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Chapter 2
Penetration of cuticles
Cuticle
- surface wax
- cuticle proper
- cuticle layer
Preexisting defense
structure
Cuticles
 Cutin –
found on aboveground (aerial) parts of all
herbaceous plants
a principal constituent of the cuticle
 Suberin –
present on underground parts
differs from cutin in that it has dicarboxylic acids,
long –chain components, and phenolic compounds.
 Waxes – associated with both cutin and suberin
Surface wax deposits
 Cutin, waxes, and suberin are made of hydrophobic
compounds.
 Cutin, suberin, and their associated waxes form
barriers between the plant and its environment that
function to keep water in and pathogen out.
 However, they do not appear to be as important in
pathogen resistance.
Fungi penetrate plant surface by mechanical means.
Others secrete cutinase to hydrolyze cutin.
 The cuticle thickness of the New Mexican-type
pepper increase from 12mm in immature green fruit
to 24mm in mature red fruit. The susceptibility of
unwounded fruit to infection of Phytophthora capsici
decreases with increased ripening.
Contradictory results for the involvement of
cutin in pathogen resistance
 Basidiospores of Uromyces vignae penetrate the
cuticle of broad bean, a nonhost, much faster and
with higher efficiency than they penetrate the
epidermis of cowpea, a host.
 The infection, however, is stopped a few hours
later within the cytoplasm of broad bean (Xu and
Mendgen, 1991).
 Thus, successful penetration of cuticle does not
ensure disease development.
Penetration of cuticle
Cutinase produced by plant pathogens
Alternaria alternata
Bortrytis cinerea
Cochliobolus heterostrophus
Colletotrichum capsici
Cryphonectria parasitica
Fusarium solani f.sp. pisi
Helminthosporium sativum
Magnaporthe grisea
Monilinia fructicola
Phytophthora capsici
Streptomyces scabies
Ulocladium consortiale
Venturia inaequalis
Molecular properties of cutinases
 Cutinase is synthesized by many fungi and
bacteria.
 Cutinase is an esterase that initially hydrolyzes
cutin to oligomers and further hydrolyze to fatty
acid monomers.
 Fungal cutinase genes are triggered by plant cutin
monomers.
Molecular properties of cutinases
 Cutinases are extracellcular enzymes.
 Cutinases are glycoproteins containing of 3 ~ 16%
carbohydrates.
 MW 18 ~ 25 kD
 Despite great similarities in amino acid
compositions, immunological heterogeneity exists
among cutinase from different fungi.
Evidence that cutinases are involved in
penetration of cuticles
 The cutinase is present at the site of penetration.
 Specific inhibition of the enzyme prevents fungal
penetration.
 Supplementing a cutinase-deficient mutant with
cutinase restores virulence.
 Inserting the cutinase gene into an avirulent
pathogen enables it to infect hosts.
Presence of cutinase at the site of penetration
 Fusarium solani f.sp. pisi vs. pea (豌豆萎凋病)
Cutinase were detected at the penetration area of
pea stems by ferritin-conjugated rabbit anticutinase antibody.
 Erysiphe graminis f.sp. hordei vs. barley (大麥白粉病)
The esterase (cutinase) released in the first stage
is constitutive and that the second one is
synthesized after initial contact (Kuno et al., 1990)
f.sp. = Formae specialis
Time course of morphogenesis of Blumeria
graminis infection structures
resistance
disease
Insertion of the cutinase gene into a nonproducing pathogen enables it to infect the host
 Mycosphaerella sp. is a parasitic fungus that
produces no cutinase.
 Cutinase gene from F. solani f.sp. pisi was
transferred into the Mycosphaerella sp. The
transformants have the capacity to infect intact
papaya fruit, and this infection was prevented by
anti-cutinase antibodies (Dickman and
Kolattukudy, 1989)
Inactivation of cutinase prevents fungal
penetration
 Infection of pea stems by spores of F. solani f.sp. pisi has
been prevented completely in the presence of either anticutinase serum or cutinase inhibitor DIFP.
 Cryphonectria parasitica vs. chestnut (西洋栗, Chestnut
blight)
 Virulent strains of the pathogen do not contain doublestranded RNA (dsRNA) in their cytoplasm.
Hypovirulent strains contain one or more dsRNAs.
 The virulent strains produced and secreted higher
amounts of cutinase than the hypovirulent strains.
 The presence of dsRNAs suppresses the expression of
the cutinase gene (Varley et al. 1992).
