Fungal Genetics and Biology 44 (2007) 10241034

www.elsevier.com/locate/yfgbi

Instability of the Magnaporthe oryzae avirulence gene

AVR-Pita alters virulence
a,b,e b,* b,c,e
Erxun Zhou , Yulin Jia , Pratibha Singh , James C. Correll d, Fleet N. Lee e

a
Department of Plant Pathology, South China Agricultural University, Guangzhou, Guangdong 510642, PR China
b
USDA-ARS, Dale Bumpers National Rice Research Center, Stuttgart, AR 72160, USA
c
Department of Plant Pathology, Cornell University, Ithaca, NY 14850, USA
d
Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA
e
Rice Research and Extension Center, University of Arkansas, Stuttgart, AR 72160, USA

Received 19 November 2006; accepted 10 February 2007

Available online 21 February 2007

Abstract

The avirulence gene AVR-Pita of Magnaporthe oryzae determines the ecacy of the resistance gene Pi-ta in rice. The structures of the
AVR-Pita alleles in 39 US isolates of M. oryzae were analyzed using polymerase chain reaction. A series of allele-specic primers were
developed from the AVR-Pita gene to examine the presence of AVR-Pita. Orthologous alleles of the AVR-Pita gene were amplied from
avirulent isolates. Sequence analysis of ve alleles revealed three introns at identical positions in the AVR-Pita gene. All ve alleles were
predicted to encode metalloprotease proteins highly similar to the AVR-Pita protein. In contrast, the same regions of the AVR-Pita
alleles were not amplied in the most virulent isolates, and signicant variations of DNA sequence at the AVR-Pita allele were veried
by Southern blot analysis. A Pot3 transposon was identied in the DNA region encoding the putative protease motif of the AVR-Pita
protein from a eld isolate B2 collected from a Pi-ta-containing cultivar Banks. These ndings show that transposons can contribute to
instability of AVR-Pita and is one molecular mechanism for defeating resistance genes in rice cultivar Banks.
Published by Elsevier Inc.

Keywords: AVR-Pita; Magnaporthe oryzae; Mutation and virulence; Pi-ta; Pot3 transposon; Rice blast; Virulent variation

1. Introduction
Rice blast disease, caused by the lamentous ascomycete
fungus Magnaporthe oryzae (anamorph Pyricularia oryzae)
(Couch and Kohn, 2002; Kato et al., 2000), is one of the
most economically devastating diseases worldwide. Resis-
tance (R) genes have been identied and incorporated into
rice cultivars for managing rice blast disease throughout
the world. Resistance conditioned by a single major R gene
is typically eective in preventing infection by races of M.
oryzae containing the corresponding avirulence (AVR)
gene (Silue et al., 1992). To date, forty R genes for blast
resistance have been described (Chauhan et al., 2002),
*
Corresponding author. Fax: +1 870 673 7581.
E-mail address: yjia@spa.ars.usda.gov (Y. Jia).
and some of them, such as Pi-b, Pi-ta, Pi-d2 and Pi9, have
been cloned (Bryan et al., 2000; Chen et al., 2006; Qu et al.,
2006; Wang et al., 1999). The Pi-b and Pi9 genes encode
putative NBS-LRR proteins, respectively (Qu et al., 2006;
Wang et al., 1999). Pi-d2 encodes a receptor-like kinase
protein with a predicted extracellular domain of a bulb-
type mannose specic binding lectin (B-lectin) and an intra-
cellular serinethreonine kinase domain (Chen et al., 2006).
The Pi-ta gene encodes a protein with a NBS and leucine
rich domain (LRD) that is located near the centromere of
rice chromosome 12 a region that is typically stable in
the rice genome (Bryan et al., 2000; Chen et al., 2002). In
the southern US, Pi-ta-based resistant cultivars including
Katy, Drew, Kaybonnet, Madison, Cybonnet, Ahrent
and Banks have been widely utilized to control the disease
since the release of the rst Pi-ta-containing cultivar Katy
in 1990 (Jia et al., 2004b; Moldenhauer et al., 1990).

