pattern ,plant stunting• All of them face three basic challenges to colonize the entire

host plant by 1. how to replicate in the cell initially infected ?2. how to move into adjacent cells & vascular bundles ?3. how to suppress host defenses ?• Each viruse encoded gene has one or more specific functions.

e.g. genome of CaMV and TMV contain 7 & 5 ORFs respectively which function in replication & movement of viral DNA, symptoms development & encapsidation

• Cont……………..

Cont…………………..• Transport of virus particles occur by the way of intracellular

(symplastic) movement through the channels between plant cells through plasmodesmata., they never cross PM like animals

• Plant virus movement proteins (MPs) in association with various components of cytoskeleton of the host cell facilitate transport of virus particles into adjacent cells by way of modified plasmodesmata

• Many viral MPs have only minimal seq. similarity-suggesting that plant viruses initially may have aquired MP function from plant genome by way of recombination.

• The process controlling long distance transport with in the phloem distinct from this measophyll one and still not clear challenge ahead is to identify host components in the phloem that interact with either with CP or the virus particle & their subsequent re-entry into the measophyll & root cortical cells

Nematodes pathogenesis -strategies

• More than 20 genera cause infection by tiny, round worms (about 1mm long),confined to plant root system,modify the metabolism of root cells, inducing the plant to form specialized feeding structures

• All are obligate biotrophs,posses a hollow feeding stylet capable of penetrating cell wall

• Ecto & Endo (Heteroderidae-cyst nematode& root knot nematode(genus Meloidogyne) parasitic in nature

• Dormant eggs receive chemical signal (Nature of this chemical signal is still not clear ) from plant roots induce hatching releasing juveniles, motile second stage juvenile then penetrate roots & migrate into vascular tissue. Cont………..

Cont………………..• Once feeding commences they loose motility & become

sedentary and push stylet into cell wall & release glandular secretions & triggers partial dissolution of cell wall leads to Syncytial feeding structure---induce both symplastic connection & finally protoplast fusion

• mitosis uncoupled from cytokinesis & DNA endo-reduplication ( nuclei with increased DNA content) leads to abnormal cortical cell growth & formation of series of giant cells in close association with phloem

• Feeding tubes- associated with stylet & unlike stylet located in plant cell cytoplasm & every time when nematode feeds a new FT is formed & by the end of pathogenesis 100 FT are present in giant cells

• 20-40 kDa protein can pass through FT( micro -injection of fluorescent labeled dextrans of various mol. masses)

Phytoplasmas ('Mycoplasma-like organisms') and spiroplasmas

Vitis vinifera with "Ca. Phytoplasma vitis" infection

Phytoplasma and Spiroplasma are a genre of bacteria that lack cell walls, and are related to the mycoplasmas which are human pathogens. Together they are referred to as the mollicutes. They also tend to have smaller genomes than true bacteria. They are normally transmitted by sap-sucking insects, being transferred into the plants phloem where it reproduces.

Arthopodes (insects) pathogenesis -strategies

• Feeding arthopodes (sap- sucking) not only damage plants directly but also act as vector to facilitate colonization by viral, bacterial & fungal pathogens by directly delivering them into the vascular tissue

• Some virus sps. also can replicate & persist inside insect vector & during subsequent feedings on plants, infected insects continue to spread viruses

• Chewing insects rarely transmit virus but the tissue damage they do frequently permits attack by necrotrophic fungal and bacterial species

Genetics of Host/Pathogen Interaction• Hundreds of specific resistance genes have been described and

mapped• Specific/Vertical resistance-effective at preventing successful

attack only by certain races of pathogen;inherited monogenically & is not durable means pthogen will evolve to overcome, e.g. resistance gene against Puccinia graminis tritici (in wheat) becomes obsolete within few years

• General/ Horizontal resistance-effective at preventing successful attack by all races of pathogen; polygenic, durable, non-host resistance

• Known Mendelian resistance genes of higher plants are genes for specific resistance while defense genes are thought to be involved in general resistance

Specific/Vertical resistance

• Cf genes for Cladosporium fulvum,• Pto (Pathovar tomato) for Pseudomonas

syringae• Mi gene for Meloidogyne incognita (a

nematode)• Tm2a for TMV

General/ Horizontal resistance

• Defense genes are isolated either via the protein products or by differential screening of cDNA libraries made from mRNA isolated from infected vs uninfected plants

• With the development of the techniques of DNA- mediated transformation now it is possible to do genetics with many pathogenic bacteria, fungi etc.

