Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice.

Abstract

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its

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960 / Molecular Plant-Microbe Interactions
MPMI Vol. 18, No. 9, 2005, pp. 960–972. DOI: 10.1094/MPMI-18-0960. © 2005 The American Phytopathological Society
Pathogen-Induced Production
of the Antifungal AFP Protein
from Aspergillus giganteus Confers Resistance
to the Blast Fungus Magnaporthe grisea
in Transgenic Rice
Ana Beatriz Moreno,1 Gisela Peñas,2 Mar Rufat,1 Juan Manuel Bravo,1 Montserrat Estopà,2
Joaquima Messeguer,2 and Blanca San Segundo1
Consorcio Laboratorio CSIC-IRTA de Genética Molecular Vegetal: 1Departamento de Genética Molecular, Instituto de
Biología Molecular de Barcelona, CSIC. Jordi Girona 18, 08034 Barcelona, Spain; 2Departamento de Genética Vegetal,
IRTA Centro de Cabrils. Carretera de Cabrils s/n, Cabrils 08348, Barcelona, Spain
Submitted 23 February 2005. Accepted 24 April 2005.
Rice blast, caused by Magnaporthe grisea, is the most im-
portant fungal disease of cultivated rice worldwide. We
have developed a strategy for creating disease resistance to
M. grisea whereby pathogen-induced expression of the afp
(antifungal protein) gene from Aspergillus giganteus occurs
in transgenic rice plants. Here, we evaluated the activity of
the promoters from three maize pathogenesis-related (PR)
genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric
gene fusions were prepared between the maize promoters
and the β β-glucuronidase reporter gene (gus A). Histochemi-
cal assays of GUS activity in transgenic rice revealed that
the ZmPR4 promoter is strongly induced in response to
fungal infection, treatment with fungal elicitors, and me-
chanical wounding. The ZmPR4 promoter is not active in
the seed endosperm. The mpi promoter also proved respon-
siveness to fungal infection and wounding but not to treat-
ment with elicitors. In contrast, no activity of the PRms
promoter in leaves of transgenic rice was observed. Trans-
genic plants expressing the afp gene under the control of
the ZmPR4 promoter were generated. Transformants
showed resistance to M. grisea at various levels. Our results
suggest that pathogen-inducible expression of the afp gene
in rice plants may be a practical way for protection against
the blast fungus.
Additional keywords: Oryza sativa, Zea mays.
Most agricultural crop species suffer from a vast array of
fungal diseases that cause severe yield losses all over the
world. Rice blast, caused by the fungus Magnaporthe grisea
(Herbert) Barr (anamorph Pyricularia grisea), is the most dev-
astating disease of cultivated rice (Oryza sativa L.), due to its
widespread distribution and destructiveness (Ou 1985). Breed-
ing of durable resistance to this fungus is a difficult problem
not only because of the high degree of pathogenic variability
of M. grisea but also because of the large number of fungal
races encountered in the field population. Conventional control
of rice blast depends on the use of chemically synthesized fun-
gicides. However, the repeated use of such chemicals has several
drawbacks, such as their lack of specificity, increased incidence
of development of resistance upon prolonged application, and
the adverse impact on human health and environment.
Genetic engineering of plants by transferring a gene of inter-
est has now been accepted as a method of choice for directional
improvement and development of disease-resistant plants. The
simplest means for genetic engineering of resistance to fungal
diseases entails the constitutive expression of antifungal genes
in transgenic plants. Thus, constitutive promoters, such as the
CaMV35S (Cauliflower mosaic virus) or the ubiquitin pro-
moter, have been successfully used to drive expression of a
spectrum of antifungal genes of both plant and nonplant origin
in transgenic plants (Coca et al. 2004; Epple et al. 1997; Datta
et al. 1999; Gao et al. 2000; Kanzaki et al. 2002; Lorito et al.
1998). These promoters may, however, be suitable for proof-
of-concept experiments to assess the effectiveness of transgene
expression but present a number of potential drawbacks for ac-
tual use in genetically improved crops. Instead, the selective
induction of the transgene at the site where the transgene prod-
uct is needed and only when needed represents a more desir-
able strategy for protection of crop species against pathogens.