1087-1845/$ - see front matter Published by Elsevier Inc.

doi:10.1016/j.fgb.2007.02.003
 E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034
Control of blast disease via conventional breeding has
achieved only short-term success due to the frequent break-
down of resistance under eld conditions. Frequent
appearance of new races (or pathotypes) of the fungus that
are virulent to previously resistant cultivars has been
proposed as the principal cause for the loss of resistance.
M. oryzae is known to reproduce asexually in nature and
instability of AVR genes was then proposed to be an adap-
tive advantage for the pathogen (Valent, 1997). To date, 25
AVR genes have been described (Dioh et al., 2000), and ve
of them [PWL1 (Kang et al., 1995), PWL2 (Sweigard et al.,
1995), AVR1-CO39 (Farman and Leong, 1998), AVR-Pita
(AVR2-YAMO) (Orbach et al., 2000) and ACE1 (Bohnert
et al., 2004)] have been molecularly characterized. The
AVR-Pita gene is located in the telomeric region of chro-
mosome 3 in M. oryzae, and encodes a putative neutral
zinc metalloprotease (Orbach et al., 2000). Pi-ta and
AVR-Pita are the rst matched pair of R/AVR genes
cloned in the rice blast system (Bryan et al., 2000; Orbach
et al., 2000), and their interactions, genetics and variations
have been studied intensively (Jia et al., 2000, 2002, 2003,
2004a,b, 2005; Kang et al., 2001).
The Pi-ta resistance gene has been eective in managing
rice blast in southeast production areas of the US since its
release in 1990 (Moldenhauer et al., 1990). Although eld
and greenhouse isolates of M. oryzae have been recovered
that have been virulent on Pi-ta-containing cultivars since
the mid 1990s (Correll et al., 2000a), no blast epidemics
had been observed on any Pi-ta-containing cultivars in
commercial production elds. However, in 2004, epidemics
were observed on Banks in several elds in several dierent
counties in Arkansas, USA (Lee et al., 2005). Banks con-
tained Pi-ta and was released to Arkansas seed growers
in 2004 (Moldenhauer et al., 2004; Lee et al., 2005). The
objectives of this study were to develop AVR-Pita-specic
primers in order to monitor the presence of AVR-Pita, to
analyze the structures of AVR-Pita alleles in virulent iso-
lates of M. oryzae, and to explain how isolates of M. oryzae
overcame the Pi-ta-based resistance used in cultivars
grown throughout the southeastern rice production areas
of the US.
2. Materials and methods
2.1. Fungal isolates, culture and pathogenicity assays
A total of 39 isolates of M. oryzae were used in the study
(Table 1). The isolates include wild-type eld isolates (WT);
race-shift isolates (RS) which were virulent isolates on
Pi-ta-containing cultivars that were originally recovered
from the susceptible lesions on Katy after inoculated with
a parental isolate which was avirulent on Katy (Correll
et al., 2000a), and nitrate (nit) and sulfate (sul) non-utilizing
mutants that were used as marked strains to conrm that
the race-shift isolate originated from the avirulent parental
isolate (Correll et al., 2000b). Isolate FL9 was recovered as
a race-shift isolate from cultivar Katy grown in greenhouse.
1025
Isolates 1188R and 1188S as well as the isolates with a B
prex were collected from the cultivar Banks from several
counties in Arkansas during severe rice blast epidemics in
commercial elds in 2004. All isolates were stored at
20 C on desiccated lter papers, and grown on plates
containing oatmeal agar for production of conidial inocula
at room temperature under blue and white uorescence
lighting, or grown in complete medium broth at 24 C for
producing mycelia to be used for DNA preparation.
The virulence of isolates of M. oryzae was evaluated on
the Pi-ta-containing cultivars Katy and Drew as well as the
non-Pi-ta-containing cultivars C101A51 and M202. Stan-
dard pathogenicity assays were performed as previously
described (Xia et al., 2000; Valent et al., 1991a). Conidial
concentration was measured with a hemacytometer, and
adjusted to 2 105 conidia per milliliter. Rice seedlings at
4-leaf stage were inoculated with 15 ml of conidial suspen-
sion ve plants per pot (6 cm diameter) using an airbrush
sprayer. After inoculation, the seedlings were incubated
in a dark dew-chamber at 21 C for 24 h, and then returned
to the greenhouse. Disease reactions were scored 7 days
post-inoculation on either a 05 (Valent et al., 1991a) or
a 09 (Xia et al., 2000) rating scale. A mean disease reac-
tion of either 02 or 03 was considered a resistant reaction
on the 05 or 09 scale, respectively, whereas a 35 or 49
disease reaction was considered susceptible.
2.2. DNA extraction, PCR, rep-PCR amplication, cloning
and sequence analysis
DNA of M. oryzae was isolated from frozen mycelia
using a Qiagen DNeasy Plant Mini Kit according to a
protocol recommended by the manufacturer (Qiagen Inc.,
Valencia, CA, USA). Three forward and ve reverse
primers from dierent regions of the AVR-Pita gene were
designed and synthesized based on the genomic DNA
sequence of AVR-Pita (GenBank Accession No.
AF207841) (Orbach et al., 2000) (Fig. 1 and Table 2). With
these forward and reverse primers, six pairs of specic
primers were used to detect the existence of AVR-Pita
(Table 3). To detect the genetic diversity in M. oryzae
race-shift isolates, another primer pair (Pot2-1 and Pot2-
2) was designed and synthesized based on an inverted
repeat of transposon in M. oryzae for the repetitive ele-
ment-based PCR (rep-PCR) experiment (Table 2, George
et al., 1998; Kachroo et al., 1994). All PCRs were per-
formed using Taq PCR Master Mix (Qiagen Inc., Valencia,
CA, USA). Each PCR consisted of the following compo-
nents: 10 ll of Taq PCR Master Mix (contains 5 U of
Taq DNA polymerase, 2 Qiagen PCR buer, 3 mM
MgCl2 and 400 lM of each dNTP), 1 ll of each 10 lM pri-
mer, 10 ng of fungal genomic DNA and distilled water
(provided by Qiagen Kit) in a nal reaction volume of
20 ll. Reactions were performed in a Peltier Thermal
Cycler (PTC-200, MJ Research, Waltham, MA, USA) with
the following PCR program: 1 cycle at 95 C for 3 min for
initial denaturation, 29 cycles at 95 C for 30 s, 5260 C
 1026
Table 1
Disease reactions of rice cultivars to isolates of M. oryzae and the presence of the AVR-Pita allele
Isolate Racea Origin MGR586 Fingerprint Isolate phenotypec Isolate referenced Disease reactionse AVR-Pita (YL169/YL149)
Groupb ampliconf

