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Plant Cell. Nov 2000; 12(11): 2033–2046.
PMCID: PMC150156

tA Single Amino Acid Difference Distinguishes Resistant and Susceptible Alleles of the Rice Blast Resistance Gene Pi-ta

Abstract

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928–amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta+ R gene together with AVR-Pita+ induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta2 gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta2 relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.

INTRODUCTION

Disease resistance (R) genes protect plants from fungal, bacterial, viral, and nematode pathogens as the first level of a complex genetic defense system. In particular, plants containing dominant (or semidominant) R genes respond to pathogens containing the corresponding avirulence (AVR) genes by initiating signal transduction pathways that activate defense systems (Bent, 1996; Hammond-Kosack and Jones, 1997; Yang et al., 1997). Numerous R genes have been cloned and characterized from dicotyledons. However, relatively few R genes have been cloned from the cereals that contribute heavily to the supply of food for humans and feed for livestock, in part because of the complex genomes of most of these crop plants. The Xa21 bacterial blight R gene from rice encodes a protein with putative extracellular receptor and cytoplasmic kinase domains (Song et al., 1995). Two other rice R genes, the Xa1 bacterial blight R gene (Yoshimura et al., 1998) and the rice blast R gene Pi-b (Wang et al., 1999), encode putative cytoplasmic receptor proteins of the nucleotide binding site (NBS) class. Characterization of an AVR/R gene pair has not been reported for a cereal plant pathogen system.

Incorporation of disease R genes into crop plants has not achieved durable resistance to highly variable fungal pathogens such as Magnaporthe grisea (Hebert) Barr, the causal agent of the devastating rice blast disease (Ou, 1985; Rossman et al., 1990). Identification of major R genes began in the early 1920s when Sasaki (Yamada, 1985) discovered physiological races of the rice blast pathogen that differ in their ability to cause disease on different rice varieties. Extensive genetic studies that followed led to the identification of 13 major blast R genes and to the development of standard rice varieties as R gene differentials (Yamada et al., 1976; Kiyosawa, 1984). Nine of these R genes were clustered, two at the Pi-ta locus on chromosome 12, five at the Pi-k locus on chromosome 11, and two at the Pi-z locus on chromosome 6. Currently, ~20 major blast R genes have been reported, with several more genes falling into R gene clusters on chromosomes 6, 11, and 12 (Mackill and Bonman, 1992; Yu et al., 1996; Chao et al., 1999).

The cluster of R genes linked to the centromere on chromosome 12 includes the allelic or tightly linked Pi-ta and Pi-ta2 genes identified by Kiyosawa and colleagues (Kiyosawa, 1971; Rybka et al., 1997). The relationship between these two indica-derived R genes appears to be complex, and some investigators have reported that Pi-ta is required for function of Pi-ta2 (Kiyosawa, 1967, 1971; Silué et al., 1992, 1993; Rybka et al., 1997). Other R genes, including Pi-4a(t), Pi-4b(t), Pi-6(t), and Pi-12(t), have been mapped to the centromeric region of chromosome 12 (Inukai et al., 1994; Yu et al., 1996; Zheng et al., 1996). One rice variety from the southern United States, called Katy, reportedly contains a tightly linked cluster of at least seven R genes that map in the same region as Pi-ta and Pi-ta2 (Moldenhauer et al., 1992; Chao et al., 1999).

Operationally, as in other gene-for-gene host pathogen systems, individual R genes can be distinguished only by the resistance they confer to corresponding avirulent strains of the pathogen (Flor, 1971). Comparisons of rice blast R genes identified in different countries have been difficult, given the regulatory prohibitions against sharing pathogen strains between geographically separated rice production regions. R gene identification also is complicated by the common occurrence of multiple AVR genes in single field isolates of the pathogen, any one of which is sufficient to trigger resistance in the presence of its corresponding R gene. Thus, the number of distinct blast R genes is uncertain, and strategies for pyramiding R genes for blast control are hampered by imprecise characterization of the genes. Cloning R genes will facilitate their incorporation into agronomically useful crop varieties, either through breeding with molecular marker–assisted selection or through transgenic strategies. Understanding the AVR gene triggers for specific R genes should provide insight into effective R gene combinations for pyramiding strategies. In the longer term, understanding the molecular basis for AVR gene triggering of resistance should lead to development of novel strategies for broad-spectrum and durable disease control that use the native plant defense system.

In a companion article, Orbach et al. (2000)(this issue) report that a pathogen gene encoding a putative zinc metalloprotease, AVR-Pita, plays a critical role in triggering Pi-ta–mediated resistance. Here, we report that Pi-ta is a member of the putative cytoplasmic NBS-receptor class of R genes (Grant et al., 1995; Bent, 1996; Jones and Jones, 1996; Salmeron et al., 1996; Wang et al., 1999). However, Pi-ta encodes a highly interrupted and relatively rudimentary leucine-rich repeat (LRR) motif, compared with that seen in other members of this class. Low constitutive expression of Pi-ta occurs in both resistant and susceptible rice varieties. The predicted protein encoded by Pi-ta differs by one amino acid from the predicted protein from a susceptible indica rice variety, and by five amino acids from the predicted protein from susceptible japonica rice varieties. Transient expression data suggest that the difference in recognition specificity for AVR-Pita hinges on a single amino acid difference between the Pi-ta proteins produced by resistant and susceptible rice. The resistant Pi-ta allele also occurs in Pi-ta2–containing rice varieties. This suggests that the Pi-ta2 blast resistance specificity is a combination of Pi-ta and at least one additional resistance gene.

