Diversification and evolution of the avirulence gene AVR-Pita1 in field isolates of Magnaporthe oryzae

Fungal Genetics and Biology 47 (2010) 973–980

Contents lists available at ScienceDirect

Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi

Diversification and evolution of the avirulence gene AVR-Pita1 in field isolates of Magnaporthe oryzae q Yuntao Dai a,b, Yulin Jia b,⇑, James Correll a, Xueyan Wang c, Yanli Wang d a

Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, United States USDA-ARS Dale Bumpers National Rice Research Center, Stuttgart, AR 72160, United States c China Jiliang University, 310018 Hangzhou, PR China d Zhejiang Academies of Agricultural Sciences, 310021 Hangzhou, PR China b

a r t i c l e

i n f o

Article history: Received 27 February 2010 Accepted 6 August 2010 Available online 16 August 2010 Keywords: Magnaporthe oryzae Effector Metalloprotease Evolution Signal recognition

a b s t r a c t Rice blast disease is the single most destructive plant disease that threatens stable rice production worldwide. Race-specific resistance to the rice blast pathogen has not been durable and the mechanism by which the resistance is overcome remains largely unknown. Here we report the molecular mechanisms of diversification and the instability of the avirulence gene AVR-Pita1 in field strains of Magnaporthe oryzae interacting with the host resistance gene Pi-ta and triggering race-specific resistance. Two-base-pair insertions resulting in frame-shift mutations and partial and complete deletions of AVR-Pita1 were identified in virulent isolates. Moreover, a total of 38 AVR-Pita1 haplotypes encoding 27 AVR-Pita1 variants were identified among 151 avirulent isolates. Most DNA sequence variation was found to occur in the exon region resulting in amino acid substitution. These findings demonstrate that AVR-Pita1 is under positive selection and mutations of AVR-Pita1 are responsible for defeating race-specific resistance in nature. Published by Elsevier Inc.

1. Introduction Race-specific plant resistance (R) genes have evolved to detect the products of the corresponding avirulence (AVR) genes and trigger effective resistance to plant pathogens. In nature, the effector molecules encoded by AVR genes were thought to facilitate disease development and were hypothesized to be under selection. It is theorized that new virulent races of the pathogen emerged by genetic modification of AVR genes using diverse mechanisms. Point mutation (Joosten et al., 1994), deletion (Schurch et al., 2004; Dodds et al., 2006), and frame-shift (Ridout et al., 2006) of AVR genes have been documented in strains of several pathogens. Further analysis of the AVR genes in naturally-occurring field isolates of a plant pathogen may yield valuable information for the deployment of R genes in field crops (Stukenbrock and McDonald, 2009). Rice blast disease, caused by the ascomycete fungal pathogen Magnaporthe oryzae [formerly Magnaporthe grisea (Hebert) Barr], is one of the most devastating plant diseases worldwide. The disease has been managed using both major and minor resistance genes integrated with effective cultural practices. The major R genes, or Pi-genes, for M. oryzae in rice are able to prevent the infection of q Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. FJ842861–FJ842898). ⇑ Corresponding author. Fax: +1 870 673 7581. E-mail address: [email protected] (Y. Jia).

1087-1845/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.fgb.2010.08.003

races of M. oryzae that contain a corresponding avirulence (AVR) gene (Silue et al., 1992). To date, more than 80 Pi genes have been reported from rice and some of them have been used to control blast disease (Ballini et al., 2008). Among these Pi genes, eleven have been cloned including Pi-ta (Bryan et al., 2000), Pi-b (Miyamoto et al., 1996; Wang et al., 1999), Pi-2/Pi-zt (Zhou et al., 2006), Pi-d2 (Chen et al., 2006), Pi-9 (Qu et al., 2006), Pi-36 (Liu et al., 2007), Pi-37 (Lin et al., 2007), Pikm (Ashikawa et al., 2008), Pi-d3 (Shang et al., 2009), Pi-5 (Lee et al., 2009b), and Pi-t (Hayashi and Yoshida, 2009). These cloned genes encode putative cytoplasmic NBS–LRR proteins, with the exception of the product of Pi-d2, which is a putative transmembrane B-Lectin-TM-Kinase (Chen et al., 2006). The fungus M. oryzae is highly variable and often can overcome the deployed resistant cultivars in a short period of time when resistance is dependent on one major R gene. The ability to defeat the R gene has been hypothesized to be due to the instability of AVR in M. oryzae (Khang et al., 2008). Thus far, 25 AVR genes in M. oryzae have been genetically mapped (Dioh et al., 2000), nine of which were recently cloned: AVR-Pita (Orbach et al., 2000), AVR1CO39 (Farman and Leong, 1998), PWL1 (Kang et al., 1995) PWL2 (Sweigard, 1995), ACE1 (Fudal et al., 2005), AVR-Pizt (Li et al., 2009), AVR-Pia, AVR-Pii, and AVR-Pik/km/kp (Yoshida et al., 2009). The AVR genes in M. oryzae are highly diversified and are predicted to be capable of rapid changes in nature (Jia et al., 2000). Farman et al. (2002) reported that three types of diversification occurred at the AVR1-CO39 locus in M. oryzae. The G types, including G1

