Genetic engineering of crop plants for fungal resistance:
role of antifungal genes
S. Antony Ceasar•S. Ignacimuthu
Received: 1 November 2011/Accepted: 2 February 2012
? Springer Science+Business Media B.V. 2012
affect agricultural production. Transgenic plants have
been produced by inserting antifungal genes to confer
resistance against fungal pathogens. Genes of fungal
cell wall-degrading enzymes, such as chitinase and
glucanase, are frequently used to produce fungal-
resistant transgenic crop plants. In this review, we
summarize the details of various transformation
studies to develop fungal resistance in crop plants.
Fungal diseases damage crop plants and
Fungal resistance ? Glucanase ? Transgenic plants
Agrobacterium-mediated ? Chitinase ?
crop plants. The production of enzymes capable of
degrading the cell walls of invading phytopathogenic
in plants. This natural host defense mechanism is
improved in fungal-resistant transgenic plants. To
improve disease-resistance genetically, plant breeding
techniques have also been employed. But this is
applicable only within sexually compatible species and
can take up to 15–20 years (Rommens and Kishore
2000). Genetic engineering has the advantage of incor-
porating genes which produce resistance proteins from
have been identified. The role of multiple genes that are
involved in immune responses after fungus infestation,
and the various pathways involved therein, have been
elucidated (Islam 2006). These defense-responsive
genes have been used to produce fungal-resistant
transgenic plants (Grover and Gowthaman 2003). The
and other antifungal genes. The details of these genes,
fungal resistant plants are given in Table 1. In some
plant for superior resistance.
Introduction of chitinase gene
Transgenic plants expressing chitinase gene showed
enhanced resistance to fungal disease in many studies.
It may be due to immediate degradation of fungal cell
wall material chitin by over expression of chitinase.
Chitinase gene (chi1) from Rhizopus oligosporus was
expressed in tobacco by Terakawa et al. (1997). A
transgenic chrysanthemum resistant to gray mold was
developed by Takatsu et al. (1999) by inserting a
chitinase gene (RCC2) of rice. Further works have
been carried out by other groups with the same
S. Antony Ceasar ? S. Ignacimuthu (&)
Division of Plant Biotechnology, Entomology Research
Institute, Loyola College, Chennai 600034, India
Table 1 Details of fungal resistant transgenic plants
Name of the gene
Terakawa et al.
Takatsu et al.
Cheong et al.
Yamamoto et al.
Xiaotian et al.
Takakura et al.
Rohini and Rao.
Datta et al.
Kishimoto et al.
Chitinase like cDNA (Chs2)
Chai et al.
b-1,3-glucanase and chitinase genes
Chang et al.
Carstens et al.
Ribosome-inactivating protein (MOD1);
RCH10 from Rice
Kim et al.
Stress-inducible b-glucanase (Gns1)
Nishizawa et al.
RCH10 from rice; ALG from
Wang et al.
Kumar et al.
Cationic peptide (msrA3)
Osusky et al.
Table 1 continued
Name of the gene
et al. (2004)
Chitinase (ech42); Chitinase (nag70);
pCAMBIA (different vectors
for each gene)
Mei et al. (2004)
Chitinase (Chi); Ribosome inactivating
Chi from bean; rip from barley
pBRC; pARIP; pBchE
Li et al. (2004)
CHIT from cucumber; GLUC
Moravcı ´kova ´
et al. (2004)
Akiyama et al.
Antifungal protein (Afp)
Aspergillus giganteus (chemically
Coca et al.
Chitinase (BjCHI1); Glucanase (HbGLU)
HbGLU from rubber tree; BjCHI1
pBj17; pBj47; HEV43
Chye et al.
Antifungal protein (AFP-PIN)
Prawn (Synthetic preparations)
pPin 35S; pBar35S
Latha et al.
Tohidfar et al.
Mitani et al.
Antifungal protein (ap24)
ch5B from Beans (Phaseolus
vulgaris); gln2 and ap24 from
pHCHI; pHGLU; pHAP24;
pHCA35; pHGA37, pHGC39
Vellicce et al.
Melander et al.
Glucanase (GLU); Antifungal protein
(alfAFP); Glucanase (GLU-AFP)
GLU from tobacco;
alfAFP from Alfalfa
pEAFP; pEGlu; pAFP-Glu
Chen et al.