E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034

State Year M202 C101A51 Katy Drew
A598 IB-49 AR 1992 A WT 1,2,3 S S R R +
A264 IC-17 AR 1993 B WT 1,2,3 S S R R +
A119 IB-49 AR 1992 C WT 1,2,3 S S R R +
A347 IC-17 AR 1992 D WT 1,2,3 S S R R +
75A49 IG-1 AR 1975 B WT 1,2,3 S S R R +
75A49/3 IG-1 B Nit 1 S S R R +
K60 IG-1 B WT-RS 2,3 S S S S 
18 IC-17 AR 1992 B WT 2,3 S S R R +
18/1 IC-17 B Sul S S R R +
18/1-2 K B Sul-RS S S S S 
18/1N IC-17 B Nit S S R R +
18/1N-13 K B Nit-RS S S S S 
24/1 IG-1 AR 1992 B Nit 1,2,3 S S R R +
24/1-2 K B Nit-RS S S S S 
25 IC-17 AR 1992 B WT 2,3 S S R R +
25/104 IC-17 B Nit S S R R +
25/104-16 K B Nit-RS S S S S 
60 IC-17 AR 1992 D WT 2,3 S S R R +
60/1 IC-17 D Nit S S R R +
60/1-5 K D Nit-RS S S S S +
S-1 K AR 1994 B WT S S S S 
94071A AR 1994 B WT 1 S S S S 
ZN4 IE-1 TX 1995 C WT 3 S S R R +
TM2 K TX B WT 1,3 S S S S 
ZN15 IB-1 WT S S R R +
FL9 IB-33 AR WT-RS S S S S 
ZN61 IB-49 AR 1992 C WT 3 S S R R +
ZN57 IC-17 AR 1992 B WT 3 S S R R +
B-C/3-1 S S S S 
1188R AR 2004 B WT S S S S 
1188S AR 2004 B WT S S S S 
B1 K AR 2004 B WT S S S S 
B2 K AR 2004 B WT S S S S 
B3 K AR 2004 B WT S S S S 
B4 K AR 2004 B WT S S S S 
B5 K AR 2004 B WT S S S S 
 B6 K AR 2004 B WT S S S S 
B7 K AR 2004 B WT S S S S 
B8 K AR 2004 B WT S S S S 
a
The race designation was determined either in the current study or part of concurrent studies (Correll et al., 2000a,b; Xia et al., 2000). The race K designation was originally used to indicate virulence
on the previously resistant cultivar Katy. In this study, race K designates virulence on Pi-ta-containing cultivars Katy and Drew.
b
The MGR586 ngerprint group was previously determined (Correll et al., 2000a,b; Xia et al., 2000).
c
Isolate phenotypes include wild-type (WT) eld isolates, race-shift (RS) isolates which were virulent on Pi-ta-containing cultivars and were originally recovered from the susceptible lesions on Katy
after being inoculated with a parental isolate which was avirulent on Katy (Correll et al., 2000a), and nitrate (nit) and sulfate (sul) non-utilizing mutants that were used as marked strains to conrm that
the race-shift isolate originated from the avirulent parental isolate (Correll et al., 2000b). Isolate FL9 was recovered as a race-shift isolate from rice cultivar Katy grown in greenhouse. Isolates 1188R
and 1188S as well as the isolates with a B prex were collected from the cultivar Banks from several counties in Arkansas during severe rice blast epidemics in commercial elds in 2004.
d
1 = Correll et al., 2000a; 2 = Correll et al., 2000b; 3 = Xia et al., 2000.
e
Cultivars Katy and Drew have the resistant Pi-ta allele, and cultivars C101A51 and M202 have the susceptible pi-ta allele (Jia et al., 2003); mean disease reactions of 02 on the 05 scale or 03 on
the 09 scale were considered as resistant reactions (R), whereas reactions of 35 or 49 were considered as susceptible reactions (S).
f
The presence (+) or absence () of a PCR amplicon with primers YL169 and YL149. The amplicon produced was 1086 bp (see Table 3).