RESULTS

Map-Based Cloning of Pi-ta

Random amplified polymorphic DNA (RAPD) markers and bulked segregant analysis were used to identify molecular markers linked to Pi-ta (Wu et al., 1995). The Pi-ta gene resides on chromosome 12 between flanking RAPD markers designated SP4B9 and SP9F3, both of which show one recombination event relative to Pi-ta in a mapping population of 990 individuals. The cosegregating RAPD marker SP7C3 was converted to a restriction fragment length polymorphism (RFLP) marker, p7C3. Chromosome walking was initiated by hybridizing this single-copy probe to filters containing a rice bacterial artificial chromosome (BAC) library (Shizuya et al., 1992) produced from a doubled haploid rice line, YT14, containing the Pi-ta gene. We assembled an overlapping set of rice BAC clones covering ~850 kb as described in Figure 1A. The left border marker SP4B9 was identified in the assembled BAC contig. However, SP9F3 has not been identified, possibly because of decreased recombination in the vicinity of a centromere.

Figure 1.
Positional Cloning of Pi-ta.

Identification of a Candidate Gene by Sequence Analysis

We adopted a sample sequencing strategy (Tarchini et al., 2000) to identify R gene candidates in the BAC contig. The region to the right of RFLP marker p7C3 in Figure 1A was relatively high in repetitive DNA sequences and low in gene density compared with the average density of one gene per 10 kb for the Adh1-Adh2 region of chromosome 11 (Tarchini et al., 2000). This result is consistent with a previous report that the region near Pi-ta is high in repetitive DNA sequences (Nakamura et al., 1997). Clones BAC77D8 and BAC142E8 contained the centromere-specific repetitive DNA sequence RCE1 (Singh et al., 1996; Dong et al., 1998) as well as regions of lesser homology with other centromere-specific repeats. Thus, sequencing analysis supported the expected centromeric location of the contig.

The Pi-ta candidate gene was identified by sequences from BAC142E8 that showed weakly significant (pLog between 1 and 10) homology with the central NBS region of NBS-LRR resistance genes. The candidate gene was fully characterized by using available small subclones and larger ones derived from the original BAC clone. DNA gel blot analysis identified RFLPs associated with this locus that correlated with resistance versus susceptibility to AVR-Pita–containing fungus (data not shown). The putative Pi-ta gene occurred near the right end of BAC142E8 in Figure 1A and was flanked by two unrelated genes. A sequence containing homology with both a hypothetical gene from Arabidopsis (AL049487) and a putative Ac-like transposase (AC006420) was identified 3 kb upstream from the putative Pi-ta open reading frame. A sequence with homology to the rice thioredoxin H gene (U92541) was identified 2 kb from the 3′ end.

A 7.5-kb fragment was subcloned from BAC142E8 as pCB1641; it contained a 2784-nucleotide coding sequence (Figure 1B), beginning at nucleotide 1256 and interrupted by a 1463- bp intron. A cDNA clone confirmed the position of the intron. The cDNA coding sequence was assembled with 2425 bp of native promoter sequence into pCB1926 for functional analysis.

Functional Analysis of Pi-ta by Stable Rice Transformation

Functional analysis was performed by transforming susceptible Nipponbare rice either with the genomic transgene in pCB1641 (Figure 1B) or with the cDNA transgene in pCB1926. The Pi-ta–containing plasmids were cotransformed along with a plasmid expressing the bacterial hygromycin B phosphotransferase (HptII) gene as a selection marker. Among 42 independent primary transformants shown to contain the genomic Pi-ta transgene, six showed complete resistance, 12 showed intermediate resistance, and 24 were susceptible. DNA gel blot analysis demonstrated that resistant primary transgenic T0 lines contained between one and four copies of the Pi-ta transgene integrated at a single locus (data not shown). Stable inheritance of specific resistance characteristic of Pi-ta was first demonstrated with a primary transformant in the intermediate class. Hygromycin-resistant T2 progeny showed resistance to M. grisea strain O-137 with AVR-Pita but were not resistant to the virulent mutant strain CP3337, which lacks a functional AVR-Pita gene.

The cDNA transgene (pCB1926) produced twice as many resistant transformants in the T0 generation and had a greater ratio of resistant to intermediate resistant lines. An example of a fully resistant primary transformant is shown in Figure 2. In a line containing a single copy of the Pi-ta cDNA transgene, T2 progeny segregated 3:1 for resistance/susceptibility when inoculated with the AVR-Pita–containing pathogen O-137, whereas all T2 progeny were susceptible to the virulent mutant strain CP3337. The resistance phenotype cosegregated with the Pi-ta transgene in DNA gel blot analysis (data not shown). These results confirm the identity of the Pi-ta gene.

Figure 2.
Functional Analysis of Pi-ta by Stable Rice Transformation.