ER-CecA; Ap-CecA (Cecropin A)
Coca et al.
Antifungal protein (Afp)
Girgi et al.
Antifungal protein (AFP-PIN)
Prawn (Synthetic preparations)
pPin 35S; pBar35S
Latha et al.
Table 1 continued
Name of the gene
a-1-purothionin; tlp-1 gene; b-1,3-glucanase
a-1-purothionin from wheat; tlp-1
& b-1,3-glucanase from barley
et al. (2006)
CHIT from cucumber; GLUC
et al. (2007)
Chitinases (RCH10 & RAC22); Glucanase
(b-Glu); Ribosome inactivating protein
RCH10 and RAC22 from rice; b-
Glu from alfalfa; B-RIP from
Zhu et al. (2007)
Chitinase (chi11); Thaumatin-like protein
Tobias et al.
Mondal et al.
He et al. (2008)
Raham et al.
Mustard defencin (BjD)
Anuradha et al.
Chitinase (chi11); Glucanase (gluc)
chi11 from rice; gluc from
Sridevi et al.
Chitinase383; Glucanase638; Cationic
Chitinase and Glucanase from
Wheat; POC1from Rice
Wally et al.
Hassan et al.
chitinase gene (RCC2) to produce fungal resistant
plants. Yamamoto et al. (2000) produced transgenic
grapevine and Kishimoto et al. (2002) developed
transgenic cucumber both expressing the same chiti-
nase (RCC2) gene. This gene (RCC2) was further used
in trifoliate orange. A fungal resistant peanut was
developed by inserting a tobacco chitinase gene (Chi)
(Rohini and Rao 2001).
Different groups produced Rhizoctonia solani
resistant rice plants by inserting various chitinase
genes. Fungal resistant rice cultivars were developed
by Datta et al. (2001) by inserting a rice chitinase gene
(RC7) from R. solani-infected rice plants. Following
this, Kumar et al. (2003) also produced R. solani
resistant rice by inserting another rice chitinase gene
(chi11). Kim et al. (1999) reported the transformation
of rice with maize ribosome-inactivating protein b-32
gene (Zmcrip3a). They found that this gene did not
confer high level of resistance to fungal disease. So in
the next approach, they used a modified maize
ribosome inactivating protein gene (MOD1) and a
rice basic chitinase gene (RCH10). These two genes
were co-expressed in rice plants; transformed plants
showed increased resistance to R. solani (Kim et al.
2003). Another chitinase gene (OsChia) isolated from
pistils of rice was used for developing fungal resistant
rice by Takakura et al. (2000).
2) from Saccharomyces cerevisiae to develop fungal-
resistant tobacco. Li et al. (2004) developed transgenic
soybean plants expressing the bean chitinase (chi) and
the barley ribosome-inactivating protein (rip) genes.
chitinase (Chi) gene by Tohidfar et al. (2005). Rice
chitinase gene (chi11) was also used for the production
of fungal-resistant barley. This gene (chi11) was used
et al. 2007); transgenic barley plants showed enhanced
fungal resistant taro by the expression of another rice
chitinase gene (ricchi11). Fungal-resistant potato was
produced in the same year using chitinase gene (ChiC)
of Streptomyces griseus (Raham et al. 2008). A fungal-
resistant common pea plant was developed by inserting
a chitinase gene (chit30) of Streptomyces olivaceovir-
idis (Hassan et al. 2009). Recently, we have also
chitinase gene (chi11) (Ignacimuthu and Ceasar 2012).
Introduction of glucanase gene
Next to chitinase, the glucanase gene has been
given the highest priority in transgenic works to
develop fungal resistant plants. In addition to func-
tioning in self-defense systems, b-1,3-glucanases are
involved in diverse physiological and developmental
processes, such as microsporogenesis, fertilization,
seed germination, flower formation, and somatic
Nishizawa et al. (2003) introduced b-1,3 and 1,4-
glucanase gene (Gns1) of rice to enhance the disease
resistance in rice. Concurrently, Akiyama et al. (2004)
used another glucanase gene of rice (OsGLN2). Resis-
tance to blast infection was confirmed by bioassay in
both these studies. Cheong et al. (2000) introduced
soyabean glucanase gene into tobacco. Wro ´bel-Kwiat-
kowska et al. (2004) introduced b-1,3-glucanase gene
Fusarium infection. Mondal et al. (2007) developed
indian mustard with glucanase gene to overcome
alternaria leaf spot disease caused by Alternaria brass-
icae. Chen et al. (2006) introduced tobacco b-1,3-
glucanase gene (GLU) into tomato. They also trans-
formed the same plant with alfalfa defensin gene
(alfAFP) and the bivalent gene GLU-AFP; the trans-
genic tomato harbouring GLU-AFP conferred higher
resistance to fungal infection (Chen et al. 2006).