E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034

A indicates an avirulent isolate and V indicates a virulent isolate.
text and Table 3) spanning the full-length 2187 bp AVR-Pita gene (in text),
from eld isolates including race-shift isolates using six pairs of primers (in
primers. (b and c) PCR amplication of AVR-Pita in dierent regions
primers and primers YL170, YL165, YL166, YL149 and Tel.3 are reverse
(Orbach et al., 2000). The primers YL150, YL169 and YL168 are forward
representation of primer locations in the full-length 2187 bp of AVR-Pita
Fig. 1. PCR primers and their amplication products. (a) Schematic
ethidium bromide, visualized and photographed using a
gels in 1 TBE (TrisBorateEDTA) buer, stained with
were separated by electrophoresis on 1.0% (w/v) agarose
and a nal extension of 72 C for 7 min. The PCR products
(varies with dierent primer pairs) for 30 s, 72 C for 30 s

1027
1028 E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034

Table 2
List of primers used in this study
Primer Sequence 5 0 to 3 0 Tm (C) Remark
YL149 TGA CCG CGA TTC CCT CCA TT 62.45 For AVR-Pita allele-specic PCR
YL150 GAC AAC TAC CAT GGA ACC C 60.16
YL165 GCC GAA ATC GCA ACG GTG TG 64.5
YL166 CTG TTA CAT TCC TTG TAA AT 52.2
YL168 GCG ATT TCG GCC TTC ACC 62.18
YL169 CGA CCC GTT TCC GCC 61.63
YL170 GGC GGA AAC GGG TCG 61.63
Tel. 3 ACC CTA ACC CTA ACC CTC TAT TGT TAG ATT GAC C 68.28
Pot2-1 CGG AAG CCC TAA AGC TGT TT 60.4 For rep-PCR
Pot2-2 CCC TCA TTC GTC ACA CGT TC 62.45

Table 3
Specic primers of AVR-Pita developed in this study and their predicted PCR products
Primer pair Location Expected PCR products (bp) and codea
YL150/YL170 128146/966980 853 A
YL169/YL165 966980/11611180 215 B
YL168/YL166 11701187/15111530 361 C
YL168/YL149 11701187/20322051 882 D
YL169/YL149 966980/20322051 1086 E
YL168/Tel.3 11701187/20472080 911 F
a
Codes AF indicate the name of each fragment presented in Fig. 1a amplied using these pairs of primers.

Table 4
List of plasmid clones in this study
Name of plasmid Isolate (race) of M. oryzae Vector/antibiotic selection Size of fragment (Kb) Primers/name of DNA polymerase
YLJ150 A119 (IB-49) pGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ151 A119 (IB-49) pGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ152 A119 (IB-49) pGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ154 A347 (IC-17) pGEMAmp 1.086 YL149/YL169/ Pfu
YLJ155 A347 (IC-17) pGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ174 ZN61 (IB-49) PGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ175 ZN57 (IC-17) pDrive/Kan 1.086 YL149/YL169/ Pfu
YLJ176 ZN57 (IC-17) pDrive/Kan 1.086 YL149/YL169/ Pfu
YLJ177 ZN57 (IC-17) pDrive/Kan 1.086 YL149/YL169/ Pfu
YLJ165 18 (IC-17) pGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ166 18 (IC-17) pGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ167 18 (IC-17) pGEM/Amp 1.086 YL149/YL169/ Pfu
YLJ298 B2 (K) pGEM/Amp 2.6 YL168/YL169/ TAG

Bio-Rad Gel Photographic System (Bio-Rad Laboratories, 2.3. Southern hybridization