Properties of the Pi-ta Protein

Pi-ta encodes the predicted 928–amino acid polypeptide shown in Figure 3, with a molecular mass of 105 kD and a pI of 7.05. A centrally located NBS region, encoding amino acids 208 to 527, shows amino acid sequence similarity to NBS domains encoded by other R genes: 31.7% identity (43.2% similarity) to the rice Pi-b NBS region (Wang et al., 1999) and 30.3% identity (44.3% similarity) to the Arabidopsis RPM1 NBS region (Grant et al., 1995). A conserved internal hydrophobic domain characteristic of other NBS-class R gene products (Bent, 1996; Jones and Jones, 1996) is present between amino acids 407 and 418. Four potential N-glycosylation sites were found, at positions 339, 556, 654, and 838.

Figure 3.
Deduced Pi-ta Protein Sequence.

The Pi-ta protein differs from those encoded by previously characterized dicot NBS-class R genes in having a unique N terminus. Pi-ta has neither a leucine zipper region nor Toll/interleukin-1 receptor homology (Bent, 1996; Jones and Jones, 1996); in addition, it lacks a classic LRR. The predicted protein contains no matches to the LxxLxxLxxLx(N/C/T)x(x)LxxIPxx motif identified in dicot R gene products with predicted cytoplasmic LRRs (Jones and Jones, 1996). Instead, the C-terminal region of Pi-ta contains a highly imperfect repeating structure with 10 repeats of various lengths (from 16 to 75 amino acids) based on the consensus LxxLxxL (Figure 3). The Pi-ta C-terminal region, amino acids 586 to 928, which corresponds approximately to the LRR of RPM1 protein (Grant et al., 1995), contained 16.4% leucine. We refer to this region of Pi-ta as a leucine-rich domain (LRD) on the basis of its structure and the functional analysis reported by Jia et al. (2000).

Pi-ta Is a Unique, Constitutively Expressed Gene

DNA gel blot analysis probing with the portion of the gene that encodes the C-terminal LRD (Figure 3) demonstrated that Pi-ta is a single-copy gene in both resistant and susceptible varieties of rice. An N-terminal probe identified a single copy against a general smear of background hybridization even under high-stringency conditions, suggesting that the rice genome may contain related genes.

RNA gel blot analysis of poly(A)+ mRNA indicated that Pi-ta was transcribed at similar levels in leaves of both resistant and susceptible rice varieties in the absence of pathogen challenge. As shown in Figure 4, use of the single-copy LRD portion of Pi-ta as a hybridization probe identified a single mRNA with an estimated transcript size of 3.3 kb in both rice varieties. Thus, sensitivity in varieties that lack the ability to recognize the AVR-Pita signal does not appear to reflect a failure to transcribe the R gene.

Figure 4.
Pi-ta Transcription in Resistant and Susceptible Rice Varieties in the Absence of Pathogen Challenge.

Low-stringency DNA gel blot analysis of other cereal and grass genomic DNAs indicated a homologous sequence in the cereals barley, maize, oat, rye, and wheat and also in crabgrass, foxtail millet, and weeping lovegrass. When stringency was increased to an intermediate value, only maize and rye contained sequences similar to the full-length Pi-ta cDNA and to the LRD region.

Structural Differences between Resistant and Susceptible Pi-ta Alleles

Additional Pi-ta alleles were sequenced to determine differences in gene sequence in resistant versus susceptible rice varieties. The Pi-ta gene from Yashiro-mochi probably is derived from an upland rice variety, Okaine, which is no longer available (Rybka et al., 1997). The allele in the second Pi-ta–differential variety, K1, has been introgressed from Tadukan, an indica variety from the Philippines (Kiyosawa, 1984). DNA sequences of the Pi-ta genes from K1 and Tadukan are identical to Pi-ta from Yashiro-mochi. In contrast, the Yashiro-mochi DNA coding sequence differs by seven nucleotides from the sequence present in susceptible japonica varieties Tsuyuake, Nipponbare, and Sariceltik. These 7-bp substitutions in the coding region result in the five–amino acid differences shown in Table 1. We also sequenced the allele from the susceptible indica variety C101A51 to eliminate sequence differences arising from the divergence of indica and japonica rices. The C101A51 allele encoded a protein identical to Pi-ta from Yashiro-mochi except for serine at amino acid residue 918. Thus, all pi-ta proteins from susceptible rice varieties share a common single amino acid difference relative to resistant Pi-ta protein: serine at residue 918 instead of alanine.

Table 1.
Amino Acid Sequence Comparison of Pi-ta Proteins from Varieties with Pi-ta, from Varieties with Pi-ta2, and from Sensitive Rice

Transient Expression Assay Confirms Pi-ta Specificity

We adapted a transient expression assay developed in dicots (Mindrinos et al., 1994; Gopalan et al., 1996; Leister et al., 1996) to confirm the importance of alanine-918 in determining the in vivo specificity of Pi-ta. In this assay, the level of activity for the enzyme β-glucuronidase (GUS) is used as an indicator of the physiological state of the transformed plant cells. High GUS activity indicates “healthy” plant cells in which the hypersensitive resistance response (HR) has not been triggered, whereas low GUS activity indicates cells undergoing HR. The assay depends on the ballistic cointroduction of the AVR-Pita gene along with the GUS reporter. Jia et al. (2000) demonstrated that an AVR-Pita176 plant expression construct, engineered to express a putative mature 176-residue form of the AVR-Pita metalloprotease, is the active form of AVR-Pita in the rice transient expression assay. As shown in Figure 5, coexpression of AVR-Pita176 with the GUS reporter makes no difference relative to expression of the GUS reporter alone in rice that lacks Pi-ta. However, coexpression of AVR-Pita176 along with the GUS reporter in Pi-ta–containing rice resulted in markedly less GUS activity. A mutant AVR-Pita176 gene with an amino acid substitution that abolishes avirulence activity in standard infection assays failed to decrease GUS activity in the transient assay (Table 2). In addition, a naturally occurring virulent avr-pita allele from TH3, a field isolate that infects rice with Pi-ta, also failed to decrease GUS activity (Table 2). Thus, particle bombardment transformation of an AVR-Pita176 transgene along with the GUS reporter produces a robust transient expression system with the in vivo specificity associated with Pi-ta–mediated defense responses.

