全 文 ：作物学报 ACTA AGRONOMICA SINICA 2012, 38(3): 381−393 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

This work is supported by USDA-ARS National Program NP301 project “Response of Diverse Rice Germplasm to Biotic and Abiotic Stresses
Project No. 6225-21000-008-00D”.
* Corresponding author: JIA Yu-Lin, E-mail: yulin.jia@ars.usda.gov, Tel: 1 870-672-9300
Received(收稿日期): 2011-09-21; Accepted(接受日期): 2011-12-19; Published online(网络出版日期): 2012-01-09.
URL: http://www.cnki.net/kcms/detail/11.1809.S.20120109.1558.003.html
DOI: 10.3724/SP.J.1006.2012.00381
Structure, Function, and Co-evolution of Rice Blast Resistance Genes
Moytri ROYCHOWDHURY1, JIA Yu-Lin2,*, and Richard D. CARTWRIGHT1,3
1 University of Arkansas, Cell and Molecular Biology Program, Fayetteville, AR 72701, USA; 2 USDA-ARS, Dale Bumpers National Rice Research
Center, Stuttgart, AR 72160, USA; 3 University of Arkansas, Division of Agriculture, Cooperative Extension Service, Little Rock, AR 72204, USA
Abstract: Rice blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the most destructive rice diseases
worldwide. Resistance (R) genes to blast encode proteins that detect pathogen signaling molecules encoded by M. oryzae viru-
lence (AVR) genes. R genes can be a single copy gene or a member of clustered gene families that have evolved through duplica-
tion and diversification. Recent advances in blast R gene cloning and subsequent characterization have provided useful insights
into R gene mediated signaling transduction pathways. This review summarizes recent advances in cloning and characterization of
blast R genes, and presents an update on evolutionary dynamics of R proteins, their interaction and co-evolution with the signaling
molecules encoded by the AVR genes, and potential implications for crop protection.
Keywords: R genes; AVR genes; Blast disease; Gene interaction; Magnaporthe oryzae
水稻抗稻瘟病基因的结构、功能和共同进化
Moytri ROYCHOWDHURY1 贾育林 2,* Richard D. CARTWRIGHT1,3
1 University of Arkansas, Cell and Molecular Biology Program, Fayetteville, AR 72701, USA; 2 USDA-ARS, Dale Bumpers National Rice
Research Center, Stuttgart, AR 72160, USA; 3 University of Arkansas, Division of Agriculture, Cooperative Extension Service, Little Rock,
AR 72204, USA
摘 要: 稻瘟病是由真菌 Magnaporthe oryzae所致, 是世界上最严重的水稻病害之一。抗病基因能够识别病原无毒蛋
白而导致抗病反应。抗病基因以单基因或基因簇的形式存在, 它是通过基因复制或基因多样性而产生的。近几年来,
由于抗病基因的不断克隆和功能分析, 使人们更好地理解和认识抗病机制。本文总结了目前抗病基因的克隆和功能
分析进展, 并对抗病基因的进化, 抗病蛋白和病原无毒因子之间的相互作用、相互影响和进化以及无毒因子的结构进
行了剖析, 同时指出这些理论对植物保护的潜在含义。
关键词: 抗病基因; 无毒基因; 稻瘟病; 基因互作; Magnaporthe oryzae
Rice (Oryza sativa L.) is the staple food for more than
half of the worlds population. Maintaining stable rice
production is extremely important to feed the constantly
growing human population. However, rice blast disease
caused by the pathogen Magnaporthe oryzae B. Couch
remains one of the most serious threats to secure global
rice production.
M. oryzae germinates when a conidium attaches to the
surface of the rice leaf, and subsequently, hyphae pro-
duce appressium for penetration, which normally occurs
within 24 h of spore germination depending on strains[1-2].
M. oryzae directly penetrates the cell membranes leading
to subsequent intracellular growth of mycelia that result
in the destruction of a living cell. Before cell death, my-
celia were thought to move to the adjacent cell via un-
known mechanisms including a possible utilization of
plasmodesmata[3]. The growth of the fungus within the
cell often results in impairment of transportation of water
and minerals in the vascular system. Once inside the
plant, the fungus produces thousands of spores on co-
nidiophores emerging from stomata that can be dispersed
by air currents to nearby rice plants for subsequent infec-
tion (Fig. 1). The fungus is highly adaptive to its host and
is capable of causing infection at any growing stage and
has also been known to overcome resistance in new rice
cultivars within a few years after commercialization.
Rice blast was first reported in Asia, and is now pre-
sent in more than 80 countries where rice is grown. Ac-
cording to the International Rice Research Institute[4],
rice blast is estimated to cause economic losses of up to
382 作 物 学 报 第 38卷

USD 60 million annually in South and Southeast Asia. At
least, more than 266 000 tons of rice is lost due to blast
disease in India annually. In Japan, the figure is similar,
with an estimated 200 000 tons of rice lost annually due
to blast disease. In China, blast disease has been an in-
creasing challenge for rice breeding programs and crop
production for the past two decades. In the United States,
blast disease occurs sporadically but causes significant
crop losses in favorable years. The destructive nature of
blast has drawn worldwide attention and intensive inves-
tigation. Today it is one of the best characterized model
host-path systems for understanding molecular mecha-
nisms of host genetic resistance.
Due to the extensive studies conducted worldwide, rice