Inc., Hercules, CA, USA). All PCRs including rep-PCR
were repeated three times.
PCR products amplied from M. oryzae using AVR-Pita
primers were puried using a QIAquick Gel Extraction Kit
(Qiagen Inc., Valencia, CA, USA), cloned into a TOPO TA
Cloning Vector (Invitrogen Corp., Carlsbad, CA, USA) or
pGEM-T Easy Vector System (Promega Corp., Madison,
WI, USA) (Table 4), and sequenced using an ABI 3700
from both directions using T7 and SP6 primers (Applied
Biosystems, Foster City, CA, USA). DNA sequences were
analyzed using Bioinformatics software Vector NTI Suite
V.6. (Invitrogen Corp., Carlsbad, CA, USA).
DNA of M. oryzae was digested with restriction
enzymes (EcoRI, EcoRV and HindIII), electrophoresed
on 1.0% agarose gels in 1 TBE buer, and transferred
onto Hybond-N+ nitrocellulose membranes (Amersham
Biosciences Corp., Piscataway, NJ, USA) followed the pro-
tocols described by Sambrook and Russell (2001). DIG-
labeled DNA probes were generated using a PCR DIG
Probe Synthesis Kit (Roche Diagnostics Corporation, Indi-
anapolis, IN, USA), hybridized to blots overnight at 42 C,
and washed under high stringency conditions according to
the manufacturers instructions.
 E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034 1029

3. Results

3.1. Variation of the AVR-Pita alleles in pathotypes of

M. oryzae from the USA

Pi-ta has been eective in preventing infections by races

of M. oryzae containing AVR-Pita. In this study, 18 of the
19 isolates examined from which AVR-Pita (E fragment)
could be amplied were avirulent on the two Pi-ta-contain-
ing cultivars tested (Table 1). Ten isolates belonged to a
MGR586 DNA ngerprint group B, and the other eight
isolates belonged to other MGR586 ngerprint groups
(Table 1). AVR-Pita could not be amplied from any of
the 20 isolates that were virulent on the two Pi-ta-contain-
ing cultivars. The one exceptional isolate, 60/1-5, belonged
to MGR586 DNA ngerprint group D; AVR-Pita could be
amplied form this isolate and it was virulent on the Pi-ta
cultivars.
Six pairs of the AVR-Pita allele-specic primers were
developed to detect AVR-Pita (Fig. 1a and Table 3). Prim-
ers YL150 and YL170 from the 5 0 leader region amplied
predicted fragments (A fragment) among all isolates
(Fig. 1a and b). The presence of a portion of 5 0 leader
region of AVR-Pita indicates this region is conserved
among all isolates tested. A series of primer pairs,
YL169/YL165, YL168/YL166, YL168/YL149 and
YL169/YL149 amplied predicted fragments (BE frag-
ments) of AVR-Pita in all avirulent isolates, not in race-
shift isolates except 60/1-5 (Fig. 1a and b; Table 2). These
results suggest that DNA sequences of AVR-Pita in race-
shift isolates except 60/1-5 are dierent from those in avir-
ulent isolates.
AVR-Pita in a Chinese isolate O-137 is at the telomere
of M. oryzae chromosome 3 (Orbach et al., 2000). A telo-
meric specic primer Tel.3 and YL168 amplied a pre-
dicted 911 bp DNA fragment (F fragment) corresponding
to the AVR-Pita gene from all avirulent isolates, whereas
the same fragment was not amplied in most virulent Fig. 2. Southern blot analysis of AVR-Pita in M. oryzae wild-type and
(race-shift) isolates (Fig. 1c). These results suggest that race-shift isolates. (a) Probe locations in the AVR-Pita gene. (b) Genomic
DNAs were digested with EcoRI and probed with coding region of AVR-
the AVR-Pita alleles may closely associated with the telo- Pita (Puried PCR products from plasmid PCB2049 containing cDNA
mere in these avirulent isolates. encoding amino acid). RS indicates a race-shift isolate and A indicates an
Southern blot analysis using two dierent probes veri- avirulent isolate. (c) Genomic DNAs of A598 and 18/1-2 were digested
ed variation of DNA sequence of AVR-Pita in race-shift with indicated restriction enzymes, electrophoresed in agarose, transferred
isolates (Fig. 2a). One or two copies of the AVR-Pita alleles to a membrane, and probed with a 5 0 fragment of AVR-Pita coding region
(YL169 and YL165 PCR product). Left, autoradiogram of Southern blot.
were detected in these eld isolates. Race-shift isolates Right, ethidium bromide-stained agarose gel.
invariably lost an expected band at 6.6 kb (K60, 18/1-2,
18/1N-13, 24/1-2 and 25/104-16) (Fig. 2b). Furthermore,
the hybridization signal of DNA of a virulent isolate 18/ protein of the Chinese isolate O-137 (Orbach et al.,
1-2 was extremely weak in comparison with that of an avir- 2000). Amino acid alignments revealed the complete
ulent isolate A598 when probed with DNA encompassing sequences of ZN61, ZN57, 18; A119 and A347 are 97.3,
the 5 0 region and rst exon amplied by primers YL169/ 97.3, 97.3, 95.5 and 95.1% identical to the AVR-Pita
YL165 indicating the DNA sequence of AVR-Pita in protein of isolate O-137, respectively (Orbach et al., 2000,
A596 is dierent than in 18/1-2 (Fig. 2c). Fig. 3). Overall, the ratio of nucleotide substitutions
The DNA sequences of AVR-Pita alleles in avirulent that lead to amino acid replacements (nonsynonymous
races/isolates ZN61, ZN57, 18, A119 and A347 were trans- substitution, Ka) and nucleotide substitutions that do not
lated into the full-length proteins. The amino acid exchange amino acids (synonymous substitutions, Ks) is
sequences were aligned and compared with AVR-Pita greater than 1.
1030 E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034

