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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
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Types of Toxins
Non Host-Specific cercosporin,trichothecenes, fumonisins
Host-Specific T-toxin, HC-toxin, ACT-toxin,Victorin, AAL-toxin
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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
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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
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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
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Sphinganine analog mycotoxins
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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
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De novo sphingolipid synthesis in plants
Brandwagt et al. PNAS 2000;97:4961-49662000 by The National Academy of Sciences
ceramidesynthase
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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)
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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?
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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
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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
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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
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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
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AKT-toxin
AF-toxin
ACT-toxin
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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
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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?
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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
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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
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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
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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
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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
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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?
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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
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Extra Slides
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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
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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
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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
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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
Non Host Specific Toxins
Trichothecenes
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Non Host Specific Toxins
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
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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)
Non Host Specific Toxins
Fumonisins
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Sphinganine
analogmycotoxins
Non Host Specific Toxins
Fumonisins
Fatty acid
Sphinganine
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Inhibition of ceramide synthase by sphinganineanalog mycotoxins
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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
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Non Host Specific Toxins
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
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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
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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
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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
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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)
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Non Host Specific Toxins
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
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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
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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)
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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
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cercosporin (contd) Additional data from Chungs newer papers?
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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
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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)
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