Figure 5.
Transient Expression of the Cloned Pi-ta Gene.
Table 2.
Effect of a Single Amino Acid Difference (A918S) on the Activity of Pi-ta in the Transient Expression Assay

The Pi-ta cDNA expression construct pCB1926 functions in place of the endogenous Pi-ta gene when cobombarded with the GUS reporter and the AVR-Pita176 gene, resulting in a marked decrease in GUS expression independent of the presence or absence of the endogenous Pi-ta gene (Figure 5). In contrast, the susceptible C101A51 pi-ta cDNA that encodes serine instead of alanine at residue 918 failed to decrease GUS expression under the same conditions (Table 2). Cobombarding the C101A51 pi-ta construct with the GUS reporter and AVR-Pita176 into Yashiro-mochi rice did not interfere with the recognition of AVR-Pita176 by the endogenous Pi-ta gene product. These data suggest that in vivo specificity in the Pi-ta gene-for-gene system results from the alteration of a single amino acid in the Pi-ta protein.

Rice with Pi-ta2 Specificity Has the Pi-ta Gene

Literature reports suggest that Pi-ta2 controls all pathogen strains controlled by Pi-ta, plus additional Pi-ta–sensitive strains (Kiyosawa, 1967, 1971; Silué et al., 1992, 1993; Rybka et al., 1997). Indica variety Tadukan, the source for the Pi-ta gene in K1, is the source for Pi-ta2 in other rice varieties. We therefore sequenced the Pi-ta allele from additional Pi-ta2–containing varieties—Tetep, Reiho, and Katy—and found that each Pi-ta2 variety contains the Pi-ta allele encoding the Pi-ta resistance protein (Table 1).

Results of infection assays, summarized in Table 3, support our sequencing result that Pi-ta2 varieties contain Pi-ta. Strain O-137 with AVR-Pita is unable to infect either Pi-ta or Pi-ta2 rice varieties, but the spontaneous O-137 avr-pita mutant CP3337 has gained the ability to infect both types of rice. This suggests that AVR-Pita in strain O-137 is responsible for triggering resistance in Pi-ta2 rice as well as in Pi-ta rice, and that O-137 lacks other AVR genes effective toward the tested Pi-ta2 varieties. Another Chinese field isolate, O-135, lacks homology with the AVR-Pita gene and fails to trigger Pi-ta–mediated resistance, as demonstrated by its ability to infect rice varieties Yashiro-mochi and K1. However, O-135 is avirulent on Pi-ta2 varieties, suggesting the presence of an AVR gene not found in strain O-137. A spontaneous mutant of O-135, CP753, was selected for virulence on rice variety Reiho. This mutant had gained the ability to infect all Pi-ta2 varieties tested but retained avirulence toward rice variety C101A51 with a different R gene. Together, these results suggest that the Pi-ta2 specificity is a combination of at least two R genes—Pi-ta plus a second, presumably linked, R gene. These results underscore the difficulty in precisely defining the R gene composition of commonly used rice varieties by using uncharacterized fungal pathogen strains.

Table 3.
Avirulent Fungus/Mutant Pairs Identify Pi-ta and a Second R Gene in Rice Varieties with Pi-ta2

DISCUSSION

The Pi-ta protein has unique features compared with other proteins of the NBS-containing class of R genes. It lacks such N-terminal features as a leucine zipper or the Toll/interleukin-1 receptor motifs characteristic of dicot R genes (Bent, 1996). The Pi-ta C-terminal LRD lacks the characteristic LRR motif found in other genes of this class. Pi-ta has 10 repeats based on the motif LxxLxxL, although they are interrupted and spread throughout a region of >300 amino acids. Pi-ta expression also differs from the two other rice NBS-LRR R genes that have been cloned, Xa1 and Pi-b, which appear to be induced in response to pathogen challenge or other environmental factors (Yoshimura et al., 1998; Wang et al., 1999). The Pi-ta R gene is expressed in healthy, unchallenged plants in readiness for pathogen attack, as predicted for R genes that would function in pathogen surveillance (Hammond-Kosack and Jones, 1997).

The most striking finding is that a difference at a single position in the predicted Pi-ta amino acid sequence alanine-918 correlates with the gene-for-gene specificity characteristic of the Pi-ta/AVR-Pita system. That is, rice varieties with the alanine-918 form of the Pi-ta protein recognize fungus with AVR-Pita+ alleles but not fungus with virulent avr-pita alleles. In contrast, the predicted pi-ta proteins encoded by susceptible indica and japonica rice varieties differ from the Pi-ta+ protein in having serine in place of alanine-918. The predicted proteins encoded by susceptible japonica varieties differ by four additional amino acids in the N-terminal region, but these do not appear to impact activity. The suggestion of a critical role for alanine-918 for the resistance phenotype was confirmed in transient expression assays, which indicated that Pi-ta+ protein with alanine-918, but not pi-ta protein with serine-918, acts with the AVR-Pita protein in cytoplasm of rice seedling cells to induce a resistance response.