Fig. 3. Amino acid alignment of AVR-Pita coding region from dierent eld isolates as compared with the O-137 isolate (single letter code was used for
amino acids).

Occasionally, multiple fragments of dierent sizes were

amplied using primers for E fragment (Fig. 1b). Sequence
analysis of these DNA fragments indicated that they are
hypothetical proteins of M. oryzae, thus it was concluded
that they result from non-specic amplication, and are
not related to the AVR-Pita gene.

3.2. A Pot3 transposon was found in the coding region of

AVR-Pita in a virulent wild-type eld isolate designated B2

A specic primer pair YL168/YL149 was successfully

used to examine AVR-Pita from Banks isolates
(Fig. 1a, Tables 2 and 3). Homologs of AVR-Pita with
similar sizes were amplied from all Banks isolates. Sig-
nicantly, a PCR product of 2.6 kb was also amplied
from genomic DNA of a virulent isolate B2, not from
other virulent isolates. Sequence analysis of the PCR
product revealed a Pot3 transposon resides in the DNA
region encoding the protease motif of the putative
AVR-Pita metalloprotease (Fig. 4). Using rep-PCR with
primers Pot2-1 and Pot2-2 eight of 12 dierent classes
(AL) of isolates were detected in 16 virulent isolates
(George et al., 1998) (Fig. 5). These results indicate that
the genomes of these virulent isolates are distinctly dier-
ent from each other.
Fig. 4. Schematic presentation of the genomic location of Pot3 transpo-
son in the AVR-Pita gene from a eld isolates B2.
4. Discussion
The Pi-ta gene has been widely used to control rice blast
disease in the southern US and is commonly found in rice
germplasm used throughout the world (Wang et al., 2007).
The eectiveness of the Pi-ta gene relies on its ability to rec-
ognize the pathogens corresponding avirulence gene AVR-
Pita. In this study, a PCR based dominant marker (YL149/
YL169) was developed from the 5 0 leader and the 3 0 trailer
regions of the AVR-Pita allele to specically detect the
AVR-Pita gene in isolates of M. oryzae. It was shown that
two Pi-ta-containing cultivars were resistant to isolates of
 E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034
Fig. 5. Genetic diversity of M. oryzae eld isolates as revealed by rep-
PCR with primers Pot2-1 and Pot2-2. PCR products were separated by
agarose gel electrophoresis and visualized with ethidium bromide.
Underlined isolates are wild-type (avirulent) isolates, and the others are
race-shift (virulent) isolates. Isolates with prex B are from Banks.
AL indicate dierent classes of isolates. M = 1 kb DNA ladder.
M. oryzae when the AVR-Pita allele could be amplied
from the isolate, whereas the Pi-ta-containing cultivars
were susceptible when an AVR-Pita allele could not be
amplied from the isolate. The control primers YL150/
YL170 amplied a portion of the 5 0 leader sequence of
the AVR-Pita allele in all isolates of M. oryzae tested and
thus could be used to rule out failed PCR amplications.
As a result, these PCR based markers for AVR-Pita can
be used to determine the existence of AVR-Pita in isolates
of M. oryzae and predict the eectiveness of blast resistance
conferred by Pi-ta.
In the past, the study of genetic diversity of populations
of M. oryzae has relied on the repetitive sequence based
probe MRG586 in the Southern blots and the use of prim-
ers designed from Pot2 PCR (rep-PCR) (Correll et al.,
2000b; George et al., 1998; Hamer et al., 1989; Kachroo
et al., 1994). These markers are useful for distinguishing
genetic groups, or families, of the pathogen. For example,
four MGR586 DNA ngerprint groups occur in the con-
temporary pathogen population in Arkansas (Xia et al.,
2000). However, the molecular markers used in population
studies are not race-specic. Similar to AVR-Pita-specic
primers developed in this study, the race-specic marker
AVR1-CO39 has been previously developed to study its
evolution (Farman et al., 2002).
The stability of resistance is determined by the genetic
changes that have occurred in the functional regions of
the avirulence gene. Disruption of AVR genes by transpo-
son insertion may be the cause of instability of resistance
conferred by an R gene. Previously, a Pot3 transposon
was found in the 5 0 non-coding region of AVR-Pita in a
laboratory strain (Kang et al., 2001). In this study, an
intact Pot3 transposon, which was identied in the coding
region of the putative protease motif of the AVR-Pita pro-
tein, was found in the virulent wild-type eld isolate desig-
