Toxins in Plant-Pathogen Interactions

Many different types of toxins produced by

microbes
Some of these may play a role in controlling the
interaction between microbes and plants
Toxins are thought to kill plant cells directly and
facilitate colonization of the plant tissue by the
pathogen - is this too simple a view?
Non host-specific - many different genotypes
are affected by toxin - even across kingdoms
Host-specific - toxin has very narrow range of
specificity - only certain genotypes within a
species
Types of Toxins
Non Host-Specific cercosporin,
trichothecenes, fumonisins

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

Host Specific Toxins in Plant-Pathogen
Interactions
1933 - discovery of AK toxin produced by Alternaria
kikuchiana (Japanese pear pathotype of Alternaria
alternata)
specific for only a few genotypes of Japanese pear
(Pyrus pyrifolia)
1947 - discovery of victorin produced by
Cochliobolus victoriae
specific for certain genotypes of oats (Avena
sativa)

First clear examples of biochemical factors controlling
host specificity in plant-microbe interactions
Host-Specific Toxins in Plant-Pathogen
Interactions
Most are low molecular weight, secondary
metabolites - few are proteins
All are produced by fungi
Reproduce disease symptoms when applied to host
agents of compatibility - contrast to avirulence
determinants
Production of toxin is required for pathogenicity -
pathogenicity determinants
Single gene conditions susceptibility in the plant -
generally only a single host genotype is susceptible
Host-Specific Toxins in Plant-Pathogen
Interactions
almost all known host-specific toxins are produced by
loculoascomycetes
Alternaria spp. and Cochliobolus spp. primarily
Scheffer & Nelson 1960s studied genetics of toxin
production by performing genetic crosses of
Cochliobolus spp.
single genetic loci control toxin production in
Cochliobolus spp.
Cochliobolus heterostrophus T-toxin TOX1
C. carbonum HC-toxin TOX2
C. victoriae Victorin TOX3
AAL-Toxin
Produced by Alternaria alternata tomato
pathotype
Disease: stem canker of tomato
Sphinganine analog mycotoxin (SAM) - similar
to fumonisins
AAL-toxin is competitive inhibitor of ceramide
synthase in plants
Sphinganine analog mycotoxins
Genetics of AAL-Toxin
Akamatsu et al. 1997
Transformed A. alternata tomato pathotype with
plasmid pAN7-1 in presence of restriction
enzymes (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 for
pathogenicity on tomato
Wang et al 1996
AAL-toxin inhibits sphinganine N-acyltransferase
(ceramide synthase) in plants and animals
AAL-toxin-induces programmed cell death (PCD)
in plant and animal cells
PCD likely involves ceramide (lipid) signalling and
disruption of the cell cycle
Sphingolipids and sphinganine bases are
secondary messengers involved in PCD
AAL-Toxin and Programmed Cell Death
De novo sphingolipid synthesis in plants
Brandwagt et al. PNAS 2000;97:4961-4966
2000 by The National Academy of Sciences
ceramide
synthase
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-aspartic
proteases)
Programmed Cell Death (PCD) in Plants
Transgenic plants expressing anti-apoptotic genes
from animals were resistant to the necrotrophic
plant pathogenic fungi Sclerotinia, Botrytis and
Cercospora (Dickman et al. 2001)
resistance appears to be the result of prevention
of PCD
does successful colonization of plants by
necrotrophic fungi depend on active host
responses like PCD?
are toxin/plant interactions much more
sophisticated than we think?
AAL Toxin
Sensitivity to AAL toxin in tomato conferred by a
single genetic locus (Gilchrist and Grogan 1976)
Named Asc (Alternaria stem canker) 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 - no
specificity at this level
Genetics of Resistance to AAL-Toxin
van der Biezen et al. 1996
Chemically mutated AAL-suceptible tomato lines
to obtain resistant mutants
mutants were as resistant as naturally-occurring
resistant tomato lines
all mutations mapped to Asc locus
Mesbah et al. 1999
Resistance mapped to chromosome 3 of tomato
based on map of RFLP markers
Obtained Yeast Artificial Chromosome clone
containing resistance locus
Brandwagt et al. 2000
cloned and characterized the Asc gene
homologous to yeast longevity assurance gene LAG -
associated with life span in yeast
LAG facilitate ER-to-Golgi transport of
glycolsylphosphtidylinositol (GPI)-anchored proteins
Asc can partially complement LAG mutations in
yeast
asc has a premature stop codon relative to Asc and
likely codes for a truncated protein (33 kD protein in
resistant plants - 10 kD in susceptible plants)
does Asc prevent apoptosis by restoring ER-to-Golgi
transport?
Genetics of Resistance to AAL-Toxin
Other Alternaria
host-specific toxins
(HSTs)
Produced by closely
related, small-spored
Alternaria spp. - are
they all one species?
each toxin is
produced by a distinct
pathotype of A.
alternata
each pathotype has
a very narrow host
range
sub-specific
designation
Alternaria toxins

