toxins plant -pathogens

8/12/2019 Toxins Plant -Pathogens

1/58

Toxins in Plant-Pathogen Interactions

Many different types of toxins produced bymicrobes

Some of these may play a role in controlling theinteraction between microbes and plants

Toxins are thought to kill plant cells directly andfacilitate colonization of the plant tissue by thepathogen - is this too simple a view?

Non host-specific - many different genotypesare affected by toxin - even across kingdoms

Host-specific - toxin has very narrow range ofspecificity - only certain genotypes within aspecies


2/58

Types of Toxins

Non Host-Specific cercosporin,trichothecenes, fumonisins

Host-Specific T-toxin, HC-toxin, ACT-toxin,Victorin, AAL-toxin


3/58

Host Specific Toxins in Plant-PathogenInteractions

1933 - discovery of AK toxin produced by Alternariakikuchiana (Japanese pear pathotype of Alternariaalternata)

specific for only a few genotypes of Japanese pear( Pyrus pyrifolia )

1947 - discovery of victorin produced byCochliobolus victoriae

specific for certain genotypes of oats ( Avenasativa )

First clear examples of biochemical factors controllinghost specificity in plant-microbe interactions


4/58


5/58

Host-Specific Toxins in Plant-PathogenInteractions

almost all known host-specific toxins are produced byloculoascomycetes

Alternaria spp. and Cochliobolus spp. primarily Scheffer & Nelson 1960s studied genetics of toxin

production by performing genetic crosses ofCochliobolus spp.

single genetic loci control toxin production inCochliobolus spp.

Cochliobolus heterostrophus T-toxin TOX1 C. carbonum HC-toxin TOX2 C. victoriae Victorin TOX3


6/58

AAL-Toxin

Produced by Alternaria alternata tomatopathotype

Disease: stem canker of tomato Sphinganine analog mycotoxin (SAM) - similar

to fumonisins AAL-toxin is competitive inhibitor of ceramide

synthase in plants


7/58

Sphinganine analog mycotoxins


8/58

Genetics of AAL-Toxin

Akamatsu et al. 1997 Transformed A. alternata tomato pathotype with

plasmid pAN7-1 in presence of restrictionenzymes (REMI)

Screened for loss of toxin production Obtained toxin-deficient mutants Mutants could not produce any disease on

tomato toxin is a pathogenicity factor - compare to

virulence factor

Conclusion: AAL-toxin is required forpathogenicity on tomato


9/58


10/58

De novo sphingolipid synthesis in plants

Brandwagt et al. PNAS 2000;97:4961-49662000 by The National Academy of Sciences

ceramidesynthase


11/58

Programmed Cell Death (PCD) in Plants

What are typical signs of PCD? loss of cell-to-cell contact cell shrinkage condensation of chromatin membrane blebbing mitochondrial dysfunction systematic DNA degradation or laddering activation of caspases (cysteine-asparticproteases)


12/58

Programmed Cell Death (PCD) in Plants

Transgenic plants expressing anti-apoptotic genesfrom animals were resistant to the necrotrophicplant pathogenic fungi Sclerotinia , Botrytis andCercospora (Dickman et al. 2001) resistance appears to be the result of preventionof PCD does successful colonization of plants by

necrotrophic fungi depend on active host

responses like PCD? are toxin/plant interactions much moresophisticated than we think?


13/58

AAL Toxin

Sensitivity to AAL toxin in tomato conferred by asingle genetic locus (Gilchrist and Grogan 1976)

Named Asc ( A lternaria s tem c anker) locus -codominant

Susceptible - asc/asc

Resistant - Asc/Asc Intermediate - Asc/asc toxin is effective against plant protoplasts as well as

intact leaf tissue ceramide synthases in resistant and susceptible

plants are both inhibited equally by AAL toxin - nospecificity at this level


14/58

Genetics of Resistance to AAL-Toxin

van der Biezen et al. 1996

Chemically mutated AAL-suceptible tomato linesto obtain resistant mutants mutants were as resistant as naturally-occurring

resistant tomato lines all mutations mapped to Asc locusMesbah et al. 1999 Resistance mapped to chromosome 3 of tomato

based on map of RFLP markers Obtained Yeast Artificial Chromosome clone

containing resistance locus


15/58


16/58

Other Alternariahost-specific toxins(HSTs)