Mackintosh et al. (2006) developed a transgenic wheat
overexpressing b-1,3-glucanase gene along with
The resultant transgenic wheat lines were tested against
Fusarium graminearum infection; over expression of
these genes enhanced the fungal resistance in wheat.
Combined introduction of chitinase and glucanase
Chitinase and glucanase genes were co-expressed in a
few transgenic projects to attain maximum fungal
resistance. Combined expression of these genes
showed higher level of resistance than expression of
either gene alone. Mei et al. (2004) made the first
approach for the combined expressions of these
transgenes. They inserted ech42 gene encoding
endochitinase, nag70 gene encoding exochitinase
and gluc78 gene encoding glucanase in rice. The rice
plants expressing ech42 gene showed superior resis-
tance to sheath blight disease.
Moravcı ´kova ´ et al. (2004) co-expressed class I
glucanase and class III chitinase genes in potato
plants using plasmid pIL12. But the transgenic
plants did not show any antifungal activity. The
same authors also co-expressed the same genes using
a different plasmid pJL06. Experiments with crude
protein extracts isolated from transgenic microtu-
bers showed growth inhibition of R. solani hyphae
(Moravcikova et al. 2007). A chitinase gene with
two chitin-binding domains (BjCHI1) from Brassica
juncea and a b-1,3-glucanase gene (HbGLU) from
Hevea brasiliensis were inserted into potato (Chye
et al. 2005). Chang et al. (2002) also transferred
chitinase and b-1,3-glucanase genes into potato.
Vellicce et al. (2006) transformed strawberry with
chitinase (ch5B) and glucanase (gln2) genes along
with thaumatin-like protein (ap24) gene. Out of
sixteen transgenic plants expressing different com-
binations of gene, two transgenic lines express-
ing only the ch5B gene displayed high levels of
resistance to fungal disease. A funga- resistant
oilseed rape was developed by introducing chitinase
and b-1,3-glucanase genes from barley (Melander
et al. 2006). Wally et al. (2009) transformed carrot
with acidic wheat class IV chitinase (383) and acidic
wheat b-1,3-glucanase (638) genes along with rice
cationic peroxidase (POC1) gene, singly or in
various combinations with each other; transgenic
plants expressing POC1 alone or in combination
with chitinase showed higher resistance to fungal
disease than other transgenic lines.
Wang et al. (2003) used rice chitinase (RCH10) and
alfalfa glucanase (ALG) genes to produce transgenic
creeping bentgrass resistant to dollar spot and brown
patch fungal pathogens. In another multigene inser-
tion study, four genes were introduced into Super
Hybrid rice. These were two chitinase genes (RCH10;
RAC22) from rice, a glucanase gene (b-Glu) from
alfalfa and a ribosome inactivating protein gene
(B-RIP) from barley (Zhu et al. 2007). Transgenic
plants and their progenies thus produced were found
to possess significant resistance to rice blast disease.
Sridevi et al. (2008) developed sheath blight resistant
transgenic rice lines with the combined expression of
rice chitinase (chi11) and tobacco b -1,3-glucanase
(glu) genes. The transgenic plants expressing these
two genes were highly resistant to sheath blight
disease compared to the control.
Introduction of other antifungal genes
Apartfromchitinase andb-1,3-glucanase,genes many
other antimicrobial proteins or peptides were also
effective in conferring disease resistance in transgenic
plants. Three different antifungal genes were intro-
duced inrice by various groups: the trichosanthin gene
(TCS) by Xiaotian et al. (2000), an antifungal protein
(afp) gene of Aspergillus giganteus by Coca et al.