The molecular mechanism by which Pi-ta with alanine-918, but not pi-ta with serine-918, responds to the AVR-Pita–produced signal molecule is of intense interest. Current data cannot rule out a negative effect of serine at residue 918 on the stability and resulting accumulation of the susceptible pi-ta protein, which could account for the in vivo specificity. However, another mechanism for specificity has received experimental support. Jia et al. (2000) have presented data from yeast two-hybrid and in vitro binding analyses that demonstrate a direct physical interaction between the Pi-ta and AVR-Pita proteins. Although Pi-ta encodes a putative cytoplasmic receptor of the NBS-LRR R gene class, the LRRs thought to be involved in protein–protein interactions are highly degenerate in Pi-ta. Nevertheless, Jia et al. (2000) present evidence that this C-terminal LRD functions as an elicitor binding domain for the AVR-Pita protein.

Our finding that rice varieties reported to have Pi-ta2 contain the Pi-ta gene is consistent with infection data from our laboratory. It is also consistent with previous reports from other laboratories that Pi-ta2, which has a broader resistance spectrum than Pi-ta, confers resistance to all strains controlled by Pi-ta (Kiyosawa, 1967; Silué et al., 1992, 1993). Neither field isolates nor laboratory strains have been found that are avirulent toward Pi-ta but virulent toward Pi-ta2 (Kiyosawa, 1967, 1971; Silué et al., 1992, 1993; Rybka et al., 1997). The straightforward explanation for these results is that the Pi-ta gene we have identified contributes to the specificity commonly known as Pi-ta2. Indeed, our results are consistent with reports that the single R gene in the Pi-ta2–containing rice variety Katy is a tightly linked cluster of at least seven genes (Moldenhauer et al.,1992; Chao et al., 1999). These results raise the possibility that other relatively broad-spectrum R genes might actually be clusters containing more than one gene.

Kiyosawa (1971)(1976) recognized the power of using an avirulent strain and a corresponding virulent mutant to identify specific R genes. Resistance to the avirulent parent and susceptibility to the mutant strongly suggest that a given variety contains the corresponding R gene. This diagnostic pair of fungal strains will be useful only if they lack AVR genes corresponding to other R genes contained in the rice varieties of interest. We now have an avirulent strain/mutant pair (O-135/CP753) that identifies a second R gene found in Pi-ta2–containing rice varieties, and these pathogen strains will be useful in further mapping and cloning this second R gene. Although random genomic sequencing in the BAC contig shown in Figure 1 so far has failed to identify classic R gene candidates in close physical proximity to Pi-ta, the centromeric location of Pi-ta is likely to increase the physical distance corresponding to genetic distance in this region. This may explain why the genetic distance of 1.5 centimorgans between markers SP7C3 and SP9F3 corresponds to >600 kb. Given that we have sequenced only 200 kb of genomic DNA to the right of Pi-ta (Figure 1), perhaps the second Pi-ta2 gene lies just outside the current BAC contig.

Both the plant R gene and the corresponding pathogen AVR gene have been isolated in only a few instances, and most of these are from bacterial disease systems (Leach and White, 1996; Laugé and De Wit, 1998). Bacterial AVR gene products are delivered into the plant cell using a type III secretion system (Galán and Collmer, 1999), which allows recognition and defense responses triggered by cytoplasmic R genes. The previously characterized fungal R/AVR gene pairs suggest that the recognition is probably extracellular. The tomato pathogen Cladosporium fulvum produces extracellular AVR gene products, and the corresponding R gene products have properties characteristic of extracellular receptors (Laugé and De Wit, 1998). Our results show that the predicted Pi-ta protein has properties characteristic of a cytoplasmic receptor, and the AVR-Pita gene product functions after production in the plant cytoplasm subsequent to ballistic transformation. Other results from our laboratory are consistent with the hypothesis that recognition of AVR-Pita occurs inside the plant cell. Recombinant AVR-Pita protein applied externally, by spray inoculation with or without the fungus or by vacuum infiltration into the extracellular spaces in leaves, failed to elicit a response in leaves of rice seedlings containing Pi-ta (Bryan et al., 2000). M. grisea grows intracellularly during colonization of host tissues. Expression of AVR-Pita has been detected only after penetration of the host cell (G.T. Bryan and B. Valent, unpublished results). Given the foregoing observations, we hypothesize that AVR-Pita is secreted into the plant cytoplasm during or after penetration and interacts directly with Pi-ta protein, leading to recognition of the pathogen and initiation of the host defense response pathway. Mechanisms whereby biotrophic or hemibiotrophic fungi, which do not immediately kill host plant cells, deliver proteins into the plant cytoplasm are currently unknown.