Fig. 1 Rice blast disease
Germinated asexual conidium (A), sporulated mycelia on disease lesion
(B), typical symptom of leaf blast disease (C), and mature rice plants
with panicle blast and leaf blast in a rice field (D).
was among the first plant species to have its complete
genome sequenced[5]. A draft genome sequence of M.
oryzae has also been determined[6]. Availability of ge-
nome sequences of rice and Magnaporthe oryzae has
expedited progress in map-based cloning of resistance
genes in rice and understanding the molecular bases of
resistance, interaction and co-evolution of rice and M.
oryzae. In this review, we describe cloned blast resistance
(R) genes, summarize the current understanding of
host-pathogen interactions and co-evolution, and discuss
the utilization of this knowledge for crop protection.
1 Blast R genes
Genetic studies of resistance to M. oryzae began when
Goto established the differential system for races of the
blast pathogen in Japan[7]. Interaction of O. sativa with M.
oryzae in most cases follows the classical gene-for-gene
interaction where an R gene named as Pyricularia (Pi) is
effective in preventing M. oryzae races that contain the
corresponding avirulence (AVR) gene[8]. The name Pi was
derived from the imperfect asexual fungus Pyricularia
oryzae (=Magnaporthe oryzae). The gene nomenclature
in rice blast resistance was introduced in 1993. New blast
R genes are designated as Pi followed by a numeral, ex-
cept for those reported before 1993. The suffix ‘(t)’ (ten-
tative) is attached until the completion of gene isolation
and allelism tests. A total of 85 major Pi genes and over
100 minor R genes have now been described[9].
To date, a total of 18 blast R genes including 16 major and
2 minor R genes have been cloned and DNA markers asso-
ciated with each R gene are summarized in Table 1. Analysis
of the cloned blast R genes thus far revealed that they are
structurally conservative and evolutionary related (Table 1).

Table 1 Summary of chromosomal location, copy number, predicted product, and expression of cloned blast R genes
R gene
cloned Chr.
Marker (locus) closely related with
the gene
Copy
number Protein type Localization Expression
Pib 2 RM138, RM166, RM266, Pib-dom Multiple CC-NBS-LRR Circadian, inducible by stress
Pita 12 OSM89, RM7102 1 CC-NBS-LRR Cytoplasm Constitutive
Pi9 6 NBS2-Pi9, NBS4Pi9 Multiple CC-NBS-LRR Constitutive
Pi2/Piz-t 6 R2123, RG64/zt56591, zt4792, zt6057 Multiple CC-NBS-LRR Constitutive
Pid2 6 RM3, RM527 1 Receptor kinase/B lectin Membrane Constitutive
Pi36 8 CRG2, CRG3, CRG4, RM5647 1 CC-NBS-LRR Constitutive
Pi37 1 RM543, FPSM1 Multiple CC-NBS-LRR Cytoplasm Constitutive
Pikm 11 K2167, K4731 Multiple CC-NBS-LRR Constitutive
Pi5 9 S04G03 Multiple CC-NBS-LRR Pi5-1 is pathogen dependentPi5-2 is constitutive
Pit 1 tdDN, tdDK, tNpB, tK59 Multiple CC-NBS-LRR Transcriptionally inactive
Pid3 dCAPS-1 Multiple CC-NBS-LRR Constitutive
pi21 4 G271, G317 Multiple Proline containing protein /CC-NBS-LRR Cytoplasm Inducible by stress
Pb1 11 NA-2, N3-2 Multiple CC-NBS-LRR Transcriptionally inactive
Pia 11 OS11gRGA4, Adh1 Multiple CC-NBS-LRR Constitutive
Pish 1 RM212, OSR3 Multiple CC-NBS-LRR Constitutive
Pik
(Pik-P) 11 K34, RM5766 Multiple CC-NBS-LRR Constitutive
第 3期 Moytri ROYCHOWDHURY et al.: Structure, Function, and Co-evolution of Rice Blast Resistance Genes 383


1.1 The Pib gene
The Pib gene on chromosome 2 was the first blast R
gene cloned[10]. This gene confers high levels of resis-
tance to most blast races in Japan (Table 2). Pib conveys
resistance to seven blast races in the US. Pib has been
introgressed into various japonica cultivars independ-
ently from two Indonesian and Malaysian cultivars. In
the US, Pib was introgressed into rice cultivar Saber
from the Chinese indica cultivar Teqing[11]. Pib is a
member of a small gene family encoding a cytoplasmic
protein with nucleotide binding sites (NBS) and
C-terminal leucine rich repeats (LRRs), but no distinct
transmembrane domain. An NBS domain is a signaling
motif shared by plant R-gene products[12]. A duplication
of the kinase 1a, 2 and 3a motifs of the NBS region was
found in the N terminal half of the Pib protein. In addi-
tion, eight cysteine residues were clustered within the
LRRs, a feature not observed in any other R proteins.
Northern blot analysis of the Pib gene family members
(Pib, PibH8, HPibH8-1, and HPibH8-2) revealed that
their expression was regulated by environmental signals
such as temperature, light, water and chemical treatments,
including jasmonic acid, salicylic acid, ethylene and
probenazole. The Pib gene family is, to the best of our
knowledge, the first plant R gene family to be investi-
gated extensively at the transcription level.

Table 2 Resistance spectrum of blast R genes to known races of Magnaporthe oryzae
R gene The US blast races International blast races/isolates
Pib IB1, IB45, IH1, IG1, IC17, IE1, IE1K 003.0
Pita IA45, IB1, IB49, IB54, IB45, IC17, ID1, IG1, IE1, IH1
Pi9 IC17
PH9, 36B23, 86061ZE39, 97-4-1, 95116AZ93, 75-49, 97-51, CHNOS,
95097AZC13, 87088ZE3, 86062ZB15, CP16-32, R01-1, KJ201, ML25,
ML8, O-249, DB-24, GUY11, ES6
Pi2/Piz-t IH1, IG1, IC17, IE1, IE1K KJ201, 81278ZB15, G2, CHE86061, G2, G11, G15, CHNOS60-2-3, ROR1
Pid2 ZB15
Pi36 CHL39, CHL273
Pi37 CHL1159
Pikm P2-b, Kyu92-22
Pi5 PO6-6, KJ105a, KJ107, KJ401, R01-1, K1215
Pit 007.0, 777.3
Pid3 Zhong-10-8-14
pi21 007.0
Pb1 003.0, MAFF101506