(2004) and synthetically-prepared antifungal genes
Ap-CecA and ER-CecA by Coca et al. (2006). Fungal-
resistant finger millet (Latha et al. 2005) and pearl
millet (Latha et al. 2006) were developed by inserting
an antifungal protein gene (PIN) of prawn. The other
antifungal genes expressed in various plants are hS2
gene encoding chitinase-like protein in creeping
bentgrass (Chai et al. 2002), N-terminally-modified
antimicrobial cationic peptide temporin-A gene in
in tobacco and peanut (Anuradha et al. 2008). These
studies suggest that in addition to chitinase and
glucanase of diverse origin, other antifungal genes
can be used for developing fungal resistant plants.
In conclusion, antifungal genes like chitinase and
glucanase have been proved to be potential candidate
genes for effective control of fungal diseases in
transgenic crop plants. These genes should be utilized
to develop more fungal resistant crop plants in future.
This will greatly help to increase the agricultural
production by protecting the crop plants from fungal
Akiyama T, Pillai MA, Sentoku N (2004) Cloning, character-
ization and expression of OsGLN2, a rice endo-1,3-beta-
glucanase gene regulated developmentally in flowers and
hormonally in germinating seeds. Planta 220:129–139
Anuradha TS, Divya K, Jami SK, Kirti PB (2008) Transgenic
tobacco and peanut plants expressing a mustard defensin
show resistance to fungal pathogens. Plant Cell Rep
cerevisiae chitinase, encoded by the CTS1–2 gene, confers
antifungal activity against Botrytis cinerea to transgenic
tobacco. Transgenic Res 12:497–508
Chai B, Maqbool SB, Hajela RK, Green D, Vargas JM, War-
kentin D, Sabzikar R, Sticklen MB (2002) Cloning of a
chitinase-like cDNA (hs2), its transfer to creeping bent-
grass (Agrostis palustris Huds.) and development of brown
patch (Rhizoctonia solani) disease resistant transgenic
lines. Plant Sci 163:183–193
Chang M, Culley D, Choi JJ, Hadwiger LA (2002) Agrobacte-
rium-mediated co-transformation of a pea b-1,3-glucanase
and chitinase genes in potato (Solanum tuberosum L. c.v.
Russet Burbank)usingasingleselectable marker.Plant Sci
Chen SC, Liu AR, Zou ZR (2006) Overexpression of glucanase
gene and defensin gene in transgenic tomato enhances
Cheong YH, Kim CY, Chun HJ, Moon BC, Park HC, Kim JK,
Lee SY, Cho MJ (2000) Molecular cloning of a soybean
class III b-1,3-glucanase gene that is regulated both
developmentally and in response to pathogen infection.
Plant Sci 154:71–81
Chye M, Zhao K, He Z, Ramalingam S, Fung K (2005) An
agglutinating chitinase with two chitin-binding domains
confers fungal protection in transgenic potato. Planta
Coca M, Bortolotti C, Rufat M, Pen ˜as G, Eritja R, Tharreau D,
Martinez del Pozo A, Messeguer J, Segundo SB (2004)
Transgenic rice plants expressing the antifungal AFP pro-
tein from Aspergillus giganteus show enhanced resistance
Coca M, Pen ˜as G, Go ´mez J, Campo S, Bortolotti C, Messeguer
J, San Segundo B (2006) Enhanced resistance to the rice
blast fungus Magnaporthe grisea conferred by expression
Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW,
Muthukrishnan S, Datta SK (2001) Enhanced resistance to
sheath blight by constitutive expression of infection- rela-
ted rice chitinase in transgenic elite indica rice cultivars.