However, the success of strategies for regulated transgene ex-
pression based on the introduction of an antifungal gene into
the plant genome under the control of a pathogen-inducible
promoter requires that the pathogen-controlled production of
the antifungal compound occurs rapidly and locally at the in-
fection site. Another undesirable side effect led by the use of
constitutive promoters comes from the accumulation of the
transgene product in plant organs used for human or animal
consumption. Thus, in addition to pathogen-inducibility, the
promoter used to direct transgene expression must not be
active in eatable organs.
Plants are known to produce numerous antimicrobial pro-
teins to defend themselves from pathogens, the induction of
the expression of pathogenesis-related (PR) genes being an
Corresponding author: B. San Segundo; E-mail: bssgmb@cid.csic.es; Fax:
34932045904.
The nucleotide sequence data is available from the EMBL nucleotide
sequence database under the accession number AJ969166 Zea mays PR4
gene promoter.
*The e-Xtra logo stands for “electronic extra” and indicates that Figures
3, 4, 6, and 7 appear in color on-line.
e-Xtra*

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ubiquitous defense response of plants to pathogen attack
(Dangl and Jones 2001; Nimchuk et al. 2003; Somssich and
Hahlbrock 1998; van Loon 1985; van Loon and van Strien
1999). There are a large number of pathogen-inducible PR
genes, and their promoters are known to contain multiple cis-
acting elements involved in the pathogen-inducibility of the
expression of these genes. Two groups of pathogen-inducible
cis-acting elements, the GCC box (AGCCGCC) and the W box
([T]TGAC[C/T]), are well studied (Eulgem et al. 2000; Lebel
et al. 1998; Rushton and Sommssich 1998; Rushton et al.
2002). Considering the pathogen-inducibility of PR genes,
their promoters are of special interest for designing protective
strategies involving inducible expression of antimicrobial
genes in crop species. Most studies on pathogen-inducible
genes have concerned dicotyledonous plants. However, pro-
moters from pathogen-inducible genes from dicotyledonous
species, usually are not useful when approaching transgene
expression in monocotyledonous plants. In maize, the induc-
tion of the expression of certain PR genes in response to fun-
gal infection has been described by our group (Bravo et al.
2003; Campo et al. 2004; Casacuberta et al. 1991; Cordero et
al. 1994a). More particularly, the fungal-inducible expression
of the ZmPR4 gene encoding a class II chitinase of the PR-4
family of PR proteins, the mpi gene encoding a proteinase in-
hibitor (PR-6 family of PR-proteins), and the PRms gene en-
coding a member of the PR-1 family of PR proteins has been
reported.
On the other hand, the production of antimicrobial proteins
is not restricted to plant species but seems to be ubiquitous in
nature (Zasloff 2002). In particular, the soil fungus Aspergillus
giganteus has been reported to produce a basic, low-molecu-
lar-weight protein (51 amino acids), the AFP protein, showing
antifungal properties against plant pathogens (Lacadena et al.
1995; Moreno et al. 2003; Vila et al. 2001). In a recent study,
we have shown that rice plants can be engineered for resistance
against the blast fungus by expression of the A. giganteus afp
gene under the control of the constitutive maize ubiquitin pro-
moter (Coca et al. 2004).
This study focused on two objectives. First, to determine the
functionality and fungal inducibility of promoters from maize
genes in stable transformed rice plants, and second, to deter-
mine whether a fungal-inducible promoter directs sufficient
levels of expression of an antifungal gene, namely the afp
gene, to protect transgenic rice from the blast fungus M.
grisea. The promoters from the ZmPR4, mpi, and PRms genes
from maize (Casacuberta et al. 1991; Cordero et al. 1994a and
b; Raventós et al. 1995) were selected for this study. The ex-
pression pattern of the three maize promoters in transgenic rice
was evaluated using the gusA reporter gene. Moreover, trans-
genic rice plants expressing the afp gene from A. giganteus
under the control of the ZmPR4 promoter were produced. The
transgenic plants obtained displayed enhanced resistance to the
blast fungus M. grisea.