Tangerine pathotype ACT-toxin
Strawberry pathotype AF-toxin
Japanese pear pathotype AKT-toxin

earliest effects are seen on plasma membrane of
susceptible hosts including electrolyte loss, membrane
invaginations (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 and
are structurally similar - share a common 9,10-epoxy-8-
hydroxy-9-methyl-decatrienoic acid backbone
AKT-toxin
AF-toxin
ACT-toxin
Alternaria toxins
Genes for AKT-, AF- and ACT-toxin production have
been cloned and characterized
Cluster of at least 5 different genes on the smallest
chromosome in the genome - conditionally dispensable
chromosomes - differ in size among pathotypes
Multiple copies of each gene in the cluster paralogs
Masunaka et al. 2000 Homologs of AKT1 and AKT2
are present in tangerine strains - approx. 90% similar
to 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 these
toxins - mode of action remains unknown
Alternaria toxins
ACRL-Toxin
produced by the rough lemon pathotype of A.
alternata specific for rough lemon
toxin was purified from culture filtrates
alpha-pyrone structure distinct from AF, ACT and
AKT-toxins but produced by a very closely related
fungus - same species?
ACRL-toxin
First visible effects of ACRL-toxin within 1 hr of
toxin treatment: swelling, vesiculation, uncoupling
of oxidative phosphorylation, changes in
membrane potentials in mitochondria
compare to ACT, AFT and AKT-toxins - effects
first seen in the plasma membrane
Effects of ACRL toxin similar to those observed
following T-toxin treatment of T-cytoplasm maize
What is mode of action of ACRL toxin at the
molecular level?
Ohtani et al. 2002 PNAS 99:2439
Cloned mitochondrial DNA from rough lemon into
E.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 a
mitochondrial tRNA gene tRNA-Ala
Type II introns self splice catalyze their own
removal from the transcript
Some Type II introns have ORFs and are translated
ACRL-Toxin

ACRL-Toxin
mitochondrial DNA from both sensitive and
resistant citrus species hybridized to ACRS - all
have the same sequence
smaller RNA transcripts from ACRS (75 bp) found
in resistant citrus compared to rough lemon (232
bp)
antibody raised to ACRS protein in E. coli
detected a protein only from rough lemon and not
from resistant citrus
post-transcriptional RNA processing confers
susceptibility to toxin
genetic control of this difference in processing is
not known - could be nuclear or organellar
Effects of deletions of ACRS on ACR-toxin sensitivity
in E. coli
Ohtani et al. PNAS 2002 99:2439-2444
2002 by The National Academy of Sciences
victoria blight of oats - caused by Cochliobolus
victoriae
disease first described in 1947 - only oats
containing Victoria-type resistance to crown rust
are susceptible
appears to be a classic gene-for-gene interaction
toxin reproduces all disease symptoms when
applied to a susceptible host alone
oats carrying Pc-2 resistance gene for crown rust
resistance (Puccinia coronata) were widely planted
in 1940s
fungus produces a host-specific toxin - family of
cyclized pentapeptides
Victorin
all isolates producing toxin are pathogenic -
mutants and segregating progeny that do not
produce toxin are non-pathogenic
toxin is pathogenicity factor
susceptibility to toxin conferred by dominant
allele at Vb locus - homozygous recessive vb plants
are toxin-insensitive and resistant
resistance to crown rust conferred by dominant
Pc-2 gene
attempts to genetically separate Pc-2 from Vb
have been unsuccessful
Victorin