Produced by closelyrelated, small-spored

Alternaria spp. - arethey all one species? each toxin isproduced by a distinct

pathotype of A.alternata

each pathotype hasa very narrow hostrange sub-specific

designation


17/58

Alternaria toxins

Tangerine pathotype ACT-toxin

Strawberry pathotype AF-toxinJapanese pear pathotype AKT-toxin

earliest effects are seen on plasma membrane ofsusceptible hosts including electrolyte loss, membraneinvaginations (Kohmoto et al. 1993) One specific genotype of each host is susceptible to

toxin - very narrow host range All are low molecular weight, secondary metabolites andare structurally similar - share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid backbone


18/58

AKT-toxin

AF-toxin

ACT-toxin


19/58

Alternaria toxins

Genes for AKT-, AF- and ACT-toxin production have

been cloned and characterized Cluster of at least 5 different genes on the smallestchromosome in the genome - conditionally dispensablechromosomes - differ in size among pathotypes

Multiple copies of each gene in the cluster paralogsMasunaka et al. 2000 Homologs of AKT1 and AKT2 are present in tangerine strains - approx. 90% similarto AKT1 and AKT2 Toxin genes are generally not present in non-pathogenic Alternaria isolates horizontal transfer?? have not been able to find a receptor for any of thesetoxins - mode of action remains unknown


20/58


21/58

ACRL-toxin First visible effects of ACRL-toxin within 1 hr oftoxin treatment: swelling, vesiculation, uncouplingof oxidative phosphorylation, changes inmembrane potentials in mitochondria compare to ACT, AFT and AKT-toxins - effects

first seen in the plasma membrane Effects of ACRL toxin similar to those observedfollowing T-toxin treatment of T-cytoplasm maize What is mode of action of ACRL toxin at themolecular level?


22/58

Ohtani et al. 2002 PNAS 99:2439

Cloned mitochondrial DNA from rough lemon intoE.coli which is normally resistant to ACRL toxin Looked for colonies which were susceptible to toxin Identified ACRS (ACR-toxin sensitivity) gene 355 bp insert with a putative 171 bp ORF ACRS was a portion of a Type II intron in amitochondrial tRNA gene tRNA-Ala Type II introns self splice catalyze their ownremoval from the transcript Some Type II introns have ORFs and are translated

ACRL-Toxin


23/58

ACRL-Toxin

mitochondrial DNA from both sensitive and

resistant citrus species hybridized to ACRS - allhave the same sequence smaller RNA transcripts from ACRS (75 bp) foundin resistant citrus compared to rough lemon (232

bp) antibody raised to ACRS protein in E. coli detected a protein only from rough lemon and notfrom resistant citrus post-transcriptional RNA processing conferssusceptibility to toxin genetic control of this difference in processing is

not known - could be nuclear or organellar


24/58

Effects of deletions of ACRS on ACR-toxin sensitivityin E. co l i

Ohtani et al. PNAS 2002 99:2439-2444

2002 by The National Academy of Sciences


25/58

victoria blight of oats - caused by Cochliobolusvictoriae disease first described in 1947 - only oatscontaining Victoria-type resistance to crown rustare susceptible appears to be a classic gene-for-gene interaction toxin reproduces all disease symptoms whenapplied to a susceptible host alone oats carrying Pc-2 resistance gene for crown rust

resistance ( Puccinia coronata ) were widely plantedin 1940s fungus produces a host-specific toxin - family ofcyclized pentapeptides

Victorin


26/58

all isolates producing toxin are pathogenic -mutants and segregating progeny that do notproduce toxin are non-pathogenic toxin is pathogenicity factor susceptibility to toxin conferred by dominantallele at Vb locus - homozygous recessive vb plantsare toxin-insensitive and resistant resistance to crown rust conferred by dominantPc-2 gene

attempts to genetically separate Pc-2 from Vbhave been unsuccessful

Victorin


27/58


28/58

Toxins and Host Death

most toxins are produced by fungi that are

considered necrotrophs accepted dogma is that these toxins kill the host inadvance of fungal colonization and derive nutritionfrom the dead host cells

at least some some HSTs elicit an active host resistance response - so are HSTs actuallyelicitors ? HSTs can elicit defense responses that are very

similar to those induced by avirulence determinants when (if at all) are toxins really toxins?


