What’s a plant? They have pathogens? And immunity?

Tobacco mosaic virus Tobacco mosaic virus (TMV)(TMV)

RNA virus discovered by Beijerinck over 100 RNA virus discovered by Beijerinck over 100 years ago, as a years ago, as a contagium vivum fluidumcontagium vivum fluidum that that was filterable and could move through agar was filterable and could move through agar media. (Bacteria were not filterable and media. (Bacteria were not filterable and remained fixed in agar).remained fixed in agar).

CTMV causes mosaic disease in tobacco and CTMV causes mosaic disease in tobacco and other plants.other plants.

Widely used in host-pathogen research.Widely used in host-pathogen research.

Why TMV?Why TMV? Mosaic disease of tobacco was detrimental to Mosaic disease of tobacco was detrimental to

the tobacco industry in the early 1900’s.the tobacco industry in the early 1900’s.

Mayer in 1886, in the Netherlands, was the first Mayer in 1886, in the Netherlands, was the first to artificially transmit a plant disease (TMV), to artificially transmit a plant disease (TMV), the causal agent of which could not be seen or the causal agent of which could not be seen or cultured.cultured.

Around the same time, Pasteur was having a Around the same time, Pasteur was having a similar problem with rabies!similar problem with rabies!

Model SystemsModel Systems Models become the driving force for Models become the driving force for

independent steps of discovery, independent steps of discovery, intervention, and development.intervention, and development.

TMV rapidly accumulates to high titres in TMV rapidly accumulates to high titres in infected plants.infected plants.

TMV is not transmitted by insects, fungi, TMV is not transmitted by insects, fungi, or nematodes, but is by rub-inoculation.or nematodes, but is by rub-inoculation.

TMV symptoms are easy to identify and TMV symptoms are easy to identify and can infect a wide range of plants.can infect a wide range of plants.

TMV in the laboratoryTMV in the laboratory

Easy, cheap, and reliable to produce.Easy, cheap, and reliable to produce.

Omega leader sequence has been Omega leader sequence has been widely used to enhance translation of widely used to enhance translation of foreign genes in transgenic plants.foreign genes in transgenic plants.

cDNA copies of TMV promoters have cDNA copies of TMV promoters have been used in gene vectors for expression been used in gene vectors for expression of foreign genes in rub-inoculated plants.of foreign genes in rub-inoculated plants.

Pathogenic Plant BacteriaPathogenic Plant Bacteria

Pseudomonas syringaePseudomonas syringae pv. pv. tomatotomato DC3000 DC3000

Bacterial speck disease caused on tomato Bacterial speck disease caused on tomato and mustard plants.and mustard plants.

>30 Type III Secretion >30 Type III Secretion

System effectors.System effectors.

Arabidopsis thalianaArabidopsis thaliana Just as animal biologists and medical researchers Just as animal biologists and medical researchers

have used model organisms such as have used model organisms such as DrosophilaDrosophila and the nematode, so plant biologists have relied and the nematode, so plant biologists have relied upon the mustard upon the mustard Arabidopsis thalianaArabidopsis thaliana to help them to help them understand the genetics, physiology, development understand the genetics, physiology, development and structure of plants in general. and structure of plants in general.

Although the efforts of Although the efforts of ArabidopsisArabidopsis sequencers sequencers have perhaps been less heralded than those of their have perhaps been less heralded than those of their colleagues working on animal genomes, the results colleagues working on animal genomes, the results they have yielded are no less scientifically they have yielded are no less scientifically interesting. interesting. ArabidopsisArabidopsis may be a simple plant (its may be a simple plant (its detractors call it a weed!) with only 50 identified detractors call it a weed!) with only 50 identified cells types and no complex nervous system or cells types and no complex nervous system or behavioural response, but its genome is surprisingly behavioural response, but its genome is surprisingly complex. Chromosomes 2 and 4 of complex. Chromosomes 2 and 4 of ArabidopsisArabidopsis were published in were published in NatureNature at the end of 1999, now at the end of 1999, now followed by chromosomes 1, 3 and 5.followed by chromosomes 1, 3 and 5.

http://www.nature.com/genomics/papers/a_thaliana.html

Plant DefensesPlant Defenses

Basal defenses:Basal defenses: Cell wall reinforcementsCell wall reinforcements Expression of defense-associated proteinsExpression of defense-associated proteins Activated when LPS or flagellin detected.Activated when LPS or flagellin detected.