In the companion article, Orbach et al. (2000)(this issue) suggest that protease activity is required for the AVR-Pita avirulence function. The possibility of direct interaction between the Pi-ta and AVR-Pita proteins raises the intriguing possibility that the putative AVR-Pita metalloprotease may cleave the Pi-ta gene product. Pi-ta apparently confers on rice the ability to recognize a putative fungal virulence factor and initiate a successful defense response. These findings again demonstrate the remarkable way in which plants have adapted to defend themselves against plant pathogens and further suggest that NBS-LRR R gene products can interact directly with pathogen AVR proteins. They also emphasize the advantage of having both components of the gene-for-gene interaction under study, the pathogen AVR gene and the host R gene.

METHODS

Fungal Strains, Rice Varieties, and Pathogenicity Assays

Field isolates O-137 (avirulent on both Pi-ta– and Pi-ta2–containing rice varieties) and O-135 (virulent on Pi-ta varieties and avirulent on Pi-ta2 varieties) were collected at the China National Rice Research Institute (Hangzhou) in 1985 by Y. Shen and B. Valent. The spontaneous mutant CP3337 was obtained from strain O-137 by isolation of a rare lesion on Yashiro-mochi, as described in the companion article (Orbach et al., 2000, this issue). Likewise, spontaneous mutant CP753 was obtained from strain O-135 as a rare lesion on rice variety Reiho during the course of other experiments (B. Valent and L. Farrall, unpublished results). Strain TH3, a field isolate from Thailand, was provided by J.-L. Notteghem (now at Ecole Nationale Supérieure Agronomique, Montpellier, France). Laboratory strains with single AVR genes, 4360-R-62 (AVR-Pita, avr-pikm) and 4360-R-67 (avr-pita, AVR-Pikm), were used in the mapping studies (Wu et al., 1995) and in analysis of transgenic plants. A mutant containing the amino acid substitution H180K, which abolishes the avirulence activity of AVR-Pita, was produced by in vitro mutagenesis.

A doubled haploid (DH) population (429 individuals) generated from reciprocal crosses between rice varieties Yashiro-mochi (Pi-ta, pi-km) and Tsuyuake (pi-ta, Pi-km) was produced by Zaida Lentini, Cesar Martinez, and Joe Tohme at the Centro Internacional de Agricultura Tropical (Cali, Colombia). An F2 population was generated by Wu et al. (1995) from crossing the DH lines YT4 (Pi-ta, pi-km) and YT10 (pi-ta, Pi-km). A DH line containing both R genes (Pi-ta, Pi-km), YT14, was used for production of the bacterial artificial chromosome (BAC) library, and a DH line containing neither R gene, YT16, was used as a susceptible control in these experiments.

Infection assays were performed as described (Valent et al., 1991). Plants were incubated at room temperature and in low light within plastic bags for 24 hr to maintain high humidity and then were transferred to an incubation chamber. Humidity in the incubation chambers was maintained between 70 and 85% to prevent sporulation and subsequent reinfection of susceptible plants.

Bacterial Strains, Plasmids, and DNA and RNA Analyses

Plasmids used in this study are listed in Table 4. Standard protocols were used for restriction digestions and DNA gel blot analysis (Sambrook et al., 1989; Ausubel et al., 1994). Blots were washed at 50 to 55°C in 2 × SSPE (1 × SSPE is 0.15 M NaCl, 10 mM sodium phosphate, and 1 mM EDTA, pH 7.4) with 0.1% SDS for low-stringency hybridizations, and at 65°C in 0.1 × SSPE with 0.1% SDS for high-stringency hybridization. Three hybridization conditions were used to determine whether homologous sequences occurred in other grasses. Blots were washed at 40°C in 2 × SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) with 0.5% SDS for low-stringency hybridization, at 55°C in 0.5 × SSC with 0.5% SDS for intermediate-stringency hybridization, or at 65°C in 0.2 × SSC with 0.5% SDS for high-stringency hybridization. The 639-bp N-terminal Pi-ta fragment (nucleotides 1401 to 2039) used as a probe for DNA gel blot analysis was amplified by using primers F8-5 (5′-TCCTCAGAG-GCGATCTCC-3′) and F12-5 (5′-CGAACGGCGCATCCAACC-3′). The C-terminal gene fragment encoding the leucine-rich domain (LRD) was subcloned for use as a hybridization probe. Primer GB47 (5′-AATGCAGAATTCACAACACCACTAGCAGGTTTG-3′) was synthesized on the basis of the sequence from nucleotides 4465 through 4497, except that residues between 4471 and 4476 were modified to incorporate an EcoRI restriction site (underlined). Likewise, primer GB46 (5′-CATTAAAGTCGACCTCAAACAATCATCAAGTCAGGT-3′) was the reverse complement of nucleotides 5519 through 5484, except that the residues corresponding to nucleotides 5512 and 5506 were modified to incorporate a SalI restriction site (underlined). The gene fragment amplified with GB46 and GB47 was subcloned into the EcoRI-SalI sites of pBD-GAL4 Cam (Stratagene) to produce pCB1645. AVR-Pita alleles from strains O-137 and TH3 were cloned by reverse transcription–polymerase chain reaction (RT-PCR) with mRNA isolated from infected leaf tissue. All subcloned fragments were sequenced to confirm structure.

Table 4.
Plasmids Used in This Study

RNA gel blot analysis with poly(A)+ mRNA was performed by probing a blot containing ~20 μg of poly(A)+ mRNA with the Pi-ta LRD sequence in pCB1645. Total RNA was prepared from 2-week-old leaves from resistant variety YT14 or those from susceptible variety YT16 by the method of Perry and Francki (1992). Poly(A)+ mRNA was prepared using a Pharmacia Poly(A)+ mRNA purification kit.