29/58

Toxins and Host Death

host cell death is common feature of both

susceptibility and resistance outcome may be dependent on type of pathogen additional evidence for this specificity from othersystems like mlo resistance in barley same gene may confer resistance to one pathogen(biotroph) and susceptibility to another (necrotroph) what is precise temporal relationship among fungalinvasion, defense response and cell death? We stilldont know this for most pathosystems may be key to understanding differences amongpathogen lifestyles


30/58

Extra Slides


31/58


32/58


33/58

13 kD protein (URF13) in inner mitochondrial membrane confers sensitivity to toxin URF13 encoded by T-urf-13 in mitochondrial genome T-urf-13 found only in mitochondrial genome of CMS-Tmaize

complex mitochondrial rearrangement chimeric ORFgenerates new protein conferring sensitivity URF13 is a ligand-gated, pore-forming T-toxin receptor E. coli cells expressing t-URF-13 are sensitive to T-toxin

easy bioassay for toxin pore-forming ability of T-toxin may be related to PCD how does T-urf-13 confer male sterility? Direct toxicityto developing anthers?

T-toxin


34/58


35/58

AAL-Toxin-Induced Cell Death in Arabidopsis

Gechev et al. 2004

Used a T-DNA knockout mutant of Arabidopsisthaliana - knocked out the Asc gene

Treated plants with AAL-Toxin and extracted andlabeled RNA for microarray analysis

Earliest upregulated genes - genes responsiveto reactive oxygen species (ROS) and ethylene

AAL-toxin appears to mediate cell death through

an oxidative burst and the production ofethylene


36/58


37/58

trichothecenes are sesquiterpenoids - inhibitprotein synthesis in animals effects on animals: reduced feed uptake, vomitingand immunosuppression does toxin play a role in plant disease? genes controlling toxin production have beencharacterized cluster of genes - very typical genomeorganization for many toxin genes in fungi - whyclustered??

Non Host Specific Toxins

Trichothecenes


38/58

Proctor et al. 2002 generated trichothecene mutants by knocking outa key gene in cluster

mutants caused less disease than wild type no differences on maize when assayed under hotand dry conditions trichothecenes are virulence factor for F.graminearum


Trichothecenes


39/58


Fumonisins

Family of amino-polyalcohol mycotoxins Produced by Fusarium verticillioides Disease: ear and stalk rot of maize

Important contaminant of cereal crops Wide host range - affect plants and animals Cause cancer in rodents and epidemiological

correlation between esophageal cancer andconsumption of contaminated grain

Cause several fatal livestock diseases


40/58

Structurally similar to the sphingoid base backboneof sphingolipids sphingolipids contain a sphinganine and a fatty acid- found in membranes structural AND signaling molecules - important inprogrammed cell death (PCD) Competitive inhibitors of sphingolipid biosynthesis Inhibit the enzyme ceramide synthase (sphinganineN-acyltransferase) Trigger PCD in animal cell lines inclusing monkeyliver cells (Wang et al. 1996)


Fumonisins


41/58

Sphinganine

analogmycotoxins


Fumonisins

Fatty acid

Sphinganine


42/58

Inhibition of ceramide synthase by sphinganineanalog mycotoxins


43/58

Fumonisin-Induced Cell Death in Arabidopsis

Asai et al. 2000 Used protoplasts from several signaling pathway

mutants of Arabidopsis thaliana Fumonisin induces PCD in intact plants and

protoplasts of wild-type plants Toxin had little effect on NahG mutant (SA pathway

knocked down) protoplasts Also reduced effect of toxin on jasmonic acid and

ethylene mutants Effect of fumonisin was light-dependent -

involvement of reactive oxygen species?

Conclusion: Fumonisin activity involves SA, JA andethylene signaling pathways


44/58


Fumonisins

what is role of fumonisins in plant disease?Desjardins et al. 2002 disrupted the FUM1 gene encoding a polyketidesynthase required for toxin production - twoindependent mutants evaluated maize ear rot using a number of assays inthe field - used marked strains to follow inoculatedstrains monitored toxin levels with HPLC no difference in virulence between mutants and wildtype

fumonisins are not virulence factors


45/58


46/58


47/58

Resistance to HC-toxin

Hm1 was the first plant resistance gene to becloned and characterized

1992 Johal & Briggs Science 258: 985 transposon mutagenesis of resistant plants

screened for susceptibility Used map-based cloning plus transposon taggingto identify mutant alleles of Hm1 Hm1 encodes a NADPH-dependent reductase (HC-

toxin reductase - HCTR) Genetic co-segregation of HCTR activity and Hm1

Meeley et al. 1992


48/58

HC-toxin genes HTS1 intronless 16 kb open reading frame encoding

for a CPS with 4 characteristic 600 amino acid CPSdomains - non-ribosomal peptide sythetase - two linkedcopies