Disease resistance is usually mediated by dominant genes, but some recessive resistance genes also exist

• Based on Mendelian hypothesis resistance to plant pathogen is inherited as a single dominant or semidominant trait(1900S)

• Later seminal genetic studies by Harold H. Flor on flax & the flax rust pathogen proposed that inheritance of not only plant resistance but also pathogen virulence leads to gene-for- gene model (1940s)

• This model predicts that plant resistance will occur only when a plant has a dominant gene (R) for pathogen detoxification

Flor’s experiment • Flor’s Advance in Plant Pathology in 1942

Back in the 1940s, before DNA was shown to be responsible for inheritance,studying flax rust, in 1942, Harold Flor deduced that a single gene of this fungal pathogen was responsible for the ability of the fungus to cause disease on the flax plant. He also determined that this gene in the fungus corresponded to a particular resistance gene in the flax plant. This interaction was called the gene-for-gene theory. It applies primarily to biotrophic plant pathogens such as rust fungi—organisms that require a living host to cause disease.

The gene for gene hypothesis• Where both plant & pathogen can be genetically manipulated• Genes (specificity) in the pathogen that determines

pathogenicity have a one-to-one corresponding match to (specificity) genes in the host that determine resistance

• The phenotype of a particular host resistance gene depends on the genotype of the pathogen at the complementing gene, and vice versa

• The particular complementing gene pairs are frequently said to interact, which must be interpreted genetically, not necessarily biochemically

• Plant resistance is usually dominant to susceptibility & pathogen avirulence is usually dominant to virulence

Proposed concepts for gene-to-gene hypothesis

• Primary gene products of a complimentary pair of resistance and avirulence genes do physically interact or to put it another way-------Plant resistance gene product ‘recognizes’ the pathogen avirulence gene product, leads to the induction of plant defense responses & hence to resistance/incompatibility

• In the –nce of recognitions, plant defenses are not induced and susceptibility occurs

• This model is consistent with the dominant alleles in both the plant and the pathogen being functional (i.e. encoding proteins), and the recessive alleles being non-functional cont………………

Cont………………………

• On the basis of the pattern of compatibility and incompatibility among potato resistance genes and races of Phytophthora infestans ,cause of late blight, one can assign a putative monogenic pathogenicity genotype to each race

• The validity of this concept is not applicable to classical genetics (based on molecular genetics)

• Concept also proposed that putative gene-for-gene interaction might have a common biochemical basis& supports possibility of the fact that many plant genes giving resistance to pathogen are allelic.,each different allele controlling resistance to a particular genotype of the pathogen

Properties of avirulence genes• Avr genes—genetic determinants of incompatibility toward particular

plant genotype• Avirulence factors- viral CP, replicase,& MP, changes in amino acids

( incompatible interaction)• First Avr gene isolated from soybean infecting Pseudomonas bacteria by

shotgun cloning, at +nt more than 30 Avr genes are cloned by this technique from Pseudomonas & Xanthomonas sps.

• Avr genes code for soluble, hydrophillic proteins• Avr generated signals- 1.Export syringolides( C- glucosides with a novel tricyclic ring) produced by

enzs. Encoded by the avr D locus of P. syringae (resistance in soy bean carry corresponding R gene –Rpg4)

2.Avr protein itself as a signal in bacteria

R-genes• Only few Avr genes known for pathogenic fungi ,colonizing

intracellular air spaces in plants & through biochemical approach small secreted peptides can be isolated ,which work as elicitors for R gene dependent defense responses in the –nce of the pathogen produces R-protein mediated plant signal perception process

• Most plant R- proteins have structural similarity & isolation of R-gene utilizes two strategies-

1.Locating R- genes on the chromosome by using populations that segregate for resistance &susceptible individuals

2. Identifying the correct seq. either by inserting a mobile genetic element ( transposon) to destroy biological activity or by using binary cosmid complementation to confer the resistance phenotype on a susceptible plant

Classes of Resistance Gene• There are several ( 6) different classes of R Genes. Out of

these 4 have LRRs• The major classes are the NBS-LRR genes and the cell surface

pattern recognition receptors (PRR).The protein products of the NBS-LRR -R genes contain a nucleotide binding site (NBS) and a leucin rich repeat (LRR). The protein products of the PRRs contain extracellular, juxtamembrane, transmembrane and intracellular non-RD kinase domains

• Within the NBS-LRR class of R genes are two subclasses: -

One subclass has an amino-terminal Toll/Interleukin receptor homology region (TIR). This includes the N resistance gene of tobacco against TMV. The other subclass does not contain a TIR and instead has a leucine zipper region at its amino terminal.