974

Y. Dai et al. / Fungal Genetics and Biology 47 (2010) 973–980

and G2, were characterized by an approximate deletion of 20 kb, while the J type was characterized by a deletion of the 50 half of AVR1-CO39 in addition to point mutations. Meanwhile, the insertion of a 1.9 kb MINE retrotransposon in the last exon of ACE1 in a virulent isolate was hypothesized to be responsible for conversion from avirulence to virulence (Fudal et al., 2005). Virulent isolates were found to exhibit either a Pot3 insertion in the promoter region or single nucleotide substitution resulting in an amino acid change in AVR-Pizt (Li et al., 2009). Transposon and telomeric sequences in the genomic regions neighboring AVR-Pia and AVR-Pii lead to the enhanced likelihood of gene loss and horizontal transfer (Silva et al., 2004; Rehmeyer et al., 2006). The processed AVR-Pita protein from the rice blast fungus was demonstrated to interact directly with the translated product of the host R gene Pi-ta in rice triggering resistance (Bryan et al., 2000; Jia et al., 2000). The AVR-Pita alleles in isolates of the M. grisea species complex, including M. oryzae, were reported to be a member of a gene family which is comprised of one to three members, and as a result, AVR-Pita from M. oryzae was renamed AVR-Pita1 (Khang et al., 2008). Many Pi-ta-containing cultivars have been effectively deployed to control rice blast disease worldwide. However, epidemics of rice blast disease have occurred on Pi-ta-containing cultivars, suggesting that the Pi-ta gene has been defeated (Lee et al., 2005). Evidence of the structural variation of AVR-Pita1 in laboratory strains that alters Pi-ta recognition specificity has been documented. Point mutations, insertions, and deletions of AVR-Pita1 detected in some lab strains rendered the fungus able to avoid triggering resistance responses mediated by Pi-ta (Orbach et al., 2000). Specific alteration of the active site from glutamic acid to aspartic acid (E177D) and substitution of methionine to tryptophan (M178 W) in the putative protease motif of the strain 4360-R-62 resulted in the loss of avirulence (Jia et al., 2000). Sequence analysis of the virulent strain CP1632 identified a Pot3 transposon in the promoter region of AVR-Pita1, suggesting that transposition is another mechanism for altering the avirulence of the AVR-Pita1 gene (Kang et al., 2001). Deletions and a 31 bp duplication of the functional copy of AVR-Pita1 were identified in the derived mutants of a single isolate (Takahashi et al., 2010). To date, the documented AVR-Pita1 mutation identified from field isolates of M. oryzae from the US has been the Pot3 transposon insertion (Zhou et al., 2007). Whereas in field isolates from Japan, a deletion of functional copies and base substitutions have occurred (Takahashi et al., 2010). In order to obtain a comprehensive understanding of the structural and functional variation of the AVR-Pita1 gene in M. oryzae, we investigated a collection of field isolates of the pathogen from major rice-producing areas around the globe (Fig. 1a). Our findings support that a functional AVR-Pita1 possesses diversified sequence structures and is under positive selection pressure in nature. We also demonstrate that frame-shift and deletions of AVR-Pita1 are responsible for the loss of the gene’s avirulence, which eventually renders the corresponding field isolates capable of avoiding the deployed Pi-ta gene in rice cultivars, resulting in disease. These results are consistent with previous findings in laboratory strains, and shed light on the molecular mechanisms of AVR-Pita1 diversification in nature. 2. Materials and methods 2.1. Rice cultivars, fungal isolates, culture, and pathogenicity assays Two rice cultivars (Katy and Drew) containing the R gene Pi-ta, and one (M202) lacking Pi-ta were used for the pathogenicity assays for the US isolates (Fig. 1b). The cultivars Tetep and K1, which contain Pi-ta, were used for pathogenicity assays for some of the non US isolates. An international set of differential rice cultivars was used for race determination and included Raminad Str #3, Zenith, NP-

Fig. 1. Isolate collection and identification. (a) A world map showing the collection sites of M. oryzae isolates used in this study. The 187 isolates were collected from rice-producing countries. The US, China, Colombia, India, Egypt and the Philippines. (b) Disease reaction of rice cultivars to the US isolates of M. oryzae. (c) A graphic presentation of the AVR-Pita1 allele showing the location of primers used in this study.

125, Usen, Dular, Kanto 51, Sha-tiao-tsao and Caloro. A collection of 187 isolates was examined (Supplemental Table S1). All isolates were stored at 20 °C on desiccated filter paper and were grown at room temperature under blue and white fluorescence lighting on plates containing oatmeal agar for producing conidial inoculum. Isolates from the US were evaluated on Katy, Drew, and M202; isolates from China were evaluated on Tetep; isolates from Colombia were evaluated on K1. Standard pathogenicity assays were performed as previously described (Valent et al., 1991). The race identification and classification method followed the methods of Ling and Ou (1969). Disease reactions were recorded 7 days post-inoculation using a 5-scale rating system (0–2: resistance; 3–5: susceptible). Each experiment was repeated three times. 2.2. DNA preparation, PCR amplification, DNA sequencing and Southern blot analysis Each isolate was grown in complete medium broth at 24 °C for approximately 7 days to produce mycelium. DNA of M. oryzae was