toxin is bound by 100 kD protein in susceptible
plants
protein is the P protein component of the glycine
decarboxylase complex (GDC)
part of an enzyme complex in the mitochondrion
involved in the photorespiratory cycle
victorin sensitivity gene (LOV1) has been
identified in Arabidopsis - typical NBS-LRR gene
Are Vb and Pc-2 the same gene?
more discussion of this next Tuesday - Sweat et
al. 2008 paper
Victorin
Toxins and Host Death
most toxins are produced by fungi that are
considered necrotrophs
accepted dogma is that these toxins kill the host in
advance of fungal colonization and derive nutrition
from the dead host cells
at least some some HSTs elicit an active host
resistance response - so are HSTs actually
elicitors?
HSTs can elicit defense responses that are very
similar to those induced by avirulence determinants
when (if at all) are toxins really toxins?
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 other
systems 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 fungal
invasion, defense response and cell death? We still
dont know this for most pathosystems
may be key to understanding differences among
pathogen lifestyles
Extra Slides
T-toxin
Polyketide toxin produced by Cochliobolus
heterostrophus
Race T of C. heterostrophus highly virulent on T-
cytoplasm corn
Caused Southern Corn Leaf Blight epidemic 1970
T-cytoplasm = Texas male-sterile cytoplasm
Used in hybrid maize seed production extensively in
the 1950s and 60s

race O doesnt produce toxin low virulence on T-
cytoplasm corn (still pathogenic though)
crosses between race O and race T segregate 1:1 for
T-toxin production and virulence
suggests that single genetic locus is involved in toxin
production of toxin sensitivity
Association of susceptibility with a cytoplasmically-
inherited trait led the hypothesis that the
mitochondrion was the site of action of the toxin
Toxin causes swelling, uncoupling of oxidative
phosphorylation, stimulation of respiration, leakage of
Ca2+ and NAD in mitochondria
HYPOTHESIS: T-toxin complexes with a protein
to form a hydrophilic pore that permeabilizes the
inner mitochondrial membrane
T-toxin
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-T
maize
complex mitochondrial rearrangement chimeric ORF
generates 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 toxicity
to developing anthers?
T-toxin

Duncan grapefruit Dancy tangerine X
(Citrus paradisi) (C. reticulata)
Minneola
Orlando
Nova Robinson Osceola
X Clementine
(C. reticulata)
X
Sunburst
Inheritance of Susceptibility to ACT-toxin in
Tangerine
AAL-Toxin-Induced Cell Death in Arabidopsis
Gechev et al. 2004
Used a T-DNA knockout mutant of Arabidopsis
thaliana - knocked out the Asc gene
Treated plants with AAL-Toxin and extracted and
labeled RNA for microarray analysis
Earliest upregulated genes - genes responsive
to reactive oxygen species (ROS) and ethylene
AAL-toxin appears to mediate cell death through
an oxidative burst and the production of
ethylene

Non Host Specific Toxins
Trichothecenes
produced by Fusarium graminearum (teleomorph:
Gibberella zeae)
disease: headblight of wheat and barley, ear rot of
maize
toxin is produced by pathogen leading to
contamination of grain
trichothecenes are sesquiterpenoids - inhibit
protein synthesis in animals
effects on animals: reduced feed uptake, vomiting
and immunosuppression
does toxin play a role in plant disease?
genes controlling toxin production have been
characterized
cluster of genes - very typical genome
organization for many toxin genes in fungi - why
clustered??
Non Host Specific Toxins
Trichothecenes
Proctor et al. 2002
generated trichothecene mutants by knocking out
a key gene in cluster
mutants caused less disease than wild type
no differences on maize when assayed under hot
and dry conditions
trichothecenes are virulence factor for F.
graminearum
Non Host Specific Toxins
Trichothecenes
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 and
consumption of contaminated grain
Cause several fatal livestock diseases
Structurally similar to the sphingoid base backbone
of sphingolipids
sphingolipids contain a sphinganine and a fatty acid
- found in membranes
structural AND signaling molecules - important in
programmed cell death (PCD)
Competitive inhibitors of sphingolipid biosynthesis
Inhibit the enzyme ceramide synthase (sphinganine
N-acyltransferase)
Trigger PCD in animal cell lines inclusing monkey
liver cells (Wang et al. 1996)
Non Host Specific Toxins
Fumonisins
Sphinganine
analog
mycotoxins
Non Host Specific Toxins
Fumonisins
Fatty acid
Sphinganine
Inhibition of ceramide synthase by sphinganine
analog mycotoxins
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 and
ethylene signaling pathways