How R AND Avr gene products activate plant defense responses

• R- genes/proteins are predicted to fulfill two basic functions-

1.To confer recognition of any Avr-gene –dependent ligand &

2.After a recognition event, to activate downstream signalling that leads to rapid induction of various defense responses

3. The various predicted R- protein structures provide some immediate clues as to how different classes of r-genes may operate as receptor and signal transducer

• Additional plant genes also participate in defense responses as are triggered by some elicitors (SA)

• Many plant pathogens can easily mutate from avirulence to virulence, enabling them to overcome the resistance mediated by a specific R gene due to extensive allelic variation at some R –gene loci

Epigenetic phenomena & environmental agents leading to disease

• In biology, and specifically genetics, epigenetics is the study of inherited changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: επί- over, above) -genetics.

• These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently.

Historical basis of epigenetics

• Epigenetics was coined by C. H. Waddington in 1942 .When Waddington coined the term the physical nature of genes and their role in heredity was not known; he used it as a conceptual model of how genes might interact with their surroundings to produce a phenotype.

• Robin Holliday defined epigenetics as "the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms.Thus epigenetic can be used to describe anything other than DNA sequence that influences the development of an organism.

Epigenetic mechanisms

Molecular basis of epigenetics

• The similarity of the word to "genetics" has generated many parallel usages. The "epigenome" is a parallel to the word "genome", and refers to the overall epigenetic state of a cell.

• The phrase "genetic code" has also been adapted—the "epigenetic code" has been used to describe the set of epigenetic features that create different phenotypes in different cells. Taken to its extreme, the "epigenetic code" could represent the total state of the cell, with the position of each molecule accounted for in an epigenomic map, a diagrammatic representation of the gene expression, DNA methylation and histone modification status of a particular genomic region. More typically, the term is used in reference to systematic efforts to measure specific, relevant forms of epigenetic information such as the histone code or DNA methylation pattern.

Cont……………….

Cont……………………….• The molecular basis of epigenetics is complex. It involves

modifications of the activation of certain genes, but not the basic structure of DNA. Additionally, the chromatin proteins associated with DNA may be activated or silenced. This accounts for why the differentiated cells in a multi-cellular organism express only the genes that are necessary for their own activity.

• Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime, but, if a mutation in the DNA has been caused in sperm or egg cell that results in fertilization, then some epigenetic changes are inherited from one generation to the next

Applications of epigenetics

Epigenetic research uses a wide range of molecular biologic techniques to enhance our understanding of epigenetic phenomena, including chromatin immunoprecipitation (together with its large-scale variants ChIP-on-chip and ChIP-seq), fluorescent in situ hybridization, methylation-sensitive restriction enzymes, DNA adenine methyltransferase identification (DamID) and bisulfite sequencing. Furthermore, the use of bioinformatic methods is playing an increasing role (computational epigenetics).

Epigenetics in microorganism- Bacteria

• Bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions.

• Bacteria make use of DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation is important in bacteria virulence in organisms such as Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella.

• In Alphaproteobacteria, methylation of adenine regulates the cell cycle and couples gene transcription to DNA replication.

• In Gammaproteobacteria, adenine methylation provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage, transposase activity and regulation of gene expression.

- Fungus

• The filamentous fungus Neurospora crassa is a prominent model system for understanding the control and function of cytosine methylation. In this organisms, DNA methylation is associated with relics of a genome defense system called RIP (repeat-induced point mutation) and silences gene expression by inhibiting transcription elongation.

- Yeast

• The yeast prion PSI is generated by a conformational change of a translation termination factor, which is then inherited by daughter cells. This can provide a survival advantage under adverse conditions. This is an example of epigenetic regulation enabling unicellular organisms to respond rapidly to environmental stress. Prions can be viewed as epigenetic agents capable of inducing a phenotypic change without modification of the genome.