Y. Dai et al. / Fungal Genetics and Biology 47 (2010) 973–980

then isolated from the frozen mycelia using a Qiagen DNeasy Plant Mini Kit according to the protocol described by the manufacturer (Qiagen Inc., Valencia, CA, USA). Four forward and five reverse primers (Fig. 1c and Table 1) from different regions of the AVRPita1 gene were designed and synthesized based on the genomic DNA sequence of AVR-Pita1 (GenBank accession no. AF207841). Primers YL169 and YL149 were used to amplify the AVR-Pita1 allele and for sequencing. For isolates from China, the primers AVR-Pita F and AVR-Pita R were used. The YT4 and YT5 primers were used to amplify the AVR-Pita1 coding region from the plasmid pCB980 (Orbach et al., 2000) and used for one of the two probes in the Southern blot analysis; primers YL169 and YL165 were used to amplify the 50 region of AVR-Pita1 from M. oryzae genomic DNA for the second probe (Fig. 2a). All PCR reactions were performed using Taq PCR Master Mix (Qiagen Inc., Valencia, CA, USA). Each PCR reaction consisted of the following components: 25 ll of Taq PCR Master Mix (contains 25U of Taq DNA polymerase, 10XQiagen PCR buffer, 15 mM MgCl2 and 200 lM of each dNTP), 1 ll of each 10 lM primer, 25 ng of fungal genomic DNA and distilled water (provided by Qiagen Kit) in 50 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, followed by 29 cycles at 95 °C for 30 s, 55–60 °C (varies with different primer pairs) for 30 s, 72 °C for 30 s, and a final extension of 72 °C for 7 min. All PCR reactions were repeated three times (12.5 ll for detection, 50 ll for sequencing). The size of the amplified fragment was estimated by GeneRuler™ Express DNA Ladder (Fermentas Inc., Glen Burnie, MD, USA). PCR products were sequenced using the same primers for PCR amplification, in addition to two AVR-Pita1 internal primers, YL166 and YL168 (Table 1), in an ABI 3730XL DNA Analyzer using 0.5 lL BigDye v3.1. at the USDA-ARS MSA Genomics Laboratory at Stoneville, MS. Each isolate was sequenced three times with separate PCR products to ensure accuracy. Restriction endonucleases, EcoRI and BamHI, were used for the digestion of genomic DNA. DIG-labeled DNA probes were generated using a PCR DIG Probe Synthesis Kit (Roche Diagnostics Corporation, Indianapolis, IN, USA). Probes were hybridized to blots overnight and then washed according to the manufacturer’s instructions. Three repeats of each experiment were applied under different stringency conditions in order to exclude any possibility of missing hybridization signals.

Table 1 List of primers used in this study. Primer name

Positiona

Nucleotide sequence (50 –30 )

Tm (°C)

YL149

2032– 2051 1161– 1180 1511– 1530 1170– 1187 966–980 977–997 2028– 2047 1134– 1147 1993– 2015

TGACCGCGATTCCCTCCATT

62.45

GCCGAAATCGCAACGGTGTG

64.5

CTGTTACATTCCTTGTAAAT

52.2

GCGATTTCGGCCTTCACC

62.18

CGACCCGTTTCCGCC CGCCTTTTATTGGTTTAATTCG0 CCTCCATTCCAACACTAACG

61.63 50.7 53.2

ACATCGATGCTTTTTTATTC

46.5

ACGGATCCTTAACAATATTTATAACGTGCAC

57.3

YL165 YL166

b

YL168

b

YL169 AVR-Pita F AVR-Pita R YT4c YT5d a

Oligonucleotide position was based on GenBank accession no. AF207841. Indicates primers only used for sequencing. c ‘‘ACATCG” is added in front of the start codon ‘‘ATG” to create the ClaI digestion site. d ‘‘ACGGATCC” is added behind the stop codon to create the BamHI digestion site. b

975

2.3. Data analysis DNA sequences of AVR-Pita1 were assembled by Vector NTI software Suite V.10 (Invitrogen, Carlsbad, CA, USA) and aligned using clustal W of the MEGA 4.0 software (Biodesign Institute, Tempe, AZ, USA). The number of DNA haplotypes, the number of polymorphic sites and the sliding window were calculated using DNasp 4.5 (Julio Rozas et al. Barcelona, Spain). Haplotype network analysis was performed using TCS1.21 (http://darwin.uvigo.es/) (Clement et al., 2000).

3. Results 3.1. Race determination and disease evaluation The race identity of the collected field isolates was determined based on disease reactions on rice differentials. Twenty-four races of M. oryzae were identified among the 126 US isolates examined based on the disease reactions (Supplementary Table S1). Among them, 33 isolates were identified as race IC17, 19 as race IB49, 16 as race IE1, and 13 as race IB1. These data suggest that IC17, IB49, IE1 and IB1 were most prevalent in the rice production areas where the samples were collected (Supplemental Table S1). Based on disease reactions, 151 out of the 187 isolates tested were avirulent to the Pi-ta-containing rice cultivars (Fig. 1b). The other 36 isolates were virulent (Supplemental Table S1). The avirulent isolates contain predicted functional AVR-Pita1 alleles with minor diversity; in contrast, major structural variation of AVR-Pita1 was detected among all the virulent isolates.