Contradictory results for the involvement of
cutinase gene in cuticle penetration
 No correlation was found between cutinase activity of
Botrytis cinerea isolates and production of lesions on
young tomato fruits (灰霉病 gray mold, leaf blight).
 Cutinase genes is not required for the fungal
pathogenicity on pea. (Stahl and Schafer. 1992. Plant
Cell)
 Cutinase-deficient mutants were obtained from the
isolate of F. solani f.sp. pisi by transformationmediated gene disruption. These mutants showed no
difference in pathogenicity and virulence on pea
compared to the wild-type. These observations
indicate that cutinase is not a virulence factor in F.
solani f.sp. pisi (Stahl et al., 1994. MPMI).
Uninoc.
WT
Mutant
Uninoc.
WT
Mutant
WT
Mutant
Figure 6. Pathogenicity Tests of the Wild-Type of N.
haematococca and a Cutinase-Deficient Mutant.
(A) Symptoms on peas grown 20 days in soil infested
with wild-type strain 77-2-3 and cutinase-deficient
mutant 77-102 are shown in pot 2 and pot 3,
respectively. Each pot was infested with 5 x 106 conidia.
Yellowing of basal foliage and stunted growth of the
above-ground plant parts were caused to the same
extent by both fungi. Uninoculated control plants are
shown in pot 1. Ten to 15 replicate plants were used per
treatment. The experiment was repeated three times
with results similar to those shown here. (B) Detailed
picture of the root and the lower stem of plants shown
in (A). The root and lower stem of an uninoculated
control plant are shown at left. The dark brown of
upper tap root and the below-ground epicotyl at center
is caused by infection with the wild-type strain. The
same symptoms are caused by infection with the
cutinase-deficient mutant 77-102, as shown at right. (C)
Lesions on etiolated pea segments, caused by
germinating conidia of wild-type strain 77-2-3 (left)
and null mutant 77-102 (right).
Correlation between cutinase production and
virulence of the pathogen
 F. solani f.sp. pisi mutants with reduced cutinase
activity are less virulent
(Rogers, Flaishman, and Kolattukudy. Plant Cell
1994).
Figure 6. Infection of Pea
Seedlings by F s. pisi 77-2-3
and Cutinase GeneDisrupted Mutant 77-102.
(A) Representative intact
seedlings of an uninoculated
control and seedlings
inoculated with 77-2-3 or its
gene-disrupted mutant 77102.
(B) All seedlings from one
experiment in which
seedlings were grown on
vermiculite inoculated with
77-2-3 or its mutant 77-102.
(C) The stems of seedlings
inoculated with 77-2-3 (left)
or its mutant 77-102 (right).
Stems inoculated with 77-23 show severe lesions, and
those inoculated with
mutant 77-102 exhibit a
limited number of lesions.
The drawbacks of Sthal & Schaffer’s experiment
 Multiple spore levels were not tested.
 Microscopic examination of the progression of
lesions.
Mechanical force as a means of direct
penetration
Many plant-pathogenic fungi form appressoria
prior to penetration of plant tissues.
Appressoria first adhere to the host surface
and then produce infection pegs that pierce the
underlying plant cuticle and cell wall.
Main function of appressoria
 To anchor the fungus firmly to the plant
 To penetrate the surface either directly through
the cuticle or through natural openings
The methods for penetration
 Physical force
Hydrophobins
Melanisation (by DHN-melanin)
Turgor pressure
 Cell wall-degrading enzymes
e book, P.38-42
Hydrophobins
 A pathogenicity gene, MPG1, is highly expressed
during appressorium formation has been identified
in Magnaporthe grisea (稻熱病).
 The gene encodes a 15 kD hydrophobin (96-187 aa)
that contains 8 cysteine residues.
 Hydrophobin interacts with the hydrophobic rice
surface and undergoes polymerisation which
provides protection against desiccation.
 acts as a developmental sensor for appressorium
formation.
 However, B. cinerea has no hydrophobin, whilst C.
fulvum has hydrophobins but does not form
appressorium.
Melanisation




Dihydroxynaphthalene (DHN)-melanin is a dark
fungal pigment that deposites between fungal cell
membrane and the appressorium cell wall and
binds to chitin.
The role of melanin is to seals the appressorium and
to retard the efflux of glycerol from the
appressorium, allowing hydrostatic turgor to build
up.
By 24-31 h after development, appressorium
maturation and turgor generation occur.
A penetration peg emerges and extend from the
pore into the cuticle.
Time course of morphogenesis of Blumeria
graminis infection structures
resistance
disease
END