Intracellular Resistance (R) Proteins:Intracellular Resistance (R) Proteins: Detect Type III effectors in plant cellsDetect Type III effectors in plant cells Hypersensitive Response (HR) induces Hypersensitive Response (HR) induces

Programmed Cell Death (PCD).Programmed Cell Death (PCD).

PseudomonasPseudomonas DC3000 DC3000

Full genome has been sequenced.Full genome has been sequenced.

DC3000 elicits HR immunity (via apoptosis) in DC3000 elicits HR immunity (via apoptosis) in plants carrying Pto R proteins (serine/threonine plants carrying Pto R proteins (serine/threonine kinase).kinase).

Plant Pto interacts with bacterial DC3000 Plant Pto interacts with bacterial DC3000 AvrPto and AvrPtoB effectors as well as Prf for AvrPto and AvrPtoB effectors as well as Prf for HR response and plant immunity.HR response and plant immunity.

Plants without Pto Plants without Pto proteinprotein

Without Pto, the effectors AvrPto and Without Pto, the effectors AvrPto and AvrPtoB promote bacterial growth!AvrPtoB promote bacterial growth!

AvrPto suppresses cell wall-based AvrPto suppresses cell wall-based defenses.defenses.

AvrPtoB has Cell Death Suppressor AvrPtoB has Cell Death Suppressor (CDS) activity to prevent HR-based PCD (CDS) activity to prevent HR-based PCD (apoptosis).(apoptosis).

AvrPtoBAvrPtoB

Belongs to VirPphA gene family.Belongs to VirPphA gene family.

VirPpha is a virulence factor located on a large VirPpha is a virulence factor located on a large Pph plasmid.Pph plasmid.

VirPpha and AvrPtoB have 51% sequence VirPpha and AvrPtoB have 51% sequence identity, so they are most likely related but not identity, so they are most likely related but not homologous proteins.homologous proteins.

AvrPtoB is bigger than AvrPto but has a similar AvrPtoB is bigger than AvrPto but has a similar interaction with Pto.interaction with Pto.

Cell Death SuppressionCell Death Suppression AvrPtoB CDS was discovered with AvrPtoB CDS was discovered with NicotianaNicotiana, ,

because of its robust cell death response.because of its robust cell death response.

AvrPto with Pto results in PCD, but AvrPtoB AvrPto with Pto results in PCD, but AvrPtoB with Pto in with Pto in NicotianaNicotiana did not result in did not result in Programmed Cell Death!Programmed Cell Death!

Mutant and pro-apoptotic experiments with Mutant and pro-apoptotic experiments with AvrPtoB supported CDS activity.AvrPtoB supported CDS activity.

Hidden R gene discovered:Hidden R gene discovered: Rsb = Rsb = RResistance esistance SSuppressed by the avrPtouppressed by the avrPtoBB C- C-

terminus involved in CDS activities.terminus involved in CDS activities.

DC3000 PathogenesisDC3000 Pathogenesis

Wild type tomato plants with AvrPtoB and Wild type tomato plants with AvrPtoB and without functional pto gene = CDS.without functional pto gene = CDS.

Mutant AvrPtoB led to PCD and immunity.Mutant AvrPtoB led to PCD and immunity.

Introducing AvrPtoB in trans restored Introducing AvrPtoB in trans restored virulence of DC3000 and CDS!virulence of DC3000 and CDS!

Rsb geneRsb gene

AvrPtoB must be recognized by at least AvrPtoB must be recognized by at least two resistance genes. Rsb is Pto related:two resistance genes. Rsb is Pto related:

Like Pto, Rsb is Prf dependent for CDS.Like Pto, Rsb is Prf dependent for CDS.