3.2. Deletions and frame-shift at AVR-Pita1 in virulent isolates Through repeated efforts, using different combinations of AVRPita1 primers, we failed to amplify the AVR-Pita1 alleles in 32 virulent isolates (data not shown). The failure of amplification of AVR-Pita1 suggested that DNA sequences at some of these primer sites may have been significantly altered. This possibility was borne out when we performed Southern blot analysis with the genomic DNA of 10 virulent isolates digested with EcoRI and BamHI, respectively (Fig. 2b and c). When hybridizing with a fragment of the AVR-Pita1 coding region, a 5 kb band was detected in the ten virulent isolates except isolate B2, which had an additional 4 kb band. However, two hybridization bands between 5 kb and 9 kb were detected in the two avirulent isolates 60 and 60/1, respectively (Fig. 2b). Similar hybridization patterns were observed when the restriction endonuclease BamHI was used (Fig. 2b). When the PCR product of AVR-Pita1 was amplified from 50 UTR, including a 50 portion of ORF, by the primer pair YL169/YL165 and used as the probe, no hybridization signal was observed among the nine virulent isolates except for B2. There was a hybridization signal in B2, suggesting that B2 has a different AVR-Pita1 allele. In contrast, two bands of the same size were observed in an avirulent check isolate 60 and 60/1 as predicted (Fig. 2c). R23 (an avirulent recombinant) being a control, further study of the remaining 22 virulent isolates by Southern blot analysis using the genomic DNA digested with BamHI revealed both similar and novel results: a single fragment was revealed in 19 of the 22 virulent isolates when using the AVRPita1 coding region as a probe while the other three isolates, IB45, 82T14 and MGS03 showed no hybridization signal at all indicating a complete deletion of AVR-Pita1 in these three isolates (Fig. 2d). When the 50 portion of AVR-Pita1 was used as a probe to hybridize the stripped membrane, none of the 22 virulent isolates had any hybridization signal (Fig. 2d). The Southern blot results

976

Y. Dai et al. / Fungal Genetics and Biology 47 (2010) 973–980

Fig. 2. Variation of AVR-Pita1 in virulent isolates. (a) A schematic presentation of AVR-Pita1 with indicated probes. (b) Genomic DNA was digested with EcoR I and BamHI, respectively, and hybridized with the coding region of AVR-Pita1. (c) Genomic DNA was digested with EcoR I and BamHI, respectively, and hybridized with the 50 portion of AVR-Pita1. (d) Genomic DNA of each isolate was digested with BamHI, and hybridized with (1) AVR-Pita1 coding region and (2) 50 portion of AVR-Pita1, respectively. (e) The alignment of a portion of amino acid sequences of avr-pita1 in four virulent isolates (China9, China47, China117, and China154) and one avirulent isolate O-137 where AVR-Pita1 was originally isolated. Frame-shift mutation occurred at the 11th amino acid, which creates a premature stop codon after the 41st amino acid. (f) A schematic presentation of mutation types in wild-type field isolates of M. oryzae resulting in gain of virulence: (1) Intact AVR-Pita1. (2) Frame-shift mutation that occurs in the first exon of AVR-Pita1 (this study). (3) Deletion of the AVR-Pita1 50 region (this study). (4) Complete deletion of AVR-Pita1 (Takahashi et al., 2010; this study). (5) Pot3 transposon insertion in the coding region corresponding to the AVR-Pita1 protease motif (Zhou et al., 2007). (6) A base substitution (Takahashi et al., 2010).

demonstrate that AVR-Pita1 was either partially or completely deleted in these virulent isolates. Among the four virulent isolates for which AVR-Pita1 could be amplified, we identified two additional nucleotides in the first exon of the AVR-Pita1 alleles of these isolates by sequencing the PCR amplified fragments. Insertion of two nucleotides in the first exon was predicted to form a premature stop codon after the 123rd nucleotide. As a result, these AVR-Pita1 alleles are predicted to produce truncated non-functional metalloproteases (Fig. 2e). These observations, together with the previous findings (Orbach et al., 2000), suggest that frame-shift mutation at the AVR-Pita1 alleles is one of the mechanisms that the fungus uses to defeat the Pita gene in nature (Fig. 2f).

3.3. AVR-Pita1 diversification in avirulent isolates The AVR-Pita1 gene was sequenced from 151 avirulent isolates (Supplementary Table S1). The resulting AVR-Pita1 sequences from the avirulent isolates were analyzed. A total of 38 AVR-Pita1 haplotypes, including the original AVR-Pita1, were identified based on the DNA sequence (Table 2). High nucleotide variation was observed, mainly in the exons of AVR-Pita1 (Fig. 3), and a haplotype network based on sequence variation was developed (Fig. 4). Among the 38 haplotypes identified, 15 haplotypes were represented by multiple isolates whereas 23 haplotypes were identified only in a single isolate (Fig. 4; Supplemental Table S1). The original AVR-Pita1 allele was found in two Chinese isolates including O-137,

977

Y. Dai et al. / Fungal Genetics and Biology 47 (2010) 973–980 Table 2 Protein variation among 38 AVR-Pita1 alleles in field isolates of M. oryzae.a Proteinb