1.2 The Pita gene
The Pita gene on chromosome 12 was the second blast
R gene cloned[13]. Pita confers resistance to a wide range
of blast races worldwide (Table 2). Katy was the first US
cultivar reported to contain the Pita gene that was de-
rived from the landrace indica variety Tetep[14]. Subse-
quently, ten rice cultivars, Drew, Madison, Kaybonnet,
Cybonnet, Ahrent, Banks, Spring, Catahoula, CL1111,
and Templeton in the US were developed. Pita encodes a
putative cytoplasmic receptor with a centrally localized
nucleotide-binding site and leucine-rich domain (LRD) at
the C-terminus[15]. It has a conserved internal hydropho-
bic domain characteristic of other NBS-class R gene pro-
teins[15] between two amino acids and four potential N
glycosylation sites. Pita differs from the dicot class of
NBS R genes in having a unique N terminus. It does not
have a leucine zipper or Toll/interleukin-1 receptor ho-
mology. The Pita carboxyl terminal domain is rich in
leucine and is thus referred to as the leucine rich domain.
It lacks the classical LRR motif found in other genes of
this class. Susceptible rice varieties were found to con-
tain a single amino acid difference relative to the Pita
resistance protein—the amino acid residue serine was in
place of alanine at the 918 position.
1.3 The Pi9 gene
The Pi9 gene on chromosome 6 is known to confer re-
sistance to the US race IC17 (Table 2)[16]. Pi9 belongs to
the NBS–LRR class of R genes. Although Pi9 has two
introns in its coding region, unlike other R genes in rice,
one of the introns is much larger (5 362 bp) than that of
the Pib gene. Whether this unique feature in the Pi9 gene
has any association with its broad resistance spectrum
will require further research. The Pi9 protein has a con-
served nT motif (WAEQIRDLSYDIEDSLDEF) which is
located 107 amino acids before the P-loop. The LRR do-
main in Pi9 is similar to that of the Pib protein and con-
sists mainly of imperfect LRR repeats. A unique struc-
tural feature of the Pi9 protein is that it contains a
57-amino-acid non-LRR region at the C terminus. In
contrast, the LRRs in both Pib and Pita extend to the end
of the C terminus. Further research is needed to investi-
gate whether this 57-amino-acid sequence at the C ter-
minus of Pi9 has any special function in regulating resis-
tance specificity to rice blast. Unlike Pib, the expression
384 作 物 学 报 第 38卷

profile of the Pi9 gene showed that Pi9 was constitu-
tively expressed in Pi9-carrying plants and was not in-
duced by blast infection.
1.4 The Pi2/Piz-t genes
The Pi2/Piz-t gene is located on chromosome 6 and is
indica-derived[17]. The structure of Pi2 and Piz-(t) in
terms of intron and exon size and position was deter-
mined by cloning the Pi2/Piz-(t) coding region. The
transcripts consisting of the entire coding region were
cloned by reverse transcriptase-PCR with the primer pair
NBS4F and NBS2R, which can amplify both Pi2 and
Piz-(t). Results showed 3 332- and 3 335-bp fragments
for Pi2 and Piz-(t), respectively, that contain a 117-bp 5′
UTR and a 116-bp 3′ UTR for both Pi2 and Piz-(t).
Aligning the sequences of the cloned Pi2 and Piz-(t)
transcripts with their genomic sequences revealed that
Pi2 and Piz-(t) contain two introns that have the same
genomic position and identical sequence to each other.
The first intron, 3 839 bp in length, is 116 bp downstream
of the start codon, corresponding to the region before the
NBS domain. The second intron, 128 bp in length, is 31
bp upstream of the stop codon, corresponding to the re-
gion after the LRR domain. A DNA fragment in the first
intron was identified which was 177 bp in length and
shared 93% sequence identity to the first 177 bp portion
of the second exon. Pi2 and Piz-t encode a 1 032- and a
1 033-amino-acid protein product, respectively, belong-
ing to an nT-NBS-LRR class of R proteins. The nT motif
is located 68 to 86 amino acids away at the N-terminal
region. The centralized NBS domain is located 153 to
460 amino acids away from the N terminal and has all
the essential motifs: kinase 1a or P-loop (193 to 202
amino acids), kinase 2 (281 to 287 amino acids), and
kinase 3a or RNBS-B (307 to315 amino acids), and
GLPL (373 to 378 amino acids). There are also 17 im-
perfect LRR repeats predicted based on the xxLxLxx
motif representing most of the 3′ portion of the protein.
Piz-(t) showed a similar expression pattern as Pi2. The
constitutive expression pattern of Pi2 and Piz-t is quite
similar to that of both Pi9[16] and Pita[13] but different
from Pib, which exhibits an induced expression pattern
after rice blast inoculation[18].
1.5 The Pid2 gene
The Pid2 gene on chromosome 6 was first identified in
the indica cultivar Digu[19]. Pid2 represents a new class
of plant R genes. Similar to Pita, Pid2 is also a single
copy gene. Pid2 confers resistance to numerous Chinese
blast races. However, there has been no report of resis-
tance to US blast races. Pid2 encodes a predicted protein
of 825 amino acids. The amino acid sequence of the Pid2
protein contains the domain characteristics of recep-
tor-like kinases (RLK), an extracellular domain, a trans-
membrane (TM) domain, and an intracellular kinase do-
main. Pid2 also contains a predicted extracellular
bulb-type mannose-specific lectin (B-lectin) binding do-
main that has not been reported in other plant R proteins.
The N-terminus of Pid2, amino acids 1–32, contains a
hydrophobic region with a predicted transit peptide func-
tion. The putative extracellular domain contains two re-
gions with known motifs. First, amino acids 48–16 en-
code a B-lectin domain (SMART 1e-19) that was pre-
dicted to mediate protein–mannose interactions or ligand
binding[20]. Additionally, amino acids 337 to 418 are pre-
dicted to encode a weak PAN domain (smart e-02) that
binds proteins or carbohydrates[19]. The core of the PAN
domain in the region of amino acids 337–403 was pre-
dicted to be connected with the formation of three disul-
phide bridges[19]. The TM-spanning region contains 23
hydrophobic amino acids (amino acids 436–458) that are
associated with a membrane-spanning helix. The cyto-
plasmic region contains a predicted serine–threonine
kinase with 11 kinase subdomains without the conserved
R in subdomain VI, suggesting that Pid2 belongs to the
non-RD class of kinases. The Pid2 protein is localized in
the plasma membrane. Similar to Pita, a single amino
acid at position 441 distinguishes resistant and suscepti-
ble alleles of Pid2. Both quantitative RT-PCR and north-
ern analysis indicated that Pid2 is constitutively ex-
pressed.
1.6 The Pi36 gene
The Pi36 gene on chromosome 8 was first identified in
the indica cultivar Kasalth[21]. Pi36 confers resistance to
a wide variety of Chinese blast races/isolates (Table 2).
Pi36 is a single-copy gene and is more closely related to
the barley powdery mildew R genes Mla1 and Mla6 than
to the rice blast R genes Pita, Pib, Pi9, and Piz-t. It has
not been reported if the gene conveys resistance to any
US blast races. The 1 056-amino acid sequence of the
Pi36 protein has six conserved motifs typical of NBS
proteins. The GMGGLGKTT sequence (beginning at
residue 206) is the kinase 1a (P loop) consensus, while
IVIDDIWD (beginning at residue 286) and GSKILVTT-
RK (beginning at residue 310) represent the kinase 2 and
kinase 3a consensus motifs[21-22], respectively. Also,
GVPLAIITIAS (beginning at residue 372) and LKNCL-
LYL (beginning at residue 427) represent the conserved
NBS domains 2 and 3 consensus motifs[22-23], respectively.
The conserved NBS motif VHD (beginning at residue
501) is similar to the conserved MHD (methion-
ine–histidine–aspartate) motif. The C-terminal region of
the protein includes 17 imperfect LRR repeats (residues
578–1 056), composed of 15% leucine. The repeats,
based on an LxxLxxLxxLxL consensus, vary in length
between 22 and 44 amino acids. The CC region contains
three perfect hxxhxxh and one hxxhxxx motif (where h
represents one of L, I, M, V, or F, and x is any residue).
Together, these findings indicate that Pi36 is a
CC–NBS–LRR type R gene. Similar to Pita and Pid2, a
single substitution event (Asp to Ser) at residue 590 was
considered to be associated with the resistant phenotype.
第 3期 Moytri ROYCHOWDHURY et al.: Structure, Function, and Co-evolution of Rice Blast Resistance Genes 385