1031
nated B2. Thus, Pot3 insertion into AVR-Pita can lead to
converting avirulent isolates into virulent isolates and
may be a distinct molecular mechanism by which the path-
ogen can defeat the Pi-ta resistance gene (Kang et al., 2001;
the present study). Genetic changes caused by transposons
have been predicted to be one of the mechanisms responsi-
ble for pathogenic variation in M. oryzae (Kang et al.,
2001; Schull and Hamer, 1994; Talbot et al., 1993; Valent
and Chumley, 1991b) and increased virulence toward the
rice cultivars with the corresponding R gene (Kang et al.,
2001; Mandel et al., 1997). Draft sequence of the M. oryzae
genome is represented by a large proportion of transpos-
able elements providing the possibility of general mecha-
nisms of transposon driven evolution (Dean et al., 2005).
Previously, diverse mutations of AVR-Pita have occurred
in several laboratory strains of M. oryzae, allowing M. ory-
zae to avoid triggering the Pi-ta-mediated defense response
(Kang et al., 2001; Orbach et al., 2000). This preclusion
results in a race-shift from avirulence to virulence on rice
cultivars previously exhibiting resistance to M. oryzae in
a gene-for-gene fashion (Flor, 1971; Correll et al., 2000a).
Our results show that insertion of Pot3 transposon into
the coding region of AVR-Pita in isolate B2 might be
responsible for inactivation of AVR-Pita function, and
leading to the loss of rice blast resistance in Pi-ta-contain-
ing cultivars including Banks.
The ratio of nucleotide substitutions Ka and Ks is infor-
mative for understanding the function and evolution of
selected coding domains. In the AVR-Pita gene, the value
of the ratio Ka/Ks is greater than 1 and provides evidence
for a positive selection for amino acid substitution (Stahl
and Bishop, 2000). It is expected that fragments of proteins
that bind a ligand will be subject to stronger adaptive selec-
tion than regions of those proteins that play a structural
function. Physical binding of the AVR-Pita protein to the
Pi-ta protein, which triggers eective resistance, suggests
a need for adaptive selection in the AVR-Pita protein to
circumvent Pi-ta recognition (Jia et al., 2000). In this study,
signicant sequence alteration of the AVR-Pita alleles was
observed in all but one isolate virulent on the two Pi-ta cul-
tivars. These alterations of sequences could be the result of
point mutations, deletions, or insertions within sequences
of the AVR gene or due to transposon insertion. All of
these events have been documented in the loss of function
of the AVR1-CO39 gene (Farman et al., 2002). The AVR-
Pita gene encodes a metalloprotease that was predicted to
act inside the plant cell and elicit Pi-ta gene-mediated resis-
tance (Bryan et al., 2000; Jia et al., 2000; Orbach et al.,
2000). Comparisons of amino acid sequence of the AVR-
Pita proteins of our isolates with the previously published
sequence of AVR-Pita showed several dierences, while
the critical zinc-binding motif was the same with only
one amino acid dierence (Orbach et al., 2000). Changes
of amino acids of the AVR-Pita protein could eventually
allow the pathogen to escape recognition by the host, while
maintaining protease activity. Maintaining an intact
protease motif of AVR-Pita may be advantageous for
1032 E. Zhou et al. / Fungal Genetics and Biology 44 (2007) 10241034
promoting the disease. Diversication of amino acid
sequence that weakens the anity of AVR-Pita for Pi-ta
would provide an adaptive advantage for isolates of M.
oryzae containing the AVR-Pita gene. Adaptive evolution
in the AVR-Pita protein is consistent with an evolutionary
arms race in the respect that pathogens, under the selection
pressure of major R genes, need to continually alter recog-
nition specicity. It would be of importance to determine
the binding anity of these AVR-Pita proteins to the Pi-
ta protein.
The presence of the AVR-Pita allele in the virulent race-
shift isolate 60/1-5 suggests a dierent mechanism may be
involved in defeating resistance mediated by Pi-ta. The fre-
quency by which race shifts from avirulence to virulence
occur appears to vary among isolates from the dierent
MGR586 DNA ngerprint groups found in Arkansas.
The highest frequency of race-shift isolates under experi-
mental conditions has been among isolates in the B nger-
print group (Correll et al., 2000a). The lowest frequency
has been among isolates in ngerprint group D whereas