Allelec

3

4

d

12

59

81

82

87

98

103

118

127

135

137

138

154

168

173e

187

191

194

197

206

AVR-Pita1 A B C D E F G H I J K L M N O P Q R S T U V W X Y Z

N/A 1 2 3,4 5,6 7 8 9,10 11 12 13 14,16 15 17 18,19 20,22,23 21 24 25 26 27,28,35 29 30 31 32,33 34 36,37

F F F F F F F F F S F F F F F F F F F F F F F F F F F

Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

– L L L L L L L L L L L L L L L L L L L L L L L L L L

V V V V V V V V V V G V V V V V V V V V V V V V V V V

S S S S S S S S S S S S S S S S C S S S C S S S S C C

N S S S S S S S S S S S S S S S S S N N N N N N N N N

D N D D D D N D D D D D D D D D D D D D D D D D D D D

R K K K K K K K K K K K K E E K K K R R R R R R E R R

Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q R Q Q

K N N N N N N N N N N N N N N N N N K N K K K K K K K

E E Q E E E E Q E E E E E E E E E E E E E E E E E E E

N N N N N N N N N N N N N N D N N N N N N N N N N N N

G E E E E E E E E E E E E E E G G G G G G G G G G G G

H H H H H H H H H H H H H H H H H H H H H H H H R H H

N N N N N N N N N N N N N N S N N N N N N N N N N N N

P P P P P P P P P P P P P G G P P P P P P P P P R P P

G G G G G G G G G G G G G G G G G G G G G G G V F G G

V I I I I I I I I I I I I I V I I V I V V V V V V V V

K K K K K K K K K K K K K K K K K K K K K K K K E K K

Y C C C C Y Y Y Y Y Y C C Y Y C C C C C C C Y Y Y C C

D D D H D D D D D D D D D D D D D D D H H H H H H H D

H H H H H H H H H P H H H H H H H H H H H H H H H H H

K R R R R R K K K K K K K K K K K K R K K K K K K R R

a Different amino acids and their positions, the letter code used for each amino acid. Number indicates a position of amino acid in the AVR-Pita1 protein (AF207841), and bold letters indicate differences in amino acids in comparison with the AVR-Pita1 protein. All isolates included in the table were avirulent toward Pi-ta containing cultivars. b Groups of AVR-Pita1 variants based on amino acid sequences. c Groups of AVR-Pita1 variants based on nucleotide; N/A = not applied. d Leucine insertion. e Amino acid in the putative protease motif.

Fig. 3. AVR-Pita1 diversification in avirulent isolates. Distribution of variation of the AVR-Pita1 alleles using sliding window. Axis, distribution of variation within the full region, including three introns and four exons of AVR-Pita1. Panes, the corresponding schematic presentation of the three introns and four exons of AVR-Pita1. Window length: 10; Step size: 2. p value corresponds with the level of variation at each site because it is the sum of pairwise differences divided by the number of pairs within the population.

and three Colombian isolates (Supplemental Table S1; Fig. 4). Based on these findings, it can be predicted that the AVR-Pita1 gene in field isolates of M. oryzae is under evolutionary process. AVR-Pita1 is known to have three introns and four exons in the open reading frame (ORF) (Orbach et al., 2000). Alignment of the

AVR-Pita1 DNA sequences assembled from the 151 avirulent isolates revealed that there were 33 polymorphic sites (excluding gaps) with 28 being in the exon region and five in the intron region; of the 28 sites in the exon region, a total of 25 led to predicted amino acid substitutions, indicating that most of the variation in

978

Y. Dai et al. / Fungal Genetics and Biology 47 (2010) 973–980

Fig. 4. The haplotype network for the 38 AVR-Pita1 alleles. The original AVR-Pita1 allele was designated as the 38th haplotype in the network. A black node in the network represents an extinct or a missing haplotype not found in the sample. Each haplotype is separated by mutational events. All haplotypes are displayed as circles. The size of the circles corresponds to the haplotype frequency. The haplotype network is highlighted by geographic characteristics of collected samples. Different colors represent different countries where isolates were collected.

the exon region would have resulted in alteration of the amino acids in the protein. Moreover, the AVR-Pita1 sequences among the 151 avirulent isolates were predicted to produce 27 functional proteins. Among these 27 proteins, amino acid variations were predicted to occur at 23 positions including a deletion/insertion. All variations occurred throughout the entire protein, including the one at position 173 of the putative protease motif (Table 2). The protease motif in the 27 putative proteins was identical except for the 173rd amino acid ‘‘V173I” in the metalloprotease motif (Orbach et al., 2000).

4. Discussion Plants and their pathogens have co-existed and co-evolved in nature (Allen et al., 2004). Host resistance, mediated by major R genes, is robust but typically not durable. However, the molecular mechanisms by which plant pathogens defeat major R genes is not well understood. The Pi-ta gene in rice is a major R gene and is effective in preventing infections by races of M. oryzae that contain the corresponding avirulence gene AVR-Pita1 because the Pi-tacontaining rice cultivars are capable of recognizing AVR-Pita1 and triggering resistance. However, breakdown of resistance can occur in commercial rice fields due to significant structural variation at the AVR-Pita1 allele among field isolates (Fig. 2f). In the present study, we have clearly demonstrated that partial deletion, complete deletion, frame-shift mutation and sequence variation have occurred in the AVR-Pita1 sequences among field isolates of M .oryzae from various rice-producing countries; our study supports the previously reported mutation types of AVR-Pita1 identified in laboratory strains of the fungus (Orbach et al., 2000). In addition, the PROSITE Dictionary of Protein Sites and Patterns predicts that either V or I at position 173 can be a critical amino acid residue for metalloprotease (Hofmann et al., 1999). Similarly we also identified V in 7 isolates, and I in 16 isolates at position 173 of AVR-Pita1 metalloprotease motif in the avirulent isolate population (Table 2).