An RT-PCR analysis showed that Pi36 is constitutively
expressed in Kasalath.
1.7 The Pi37 gene
The Pi37 gene on chromosome 1 was identified in the
rice cultivar St. No. 1[24]. Pi37 confers resistance to a
wide range of Chinese rice blast races but not the Japa-
nese rice blast races (Table 2). No reports were available
with regards to the response to US blast races. Pi37 was
considered to be the first representative of a cereal
NBS–LRR gene lacking an intron. Pi37 encodes a cyto-
plasmic protein with NBS–LRR domains. In the NBS
region, two substitutions (V239A and I247M) were
shown to be associated with the resistance phenotype.
The Pi37 open reading frame encoded a 1 291-residue
polypeptide. The N-terminal section contained three
typical NBS family motifs, specifically GGAGKS (be-
ginning at residue 222), LLVLDDV (beginning at residue
297), and GSRVLVTSRR (beginning at residue 327).
These corresponded to the kinase 1a (P-loop), the kinase
2, and the kinase 3a consensus motifs, respectively. The
C-terminal region of the protein has 25 irregular LRRs
between residues 590 and 1 290. Semi-quantitative ex-
pression analysis showed that in St. No. 1, Pi-37 was
constitutively expressed and only slightly induced by
blast infection. Transient expression experiments indi-
cated that the Pi37 product was restricted to the cyto-
plasm.
1.8 The Pikm gene
The Pikm gene on chromosome 11 was first identified
in the Chinese japonica cultivar Hokushi Tami. Complete
resistance of Pikm requires functions of two family
members Pikm1-TS and Pikm2-TS[25]. Pikm1-TS and
Pikm2-TS reside adjacently and encode non-TIR NBS-
LRR-class proteins. Although Pikm1-TS and Pikm2-TS
reside adjacently at the Pikm locus as a cluster, their
structures differ. First, they differed in the number and
position of introns: both Pikm1-TS and Pikm2-TS con-
tained an intron at the N-terminal side of the sequence
encoding the kinase 2 motif in the NBS domain. In addi-
tion, Pikm1-TS also contained another intron upstream of
the sequence encoding the NBS domain. Second, the
Pikm1-TS product contained a C-terminal non-LRR re-
gion; however, Pikm2-TS did not. Finally, Pikm1-TS
contained well conserved repeat units matching a con-
sensus sequence in its LRR domain, whereas Pikm2-TS
did not. All of the above-mentioned structural differences
indicate that these two genes did not evolve from one
another by a simple duplication event.
Expression of both Pikm1-TSand Pikm2-TS was de-
tected in uninoculated plants. Following blast inocula-
tion, expression of Pikm1-TS increased from 0.5 to 3
days after inoculation (DAI), and declined toward the
original level by 5 DAI. Therefore, the induced expres-
sion in Pikm1-TS was not detected in the negative
control inoculations, indicating that the observed induc-
tion of Pikm1-TS expression was due to the challenge of
blast infection. In contrast, although expression of
Pikm2-TS appeared to increase slightly from 0.5 to 3.0
DAI, the extent of the induction was relatively minor.
1.9 The Pi5 gene
The Pi5 gene on the short arm of chromosome 9 con-
fers resistance to numerous Korean and Philippines blast
races[26] (Table 2). Similar to Pikm, two members, Pi5-1
and Pi5-2 are required for resistance. Both members were
predicted to encode an N-terminal CC, a centrally located
NB and LRR, and C-terminal regions. Residues 109–576
of Pi5-1 and 109–567 of Pi5-2 have an NB domain. The
conserved internal domains characteristic of NB-
containing R-gene products were also identified in Pi5-1
and Pi5-2, including the P-loop, kinase-2, RNBS-B,
GLPL, RNBS-D, and MHDV domains. The Pi5-1 and
Pi5-2 proteins harbor a unique C terminus that is distinct
from those of other NB–LRR proteins[19] and that does
not match any known protein motif. The position of in-
trons in the NB domain of the Pi5 protein was studied to
better understand the phylogenetic relationship between
Pi5 and other cloned rice blast resistance genes. Notably,
Pi5-1 and Pi5-2 harbor an intron between their RNBS-D
and MHDV domains[27]. In addition, Pi5-1 and Pi5-2
appear to have more introns (four and five, respectively)
compared to other identified blast R genes. The distinc-
tive number of introns and the genomic positions of Pi5-1
and Pi5-2 further validate that they belong to the same
clade and are different from other NBS–LRR genes[26].
Gene expression results indicated that Pi5-1 transcripts
accumulate after pathogen challenge, whereas the Pi5-2
gene is constitutively expressed.
1.10 The Pit gene
The Pit gene, located on chromosome 1[28], was ini-
tially reported in the Indonesian rice variety Tjahaja[29].
Pit confers resistance to a broad spectrum of Japanese
blast races[28] (Table 2). The structure of the Pit protein is
typical of NBS-LRR proteins. Pit contains conserved
motifs indicative of an NBS domain, and the putative
LRR domain (with 18 imperfect repeats) matches the
cytoplasmic LRR consensus sequence. A COILS analysis
of the Pit protein sequence detected two CC regions, lo-
cated between the 27th and 54th (maximum probability:
52%), and between the 112th and 147th, amino acid posi-
tions (maximum probability: 86%). In the N-terminal
region of the protein, an nT motif located between the
68th and 81st amino acid position was identified between
the two CC regions. These results indicate that Pit is a
CC-NBS-LRR-type R gene.
Expression of Pit-K59 and Pit-Npb was compared by
RT-PCR at 0, 8, 16, 24, and 48 h after inoculation with
M. oryzae or water (mock inoculation). In Nipponbare
leaves, a constitutive but low level of Pit expression was
detected. The decline in transcription in K59 was proba-
bly not a result of the inoculation, but a result of the ex-
perimental conditions (dark treatment with high humi-
386 作 物 学 报 第 38卷