Plant Sci 160:405–414
mildew resistance in pearl millet (Pennisetum glaucum)
mediated by heterologous expression of the afp gene from
Aspergillus giganteus. Transgenic Res 15:313–324
Grover A, Gowthaman R (2003) Strategies for development of
fungus-resistant transgenic plants. Curr Sci 84:330–340
Hassan F, Meens J, Jacobsen H, Kiesecker H (2009) A family 19
T. harzianum in vitro. J Biotechnol 143:302–330
He X, Miyasaka SC, Fitch MM, Moore PH, Zhu YJ (2008)
Agrobacterium tumefaciens-mediated transformation of
taro (Colocasia esculenta (L.) Schott) with a rice chitinase
gene for improved tolerance to a fungal pathogen Sclero-
tium rolfsii. Plant Cell Rep 27:903–909
Ignacimuthu S, Ceasar SA (2012) Development of transgenic
blast disease. J Biosci. doi:10.1007/s12038-011-9178-y
Islam A (2006) Fungus resistant transgenic plants: strategies,
progress and lessons learnt. Plant Tissue Cult Biotechnol
Kim JK, Duan X, Wu R, Seok SJ, Boston RS, Jang I-C (1999)
Molecular and genetic analysis of transgenic rice plants
expressing the maize ribosome-inactivating protein b-32
gene and the herbicide resistance bar gene. Mol Breed
Kim JK, Jang IC, Wu R, Zuo WN, Boston RS, Lee YH, Ahn IP,
Nahm BH (2003) Co-expression of a modified maize
ribosome-inactivating protein and a rice basic chitinase
gene in transgenic rice plants confers enhanced resistance
to sheath blight. Transgenic Res 12:475–484
Kishimoto K, Nishizawa Y, Tabei Y, Hibi T, Nakajima M,
Akutsu K (2002) Detailed analysis of rice chitinase gene
expression in transgenic cucumber plants showing differ-
ent levels of disease resistance to gray mold (Botrytis
cinerea). Plant Sci 162:655–662
Kumar KK, Poovannan K, Nandakumar R, Thamilarasi K,
Geetha C, Jayashree N, Kokiladevi E, Raja JAJ, Sami-
yappan R, Sudhakar D, Balasubramanian P (2003) A high
throughput functional expression assay system for a
defense gene conferring transgenic resistance on rice
against the sheath blight pathogen, Rhizoctonia solani.
Plant Sci 165:969–976
Rao K (2005) Production of transgenic plants resistant to
leaf blast disease in finger millet (Eleusine coracana (L.)
Gaertn.). Plant Sci 169:657–667
Latha MA, Rao KV, Reddy TP, Reddy VD (2006)Development
of transgenic pearl millet(Pennisetum glaucum (L.) R. Br.)
plants resistant to downy mildew. Plant Cell Rep
Li HY, Zhu YM, Chen Q, Conner RL, Ding XD, Zhang BB
(2004) Production of transgenic soybean plants with two
anti-fungal protein genes via Agrobacterium and particle
bombardment. Biologia Plant 48:367–374
Mackintosh CA, Garvin DF, Radmer LE, Heinen SJ,
Muehlbauer GJ (2006) A model wheat cultivar for trans-
formation to improve resistance to fusarium head blight.
Plant Cell Rep 25:313–319
Mei L, Zong-xiu S, Jei Z, Tong X, Gary EH, Matteo L (2004)
Enhancing rice resistance to fungal pathogens by trans-
formation with cell degrading enzyme genes from Trich-
oderma atroviride. J Zhejiang Uni Sci 5:133–136
T (2006) Stability of transgene integration and expression
in subsequent generations of doubled haploid oilseed rape
transformed with chitinase and b-1,3-glucanase genes in a
double-gene construct. Plant Cell Rep 25:942–952
Mitani N, Kobayashi S, Nishizawa Y, Kuniga T, Matsumoto R
(2006) Transformation of trifoliate orange with rice chiti-
nase gene and the use of the transformed plant as a root-
stock. Sci Hortic 108:439–441
Mondal KK, Bhattacharya RC, Koundal KR, Chatterjee SC
(2007) Transgenic Indian mustard (Brassica juncea)
expressing tomato glucanase leads to arrested growth of
Alternaria brassicae. Plant Cell Rep 26:247–252
Moravcıkova J, Libantova J, Heldak J, Salaj JM, Matusıkova I,
Galova Z, Mlynarov L (2007) Stress-induced expression of
cucumber chitinase and Nicotiana plumbaginifolia b-1,3-
glucanase genes in transgenic potato plants. Acta Physiol
Moravc ˇı ´kova ´ J,Matus ˇı ´kova ´ I,Libantova ´ J,BauerM,Mlyna ´rova ´
L (2004) Expression of cucumber class III chitinase and
Nicotiana plumbaginifolia class I glucanase genes in
transgenic potato plants. Plant Cell Tissue Organ Cult
Nishizawa Y, Saruta M, Nakazono K, Nishio Z, Soma M, Download full-text
Yoshida T, Nakajima E, Hibi T (2003) Characterization of
transgenic rice plants over-expressing the stress-inducible
b-glucanase gene Gns1. Plant Mol Biol 51:143–152
Osusky M, Osuska L, Hancock RE, Kay W, Misra S (2004)
resistant to late blight and pink rot. Trans Res 13:181–190
transgenic potato exhibiting enhanced resistance to fungal
Rohini VK, Rao KS (2001) Transformation of peanut (Arachis
hypogaea L.) with tobacco chitinase gene: variable
response of transformants to leaf spot disease. Plant Sci
Rommens CM, Kishore GM (2000) Exploiting the full potential
of disease- esistance genes for agricultural use. Curr Opin
Sridevi G, Parameswari C, Sabapathi N, Raghupathy V,
Veluthambi K (2008) Combined expression of chitinase
and b-1,3-glucanase genes in indica rice (Oryza sativa L.)