Considering the co-evolutionary relationship between AVRPita1 and Pi-ta, it is difficult to surmise how the Pi-ta gene has remained effective to all of the AVR-Pita1 variants identified from the present study if indeed they all produce active metalloproteases. There are two predominant hypotheses for the co-evolution of R and AVR genes in plants. The foremost hypothesis is the ‘‘arms race” that predicts that both R and AVR genes are under diversified selection whereas a ‘‘trench warfare” hypothesis predicts that either R or AVR is under balanced selection, and the other is under diversified selection. Patterns potentially consistent with recent directional selection were found in the Pi-ta region in accessions of Oryza rufipogon, while significant deviation from neutral evolution was not found in accessions of Oryza sativa (Huang et al., 2008; Lee et al., 2009a). Sequence variation observed in flanking regions around Pi-ta in O. sativa suggest that the size of the resistance Pi-ta introgressed block was at least 5.4 Mb in all elite resistant cultivars, but not in the cultivars without Pi-ta. Despite that, it is still difficult to determine if a balanced or an unbalanced selection had occurred at the Pi-ta region. These findings do suggest that the Pi-ta region has evolved under extensive selection pressure during crop breeding for resistance (Lee et al., 2009a; Jia, 2009). AVR-Pita1 is known to reside near the highly unstable telomeric region (Orbach et al., 2000; Khang et al., 2008). The presence of multiple AVR-Pita1 variants in avirulent field isolates appears to suggest the ‘‘arms race” occurs between Pi-ta and AVR-Pita1, if similar numbers of resistant and susceptible alleles can be found at the Pi-ta locus. However, only one resistant Pi-ta haplotype has been identified in several extensive surveys of rice germplasm (Wang et al., 2008; Huang et al., 2008; Lee et al., 2009a) that does not support the ‘‘arms race”. Pi-ta is known to confer resistance in up to 10 US races of M. oryzae (Jia et al., 2009). It is also known that Pi-ta is located near the centromere in rice, a region that is relatively stable (Bryan et al., 2000). In addition, Costanzo and Jia (2009) recently demonstrated that Pi-ta is capable of producing a total of 12 transcripts, most of which encode proteins with NBS–LRR domains, indicating their roles as the R proteins. The location of the Pi-ta gene at the centromere region was somewhat surprising since centromeres in most eukaryotes only contain repetitive DNA sequences. Discovery of a transposon at the Pi-ta promoter region in resistant rice germplasm may explain the molecular mechanisms of gene activation (Lee et al., 2009a). These findings suggest that Pi-ta engages in ‘‘trench warfare” with AVR-Pita1 with both genes maintaining an array of strategies to prevent/cause disease. Further examination of the resistant function of Pi-ta proteins encoded by alternative transcripts and natural Pi-ta haplotypes should help elucidate this interaction. In addition, recent identification of another critical gene Ptr(t) in the Pi-ta-mediated disease resistance (Jia and Martin, 2008) is a significant advancement in further uncovering the molecular mechanisms of Pi-ta and AVR-Pita1 interactions. Cloning and examination of physical interactions among Pi-ta, AVR-Pita1 and Ptr(t) may reveal the roles of Pi-ta, AVR-Pita1 and Ptr(t) in signaling recognition and transduction. In conclusion, we present evidence to support the hypothesis that AVR-Pita1 has been evolving under the pressure of positive selection in nature; major variation at the AVR-Pita1 locus is responsible for the inactivation of Pi-ta-mediated resistance in commercial rice cultivars. These findings illustrate the fact that these AVR-Pita1 variants are both ecologically fit and capable of avoiding host recognition to trigger host resistance. Therefore, it is fully possible that these AVR-Pita1 variants would evolve pathogenicity factors for a future epidemic of the rice blast disease in rice-producing areas. On the other hand, the loss of host resistance by a major R gene can be restored by reintroducing AVR-Pita1 into virulent field isolates (Orbach et al., 2000). This new knowledge