RESULTS
Isolation and characterization of the ZmPR4 gene.
A genomic clone containing the ZmPR4 gene sequence was
isolated from a lambda EMBL3 library of Zea mays, using the
ZmPR4 cDNA fragment described by Bravo and associates
(2003) as probe. The ZmPR4 gene encodes a class II chitinase
protein from maize, a member of the PR4 family of PR pro-
teins. Nucleotide sequence analysis of the genomic clone
revealed that it contained the entire coding as well as 5′and 3′
flanking regions of the ZmPR4 gene. In comparing the ge-
nomic and the cDNA sequences, an intron of 95 nt in length is
found within the coding sequence of the ZmPR4 gene (Fig.
1A). Members of the PR4 family of PR proteins, the class I
and II PR4 chitinases, all have an intron at this position. The
assignment of the transcription start site, which locates 81 nt
upstream of the translation start site, was made by taking into
account the longest ZmPR4 cDNA sequence previously reported
(Bravo et al. 2003) and also by homology with the consensus
sequences for the transcription initiation sites described for
plant genes (Joshi 1987).
Sequence analysis of the ZmPR4, mpi, and PRms promoters.
Close inspection of the 5′-flanking nucleotide sequence of
the ZmPR4 gene revealed the presence of sequences identical
to previously identified cis elements, which are known to be
involved in stress-induced expression of plant defense genes.
Location of these elements is shown in Figure 1A. In the
ZmPR4 promoter, a W-box element, TTTGACT, in reverse ori-
entation was found at position –864 to –858 bp upstream of
the putative transcription start site. Of interest, an elicitor-
responsive element (ERE) ATTGACC was located in reverse
orientation at position –285 to –279 of the ZmPR4 promoter
sequence. This ERE is also found at position –246 in the PRms
promoter (Raventós et al. 1995). In addition, multiple W box
TGAC core sequences, either in direct or reverse orientation,
are present in the ZmPR4 promoter (TGAC/GTCA). W-box
elements have been shown to be important cis-acting elements
in many pathogen-activated genes. Finally, several regulatory
sequence motifs that are known to mediate the transcriptional
activation of wound- and methyl jasmonate (MeJA)-inducible
genes were identified in the ZmPR4 promoter (GAGTA,
TGACG, and CGTCA).
Concerning the mpi promoter, a genomic clone containing
the mpi gene from maize had been previously identified by our
group (Cordero et al. 1994b). Moreover, the –689 to +197 pro-
moter fragment of the mpi gene, named the C1 mpi promoter,
has been shown to confer wound-inducible expression to the
gus reporter gene in transgenic rice (Breitler et al. 2001). To
identify motifs potentially involved in fungal-responsive ex-
pression of mpi, the nucleotide sequence of the mpi promoter
region extending to nucleotide –1,872 upstream of the transla-
tion start site was obtained. Interestingly, two W-boxes
(CTTGACT) were identified at positions –1,471 and –786 of
the mpi promoter (Fig. 1A). Multiple copies of either MeJA,
wound-responsive elements, or both in either direct or reverse
orientation (GAGTA and TACTC) were found in the –1,872 to
+197 mpi promoter fragment (Fig. 1A).
Finally, the –736 to +53 fragment of the PRms promoter was
also used in this study. This PRms promoter region is known to
contain the ATTGACC element (ERE) located at position –246
(Fig. 1A). This cis-acting element was found to be necessary
and sufficient to mediate elicitor responsiveness to the maize
PRms gene in barley aleurone protoplasts (Raventós et al.
1995). An additional regulatory sequence motif, a W-box
(TTTGACT) is present at position –286 of the PRms promoter.
Production of transgenic rice
containing the promoter-gus gene fusions.
For functional characterization of the three maize promoters
in transgenic rice, transcriptional fusions between each maize
promoter and the β-glucuronidase reporter gene (gus A) were
prepared. A schematic representation of the plant expression
vectors used for production of promoter-gusA lines is presented
in Figure 1B. Rice plants were transformed either by Agrobac-
terium-mediated transformation (ZmPR4-gus gene fusion) or
by particle bombardment (mpi:gus and PRms:gus gene fusions).