enhances resistance against Rhizoctonia solani. Plant Sci
Flower-predominant expression ofagene encoding anovel
class I chitinase in rice (Oryza sativa L.). Plant Mol Biol
Takatsu Y, Nishizawa Y, Hibi T, Akutsu K (1999) Transgenic
chrysanthemum (Dendranthema grandiflorum (Ramat.)
resistance to gray mold (Botrytis cinerea). Sci Hortic
Terakawa T, Takaya N, Horiuchi H, Koike M, Takagi M (1997)
Afungal chitinase gene from Rhizopus oligosporus confers
antifungal activity to transgenic tobacco. Plant Cell Rep
Tobias DJ, Manoharan M, Pritsch C, Dahleen LS (2007) Co-
bombardment, integration and expression of rice chitinase
and thaumatin-like protein genes in barley (Hordeum
vulgare cv.Conlon). Plant Cell Rep 26:631–639
Tohidfar MM, Mohammadi T, Ghareyazie B (2005) Agrobac-
terium-mediated transformation of cotton (Gossypium
hirsutum) using a heterologous bean chitinase gene. Plant
Cell Tissue Organ Cult 83:83–96
van der Biezen EA (2001) Quest for antimicrobial genes to
engineer disease-resistant crops. Trends Plant Sci 6:89–91
Vellicce GR, Ricci JCD, Herna ´ndez L, Castagnaro AP (2006)
Enhanced resistance to Botrytis cinerea mediated by the
transgenic expression of the chitinase gene ch5B in
strawberry. Transgen Res 15:57–68
Wally O, Jayaraj J, Punja Z (2009) Comparative resistance to
foliar fungal pathogens in transgenic carrot plants
expressing genes encoding for chitinase, b-1,3-glucanase
and peroxidise. Eur J Plant Pathol 123:331–342
Wang Y, Kausch AP, Chandlee JM, Luo H, Ruemmele BA,
Co-transfer and expression of chitinase, glucanase, and bar
genes in creeping bentgrass for conferring fungal disease
resistance. Plant Sci 106:497–506
Wro ´bel-Kwiatkowska M, Lorenc-Kukula K, Starzycki M,
Oszmian ˜skiJ,Kepczyn ˜skaE,SzopaJ(2004)Expressionof
a ˆ-1, 3-glucanase in flax causes increased resistance to
fungi: Physiol Mol. Plant Pathol 65:245–256
Xiaotian M, Lijiang W, Chengcai AN, Huayi Y, Zhangliang C
(2000) Resistance to rice blast (Pyricularia oryzae) caused
by the expression of trichosanthin gene in transgenic rice
plants transferred through Agrobacterium method. Chinese
Sci Bulet 45:1774–1778
Yamamoto T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hibi
T, Hayashi T, Matsuta N (2000) Transgenic grapevine
plants expressing a rice chitinase with enhanced resistance
to fungal pathogens. Plant Cell Rep 19:639–646
Zhu H, Xu X, Xiao G, Yuan L, Li B (2007) Enhancing disease
resistances of super hybrid rice with four antifungal genes.
Sci China C Life Sci 50:31–39
N, GoldsmithMR (2003)