Y. Dai et al. / Fungal Genetics and Biology 47 (2010) 973–980

should assist in the development of both novel and conventional strategies to achieve effective and durable blast resistance worldwide. Acknowledgments We thank the Arkansas Rice Research and Promotion Board for their financial support to Yuntao Dai, Asia Rice Travel Grant Foundation, USA for supporting Yuntao Dai’s traveling expenses, and all supporting staff members of DB NRRC and cooperators for technical assistance and useful discussions.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fgb.2010.08.003. References Allen, R.L., Bittner-Eddy, P.D., Grenville-Briggs, L.J., Meith, J.C., Rehmany, A.P., Rose, L.E., Beynonm, J.L., 2004. Host-parasite coevolutionary conflict between Arabidopsis and downy mildew. Science 306, 1957–1960. Ashikawa, I., Hayashi, N., Yamane, H., Kanamori, H., Wu, J., Matsumoto, T., Ono, K., Yano, M., 2008. Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance. Genetics 180, 2267–2276. Ballini, E., Morel, J.B., Droc, G., Price, A., Courtois, B., Notteghem, J.L., Tharreau, D., 2008. A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance. Mol. Plant Microbe Interact. 21, 859–868. Bryan, G.T., Wu, K., Farrall, L., Jia, Y., Hershey, H.P., McAdams, S.A., Faulk, K.N., Donaldson, G.K., Tarchini, R., Valent, B., 2000. A single amino acid difference distinguishes resistant and susceptible alleles of rice blast resistance gene Pi-ta. Plant Cell 12, 2033–2045. Chen, X., Shang, J., Chen, D., Lei, C., Zou, Y., Zhai, W., Liu, G., Xu, J., Ling, Z., Cao, G., Ma, B., Wang, Y., Zhao, X., Li, S., Zhu, L., 2006. A B-lectin receptor kinase gene conferring rice blast resistance. Plant J. 46, 794–804. Clement, M., Posada, D., Crandall, K., 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9, 1657–1659. Costanzo, S., Jia, Y., 2009. Alternatively spliced transcripts of Pi-ta blast resistance gene in Oryza sativa. Plant Sci. 177, 468–478. Dioh, W., Tharreau, D., Notteghem, J.L., Orbach, M., Lebrun, M.H., 2000. Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers. Mol. Plant Microbe Interact. 13, 217–227. Dodds, P.N., Lawrence, G.J., Catanzariti, A-.M., Teh, T., Wang, C.-I., Ayliffe, M.A., Kobe, B., Ellis, J.G., 2006. Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes. Proc. Natl. Acad. Sci. USA 103, 8888–8893. Farman, M.L., Eto, Y., Nakao, T., Tosa, Y., Nakayashiki, H., Mayama, S., Leong, S.A., 2002. Analysis of the structure of the AVR1-CO39 avirulence locus in virulent rice-infecting isolates of Magnaporthe grisea. Mol. Plant Microbe Interact. 15, 6– 16. Farman, M.L., Leong, S.A., 1998. Chromosome walking to the AVR1-CO39 avirulence gene of Magnaporthe grisea: discrepancy between the physical and genetic maps. Genetics 150, 1049–1058. Fudal, I., Bohnert, H.U., Tharreau, D., Lebrun, M.H., 2005. Transposition of MINE, a composite retrotransposon, in the avirulence gene ACE1 of the rice blast fungus Magnaporthe grisea. Fungal Genet. Biol. 42, 761–772. Hayashi, K., Yoshida, H., 2009. Refunctionalization of the ancient rice blast disease resistance gene Pit by the recruitment of a retrotransposon as a promoter. Plant J. 57, 413–425. Hofmann, K., Bucher, P., Falquet, L., Bairoch, A., 1999. The PROSITE database, its status in 1999. Nucleic Acids Res. 27, 215–219. Huang, C.L., Hwang, S.Y., Chiang, Y.C., Lin, T.P., 2008. Molecular evolution of the Pi-ta gene resistant to rice blast in wild rice (Oryza rufipogon). Genetics 179, 1527– 1538. Jia, Y., 2009. Artificial introgression of a large fragment around the Pi-ta rice blast resistance gene in backcross progenies and several elite rice cultivars. Heredity 103, 333–339. Jia, Y., Martin, R., 2008. Identification of a new locus, Ptr(t), required for rice blast resistance gene Pi-ta-mediated resistance. Mol. Plant Microbe Interact. 21, 396– 403. Jia, Y., McAdams, S., Bryan, G., Hershey, H., Valent, B., 2000. Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. EMBO J. 19, 4004–4014. Jia, Y., Lee, F., McClung, A., 2009. Determination of resistance spectra to US races of Magnaporthe oryzae causing blast in a recombinant inbred line population. Plant Dis. 93, 639–644.