TOXA membrane transporter clustered with HTS1 two linked copies

TOXC fatty acid synthase linked to HTS1 and TOXA 3 copies

TOXD no known function linked to HTS1

TOXE unusual regulatory gene regulates other toxinsynthesis genes Pedley & Walton 2001

Over 120 kb of unique Tox2 DNA


49/58

HC- toxin (contd) Dominant gene identified in 1941 ( Hm1 )

provided complete protection from race 1 Ullstrup 1941

Another gene ( Hm2 ) provides partial, adultplant resistance - linked to Hm1

How did this disease evolve? Multani et al. 1998 PNAS 95: 1686 Used sequence information from cloned Hm1

to look at sequences from different lines andother plants

hm1 allele has a transposable elementinsertion in exon 4


50/58

transposon disrupted the function of Hm1

conferred susceptibility to HC-toxin Hm2 is largely deleted in susceptible lines onlypart of the sequence is present Hm1 and Hm2 alleles are most common in maize most maize lines are resistant mutant hm1 and hm2 alleles are found in othersusceptible maize lines same mutations Hm1 also found in barley, rice and sorghum indicates that Hm -encoded resistance is ancient Hm1 and Hm2 in rice and sorghum are found insynteny with maize

HC- toxin (contd)


51/58


52/58


Cercosporin produced by Cercospora spp. perylenequinone toxins photosensitizing compounds transfer light

energy to oxygen form activated oxygenspecies

produces superoxide, hydrogen peroxide,hydroxyl radicals, singlet oxygen

lipid peroxidation, membrane damage broad spectrum of activity bacteria, mice, fungi demonstrated virulence factor in plant

pathogenesis


53/58

cercosporin (contd) cercosporin produces primarily singlet oxygen

Cercospora spp. are resistant to toxin How are these fungi resistant to the toxins they

produce? cercosporin-sensitive mutants were generated genetic complementation of mutants looked for genes which could restore resistance restored singlet oxygen resistance to mutants

which are sensitive to cercosporin AND othersinglet oxygen generators

Similar approaches have been undertaken withCochliobolus carbonum to understand how fungiare resistant to the toxins they produce


54/58

b) crg1 (Chung et al. 1999) cloned by complementation of a cercosporin-sensitive mutant crg1 codes for putative 550 aa protein 4transmembrane domains suggests role in membrane transport targeted disruption of crg1 in wild type becomessensitive to toxin complemented a mutant which was sensitive tocercosporin BUT resistant to other singlet oxygengenerators more specific for cercosporin than sor1

cercosporin (contd)


55/58

cercosporin (contd) Callahan et al. 1999 MPMI 12: 901-910

identified several cDNA clones that wereenhanced in light targeted disruption of genomic copy of one clone

( CFP cercosporin facilitator protein) led to:

large reduction in virulence on soybean large reduction in toxin production increased sensitivity to exogenous cercosporin 65 kDa protein similar to members of the major

facilitator superfamily (MFS) membranetransporter not related to crg1 Cercopsora sp. pump toxin from their cells


56/58

cercosporin (contd) Additional data from Chungs newer papers?


57/58

Non-Specific Toxins

1) Trichothecenes produced by Fusarium spp.

- family of related sesquiterpenoid toxins

- mycotoxins

- inhibit protein synthesis

- broad spectrum of activity plants, animals,fungi

- toxin production - at least 10 genes involvedwhich are clustered in a 25 kb region of the

genome- review paper: Desjardins & Hohn 1997 MPMI10:147


58/58

research on genetics of host-specific toxins(HC-toxin,T-toxin etc.) has provided impetusfor investigation of role of these toxins inplant pathogenesis

gene disruption experiments haveconclusively demonstrated role as virulencefactor:

Gibberella pulicaris on parsnip - Desjardins etal.1992, MPMI 5: 214

Gibberella zeae on wheat - Proctor et al. 1995,MPMI 8:593

Trichothecenes (contd)

toxins plant -pathogens

Documents