dity). The increased level of Pit transcription in K59
compared with Nipponbare was suggested to be due to
the LTR retrotransposon Renovator. Renovator is known
to contain a promoter in its long terminal repeat (LTR)
which enhances expression, in this case of Pit. Renovator
belongs to a family of Ty1/copia-like retrotransposons
classified as rn_44 in the RetrOryza database[29]. Reno-
vator is 5.5-kb long and is composed of two identical
114-bp LTRs, bordered by a 5-bp target site duplica-
tion[29].
Many plants have a large number of NBS-LRR-type R
gene analogs (RGAs), of which only a few have been
assigned functions as disease resistance genes. RGA su-
per families are thought to have been generated by tan-
dem or segmental duplication of ancestral genes during
evolution. For an RGA to function as an R gene, it must
be expressed in an appropriate temporal and spatial
manner. Therefore, duplication of the coding sequence
alone is not sufficient for multiplying R genes. New R
genes could be generated by duplication of a transcrip-
tionally active R gene as a unit, including transcriptional
regulatory sequences as well as coding sequences, fol-
lowed by sequence diversification.
Another mechanism could be the transcriptional acti-
vation of otherwise transcriptionally-inactive RGAs
through acquisition of promoter sequences. It was re-
ported that Pit was created as a result of transcriptional
activation of an inactive sleeping RGA[29]. The functional
Pit allele was formed as a result of insertion of Renovator
upstream of the Pit sequence. The acquisition of pro-
moter sequences therefore seems to be a general mecha-
nism that generates R genes.
1.11 The Pid3 gene
The Pid3 gene was first reported in indica variety
Digu[30]. The gene is known to confer resistance to indica
and japonica races collected from China (Table 2). There
is no report if this gene confers resistance to US blast
races. The Pid3 gene encodes a 924-amino-acid polypep-
tide that contains a conserved NBS domain in positions
158–466 from the translation initiation site. The NBS
domain has four sequence motifs, GMGGIGKTA (posi-
tions 202–210), KRYVLVLDDVW (positions 280–290),
IGRIILTSRNYDV (positions 307–319), and GLPIAI
(positions 373–378), corresponding to kinase 1a (p-loop),
kinase 2, kinase 3a, and GLPL motifs, respectively. At
the C terminus is the LRR region that comprises 13 im-
perfect LRR repeats. The MHD motif, MHDILRV (posi-
tions 502–508), and the NBS–LRR linker motif,
EQNFCIVVNHS (positions 516–526), are present be-
tween the NBS domain and the LRR region. At the N
terminus, there is a conserved motif, RSLALSIEDVVD
(positions 78–89), but no TIR or coiled-coil motif is
found. Expression studies indicated that the gene was
constitutively expressed. It is worth to note that the Pi25
gene in a resistant cultivar Gumei 2 was an allele of Pid3
with a single nucleotide substitution resulting in no
changes of the Pid3 protein[31].
1.12 The pi21 gene
The pi21 locus was originally identified as a major
QTL that was mapped on chromosome 4[32]. The resistant
pi21 allele was first identified in the japonica cultivar
Owarihatamopchi. This recessive pi21 gene is known to
confer non-race-specific resistance. The dominant Pi21
gene encodes a proline-rich protein that has a putative
heavy metal binding domain and putative protein-protein
interaction motifs. Wild type Pi21 appears to slow down
defense responses[32]. However, deletions in the proline-
rich motif inhibit slowing of defense responses. It was
reported that the deletion was in 18- and 48-bp sequences
and the resistant pi21 allele carrying the 18 and 48 bp
deletion was only observed in a japonica cultivar. Hence
it was hypothesized that the deletion of both the 18- and
48-bp sequences resulted in a defect of the pi21 function,
which represents the consensus sequence motif PxxPxxP,
the core motif for protein-protein interaction in multicel-
lular organisms[32]. The proline rich motifs (PRMs) were
thought to be associated with host defense, possibly
through competitive inhibition of protein-protein interac-
tion of the proline rich motif and its counterpart. The
PRMs contain several proline residues, most of which are
organized in repeats of three. The heavy metal trans-
port/detoxification protein domain has two conserved
cysteines involved in metal binding. Although it con-
ferred resistance to blast, the pi21 allele was associated
with poor flavor in cooked rice, and thus was not used in
commercial cultivars. Expression results indicated that
the gene is not constitutively expressed, but dependent on
stress factors including humidity.
1.13 The Pb1 gene
The panicle blast R gene 1 (Pb1) was derived from the
indica cultivar Modan[33]. Pb1 is located in the middle of
the long arm of chromosome 11[34]. The gene confers
resistance to a wide variety of Japanese blast races and a
few races from Indonesia, China, the Philippines, Brazil,
and Thailand. There is no report of its resistance to US
blast races (Table 2). The gene is partially resistant to leaf
blast but is more efficient in conferring resistance to
panicle blast, and resistance is quantitative.
Pb1 has two putative CC domains, CC1 and CC2, lo-
cated in its N-terminus, with an nT motif-like sequence
intervening them[33]. The COILS analysis did not con-
sider these sequences as CC domains, and the periodical
occurrence of leucine, or other hydrophobic amino acids,
was observed in these regions. In addition, this region
shared amino acid sequence similarity with CC domains
of other CC-NBS-LRR proteins, including barley
MLA10[35] and Arabidopsis RPM1[22]. The EDVID motif
is associated with intramolecular interaction with other
parts of CC-NBS-LRR R proteins, and is needed for in-
ducing the hypersensitive response (HR) phenotype[36].
第 3期 Moytri ROYCHOWDHURY et al.: Structure, Function, and Co-evolution of Rice Blast Resistance Genes 387