979

Joosten, M.H.A.J., Cozijnsen, A.J., de Wit, P.J.G.M., 1994. Host resistance to a fungal tomato pathogen lost by a single base-pair change in an avirulence gene. Nature 367, 384–386. Kang, S., Lebrun, M.H., Farrall, L., Valent, B., 2001. Gain of virulence caused by insertion of a Pot3 transposon in a Magnaporthe grisea avirulence gene. Mol. Plant Microbe Interact. 14, 671–674. Kang, S., Sweigard, J.A., Valent, B., 1995. The PWL host specificity gene family in the blast fungus Magnaporthe grisea. Mol. Plant Microbe Interact. 8, 939–948. Khang, C.H., Park, S.Y., Lee, Y.H., Valent, B., Kang, S., 2008. Genome organization and evolution of the AVR-Pita avirulence gene family in the Magnaporthe grisea species complex. Mol. Plant Microbe Interact. 21, 658–670. Lee, F.N., Cartwright, R.D., Jia, Y., Correll, J.C., Moldenhauer, K.A., Gibbons, J.W., Boyett, V., Zhou, E., Boza, E., Seyran, E. 2005. A preliminary characterization of the rice blast fungus on ‘Banks’ rice. In: Norman, R.J., Meullenet, J.-F., Moldenhauer, K.A.K., (Eds.), B.R. Wells Rice Research Studies 2004, Arkansas Agricultural Experiment Station Research Series 529. pp. 103–110. Lee, S., Costanzo, S., Jia, Y., Olsen, K.M., Caicedo, A.L., 2009a. Evolutionary dynamics of the genomic region around the blast resistance gene Pi-ta in AA genome Oryza species. Genetics 183, 1315–1325. Lee, S.K., Song, M.Y., Seo, Y.S., Kim, H.K., Ko, S., Cao, P.J., Suh, J.P., Yi, G., Roh, J.H., Lee, S., An, G., Hahn, T.R., Wang, G.L., Ronald, P., Jeon, J.S., 2009b. Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil– nucleotide-binding-leucine-rich repeat genes. Genetics 181, 1627–1638. Li, W., Wang, B., Wu, J., Lu, G., Hu, Y., Zhang, X., Zhang, Z., Zhao, Q., Feng, Q., Zhang, H., Wang, Z., Wang, G.L., Han, B., Wang, Z., Zhou, B., 2009. The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t. Mol. Plant Microbe Interact. 22, 411–420. Lin, F., Chen, S., Que, Z., Wang, L., Liu, X., Pan, Q., 2007. The blast resistance gene Pi37 encodes a nucleotide binding site-leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1. Genetics 177, 1871–1880. Ling, K.C., Ou, S.H., 1969. Standardization of the international race numbers of Pyricularia oryzae. Phytopathology 59, 339–342. Liu, X., Lin, F., Wang, L., Pan, Q., 2007. The in silico map-based cloning of Pi36, a rice coiled-coil–nucleotide-binding site–leucine-rich repeat gene that confers racespecific resistance to the blast fungus. Genetics 176, 2541–2549. Miyamoto, M., Ando, I., Rybka, K., Kodama, O., Kawasaki, S., 1996. High resolution mapping of the indica-derived rice blast resistance genes. 1. Pi-b. Mol. Plant Microbe Interact. 9, 6–13. Orbach, M.J., Farrall, L., Sweigard, J.A., Chumley, F.G., Valent, B., 2000. A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta. Plant Cell 12, 2019–2032. Qu, S., Liu, G., Zhou, B., Bellizzi, M., Zeng, L., Dai, L., Han, B., Wang, G.L., 2006. The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site– leucine-rich repeat protein and is a member of a multigene family in rice. Genetics 172, 1901–1914. Rehmeyer, C., Li, W., Kusaba, M., Kim, Y.S., Brown, D., Staben, C., Dean, R., Farman, M., 2006. Organization of chromosome ends in the rice blast fungus, Magnaporthe oryzae. Nucleic Acids Res. 34, 4685–4701. Ridout, C.J., Skamnioti, P., Porritt, O., Sacristan, S., Jones, J.D.G., Brown, J.K.M., 2006. Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance. Plant Cell 18, 2402–2414. Schurch, S., Linde, C.C., Knogge, W., Jackson, L.F., Mcdonald, B.A., 2004. Molecular population genetic analysis differentiates two virulence mechanisms of the fungal avirulence gene NIP1. Mol. Plant Microbe Interact. 17, 1114–1125. Shang, J., Tao, Y., Chen, X., Zou, Y., Lei, C., Wang, J., Li, X., Zhao, X., Zhang, M., Lu, Z., Xu, J., Cheng, Z., Wan, J., Zhu, L., 2009. Identification of a new rice blast resistance gene, Pid3, by genomewide comparison of paired nucleotide-binding site-leucine-rich repeat genes and their pseudogene alleles between the two sequenced rice genomes. Genetics 182, 1303–1311. Silue, D., Notteghem, J.L., Tharreau, D., 1992. Evidence for a gene for gene relationship in the Oryza sativa-Magnaporthe grisea pathosystem. Phytopathology 82, 577–582. Silva, J.C., Loreto, E.L., Clark, J.B., 2004. Factors that affect the horizontal transfer of transposable elements. Curr. Issues Mol. Biol 6, 57–72. Stukenbrock, E.H., McDonald, B.A., 2009. Population genetics of fungal and oomycete effectors involved in gene-for-gene interactions. Mol. Plant-Microbe Interact. 22, 371–380. Sweigard, J.A., 1995. Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus. Plant Cell 7, 1221–1233. Takahashi, M., Ashizawa, T., Hiravae, K., Moriwaki, J., Sone, T., Sonoda, R., Noguchi, M.T., Nagashima, S., Ishikawa, K., Arai, M., 2010. One of two major paralogs of AVRPita1 is functional in Japanese rice blast isolates. Phytopathology 100, 612–618. Valent, B., Farrall, L., Chumley, F.G., 1991. Magnaporthe grisea genes for pathogenicity and virulence identified through a series of backcrosses. Genetics 127, 87–101. Wang, X., Jia, Y., Shu, Q., Wu, D., 2008. Haplotype diversity at the Pi-ta locus in cultivated rice and its wild relatives. Phytopathology 98, 1305–1311. Wang, X., Yano, M., Yamanouchi, U., Iwamoto, M., Monna, L., Hayasaka, H., Katayose, Y., Sasaki, T., 1999. The Pi-b gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes. The Plant J. 19, 55–64. Yoshida, K., Saitoh, H., Fujisawa, S., Kanzaki, H., Matsumura, H., Yoshida, K., Tosa, Y., Chuma, L., Takano, Y., Win, J., Kamoun, S., Terauchi, R., 2009. Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae. Plant Cell 21, 1573–1591.

980

Y. Dai et al. / Fungal Genetics and Biology 47 (2010) 973–980

Zhou, B., Qu, S., Liu, G., Dolan, M., Sakai, H., Lu, G., Bellizzi, M., Wang, G., 2006. The eight amino acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea. Mol. Plant Microbe Interact. 19, 1216– 1228.

Zhou, E., Jia, Y., Singh, P., Correll, J.C., Lee, F.N., 2007. Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence. Fung. Genet. Biol. 44, 1024– 1034.