BAC Library Construction

A BAC library was prepared from the Pi-ta–containing DH line YT14 in pBeloBAC11 (Shizuya et al., 1992). Protoplasts isolated from young leaves and sheaths were used for genomic DNA isolation as previously described (Wu and Tanksley, 1993). The DNA was partially digested with HindIII in agarose plugs and fractionated on CHEF gels (Bio-Rad, Hercules, CA). DNA fragments ranging from 100 to 150 kb were eluted and subjected to a second size-selection procedure to remove small molecules. Size-selected DNA then was purified from agarose by using GELase (Epicentre Technologies, Madison, WI) and was ligated into the HindIII site of pBeloBAC11. Transformation of Escherichia coli DH10B cells was performed by electroporation with the Cell Porator Electroporation System I (Gibco BRL). BAC colonies were selected on Luria Broth (LB) plates containing 12.5 μg/mL chloramphenicol. A total of 20,160 independent colonies were picked and stored in 96-well microtiter plates containing 100 μL of LB with chloramphenicol and 10% glycerol. The average insert size was 110 kb, as determined by analysis of 28 random colonies. The library contained approximately five haploid genome equivalents of rice DNA.

The BAC library, contained in 210 microtiter plates, was gridded onto fourteen 8 × 12–cm filters in a 4 × 4 pattern by using an HDR 96-pin tool for the Beckman Biomek 1000 (Beckman Instruments, Fullerton, CA). Filters were handled as recommended by Olsen et al. (1993).

BAC Contig Assembly

The Pi-ta gene was most closely flanked by random amplified polymorphic DNA (RAPD) markers SP4B9 (5′-AGGCGTCTTC-3′) and SP9F3 (5′-AAAGGCAGTG-3′), and it cosegregated with RAPD marker SP7C3 (5′-ATGGCAGATG-3′). Chromosome walking experiments were initiated with the single-copy polymorphic sequence p7C3, which was cloned as the linked DNA fragment identified with RAPD marker SP7C3. Cloned BAC ends were used to reprobe the filters, or BAC ends were sequenced and new overlapping BACs were identified by PCR screening of pooled BAC clones. If both the right and left border fragments of a BAC contained repetitive DNA sequences, a single-copy fragment internal to the BAC insert was cloned and used to probe the library (e.g., 77D8-12 from BAC77D8). One step to the left identified one recombination border, that is, BAC clones containing the marker SP4B9, which was separated from the R gene by a recombination event in the F2 progeny line K25. Using the specific PCR primers GB24 (5′-AGGCGTCTTCAGTTTTGTAATA-3′) and GB25 (5′-AGGCGTCTTCCGGAAAGCAGCG-3′) showed that the SP4B9 sequence resided in overlapping BAC clones 31H3, 107F10, 157A9, 70F1, and 147G7. The primers had been produced by subcloning and sequencing the linked SP4B9 PCR fragment from an agarose gel. Marker SP9F3, which defined the right border, showed recombination with the R gene in one DH line, YT171.

Genomic Sequencing for Candidate Gene Identification

Minimally overlapping BAC clones (Figure 1A) were chosen for random genomic sequencing. Cesium chloride–purified DNA was sheared by nebulization. The ends of the sheared and reprecipitated DNA fragments were blunt-ended by using a fill-in reaction mediated by high-fidelity DNA polymerase (PFU; Stratagene). DNA fragments in the size range of 1000 to 2000 bp were isolated from agarose gels by using the QIAEX II Gel Extraction Kit (Qiagen), subcloned into the SmaI site of pUC18, and transformed into DH10B by electroporation. Clones containing BAC DNA inserts were sequenced in one direction by using the universal M13 reverse primer. DNA templates for sequencing were isolated with the 96-well Alkaline Lysis Miniprep kit (Advanced Genetic Technologies, Gaithersburg, MD). Sequencing reactions were performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA) with FS AmpliTaq DNA polymerase (Perkin-Elmer) and analyzed on ABI 377 sequencing gels. For each BAC, 300 to 400 clones were sequenced, providing an average twofold coverage of the region and an associated probability of 86.5% for sequencing any base (Fleischmann et al., 1995). Similarity searches were performed using the BlastX program (Gish and States, 1993). The clones were divided into three groups: those with highly significant hits (pLog > 10), those with weakly significant hits (1 [less-than-or-eq, slant] [highest pLog] [less-than-or-eq, slant] 10), and those with no hits (pLog < 1).