This motif is degenerated in Pb1 so it is likely not func-
tionally conserved in Pb1. In many R proteins the pen-
tapeptide EDVID motif is conserved within the nt motif.
Adjacent to the CC region is the NBS domain-like region,
followed by an LRR domain consisting of 14 imperfect
leucine-rich repeats (residues 928–1 296). The Pb1 pro-
tein differed from the previously reported R proteins,
particularly in the NBS domain, which is different from
the typical NBS-LRR proteins because it lacks the P loop.
A long stretch of peptides with no significant sequence
similarity to other sequences is located after the CC re-
gion, and a walker-like sequence is present at amino acid
position 641. The NBS domain also has RNBS-B and
GLPL motif-like sequences. The RNBS-D and MHD
motifs near the C-terminal ends of the NBS domain,
which transduce pathogen perception by LRR into R
protein, are highly conserved in the Pb1 and R protein.
Therefore, the NBS domain of Pb1 is homologous to
those in R proteins in its C-terminal region, but its ho-
mology becomes weaker towards the N-terminus. The
local genome duplication of a 60-kb region was predicted
to place a promoter sequence just upstream of a tran-
scriptionally inactive ‘sleeping’ RGA, resulting in activa-
tion of the RGA and generation of Pb1. The structure of
the Pb1 locus indicates that the coding and the promoter
sequences were located at the 5′ and 3′-termini, respec-
tively, of the ancestral 60-kb region before the duplica-
tion occurred. The genome duplication at this specific
site was therefore critical for the generation of Pb1. The
acquisition of promoter sequences therefore seems to be
a general mechanism that generates R genes, and is of
particular importance in the case of Pb1. The characteris-
tic temporal and spatial pattern of Pb1 promoter activity
is likely to be one of key factors contributing to the dura-
bility of its resistance and therefore its practical useful-
ness as a panicle resistance gene.
1.14 The Pia gene
The Pia gene has not been reported in US cultivars,
however, has been found in numerous japonica cultivars
in Japan that was isolated using a combination of candi-
date gene, association of DNA sequence variation with
phenotype of wild and induced mutants, protoplast assays
using both candidate and the corresponding avirulence
gene system and verified by stable transformation[37].
Two NBS-LRR types located in proximal to each other
on chromosome 11 were found to be essential for com-
plete resistance to the fungal strains that carry the corre-
sponding avirulence gene AVR-Pia both in transient and
stable assays. Limited partial DNA sequence analysis
suggests that Pia-1, Os11gRGA4 was much more con-
served than Pia-2, Os11gRGA5 where more non-
synonymous substitutions than synonymous substitution
was observed. In Pia1, most nucleotide variation was
observed at 5′ region that encode CC and NBS domains
where in Pia-2, most nucleotide variation was observed
at 3′ region that encode LRR domain. AVR-Pia was
demonstrated to recognize Pia inside plant cell[38], and
location of the product of Pia was also predicted to be in
the cytoplasm of plant cell. It would be of significant to
determine how these NBS-LRR proteins interact with
product of AVR-Pia inside plant cells in activating effec-
tive defense responses. Despite genome organization and
expression of Pia have not been clearly demonstrated,
cloning of blast R gene using candidate gene approach
has been approved to be one of the fastest methods for
positional cloning of blast R genes.
1.15 The Pik/Pik-p genes
The Pik locus on the long arm of chromosome 11 is a
complex locus where allelic specificity has been well
documented. Thus far, Pik-p, Pikh, Piks, Pikm, Pik alleles
were found in diverse rice germplasm. Among them,
Pikh(Pikm) and Piks have been reported in US cultivars
that provide resistance to the blast races, IB54, IB45, IH1
and IG1, and IB54 in the US respectively [39]. The Pik
gene in cutlivar Kusabue was isolated using classical
map-based cloning and candidate gene approach. Two
NBS-LRR type genes in cluster were demonstrated to be
essential for completed resistance using stable transfor-
mation[40]. RNA interference assays were also used to
demonstrate the contribution of each gene to the resistant
phenotype.
An allele of Pik, Pik-p in cultivar K60 was also iso-
lated and their predicted structures are highly similar to
that of Pik[41]. Again two NBS-LRR type genes are re-
quired for resistant function. It is worth to note that Pik-p
was the only allele that was found in wild relatives of
rice in recent surveys using allele specific marker. This
result suggests that Pik-p was the ancestral allele and
other Pik alleles were evolved after rice domestication.
Further germplasm survey will definite provide more
clues into their introductions and evolution.
1.16 The Pish gene
The Pish gene in Nipponbare was previously mapped
at a quantitative locus on chromosome 1, and was diffi-
cult to clone. Recently, Pish was isolated using Tos 17
transposon mediated mutant screening and DNA se-
quence analysis of candidate genes[42]. A total of 58 mu-
tants occurred in a candidate NBS-LRR type gene have
been detected that were associated with the loss of resis-
tance and 14 mutations occurred at other genes suggest-
ing that mutations occurred in other plant partner for Pish
mediated resistance. Using real time PCR it was demon-
strated that Pish was constitutively expressed and no ob-
vious changes of transcripts were observed upon inocula-
tion with both compatible and incompatible blast races.
Expression of Pish is similar to that of the most cloned
plant R gene.
This candidate NBS-LRR gene was confirmed to be
Pish by stable transformation. Interestingly three other
NBS-LRR type genes were also found within a 55 kb
388 作 物 学 报 第 38卷