For production of transgenic rice containing the ZmPR4:gus
gene fusion, 300 calli from cv. Senia were cocultured with the
EAH105 Agrobacterium strain bearing the pCZmPR4::gus::nos

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Fig. 1. Structural features of the promoters from the maize ZmPR4, mpi, and PRms genes and constructs used for rice transformation. A, Diagrammatic represen-
tation of the ZmPR4, mpi, and PRms maize promoters. Striped and open boxes in the ZmPR4 gene denote the intron and coding regions, respectively. The
shaded areas represent the promoter sequence. Location of the putative pathogen- and wound-inducible cis-acting elements identified in the ZmPR4, mpi, and
PRms promoters are indicated as follows: the rectangular dark gray boxes indicate the W box (C/TTTGACT/C), the dark gray diamonds indicate ERE (ATT
GACC), and the dark gray ovals indicate the? methyl jasmonate- and wound-responsive elements (GAGTA; TGACG; CGTCA). Arrows at the bottom indicate
the orientation, direct or reverse, of the cis-element. B, Components of the various constructs used for functional analysis of maize promoters, the ZmPR4:gus,
mpi:gus, and PRms:gus gene fusions. The construct used for fungal-inducible expression of the afp gene in transgenic rice, pCZmPR4::afp::nos, is shown at
the botton. hptII = hygromycin phosphotransferase; RB = right border; LB = left border; 35SCaMV prom = 35S cauliflower mosaic virus promoter; 3′CaMV =
cauliflower mosaic virus terminator; 3′nos = nopaline synthase terminator.

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Vol. 18, No. 9, 2005 / 963
construct. The hptII gene encoding resistance to hygromycin
that is present in the parent pCAMBIA 1381Z vector served as
the selectable marker in rice transformation. A total of 86
ZmPR4:gus plants were regenerated from hygromycin-resistant
cell lines.
For production of transgenic rice harboring the mpi:gus gene
fusion, a total of 300 embryogenic calli were cobombarded
with the pBmpi::gus::nos and pILTAB227 plasmids and 70
plants were regenerated from hygromycin-resistant cell lines.
Finally, 200 embryogenic calli were cobombarded with the
pBPRms:gus:nos and pILTAB plasmids and 23 transformants
were regenerated for this construct.
All transgenic rice plants harboring promoter:gus fusions,
the ZmPR4:gus, mpi:gus, and PRms:gus lines, exhibited wild-
type morphology and growth when compared with nontrans-
genic plants.
Southern blot analyses were carried out to determine the
copy number and integration patterns of the ZmPR4:gus,
mpi:gus, and PRms:gus transgenes. Transgenic T0 ZmPR4:gus
lines, which were generated by Agrobacterium-mediated trans-
formation, had one or two copies of the transgene inserted into
their genomes (results not shown; Southern blot analysis of T2
transgenic lines is shown in Figure 2). Those T0 lines showing
one or two integration events were selected to obtain homozy-
gous transgenic lines in the T2 generation. On the other hand,
multiple copies of the transgene integrated in the genome of
the mpi:gus and PRms:gus lines, as it is usually observed in
transgenic plants produced by particle bombardment. Stability
of integration and inheritance of the transgenes was monitored
by Southern blot analysis of the progeny plants (up to the T2
generation). Figure 2 shows the results obtained on the South-
ern blot analysis of representative T2 homozygous ZmPR4:gus
(R32-17, R32-2, and R32-14) and mpi:gus (M17-8 and M16-
4) lines.
Histochemical analysis of GUS activity
in tissues of transgenic rice.
GUS histochemical assays were conducted on vegetative
and reproductive organs of T2 homozygous transgenic rice
plants harboring promoter-gus A gene fusions. As shown in
Figure 3A, the ZmPR4 promoter had no background expres-
sion in leaves, but GUS staining was observed at the cutting
sites of the leaf blades. Equally, no GUS activity was detected
in the stem of the ZmPR4:gus lines, except at the cutting sites
(Fig. 3B). In the root system of ZmPR4:gus rice plants, GUS
staining was observed at the locus of initiation of lateral roots
(Fig. 3C).