Environmental factors leading to plant diseases

• There are three types of responsess-• 1. Immediate response of invaded cells: 2.local response and

gene inactivation: 3.Systemic response and gene inactivation• 1. Immediate response of invaded cells:• (a) Generation of reactive oxygen species• (b) Nitric oxide synthesis• ( c) Opening of ion channels• (d)Protein phosphorylation/dephosphorylation• (e)Cytoskeletal rearrangements• (f) Hypersensitive cell death/response (HR)-necrosis• (g) Gene induction

:

• 2.local response and gene inactivation:• Alterations in secondary metabolites/pathways• Cessation of cell cycle• Synthesis of pathogen related proteins• Accumulation of benzoic and salicylic acid • Production of ethylene and jasmonic acid• Fortification of cell-walls( lignin,PGIPs,HRGPs)

• 3.Systemic response and gene inactivation: Glucanase,Chitinase,Peroxidases,Synthesis of other PR proteins

(a) Generation of reactive oxygen species

• Production of ROS (incompatible interactions)• Typical ROS are super oxide (O2

•-) and hydrogen peroxide(H2O2)

• Production of superoxide from molecular oxygen involves plasma-membrane associated NADPH oxidase leads to production of H2O2 which is permeable to PM and highly toxic

• Eventually H2O2 is converted into H2O by antioxidant enz.,

• Plays a vital role in plant defense.

ROS-defense-mechanism• H2O2 is directly toxic to pathogen & in presence of iron give

rise to highly reactive hydroxyl radicle or may contribute to structural reinforcement of plant cell walls either by cross- linking various hydroxy-proline and proline- rich glycoproteins to the polysaccharide matrix or by increasing the rate of lignin polymerization by peroxidase enzyme activity and both would make the plant cell wall more resistant to microbial penetration and enzymatic degradation

• Signaling role of ROS: H2O2 induces benzoic acid 2- hydroxylase(BA 2-H) enzyme activity for SA biosynthesis

• H2O2 also induces genes for protein involved in certain cell protection mechanism(e.g.glutathione –s- reductase.

• Cont……

Cont………………………..

• ROS production also alter the redox balance in responding cells (e.g.-specific plant transcription factors are redox regulated

Responses to abiotic stresses-excess or deficit in physical/chemical environment

• Environmental conditions- water logging, drought, high or low temperature,

excessive soil salinity, inadequate mineral nutrients in the soil and too much or too little light,

phytotoxic agents like ozone • Plant responses to abiotic stress- --Plant stresses greatly diminish crop yields ---Resistance mechanisms allow to avoid or tolerate stress ---Gene expression patterns often change in response to

stress

Stresses involving water deficit

• Water deficit can induced by many environmental conditions

• Two parameters that describe the water status of plants are water potential and relative water content

Impact of water deficit and salinity on transport across plant membrane

• Carriers,pumps and channels operate ti minimize the impact of perturbing ions on cell metabolism

• Synthesis and activity of aquaporin may be up-regulated in response to drought

Additional genes induced by water stress

• Some seed proteins may protect vegetative tissue from stress

• Osmotin,a tobacco protein with antifungal activity ,accumulates during water deficit

• Some genes induced by water stress are responsive to ABA

• Specific cis-elements and trans- acting factors promote transcription in response to ABA and water deficit

Freezing stress

• Some plants can acclimate to subfreezing temperatures

• A primary function of freeze- tolerance mechanism is membrane stabilization

• Roles of the osmolytes and antifreeze proteins that accumulate in promoting freezing tolerance remain poorly understood

• Freezing tolerance involves changes in gene expression

Flooding and oxygen deficit

• Plants vary in ability to tolerate flooding• During short – term acclimation to anoxic conditions,plants

generate ATP through glycolysis and fermentation• Shifting from aerobic metabolism to glycolytic fermentation

involves changes in gene expression• The plant hormone ethylene promotes long-term acclimative

responses,including formation of aerenchyma and stem elongation,in wetland and flood- tolerant species

• Ethylene triggers epinasty in some flood sensitive species• How do plant sense oxygen deprivation

Oxidative stress

• Tropospheric ozone is linked to oxidative stress in plants

• Ozone causes oxidative damage to biomolecules• Chloroplasts are susceptible to ozone – induced

damage• Increased synthesis of antioxidants and antioxidant

enzymes can improve tolerance to oxidative stress• Oxidative stress or ozone can interact with plant

hormones such as SA and ethylene to produce plant responses

Heat Stress

• Heat stress alters cellular functions• Plants can acclimate to heat stress• HSPs are conserved among different

organisms• Five classes of HSPs are defined according to

size• Expression of many HSPs is controlled by a

transcription factor that recognizes a conserved promotor sequences