genomic region with Pish. One of which was highly
similar to Pi37 with putative product differing with two
amino acids (V239 and I247) in the NBS domain. This
analysis suggests that V239A and I247M may determine
recognition specificity of Pi37-mediated blast resistance.
2 R-AVR Recognition
A simple explanation of gene for gene interaction is
products of both R and AVR interact directly. The
Pita/AVR-Pita interaction has been the only well charac-
terized R/AVR interaction demonstrated in the rice blast
system to date[43]. Transient expression in rice cells of the
Pita gene together with AVR-Pita induces a resistance
response. The AVR-Pita176 protein was demonstrated to
bind specifically to the LRD of the Pita protein, both in
the yeast two-hybrid system and in an in vitro binding
assay, suggesting that the AVR-Pita176 protein binds di-
rectly to the Pita LRD region inside the plant cell to initi-
ate a Pita-mediated defense response. Identification of
genes downstream of R genes has become critical for
understanding the R–AVR recognition and transduction
pathway. The absence of efficient NSB-LRR R pro-
tein-protein assay, gene function redundancy and lethality
when mutagenesis is used that all contribute to an ineffi-
cient R-AVR recognition. Using a mutagenesis approach,
a Pita-susceptible mutant referred to as Ptr(t) was identi-
fied which was required for Pita resistance. Ptr(t) is
probably specific to Pita-mediated signal recognition
because it is not required by other R genes. Genetic
analysis results showed that Ptr(t) segregated as a single
dominant nuclear gene linked with Pita within in a nine
megabase region[44]. Cloning this gene will improve un-
derstanding of Pita-mediated signal recognition and
transduction.
AVR proteins serve as R-protein associated effectors
that activate host defense responses. Thus far, 40 AVR
genes of M. oryzae have been identified[38, 45-48], nine of
which have been cloned (Table 3).

Table 3 Summary of the corresponding R gene and predicted product of cloned AVR gene in Magnaporthe oryzae
AVR gene R gene Encoding protein
PWL1 Glycine rich, hydrophilic protein, secreted protein
PWL2 Glycine rich, hydrophilic protein, secreted protein
Avr Pi-ta Pi-ta Secreted protein
Avr1-CO39 Pi-CO39 Secreted protein
ACE1 Pi33 Polyketide synthase/peptide synthetase
AvrPiz-t Piz-t Secreted protein
AvrPia Pia Secreted protein
AvrPii Pii Secreted protein
AvrPik/km/kp Pik/km/kp Secreted protein

Effectors are protein molecules secreted by the patho-
gen and commonly located in unstable genomic regions.
Multiple genetic mutation events, like deletion[48], point
mutations[48], and transposon insertion[49-50], have been
found to be major driving forces in the creation of new
virulent races that break major R genes. Orbach et al.[48]
found that a fragment deletion from intron 3 to exon 4 in
the avr-pita- mutant strain CP983; several nonsense mu-
tations (e.g. TGG1487TAG in the mutant strain CP918
and TTA1736TGA in the mutant strain CP1615); and a
missense mutation (e.g. GAA1718GGA in the mutant
strain CP1635) increased the virulence of the Avr-Pita
gene. In addition, transposon insertion usually leads to
loss of function of Avr genes in the pathogen through a
change in gene expression level or pattern. Fudal found
that an insertion of a 1.9-kb MINE retrotransposon in the
last ACE1 exon led to a loss of ACE1 avirulence and ac-
tivated its virulence in the virulent isolate 2/0/3. Similar
events were also reported in AvrPiz[51]. This analysis
suggests that AVR genes in M. oryzae are not stable.
3 Co-evolution of R and AVR genes
The instability of AVR in M. oryzae can allow the fun-
gus to escape resistance in a very short time. As reviewed
in a previous section, Pita, Pid2, Pi36 are single copy
genes with a single amino acid determining resistance
specificity. Other blast R genes are members of small
gene families that can evolve specificity during unequal
crossing over. Most cloned blast R genes are predicted to
encode highly similar cytoplasmic proteins with
NBS-LRR domains[52] (Fig. 2, Fig. 3). AVR genes encode
effector molecules which favor disease development and
are under constant selection. The fundamental question is
how plant R genes have evolved the ability to detect
pathogen signals (Fig.4).
Recent studies suggest that evolution of blast R gene
may involve in transposons. Pit was demonstrated to be
activated by a transposon in the promoter region[29]. A
different transposon at the promoter region of the Pita
gene was found to be highly associated with blast resis-
tance[53]. Pita is located near the centromere, a region that
is relatively stable with few active genes[13]. Both Pit and
Pita cases led to a hypothesis that transposon could play
a positive role in regulating blast R genes. Additionally,
Pita was predicted to encode 12 distinct products be-
tween 315 and 1 033 amino acids that can function as
resistance proteins[54], suggesting that posttranscriptional
modifications through exon skipping and alternative
第 3期 Moytri ROYCHOWDHURY et al.: Structure, Function, and Co-evolution of Rice Blast Resistance Genes 389