Gene silencing experiments of Pto family Gene silencing experiments of Pto family members eliminates Rsb resistance in members eliminates Rsb resistance in tomatoes and PCD in tomatoes and PCD in Nicotiana Nicotiana plants.plants.

Other DC3000 EffectorsOther DC3000 Effectors

HoPtoD2, HopPtoE, HopPtoF, HopPtoN, HoPtoD2, HopPtoE, HopPtoF, HopPtoN, AvrPphE, AvrPpiB1…AvrPphE, AvrPpiB1…

Cell Death Suppression activity is not Cell Death Suppression activity is not specific and can inhibit various R proteins.specific and can inhibit various R proteins.

Most effectors were discovered based on Most effectors were discovered based on eliciting the HR immune response, but they eliciting the HR immune response, but they also have the potential for CDS activity.also have the potential for CDS activity.

Trump ModelTrump Model

Not the Donald, thank goodness!Not the Donald, thank goodness!

The host-pathogen interaction may The host-pathogen interaction may involve an R protein as well as a Trump involve an R protein as well as a Trump factor that can suppress the CDS activity.factor that can suppress the CDS activity.

This model accounts for the ‘hidden This model accounts for the ‘hidden resistance genes’ and complex plant-resistance genes’ and complex plant-bacteria interactions.bacteria interactions.

Trump Model Version 1Trump Model Version 1

The CDS effector is dominantly expressed if The CDS effector is dominantly expressed if R protein but no T factor is present.R protein but no T factor is present.

Trump Model Version 2Trump Model Version 2

CDS effector is suppressed if both R CDS effector is suppressed if both R protein and T factor are present.protein and T factor are present.

Trump Model Version 3Trump Model Version 3

CDS effector is dominantly expressed if T CDS effector is dominantly expressed if T factor but no R protein present. factor but no R protein present.

Other Virulence ActivitiesOther Virulence Activities

AvrPtoBAvrPtoB DC3000 mutants without CDS activity cause DC3000 mutants without CDS activity cause

a 10-fold growth decrease in tomato plants.a 10-fold growth decrease in tomato plants.

Plants with prf mutations cannot mount Pto- Plants with prf mutations cannot mount Pto- or Rsb-mediated immunity.or Rsb-mediated immunity.

AvrPtoB can activate ethlyene production in AvrPtoB can activate ethlyene production in tomato plants, increasing cell death.tomato plants, increasing cell death.

Why would bacteria Why would bacteria evolve to both enhance evolve to both enhance and suppress cell death?and suppress cell death? Perhaps early in infection it is Perhaps early in infection it is

advantageous to suppress PCD to advantageous to suppress PCD to escape the immune system and establish escape the immune system and establish the infection.the infection.

Later in the infection it may be Later in the infection it may be advantageous to enhance PCD to gain advantageous to enhance PCD to gain access to new cells and nutrients.access to new cells and nutrients.

Phytophthora ramorumPhytophthora ramorum

First observed causing bleeding stem First observed causing bleeding stem cankers and mortality of tanoak and coast cankers and mortality of tanoak and coast live oak in coastal CA in the 1990’s, it has live oak in coastal CA in the 1990’s, it has now been identified in North America and now been identified in North America and Europe.Europe.

Mortality associated with Fagus genus but Mortality associated with Fagus genus but foliar and twig infections of other species foliar and twig infections of other species are important for pathogen spread.are important for pathogen spread.

Sudden Oak Death Sudden Oak Death ResearchResearch Individual tree levelIndividual tree level

Forest levelForest level

Regional levelRegional level

Forest SuccessionForest Succession

Mosaic landscapes provide a variety of Mosaic landscapes provide a variety of niches and microclimates.niches and microclimates.

Successional stages follow disturbances Successional stages follow disturbances such as fire or logging.such as fire or logging. Tanoak sprouts prolifically following fire, and Tanoak sprouts prolifically following fire, and

is later replaced by Douglas-fir or coast is later replaced by Douglas-fir or coast redwood trees.redwood trees.

The End!