A more detailed study on the wound-inducibility of the
ZmPR4 promoter in tissues of transgenic rice plants was car-
ried out. Towards this end, leaves of soil-grown ZmPR4:gus
plants were wounded by making perpendicular cuts on the leaf
blade and were subjected to GUS histochemical assays at dif-
ferent times after wounding. Representative results of GUS
activity in wounded leaves of the ZmPR4:gus rice lines are
shown in Figure 3D. At 10 min after wounding, intense stain-
ing at the cut edge of the wound (approximately 0.2 mm) was
observed, suggesting strong local wound-response of the
ZmPR4 promoter in rice plants. Responsiveness to wounding
was observed in both young and mature leaves of ZmPR4:gus
rice lines. Roots, when cut longitudinally, also showed strong
activity of this promoter (Fig. 3E). Most probably, the GUS
activity that is observed in unwounded roots at the sites of lat-
eral root emergence (Fig. 3C) is the consequence of the wound
produced into the root tissue by the emerging lateral root. A
similar pattern of wound-induced GUS activity was observed
in three independent ZmPR4:gus lines.
Histochemical GUS analysis was also performed in mature
seeds from ZmPR4:gus plants (Fig. 3F). No activity was de-
tected in seeds that were manually dehulled and incubated in
the staining solution, even after an overnight period of incuba-
tion in the X-gluc substrate (Fig. 3F, left panel). In seeds that
were longitudinally cut before incubation in staining solution,
a low level of GUS activity was detected at the epithelium of
the embryo scutellum (Fig. 3F, middle panel). Finally, intact
dehulled seeds that had been heavily damaged by brushing and
by making small cuts before staining for GUS activity, stained
faintly only in the seed tegument. Of interest, ZmPR4 pro-
moter activity was not observed in the endosperm tissue of the
mature rice seed from ZmPR4:gus lines under any of the
wound regimes here assayed. For a comparison, seeds from
transgenic plants harboring the 35SCaMV:gus construct were
also longitudinally cut and stained for GUS activity. Endo-
sperms of seeds from 35SCaMV:gus plants stained deeply in
blue.

Fig. 2. Southern blot analysis of the ZmPR4:gus and mpi:gus rice plants. Genomic DNA (10 µg) from T2 transgenic lines was digested with restriction
enzymes and was subjected to electrophoresis through a 0.8% wt/vol agarose gel. DNA samples were transferred to nylon membrane and were hybridized
with the 32P-labeled DNA probes. The number of bands reflects the number of transgene insertions. A, ZmPR4:gus plants (lines R32-17, R32-2, and R32-14
lines) and control Senia plants (SC). The ZmPR4 promoter sequence was used as the hybridization probe. B, mpi:gus plants (lines M17-8, M16-4) and
control Senia plants (SC). The gus gene was used as the hybridization probe. B = BamHI, E = EcoRI, H = HindIII, and P = PstI.

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Fig. 3. Histochemical localization of β-glucuronidase (GUS) activity in representative transgenic rice plants containing promoter:gus constructs. The GUS
expression patterns directed by the ZmPR4, mpi, and 35SCaMV promoters are shown. A through G, Representative examples of the pattern of GUS
expression in vegetative and reproductive tissues of rice plants harboring the ZmPR4:gus (T2 homozygous lines). A, Leaf, B, stem; C, root (inset, site of
lateral root emergence); D, wounded leaf; E, wounded roots; F, seeds (unwounded, longitudinally cut, and mechanically damaged); and G, flowers (inset,
pollen grains). H and I, Histochemical analysis of expression of the mpi:gus fusion in tissues of transgenic rice plants. H, Leaf, stem, and roots; and I,
wounded leaf. J through M, Histochemical analysis of expression of the 35SCaMV:gus fusion in tissues of transgenic rice plants. J, Wounded leaf; K, stem;
L, root (left panel) and wounded root (right panel) (arrows denote sites of injury), and M, seeds (longitudinally cut).