Non Host Specific Toxins
Fumonisins
what is role of fumonisins in plant disease?
Desjardins et al. 2002
disrupted the FUM1 gene encoding a polyketide
synthase required for toxin production - two
independent mutants
evaluated maize ear rot using a number of assays in
the field - used marked strains to follow inoculated
strains
monitored toxin levels with HPLC
no difference in virulence between mutants and wild
type
fumonisins are not virulence factors
HC-toxin
Cyclic peptide produced by Cochliobolus
carbonum causes Northern Leaf Spot of Corn







Disease first reported in 1938 on inbred line Pr
Specific interaction between race 1 of C. carbonum
and maize genotype hm/hm
single genetic locus (Tox2) controls toxin
production in the fungus AND pathogenicity on Pr
toxin does not kill plant cells cytostatic
toxin inhibits histone deacetylases of several
organisms
histone deacetylation is known to have a major
effect on inducible gene expression defense
genes?
mode of action may be different than T-toxin -
inhibiting defense reactions rather than causing cell
death
can HC-toxin be considered a suppressor rather
than a toxin?
HC-toxin
Resistance to HC-toxin
Hm1 was the first plant resistance gene to be
cloned and characterized
1992 Johal & Briggs Science 258: 985
transposon mutagenesis of resistant plants
screened for susceptibility
Used map-based cloning plus transposon tagging
to 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
HC-toxin genes
HTS1 intronless 16 kb open reading frame encoding
for a CPS with 4 characteristic 600 amino acid CPS
domains - non-ribosomal peptide sythetase - two linked
copies
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 toxin
synthesis genes Pedley & Walton 2001
Over 120 kb of unique Tox2 DNA
HC-toxin (contd)
Dominant gene identified in 1941 (Hm1)
provided complete protection from race 1
Ullstrup 1941
Another gene (Hm2) provides partial, adult
plant 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 and
other plants
hm1 allele has a transposable element
insertion in exon 4
transposon disrupted the function of Hm1
conferred susceptibility to HC-toxin
Hm2 is largely deleted in susceptible lines only
part 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 other
susceptible 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 in
synteny with maize
HC-toxin (contd)
Non Host Specific Toxins
Cercosporin
produced by Cercospora spp.
perylenequinone toxins
photosensitizing compounds transfer light
energy to oxygen form activated oxygen
species
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
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 other
singlet oxygen generators
Similar approaches have been undertaken with
Cochliobolus carbonum to understand how fungi
are resistant to the toxins they produce
b) crg1 (Chung et al. 1999)
cloned by complementation of a cercosporin-
sensitive mutant
crg1 codes for putative 550 aa protein 4
transmembrane domains
suggests role in membrane transport
targeted disruption of crg1 in wild type becomes
sensitive to toxin
complemented a mutant which was sensitive to
cercosporin BUT resistant to other singlet oxygen
generators
more specific for cercosporin than sor1
cercosporin (contd)
cercosporin (contd)
Callahan et al. 1999 MPMI 12: 901-910
identified several cDNA clones that were
enhanced 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) membrane
transporter not related to crg1
Cercopsora sp. pump toxin from their cells
cercosporin (contd)
Additional data from Chungs newer papers?
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 involved
which are clustered in a 25 kb region of the
genome
- review paper: Desjardins & Hohn 1997 MPMI
10:147
research on genetics of host-specific toxins
(HC-toxin,T-toxin etc.) has provided impetus
for investigation of role of these toxins in
plant pathogenesis
gene disruption experiments have
conclusively demonstrated role as virulence
factor:
Gibberella pulicaris on parsnip - Desjardins et
al.1992, MPMI 5: 214
Gibberella zeae on wheat - Proctor et al. 1995,
MPMI 8:593
Trichothecenes (contd)