splicing may play an important role in evolution of a
blast R gene. Despite convincing experimental evidence
is still unavailable, AVR gene products were predicted to
be involved in promoting pathogen virulence and fitness.
In fact, diversification of AVR genes was thought to be
one of the strategies that the pathogen can develop for
survival[55-56]. Diversification of AVR genes has been
found through partial, complete deletions, frame-shift
mutations, and/or transposon insertions. Further charac-
terization of matched pairs of blast Pi and AVR genes
should help determine if these genomic rearrangement
and gene regulation are key strategies that host and
pathogen have employed during co-evolution of plant
and pathogen.

Fig. 2 Phylogenetic tree of selected cloned blast R genes
This tree was constructed using Vector NTI using genomic sequence (A) and coding region (B).

4 Strategies for preventing blast disease
R gene-mediated resistance is attractive for disease
control for farmers with special benefit for environment.
When resistance mediated by R genes induced in a
timely manner, the responses can efficiently stop patho-
gen growth with minimal collateral damage to the
plant[57]. No input will be required from the farmer and
there are no adverse environmental effects. One approach
for R gene deployment is to sow a mixture of lines each
expressing a different R gene(s) in the same field[57-58]. A
susceptible line can be included in the mixture to reduce
the selection pressure for mutations in Avr genes[58]. A
multiline protocol was tested with striking success ex-
perimentally[57]; however, multiline resistance has not
been widely extended because of logistic difficulties in
its deployment in different rice production systems
worldwide. Many R genes lack durability because they
can be defeated by a single loss-of-function mutation in
the corresponding AVR gene. Because individual Avr
genes often make only incremental contributions to viru-
lence, pathogens can alter or discard an Avr gene with
little or no fitness penalty. Traditional breeding strategies
have used R genes “one at a time” in crop monocultures.
Such homogeneous host populations exert strong selec-
tion for mutation of the relevant Avr gene, and then be-
come extremely vulnerable to the emerging pathogen. As
an alternative to single-gene deployment, multiple R
genes (“pyramids”) with overlapped resistance spectra to
different races/isolates can be bred into individual plant
lines. In reality, these pyramids require the pathogen to
accumulate mutations in multiple Avr genes to escape
detection, which is not likely to occur if the mutations
have a strong cumulative effect on virulence. This analy-
sis suggests that stacking major blast R genes into elite
rice breeding lines should continue to be an important
strategy to manage rice blast disease.
第 3期 Moytri ROYCHOWDHURY et al.: Structure, Function, and Co-evolution of Rice Blast Resistance Genes 391


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390 作 物 学 报 第 38卷

5 Future perspectives
Plants do not have the benefit of a circulating antibody
system like animals so plant cells autonomously maintain
constant monitoring against pathogens by expressing
large arrays of R genes[60-62]. Despite plant R genes have
been used in breeding programs for decades the global
food supply is still suffering severe losses incurred by
rapid changes of the pathogens under unpredicted global
climate. Much effort in the future should be engaged
towards understanding innate resistance mechanisms in
order to develop innate immunity in plants.


Fig. 3 Schematic presentation of products of selected blast R genes showing predicted functional motifs.



Fig. 4 Molecular mechanisms of blast R gene mediated responses
in compatible and incompatible interactions
R indicates resistance and S indicates susceptible.
Acknowledgements
The authors would like to thank Dr. Peter Horevaj of
Department of Plant Pathology, University of Arkansas,
and Dr. Pesach Lubinsky of USDA for critical reviews
and Ellen McWhirter of DB NRRC for proof reading,
and Justin Owens and other staff members of DB NRRC
for their technical assistance. USDA is an equal opportu-
nity provider and employer.
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会议消息
作物杂种优势利用国际学术大会
为了更好地总结作物杂种优势研究与利用的成果与经验, 由中国工程院、国家外国专家局和陕西省人民
政府主办, 西北农林科技大学承办的“作物杂种优势利用国际学术大会”将于 2012 年 8 月 20~22 日在陕西西
安召开。
会议宗旨: 积极促进作物杂种优势利用领域国际间的学术交流与合作, 着力推动作物杂种优势利用科
学技术在全球的发展, 全面推进作物杂种优势利用产业化进程, 为全世界粮食安全和农产品有效供给做出
新的更大的贡献。
会议主题: 开创作物杂种优势利用新时代。会议议题分为三个方面: 作物杂种优势机理与基础研究, 作
物杂交种选育理论与技术, 杂种作物亲本繁殖与杂交种生产的理论与技术。会议将邀请国内外著名专家作大
会报告, 并设有高端论坛和分组专题报告与墙报展示。
会议主席: 袁隆平院士
网站: http://icuhc.nwsuaf.edu.cn; E-mail: icuhc2012@gmail.com
联系人: 郭东伟; 电话: 029-87082942(O); 18792737659(Mobile)
热诚欢迎各位同仁莅临本次盛会。
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