Genomic Pi-ta Clone

The 2-kb BAC142E8 subclone bac142e8.pk0001.f8 obtained from the genomic sequencing experiments contained 1255 bp of promoter sequence before the putative initiation codon. The insert from this clone was used to identify a 5.3-kb BamHI fragment from BAC142E8 that contained the remainder of the gene plus ~2 kb of 3′ untranslated region. This 5.3-kb BamHI fragment was subcloned from the BAC DNA into the vector pBluescript II SK+ (Stratagene) and was designated clone 7. The entire gene was assembled in pCB1641 (Figure 1B) by digesting both bac142e8.pk0001.f8 and clone 7 with EcoRI and then ligating the 5.3-kb EcoRI fragment from clone 7 to replace the small EcoRI fragment in bac142e8.pk0001.f8. The sequence from this genomic fragment is included in GenBank accession number AF207842, which spans a 2425-bp native promoter fragment through 255 bp of 3′ untranslated sequence. Subclone pCB1649 was produced by inserting a 4.8-kb HindIII-SacII subclone of Bac142E8 into the HindIII and SacII sites of pCB1641, which added an additional 3 kb of promoter region to the Pi-ta clone.

cDNA

Plasmid pCB1926, containing a full-length Pi-ta cDNA coding sequence, was obtained as follows. An incomplete cDNA was first obtained by reverse transcription–PCR. mRNA was isolated from transgenic Nipponbare line 27-4-8-1, which was shown by RNA gel blot analysis to overexpress Pi-ta behind the 35S promoter (data not shown). A 2.1-kb Pi-ta cDNA was amplified from first-strand cDNA with use of primers GB67 (5′-CCATTAAGCTTGGTTTCAAACAATC-3′) and F12-1 (5′-GTGGCTTCCATTG TTGGATC-3′), and the product was cloned into pSL1180 (Pharmacia) by using the BamHI site (partially represented in F12-1 and present in the Pi-ta fragment) and HindIII (restriction site underlined in GB67 sequence) cloning sites. A full-length synthetic cDNA coding sequence (pCB1906) was obtained by cloning a 706-bp NcoI-BamHI fragment containing the 5′ end of Pi-ta from pCB1649 upstream of the 2090-bp BamHI-HindIII partial cDNA. A native Pi-ta promoter fragment (2425 bp) was added by cloning a 3173-bp EcoRI fragment from pCB1649 into the EcoRI sites of pCB1906, resulting in pCB1926. The final Pi-ta cDNA construct was verified by sequencing.

Stable Transformation of Rice

Stable transformation of rice was performed using the bacterial hygromycin B phosphotransferase (HptII) gene from Streptomyces hygroscopicus as the selectable marker. Embryogenic calli were initiated from scutellar tissue of mature seed of the japonica cultivar Nipponbare. The protocol of Li et al. (1993) was used with the following modifications. Month-old embryogenic callus was bombarded with the Bio-Rad 1000/He Biolistic gun at 1100 p.s.i. as described in the Bio-Rad manual. Three milligrams of gold microcarriers, 0.6 μm in diameter, were coated with 2 μg of each plasmid DNA and used for six shots. Each plate of callus was bombarded two times at a distance of 8 cm beneath the stopping plate. After bombardment, plates were placed in the dark at 28°C. After 3 days, callus was transferred to fresh medium containing 50 mg/L hygromycin B. After 4 weeks of selection, surviving calli were transferred to regeneration medium (containing 1% sucrose, 3% sorbitol, 2 mg/L zeatin, and 1 mg/L indoleacetic acid) and incubated at 25°C with a 16-hr photoperiod for 2 to 4 weeks until plantlets emerged. Plantlets were cultured for another 2 weeks on 0.5 × Murashige and Skoog (1962) medium containing 1% sucrose before being transferred to soil.

The genomic clone, plasmid pCB1641 (Figure 1B), or the cDNA clone, plasmid pCB1926, was cotransformed along with plasmid pML18 containing the cauliflower mosaic virus (CaMV) 35S::HptII gene. DNA gel blot analysis and allele-specific PCR with primers GB58 (5′GTCAGGTTGAAGATGCATAGC-3′) and GB60 (5′-CAATGCCGAGTGTGCAAAGG-3′) were used to confirm the presence of the Pi-ta transgene in HygR plants.

Transient Expression Assay

For the transient expression assay, intact 1-week-old rice seedlings are grown on agarose medium and ballistically transformed with plasmid pML63 containing a β-glucuronidase (GUS) reporter gene (Jia et al., 2000). Rice varieties used included Nipponbare (pi-ta), YT16 (pi-ta), Yashiro-mochi (Pi-ta), and YT14 (Pi-ta). For expression of AVR-Pita176 in plants, the 35S promoter with the Adh1 intron was fused to a truncated AVR-Pita cDNA encoding AVR-Pita176 (pCB1947). This plasmid also contained 3′ nos terminator sequences. As a control, an equivalent construct was produced that encoded an avr-pita176 protein in which a single amino acid substitution (H180K) had been introduced by in vitro mutagenesis (pCB2011). To test in vivo specificity, we produced an avr-pita176 expression construct from a naturally occurring avr-pita allele from virulent pathogen strain TH3 (pCB2148).

Acknowledgments

We acknowledge Forrest Chumley (DuPont) for his role in initiating this project and for continuing valuable discussions. We are indebted to Zaida Lentini, Cesar Martinez, and Joe Tohme (Centro Internacional de Agricultura Tropical, Colombia) for their critical contribution of producing the doubled haploid mapping population. We are grateful to Phil Smith, Kathy Kline-Smith, Nancy Glisson, and Paul Owen for their excellent assistance in caring for the transgenic rice plants used in this study. We are indebted to our DuPont colleagues Maureen Dolan, Mike Hanafey, Scott Tingey, and members of the Delaware Technology Park Sequencing Group for genomic sequencing of our BAC contig; and we thank Antoni Rafalski for valuable discussions.

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