no race-shift isolates that can overcome Pi-ta have been
recovered from group A or C isolates. Isolates 60/1-5
belongs to DNA ngerprint group D whereas all other iso-
lates examined, including all of the wild-type eld isolates
from Banks and virulent on Banks are in group B. Thus,
future sequence analysis of the promoter region of AVR-
Pita will help to determine if there is a transposon or other
changes in the promoter region in 60/1-5.
Pot3 was not detected in the coding region of AVR-Pita
from other virulent isolates. This could be due to the fact
that the transposon insertion may have moved the primers
too far apart for successful amplication or perhaps caused
signicant chromatin rearrangement. Nevertheless, the
inability in detecting Pot3 in other virulent isolates is con-
sistent with the hypothesis that mutation at the AVR-Pita
locus is responsible for the altered recognition specicity
of isolate B2 by rice cultivars carrying the Pi-ta gene. These
dierent interaction mechanisms between Pi-ta and AVR-
Pita seem to be the outcome of co-evolution between rice
and M. oryzae over time. Further sequence analysis of iso-
lates of M. oryzae from dierent geographic regions of rice
production will provide further insight into the evolution
of the AVR-Pita gene.
The pathogen AVR genes playing crucial roles in patho-
genictiy and tness are often recognized by host durable R
genes. Besides AVR-Pita two other race-specic AVR genes
of M. oryzae have been molecularly characterized. AVR
genes AVR1-CO39 and ACE1 determine the ecacies of
the R genes Pi-CO39(t) and Pi 33, respectively (Berruyer
et al., 2003; Farman et al., 2002; Orbach et al., 2000).
AVR1-CO39 apparently has been lost in the rice-infecting
population of M. grisea (renamed as M. oryzae) suggesting
its function was not fundamental for pathogen survival
(Farman et al., 2002). ACE1 is more commonly found in
rice pathogens worldwide suggesting its usefulness for the
pathogen (Berruyer et al., 2003). In a natural pathosystem,
plants continue to develop new resistances against their
pathogens. Simultaneously, the pathogen also evolves to
overcome the host resistance, resulting in the dynamic co-
evolution between host and pathogen (Lauge and De
Wit, 1998). Tosa et al. (2005) evaluated the signicance
of the AVR1-CO39 and Pi-CO39(t) interaction during
the evolution, suggesting that AVR1-CO39 appeared dur-
ing the early stage of evolution of M. oryzae. The durabil-
ity of Pi-ta-based cultivars suggests that the AVR-Pita gene
may be important for tness and pathogenicity. The AVR-
Pita alleles have been detected in M. oryzae isolates/races
(contemporary and archived) worldwide indicating its uni-
versal need for reproduction and survival of the pathogen
(Jia and Correll, unpublished data). Further studies of
the role of AVR-Pita in pathogenicity and tness will lead
to a better understanding of the mechanisms of host sup-
pression mediated by AVR-Pita.
In the current study, it has been shown that a transposi-
tion mutation in M. oryzae is sucient to defeat Pi-ta-
based resistance. It was also observed that additional
virulent wild-type eld isolates that were virulent on Pi-ta
cultivars did not have the transposon insertion. As these
isolates belonged to dierent rep-PCR classes and were
therefore not likely to be clonal, multiple molecular mech-
anisms may be operative among eld isolates to defeat
Pi-ta. These ndings could serve as a foundation for devel-
oping strategies for pyramiding dierent R genes that rec-
ognize dierent pathogen avirulence genes for improving
the durability of resistance in rice breeding programs.
Acknowledgments
The authors are thankful for the Arkansas Rice
Research and Promotion Board and USDA-ARS for fund-
ing this project, and South China Agricultural University
for supporting sabbatical leave for E. Zhou. The authors
are grateful to B. Reyes, M.J. Orbach, G.-L. Wang, S.A.
Leong, M.H. Jia and S. Brooks for critical reading and
insightful discussion of this manuscript, and for C. Flow-
ers, M. Lin and M.H. Jia for their excellent technical
support. Financial support for this work came from the
projects Development of molecular strategies to control
rice diseases and Examining stability of rice blast resis-
tance funded by the University of Arkansas Rice Research
and Promotion Board and the USDA-ARS Project:
Genome characterization of rice germplasm.
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